Finger ring for delivery of secretory treatment modalities

文档序号：1821191 发布日期：2021-11-09 浏览：20次中文

阅读说明：本技术 用于递送分泌型治疗方式的指环体 (Finger ring for delivery of secretory treatment modalities ) 是由 E.G.韦因斯坦 A.卡韦吉安 S.德拉格雷夫 N.L.尤兹维亚克 K.J.莱博 F.M. 于 2019-12-12 设计创作，主要内容包括：本发明总体上涉及指环体及其组合物和用途。(The present invention relates generally to finger rings and compositions and uses thereof.)

1. A synthetic finger ring comprising:

(I) a genetic element comprising:

(a) a promoter element which is capable of expressing a promoter sequence,

(b) a nucleic acid sequence encoding an exogenous effector, wherein the nucleic acid sequence is operably linked to the promoter element, and wherein the exogenous effector is a growth hormone (e.g., hGH), and

(c) a 5' UTR domain comprising the nucleic acid sequence of nucleotide 323-393 of SEQ ID NO:54 or a nucleic acid sequence at least 90% identical thereto;

(II) an outer portion of a protein comprising a polypeptide encoded by an ORF1 nucleic acid from a dactylovirus, e.g., the ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the synthetic finger loop body is capable of delivering the genetic element into a human cell.

2. The synthetic finger ring body of claim 1, wherein the ORF1 molecule comprises the amino acid sequence of SEQ ID NO 217 or an amino acid sequence having at least 90% identity thereto.

3. The synthetic finger ring body of any one of the preceding claims, wherein the ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO 54.

4. The synthetic finger ring of any one of the preceding claims, wherein the genetic element comprises the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID No. 54, or a nucleic acid sequence having at least 85% sequence identity thereto.

5. The synthetic finger ring body of any one of the preceding claims, wherein the ORF1 molecule comprises an amino acid sequence comprising one or more of: an amino acid sequence of an arginine-rich region, a jelly roll domain, a hypervariable domain, an N22 domain, and/or a C-terminal domain as set forth in table 16, or an amino acid sequence having at least 85% identity thereto.

6. The synthetic finger ring body of any one of the preceding claims, wherein the ORF1 molecule comprises the amino acid sequence of SEQ ID NO:58, or a nucleic acid sequence having at least 85% sequence identity thereto.

7. The synthetic finger ring of any one of the preceding claims, further comprising a polypeptide comprising the amino acid sequence of ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2, or an amino acid sequence at least 85% identical thereto, as set forth in table 16.

8. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element encodes the amino acid sequence of, or an amino acid sequence having at least 85% identity to, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16.

9. The synthetic finger ring body of any one of the preceding claims, wherein the synthetic finger ring body does not include a polypeptide comprising the amino acid sequence of ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2, or an amino acid sequence at least 85% identical thereto, as set forth in table 16.

10. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element does not encode an amino acid sequence of, or has at least 85% identity with, an amino acid sequence of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16.

11. The synthetic finger ring body of any one of the preceding claims, wherein the ORF1 molecule comprises the amino acid sequence YNPX²DXGX²N (SEQ ID NO:829), wherein XⁿEach independently is a contiguous sequence of any n amino acids.

12. The synthetic finger ring body of claim 11, wherein the ORF1 molecule further comprises a nucleotide sequence located in the amino acid sequence YNPX²DXGX²N (SEQ ID NO:829) flanking a first beta-strand and a second beta-strand, e.g., wherein the first beta-strand comprises the amino acid sequence YNPX²DXGX²N (SEQ ID NO:829) and/or wherein the second beta-chain comprises the amino acid sequence YNPX²DXGX²The second asparagine (N) residue (from N to C) of N (SEQ ID NO: 829).

13. The synthetic finger ring body of any one of the preceding claims, wherein the ORF1 molecule comprises, in order from N-terminus to C-terminus, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and a ninth beta strand.

14. The synthetic finger ring body of any of the preceding claims, wherein the genetic element is capable of being amplified in a host cell by rolling circle replication, e.g., producing at least 8 copies.

15. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element is single-stranded.

16. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element is circular.

17. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element is DNA.

18. The synthetic finger ring body of any of the preceding claims, wherein the genetic element is minus-strand DNA.

19. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element is integrated at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of the finger ring body entering the cell, e.g., wherein the synthetic finger ring body is non-integrated.

20. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element comprises a sequence of a consensus 5' UTR nucleic acid sequence set forth in table 16-1.

21. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element comprises the sequence of a consensus GC-rich region shown in table 16-2.

22. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element comprises a sequence of at least 100 nucleotides in length, at least 70% (e.g., about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90%) of the sequence consisting of a G or a C at a position.

23. The synthetic finger ring body of any one of the preceding claims, wherein the genetic element comprises the nucleic acid sequence of SEQ ID No. 120.

24. The synthetic finger ring body of any one of the preceding claims, wherein the promoter element is exogenous to a wild-type finger ring virus.

25. The synthetic finger ring body of any one of the preceding claims, wherein the promoter element is endogenous to a wild-type finger ring virus.

26. The synthetic finger loop body of any one of the preceding claims, wherein the genetic element has a length of about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0 kb.

27. The synthetic ring body of any of the preceding claims, wherein the synthetic ring body is capable of infecting human cells, such as blood cells, skin cells, muscle cells, nerve cells, adipocytes, endothelial cells, immune cells, hepatocytes, lung epithelial cells, e.g., in vitro.

28. The synthetic finger ring of any one of the preceding claims, which is substantially non-immunogenic, e.g., does not induce a detectable and/or unwanted immune response, e.g., detected according to the method described in example 4.

29. The synthetic finger ring body of claim 28, wherein the substantially non-immunogenic finger ring body has a potency in a subject that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the potency in a reference subject that lacks an immune response.

30. The synthetic finger ring of any one of the preceding claims, wherein a population having at least 1000 of the finger rings is capable of delivering at least about 100 copies (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies) of the genetic element into one or more human cells.

31. A synthetic finger ring comprising:

(I) a genetic element comprising:

(a) a promoter element which is capable of expressing a promoter sequence,

(b) a nucleic acid sequence encoding an exogenous effector, wherein the nucleic acid sequence is operably linked to the promoter element,

wherein the exogenous effector is a secreted therapeutic agent selected from the group consisting of:

(i) an antibody molecule that binds to a growth factor or a growth factor receptor, or a cytokine receptor;

(ii) an enzyme or a functional variant thereof;

(iii) a hormone or a functional variant thereof;

(iv) a cytokine or functional variant thereof);

(v) a complement inhibitor;

(vi) the growth factors are selected from the group consisting of,

(vi) an inhibitor of a growth factor, which is,

(vii) a blood coagulation factor, or

(viii) Modulators of STING/cGAS signaling;

(c) a 5' UTR domain comprising the nucleic acid sequence of nucleotide 323-393 of SEQ ID NO:54 or a nucleic acid sequence at least 85% identical thereto;

(II) an outer protein portion comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., an ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the synthetic finger loop body is capable of delivering the genetic element into a human cell.

32. A pharmaceutical composition comprising the synthetic finger ring of any one of the preceding claims, and a pharmaceutically acceptable carrier or excipient.

33. The pharmaceutical composition of claim 32, comprising at least 10³、10⁴、105、10⁶、10⁷、10⁸Or 10⁹A synthetic ring finger.

34. The pharmaceutical composition of claim 32 or 33, wherein the pharmaceutical composition has a predetermined ratio of particles to infectious units (e.g., <300:1, <200:1, <100:1, or <50: 1).

35. A reaction mixture, comprising:

(i) a first nucleic acid (e.g., double-stranded or single-stranded circular DNA) comprising the sequence of the genetic element of the synthetic finger loop of any one of the preceding claims, and

(ii) a second nucleic acid sequence encoding one or more amino acid sequences selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16, or an amino acid sequence having at least 85% sequence identity thereto.

36. The reaction mixture of claim 35, wherein the first nucleic acid and the second nucleic acid are in the same nucleic acid molecule.

37. The reaction mixture of claim 35, wherein the first nucleic acid and the second nucleic acid are different nucleic acid molecules.

38. The reaction mixture of claim 35, wherein the first nucleic acid and the second nucleic acid are different nucleic acid molecules and wherein the second nucleic acid is provided as a double-stranded circular DNA.

39. The reaction mixture of claim 35, wherein the first nucleic acid and the second nucleic acid are different nucleic acid molecules and wherein the first nucleic acid and the second nucleic acid are provided as double-stranded circular DNA.

40. The reaction mixture of claim 37, wherein the second nucleic acid sequence is contained in a helper cell or helper virus.

41. A method of making a synthetic finger ring, the method comprising:

a) providing a host cell comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence of the genetic element of the synthetic finger loop of any one of the preceding claims, and

(ii) a second nucleic acid molecule encoding one or more amino acid sequences selected from, for example, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in any one of table 16, or an amino acid sequence having at least 85% sequence identity thereto; and is

b) Incubating the host cell under conditions suitable for the preparation of a synthetic finger ring body;

thereby preparing the synthetic finger ring.

42. The method of claim 41, further comprising introducing the first nucleic acid molecule and/or the second nucleic acid molecule into the cell prior to step (a).

43. The method of claim 42, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

44. The method of any one of claims 41 or 42, wherein the second nucleic acid molecule is integrated into the genome of the host cell.

45. The method of any one of claims 41-44, wherein the second nucleic acid molecule is a helper (e.g., a helper plasmid or the genome of a helper virus).

46. The method of any one of claims 41-44, wherein the second nucleic acid molecule encodes a polypeptide comprising the amino acid sequence [ W/F ™]X⁷HX³CX¹CX⁵ORF2 molecule of H (SEQ ID NO:949), wherein XⁿIs a contiguous sequence of any n amino acids.

47. A method of manufacturing a synthetic finger ring formulation, the method comprising:

a) providing a plurality of the synthetic finger ring body of claims 1-31, the pharmaceutical composition of any one of claims 32-34, or the reaction mixture of any one of claims 35-40;

b) optionally evaluating the plurality of synthetic finger rings of claims 1-31 for one or more of: contaminants, optical density measurements (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio) as described herein; and is

c) For example, if one or more parameters of (b) meet a specified threshold, then the plurality of synthetic ring bodies are formulated, e.g., as a pharmaceutical composition suitable for administration to a subject.

48. A host cell, comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence of the genetic element of the synthetic finger loop of any one of the preceding claims, and

(ii) optionally, a second nucleic acid molecule encoding one or more amino acid sequences selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in any one of table 16, or an amino acid sequence having at least 85% sequence identity thereto.

49. A method of delivering an exogenous effector (e.g., a therapeutic exogenous effector) to a mammalian cell, the method comprising:

(a) providing a synthetic ring body of any one of the preceding claims; and

(b) contacting a mammalian cell with the synthetic finger ring;

wherein the synthetic finger ring body is capable of delivering the genetic element into the mammalian cell; and

optionally, wherein the synthetic finger ring is produced by introducing the genetic element into a host cell under conditions suitable to enclose the genetic element within the protein exterior in the host cell;

Thereby delivering the therapeutic exogenous effector to the mammalian cell.

50. Use of the synthetic finger ring body of any one of claims 1-31 or the pharmaceutical composition of any one of claims 32-34 for delivering the genetic element to a host cell.

51. Use of the synthetic finger ring body of any one of claims 1-31 or the pharmaceutical composition of any one of claims 32-34 for treating a disease or disorder in a subject.

52. The synthetic finger ring body of any one of claims 1-31 or the pharmaceutical composition of any one of claims 32-34 for use in treating a disease or disorder in a subject.

53. A method of treating a disease or disorder in a subject, the method comprising administering to the subject the synthetic finger ring body of any one of claims 1-31 or the pharmaceutical composition of any one of claims 32-34.

54. Use of the synthetic finger ring body of any one of claims 1-31 or the pharmaceutical composition of any one of claims 32-34 in the manufacture of a medicament for treating a disease or disorder in a subject.

Background

There is a continuing need to develop suitable vectors to deliver therapeutic genetic material to patients.

Disclosure of Invention

The present disclosure provides a finger ring body (anellosome), e.g., a synthetic finger ring body, that can be used as a delivery vehicle, e.g., for delivering genetic material to eukaryotic cells (e.g., human cells or human tissue), for delivering an effector (e.g., a payload), or for delivering a therapeutic agent or therapeutic effector (e.g., a secreted protein). Exemplary secreted therapeutic agents that can be delivered using the finger ring body include, for example, antibody molecules, enzymes, hormones, cytokine molecules, complement inhibitors, growth factors, or growth factor inhibitors.

In some embodiments, a ring (e.g., a particle, e.g., a viral particle, e.g., a ring virus (anoviridus) particle) comprises a genetic element (e.g., a genetic element comprising a therapeutic DNA sequence) encapsulated outside of a protein (e.g., outside of a protein comprising a ring virus capsid protein, e.g., ring virus ORF1 protein or a polypeptide encoded by a ring virus ORF1 nucleic acid, e.g., as described herein) that is capable of introducing the genetic element into a cell (e.g., a mammalian cell, e.g., a human cell). In some embodiments, the ring is a particle comprising an outer portion of a protein comprising a polypeptide encoded by: an ORF1 nucleic acid from a finger virus (e.g., an ORF1 nucleic acid from a type a, b, or c ringvirus, e.g., ORF1 from a type a ringvirus clade 1, a type a ringvirus clade 2, a type a ringvirus clade 3, a type a ringvirus clade 4, a type a ringvirus clade 5, a type a ringvirus clade 6, or a type a ringvirus clade 7, e.g., as described herein). The genetic elements of the finger loops of the present disclosure can be circular and/or single-stranded DNA molecules (e.g., circular and single-stranded), and typically include a protein-binding sequence enclosed by the protein exterior or a polypeptide attached thereto that can facilitate the closure of the genetic element within the protein exterior and/or enrichment of the genetic element within the protein exterior relative to other nucleic acids. In some cases, the genetic element is circular or linear. In some cases, the genetic element comprises or encodes an effector (e.g., a nucleic acid effector, such as a non-coding RNA, or a polypeptide effector, such as a protein), for example, which can be expressed in a cell. In some embodiments, the effector is a therapeutic agent or therapeutic effector (e.g., a secreted therapeutic agent, e.g., a secreted polypeptide), e.g., as described herein. In some cases, the effector is an endogenous effector or an exogenous effector, e.g., for a wild-type dactylvirus or target cell. In some embodiments, the effector is exogenous to the wild-type dactylvirus or the target cell. In some embodiments, the ring body can deliver the effector into the cell by contacting the cell and introducing a genetic element encoding an external effector into the cell, such that the effector is produced or expressed by the cell. In some cases, the effector is an endogenous effector (e.g., endogenous to the target cell, but provided in an increased amount, e.g., by the finger ring body). In other cases, the effector is an exogenous effector. In some cases, the exogenous effector can modulate a function of the cell or modulate the activity or level of a target molecule in the cell. For example, an effector can reduce the level of a target protein in a cell (e.g., as described in examples 3 and 4). In another example, the ring can deliver and express an effector, such as a foreign protein, in vivo (e.g., as described in examples 19 and 28). The ring body can be used, for example, to deliver genetic material to a target cell, tissue, or subject; delivering an effector to a target cell, tissue, or subject; or for the treatment of diseases and disorders, for example by delivering effectors that may function as therapeutic agents to a desired cell, tissue, or subject.

The invention further provides synthetic finger ring bodies. The synthetic finger ring virus has at least one structural difference compared to a wild-type virus (e.g., a wild-type finger ring virus, e.g., as described herein), e.g., a deletion, insertion, substitution, modification (e.g., enzymatic modification) relative to the wild-type virus. Typically, a synthetic ring body includes an exogenous genetic element enclosed within an outer portion of a protein that can be used to deliver the genetic element, or an effector (e.g., an exogenous effector or an endogenous effector) (e.g., a polypeptide or nucleic acid effector) encoded therein, into a eukaryotic cell (e.g., a human cell). In embodiments, the ring does not elicit a detectable and/or unwanted immune or inflammatory response, e.g., does not elicit an increase in one or more molecular markers of inflammation (e.g., TNF-a, IL-6, IL-12, IFN) by more than 1%, 5%, 10%, 15%, and does not elicit a B cell response, e.g., a reactive or neutralizing antibody, e.g., the ring may be substantially non-immunogenic to a target cell, tissue, or subject.

In one aspect, the invention features a finger ring including: (i) a proteinaceous outer portion; (b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) and a protein-binding sequence (e.g., an external protein-binding sequence) encoding an exogenous effector; wherein the exogenous effector comprises a secreted polypeptide selected from the group consisting of: an antibody molecule, enzyme, hormone, cytokine molecule, complement inhibitor, growth factor or growth factor inhibitor, or a functional variant of any of the foregoing; wherein the genetic element is enclosed within the protein exterior; and wherein the ring is configured to deliver the genetic element into a eukaryotic cell. Optionally, the genetic element comprises at least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, modification (e.g., enzymatic modification), and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain or portion thereof (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region).

In one aspect, the invention features a finger ring including: (i) genetic elements comprising a promoter element and sequences encoding an effector (e.g., an endogenous or exogenous effector) and a protein binding sequence (e.g., an external protein binding sequence, such as a packaging signal); and (ii) a proteinaceous outer portion; wherein the genetic element is enclosed within the outer portion (e.g., capsid) of the protein; and wherein the finger ring body is capable of delivering the genetic element into a eukaryotic (e.g., mammalian, e.g., human) cell. In some embodiments, the genetic element is single-stranded and/or circular DNA. Alternatively or in combination, the genetic element has one, two, three or all of the following properties: is circular, is single-stranded, integrates into the genome of the cell at less than about 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the frequency of the genetic element entering the cell, and/or integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies/genome. In some embodiments, the integration frequency is determined as described in Wang et al (2004, Gene Therapy [ 11: 711-) -721, incorporated herein by reference in its entirety). In some embodiments, the genetic element is encapsulated within the exterior of the protein. In some embodiments, the finger ring is capable of delivering a genetic element into a eukaryotic cell. In some embodiments, the genetic element comprises a nucleic acid sequence (e.g., a 300-nucleotide 4000 nucleic acid sequence, e.g., 300-nucleotide, 300-3000 nucleic acid, 300-nucleotide, 2500-nucleotide, 300-nucleotide, 2000-nucleotide, 300-nucleotide, 3500-nucleotide, 300-nucleotide) having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99, or 100%) sequence identity to a wild-type dacryovirus sequence (e.g., a wild-Type Torque (TTV), a parvovirus (TTMV), or a TTMDV sequence, e.g., a wild-type dacryovirus sequence listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17). In some embodiments, the genetic element comprises a nucleic acid sequence (e.g., a nucleic acid sequence of at least 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides or more) having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 or 100%) sequence identity to a wild-type ring virus sequence (e.g., a wild-type ring virus sequence as described herein, e.g., listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15 or 17). In some embodiments, the nucleic acid sequence is codon optimized, e.g., for expression in a mammalian (e.g., human) cell. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the nucleic acid sequence are codon optimized, e.g., for expression in a mammalian (e.g., human) cell.

In one aspect, the invention features an infectious (to human cells) particle comprising a dactylovirus capsid (e.g., a capsid comprising a dactylovirus ORF, e.g., ORF1, polypeptide) that encapsulates a genetic element comprising a capsid-associated protein binding sequence and a heterologous (to the dactylovirus) sequence encoding a therapeutic effector. In embodiments, the particle is capable of delivering a genetic element into a cell of a mammal, such as a human. In some embodiments, the genetic element is less than about 6% (e.g., less than 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or less) identical to a wild-type finger ring virus. In some embodiments, the genetic element is no more than 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6% identical to a wild-type finger ring virus. In some embodiments, the genetic element is at least about 2% to at least about 5.5% (e.g., 2 to 5%, 3% to 5%, 4% to 5%) identical to the wild-type finger ring virus. In some embodiments, the genetic element has a non-viral sequence (e.g., a non-ring virus genomic sequence) of greater than about 2000, 3000, 4000, 4500, or 5000 nucleotides. In some embodiments, the genetic element has a non-viral sequence (e.g., a non-ring virus genomic sequence) greater than about 2000 to 5000, 2500 to 4500, 3000 to 4500, 2500 to 4500, 3500, or 4000, 4500 (e.g., about 3000 to 4500) nucleotides. In some embodiments, the genetic element is single-stranded, circular DNA. Alternatively or in combination, the genetic element has one, two or 3 of the following properties: is circular, is single-stranded, integrates into the genome of the cell at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell, integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies/genome or integrates at a frequency of less than about 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell. In some embodiments, the integration frequency is determined as described in Wang et al (2004, Gene Therapy [ 11: 711-) -721, incorporated herein by reference in its entirety).

Also described herein are finger-ring virus-based viral vectors and viral particles that can be used to deliver an agent (e.g., an exogenous effector or an endogenous effector, e.g., a therapeutic effector) to a cell (e.g., a cell in a subject to be treated). In some embodiments, the ring virus can be used as an effective delivery vehicle to introduce an agent (e.g., an effector described herein) into a target cell, e.g., a target cell in a subject for therapeutic or prophylactic treatment.

In one aspect, the invention features a polypeptide (e.g., a synthetic polypeptide, such as an ORF1 molecule) comprising (e.g., in tandem):

(i) a first region comprising an arginine-rich region, an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence described herein, or a sequence of at least about 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof),

(ii) a second region comprising a jelly-roll (jelly-roll) domain, e.g., an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a jelly-roll region sequence described herein, or a sequence comprising at least 6 beta strands,

(iii) A third region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an N22 domain sequence described herein,

(iv) a fourth region comprising an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a C-terminal domain (CTD) sequence of ORF1 of an dactylovirus as described herein, and

(v) optionally, wherein the polypeptide has an amino acid sequence with less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type finger ring virus ORF1 protein described herein.

In some embodiments, the polypeptide comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99, or 100% sequence identity to an ring virus ORF1 molecule described herein (e.g., as set forth in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10). In some embodiments, the polypeptide comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a subsequence (e.g., an arginine (Arg) -rich domain, a jellyroll domain, High Variable Region (HVR), N22 domain, or C-terminal domain (CTD)) of an dactylovirus ORF1 molecule described herein (e.g., as listed in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10). In one embodiment, the amino acid sequences of regions (i), (ii), (iii) and (iv) have at least 90% sequence identity to their respective references, and wherein the polypeptide has an amino acid sequence with less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to the wild-type finger ring virus ORF1 protein described herein.

In one aspect, the invention features a complex comprising a polypeptide as described herein (e.g., a finger ring virus ORF1 molecule as described herein) and a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and a protein binding sequence.

The present disclosure also provides nucleic acid molecules (e.g., nucleic acid molecules comprising genetic elements as described herein, or nucleic acid molecules comprising sequences encoding protein external proteins as described herein). The nucleic acid molecules of the invention may comprise one or both of (a) a genetic element as described herein and (b) a nucleic acid sequence encoding a protein external protein as described herein.

In one aspect, the invention features an isolated nucleic acid molecule comprising a genetic element comprising a promoter element and an external protein binding sequence operably linked to a sequence encoding an effector, e.g., a payload. In some embodiments, the external protein binding sequence comprises a sequence that is at least 75% (80%, 85%, 90%, 95%, 97%, 100%) identical to a 5' UTR sequence of a finger ring virus disclosed herein. In embodiments, the genetic element is a single-stranded DNA, is circular, integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell, and/or integrates at a frequency of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome into the genome of the target cell or integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell. In some embodiments, the integration frequency is determined as described in Wang et al (2004, Gene Therapy [ 11: 711-) -721, incorporated herein by reference in its entirety). In embodiments, the effector is not derived from TTV, and is not SV 40-miR-S1. In an embodiment, the nucleic acid molecule does not comprise the polynucleotide sequence of TTMV-LY 2. In embodiments, the promoter element is capable of directing expression of the effector in a eukaryotic (e.g., mammalian, e.g., human) cell.

In some embodiments, the nucleic acid molecule is circular. In some embodiments, the nucleic acid molecule is linear. In some embodiments, a nucleic acid molecule described herein comprises one or more modified nucleotides (e.g., base modifications, sugar modifications, or backbone modifications).

In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF1 molecule (e.g., an ORF1 protein of a finger virus, e.g., as described herein). In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF2 molecule (e.g., an ORF2 protein of a finger virus, e.g., as described herein). In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF3 molecule (e.g., an ORF3 protein of a finger virus, e.g., as described herein). In one aspect, the invention features a genetic element that includes one, two, or three of: (i) promoter elements and effector-encoding sequences, such as exogenous or endogenous effectors; (ii) at least 72 contiguous nucleotides (e.g., at least 72, 73, 74, 75, 76, 77, 78, 79%, 80%, 90, 91, 92, 93%, 94, 95, 96%, 97%, 98, 99%, or 100%) having at least 75% (e.g., at least 75%, 76%, 78%, 79, 80, 90, 100, or 150 nucleotides) sequence identity to a wild-type finger ring virus sequence; or at least 100 (e.g., at least 300, 500, 1000, 1500) contiguous nucleotides having at least 72% (e.g., at least 72, 73, 74, 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a wild-type finger ring virus sequence; and (iii) a protein binding sequence, such as an external protein binding sequence, wherein the nucleic acid construct is a single stranded DNA; and wherein the nucleic acid construct is circular, integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell, and/or integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies/genome. In some embodiments, a genetic element encoding an effector (e.g., an exogenous or endogenous effector, e.g., as described herein) is codon optimized. In some embodiments, the genetic element is circular. In some embodiments, the genetic element is linear. In some embodiments, the genetic element comprises a ring vector, e.g., as described herein. In some embodiments, a genetic element described herein comprises one or more modified nucleotides (e.g., base modifications, sugar modifications, or backbone modifications). In some embodiments, the genetic element comprises a sequence encoding an ORF1 molecule (e.g., an ORF1 protein of a finger virus, e.g., as described herein). In some embodiments, the genetic element comprises a sequence encoding an ORF2 molecule (e.g., an ORF2 protein of a finger virus, e.g., as described herein). In some embodiments, the genetic element comprises a sequence encoding an ORF3 molecule (e.g., an ORF3 protein of a finger virus, e.g., as described herein).

In one aspect, the invention features a host cell or helper cell comprising: (a) a nucleic acid comprising a sequence encoding one or more of an ORF1 molecule, an ORF2 molecule, or an ORF3 molecule (e.g., a sequence encoding a ring virus ORF1 polypeptide described herein), wherein the nucleic acid is a plasmid, is a viral nucleic acid, or is integrated into a helper cell chromosome; and (b) a genetic element, wherein the genetic element comprises (i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and (ii) a protein binding sequence that binds to the polypeptide of (a), wherein optionally the genetic element does not encode an ORF1 polypeptide (e.g., an ORF1 protein). For example, a host cell or helper cell comprises (a) and (b) in cis (two parts of the same nucleic acid molecule) or in trans (each part of a different nucleic acid molecule). In embodiments, the genetic element of (b) is a circular single-stranded DNA. In some embodiments, the host cell is a producer cell line. In some embodiments, the host cell or helper cell is adherent or suspended, or both. In some embodiments, the host cell or helper cell is grown in a microcarrier. In some embodiments, the host cell or helper cell is compatible with cGMP production practices. In some embodiments, the host cell or helper cell is grown in a medium suitable to promote cell growth. In certain embodiments, once the host cells or helper cells have sufficiently grown (e.g., to achieve a suitable cell density), the culture medium can be changed to a medium suitable for the host cell or helper cell to produce the finger loop.

In one aspect, the invention features a pharmaceutical composition that includes a ring body described herein (e.g., a synthetic ring body). In embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In an embodiment, the pharmaceutical composition comprises a unit dose comprising about 10⁵-10¹⁴Genome equivalent finger ring/kg target subject. In some embodiments, the pharmaceutical composition comprising the formulation is stable over an acceptable time and temperature range, and/or is compatible with the desired route of administration and/or any device (e.g., needle or syringe) required for that route of administration. In some embodiments, the pharmaceutical composition is formulated for administration as a single dose or multiple doses. In some embodiments, the pharmaceutical composition is formulated at the site of administration, e.g., by a healthcare professional. In some embodiments, the pharmaceutical composition comprises a desired concentration of the ring genome or genome equivalent (e.g., as defined by the number of genomes/volume).

In one aspect, the invention features a method of treating a disease or disorder in a subject, the method including administering to the subject a finger ring, e.g., a synthetic finger ring, e.g., as described herein.

In one aspect, the invention features a method of delivering an effector or payload (e.g., an endogenous or exogenous effector) to a cell, tissue, or subject, the method comprising administering to the subject a finger ring, e.g., a synthetic finger ring, e.g., as described herein, wherein the finger ring comprises a nucleic acid sequence encoding an effector. In embodiments, the payload is a nucleic acid. In embodiments, the payload is a polypeptide.

In one aspect, the invention features a method of delivering a finger ring to a cell, the method including contacting, e.g., in vivo or ex vivo, a finger ring, e.g., a synthetic finger ring (e.g., as described herein), with a cell (e.g., a eukaryotic cell, e.g., a mammalian cell).

In one aspect, the invention features a method of making a finger ring body, e.g., a synthetic finger ring body. The method comprises the following steps:

a) providing a host cell comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of a finger loop, e.g., a synthetic finger loop, as described herein, and

(ii) the first nucleic acid or a second nucleic acid molecule encoding one or more of: an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 (e.g., as set forth in any one of table 16), or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto; and

b) Incubating the host cell under conditions suitable for preparation of the finger ring body.

In some embodiments, the method further comprises, prior to step (a), introducing the first nucleic acid molecule and/or the second nucleic acid molecule into the host cell. In some embodiments, the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. In other embodiments, the second nucleic acid molecule is integrated into the genome of the host cell. In some embodiments, the second nucleic acid molecule is a helper (e.g., a helper plasmid or the genome of a helper virus). In another aspect, the invention features a method of making a finger ring body composition, the method including:

a) a host cell is provided that comprises, e.g., expresses, one or more components (e.g., all components) of a finger ring (e.g., a synthetic finger ring) such as described herein. For example, the host cell comprises (a) a nucleic acid comprising a sequence encoding an ORF1 polypeptide of a finger virus described herein, wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into a helper cell chromosome; (b) a genetic element, wherein the genetic element comprises (i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and (i) a protein binding sequence (e.g., a packaging sequence) that binds to a polypeptide of (a), wherein the host cell or helper cell comprises (a) and (b) in cis or in trans. In embodiments, the genetic element of (b) is a circular single-stranded DNA. In some embodiments, the host cell is a production cell line;

b) Culturing the host cell under conditions suitable for producing a finger ring formulation from the host cell, wherein the finger ring of the formulation comprises a proteinaceous outer portion (e.g., comprising an ORF1 molecule) that encapsulates the genetic element (e.g., as described herein), thereby producing the finger ring formulation; and

optionally, c) formulating the formulation of the finger ring body, e.g., as a pharmaceutical composition suitable for administration to a subject.

In some embodiments, the components of the finger loop are introduced into the host cell at the time of production (e.g., by transient transfection). In some embodiments, the components of the finger loop are stably expressed by the host cell (e.g., wherein one or more nucleic acids encoding the components of the finger loop are introduced into the host cell or a progenitor thereof, e.g., by stable transfection).

In some embodiments, the method further comprises one or more purification steps (e.g., purification by sedimentation, chromatography, and/or ultrafiltration). In some embodiments, the purification step comprises removing one or more of serum, host cell DNA, host cell proteins, particles lacking genetic elements, and/or phenol red from the preparation. In some embodiments, the resulting formulation or pharmaceutical composition comprising the formulation is stable over an acceptable time and temperature range, and/or is compatible with the desired route of administration and/or any device (e.g., needle or syringe) required for that route of administration.

In one aspect, the invention features a method of making a finger ring body composition, the method including: a) providing a plurality of finger ring bodies described herein, or a formulation of finger ring bodies described herein; b) the finger ring bodies or formulations thereof are formulated, for example, as a pharmaceutical composition suitable for administration to a subject.

In one aspect, the invention features a method of making a host cell, e.g., a first host cell or a producer cell (e.g., as shown in fig. 12), e.g., a population of first host cells, comprising introducing a genetic element, e.g., as described herein, into a host cell and culturing the host cell under conditions suitable for production of a finger ring. In embodiments, the method further comprises introducing an adjuvant, such as a helper virus, into the host cell. In embodiments, the introducing comprises transfecting (e.g., chemically transfecting) or electroporating the host cell with the finger ring body.

In one aspect, the invention features a method of making a finger ring body, including providing a host cell, such as a first host cell or a producer cell (e.g., as shown in fig. 12), comprising a finger ring body, such as described herein, and purifying the finger ring body from the host cell. In some embodiments, the method further comprises, prior to the providing step, contacting the host cell with a finger ring body, e.g., as described herein, and incubating the host cell under conditions suitable for production of the finger ring body. In embodiments, the host cell is the first host cell or producer cell described in the methods of making host cells described above. In an embodiment, purifying the finger ring from the host cell comprises lysing the host cell.

In some embodiments, the method further comprises a second step of contacting the finger ring body produced by the first host cell or the producer cell with a second host cell, e.g., a permissive cell (e.g., as shown in fig. 12), e.g., a second population of host cells. In some embodiments, the method further comprises incubating the second host cell under conditions suitable for production of the finger ring body. In some embodiments, the method further comprises purifying the finger ring body from the second host cell, e.g., to produce a population of finger ring body seeds. In embodiments, the second host cell population produces at least about 2-100 times more ring bodies than the first host cell population. In an embodiment, purifying the finger ring from the second host cell comprises lysing the second host cell. In some embodiments, the method further comprises a second step of contacting the finger ring produced by the second host cell with a third host cell, e.g., a permissive cell (e.g., as shown in fig. 12), e.g., a third population of host cells. In some embodiments, the method further comprises incubating the third host cell under conditions suitable for production of the finger ring body. In some embodiments, the method further comprises purifying the finger ring from a third host cell, e.g., to produce a stock population of finger rings. In an embodiment, purifying the finger ring from the third host cell comprises lysing the third host cell. In embodiments, the third population of host cells produces at least about 2-100 times more finger rings than the second population of host cells.

In some embodiments, the host cell is grown in a medium suitable to promote cell growth. In certain embodiments, once the host cell has been sufficiently grown (e.g., to a suitable cell density), the medium can be changed to a medium suitable for production of the finger loop by the host cell. In some embodiments, the finger ring produced by the host cell is isolated from the host cell (e.g., by lysing the host cell) prior to contact with the second host cell. In some embodiments, the finger ring bodies produced by the host cell are contacted with a second host cell without an intervening purification step.

In one aspect, the invention features a method of making a pharmaceutical finger ring formulation. The method comprises (a) preparing a ring formulation as described herein, (b) evaluating the formulation (e.g., a pharmaceutical ring formulation, a population of ring seeds, or a population of ring stock) for one or more of the following pharmaceutical quality control parameters: identity, purity, potency (e.g., in genomic equivalents per finger ring particle) and/or nucleic acid sequence, e.g., from a genetic element comprised by a finger ring, and (c) formulation formulated for evaluated pharmaceutical use meets predetermined criteria, e.g., meets pharmaceutical specifications.

In some embodiments, assessing identity comprises assessing (e.g., confirming) the sequence of a genetic element, e.g., a sequence encoding an effector, of a finger loop. In some embodiments, assessing purity comprises assessing the amount of impurities, such as mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and/or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replication-competent factors (RCA), such as replication-competent viruses or unwanted finger rings (e.g., finger rings other than the desired finger ring, e.g., a synthetic finger ring described herein), free viral capsid proteins, exogenous factors, and aggregates. In some embodiments, assessing potency comprises assessing the ratio of functional versus non-functional (e.g., infectious versus non-infectious) finger rings in a formulation (e.g., as assessed by HPLC). In some embodiments, assessing potency comprises assessing the level of detectable ring function in the formulation (e.g., expression and/or function of the effector encoded therein or a genomic equivalent).

In embodiments, the formulated formulation is substantially free of pathogens, host cell contaminants, or impurities; non-infectious particles having a predetermined level or a predetermined ratio of particles: infectious units (e.g., <300:1, <200:1, <100:1, or <50: 1). In some embodiments, multiple ring bodies can be produced in a single batch. In an embodiment, the level of ring bodies produced in a batch can be evaluated (e.g., individually or together).

In one aspect, the invention features a host cell comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence as described herein referring to the genetic element of the loop body, and

(ii) optionally, a second nucleic acid molecule encoding one or more of: an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 (as set forth in any one of table 16), or an amino acid sequence having at least about 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto.

In one aspect, the invention features a reaction mixture that includes a finger loop as described herein and a helper virus, wherein the helper virus comprises a polynucleotide, such as a polynucleotide that encodes an external protein (e.g., an external protein that is capable of binding to an external protein binding sequence and optionally a lipid envelope), a polynucleotide that encodes a replication protein (e.g., a polymerase), or any combination thereof.

In some embodiments, the finger loop body is isolated (e.g., a synthetic finger loop body), e.g., from a host cell and/or from other components in a solution (e.g., a supernatant). In some embodiments, the finger ring body (e.g., a synthetic finger ring body) is purified, for example, from a solution (e.g., a supernatant). In some embodiments, the finger ring bodies in the solution are enriched relative to other components in the solution.

In some embodiments of any of the foregoing finger rings, finger ring vectors, compositions, or methods, the genetic element comprises a finger ring genome, e.g., identified according to the method described in example 9. In an example, the genome of the finger ring comprises a TTV-tth8 nucleic acid sequence, such as the TTV-tth8 nucleic acid sequence shown in Table 5, having a deletion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of nucleotides 3436-3707 of the TTV-tth8 nucleic acid sequence. In embodiments, the genome of the finger genome comprises a TTMV-LY2 nucleic acid sequence, such as the TTMV-LY2 nucleic acid sequence shown in Table 15, having a deletion of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of nucleotides 574-. In embodiments, the genome of a finger ring is one that is capable of self-replication and/or self-amplification. In embodiments, the genome of the ring is not capable of self-replication and/or self-amplification. In embodiments, the genome of the finger ring is capable of replicating and/or amplifying in trans, e.g., in the presence of an auxiliary, e.g., a helper virus.

Additional features of any of the foregoing ring bodies, ring carriers, compositions, or methods include one or more of the following enumerated embodiments.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalent embodiments are intended to be encompassed by the embodiments listed below.

Illustrative examples

1. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) and a protein-binding sequence (e.g., an external protein-binding sequence) encoding an exogenous effector;

wherein the exogenous effector comprises a secreted polypeptide selected from the group consisting of: an antibody molecule, enzyme, hormone, cytokine molecule, complement inhibitor, growth factor or growth factor inhibitor, or a functional variant of any of the foregoing;

wherein the genetic element is enclosed within the protein exterior;

wherein the ring body is configured to deliver the genetic element into a eukaryotic cell;

optionally, wherein the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region).

2. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element operably linked to a heterologous nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector);

wherein the exogenous effector comprises a secreted polypeptide selected from the group consisting of: an antibody molecule, enzyme, hormone, cytokine, complement inhibitor, growth factor or growth factor inhibitor, or a functional variant of any of the foregoing;

wherein the genetic element is enclosed within the protein exterior;

3. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

Wherein the exogenous effector comprises a secreted polypeptide;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell;

4. The finger ring body of any one of the preceding embodiments, wherein the antibody molecule binds a cytokine, e.g., a cytokine of table a, e.g., IL-6, or wherein the antibody molecule binds a cytokine receptor, e.g., a receptor of table a, e.g., IL-6R.

5. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a cytokine of table a or a functional variant thereof.

6. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a hormone of table B or a functional variant thereof.

7. The finger ring body of any one of the preceding embodiments, wherein the antibody molecule binds a growth factor, e.g., a growth factor of table C, e.g., VEGF.

8. The finger ring body of any one of the preceding embodiments, wherein the antibody molecule binds to a growth factor receptor, e.g., a growth factor receptor of table C, e.g., VEGFR.

9. The finger ring body of any one of the preceding embodiments, wherein the effector comprises:

(i) antibody molecules, such as anti-VEGFR antibody molecules, anti-VEGF antibody molecules, anti-cytokine antibody molecules (e.g., anti-IL 6 antibody molecules), antibody molecules that bind cytokine receptors (e.g., anti-IL 6R antibody molecules), or anti-TNF α antibody molecules;

(ii) an enzyme, such as ADAMTS13 or a functional variant thereof;

(iii) hormones (e.g., peptide hormones, such as atrial peptides or functional variants thereof);

(iv) cytokines (e.g., IL2 or TNF- α or functional variants thereof);

(v) complement inhibitors (e.g. C3 inhibitors, e.g. compstatin or pan-complement inhibitors, e.g. PgtE), or

(vi) Growth factor inhibitors, such as angiogenin binding peptides, e.g. the A11 angiogenin inhibitor peptide.

10. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a ring body composition, wherein the ring body composition comprises a plurality of ring bodies comprising:

(a) A proteinaceous outer portion;

(b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence (e.g., an external protein binding sequence);

wherein the effector comprises a secreted polypeptide selected from an antibody molecule, an enzyme, a hormone, a cytokine, a complement inhibitor, a growth factor or a growth factor inhibitor, or a functional variant of any of the foregoing;

wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region).

11. The method of embodiment 10, wherein the disease or disorder is a cancer, a hematologic disorder, an inflammatory disorder (e.g., rheumatoid arthritis), a cardiovascular and/or metabolic disease, an autoimmune disease or disorder, or a fibrotic disease or disorder.

12. A method of delivering an effector to a subject, the method comprising administering to the subject an effective amount of a finger ring composition,

wherein the ring body composition comprises a plurality of ring bodies comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector), and a protein binding sequence (e.g., an external protein binding sequence);

optionally, wherein the genetic element comprises at least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type ring virus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region); and is

Wherein the genetic element is enclosed within the protein exterior;

thereby delivering the effector to the subject.

13. A method of modulating, e.g., inhibiting or enhancing, a biological function in a subject, e.g., a subject having a disease or disorder that is treatable by modulating a biological function in a subject, the method comprising administering an effective amount of a ring composition, e.g., as described herein, to the subject,

wherein the ring body composition comprises a plurality of ring bodies comprising:

(a) a proteinaceous outer portion;

Wherein the genetic element is enclosed within the protein exterior; and is

Thereby modulating, e.g., inhibiting or enhancing, a biological function of the subject.

14. The finger ring body of any one of embodiments 10-13, wherein the antibody molecule binds a cytokine, e.g., a cytokine of table a, e.g., IL-6.

15. The finger ring body of any one of embodiments 10-14, wherein the antibody molecule binds to a cytokine receptor, e.g., a receptor of table a, e.g., IL-6R.

16. The finger ring body of any one of embodiments 10-15, wherein the effector comprises a cytokine of table a or a functional variant thereof.

17. The finger ring body according to any one of embodiments 10-16, wherein the effector comprises a hormone of table B or a functional variant thereof.

18. The finger ring body of any one of embodiments 10-17, wherein the antibody molecule binds a growth factor, e.g., a growth factor of table C, e.g., VEGF.

19. The finger ring body of any one of embodiments 10-18, wherein the antibody molecule binds to a growth factor receptor, e.g., a growth factor receptor of table C, e.g., VEGFR.

20. The method of any one of embodiments 10-19, wherein the effector comprises:

(i) Antibody molecules, such as anti-VEGFR antibody molecules, anti-VEGF antibody molecules, anti-cytokine antibody molecules (e.g., anti-IL 6 antibody molecules), antibody molecules that bind cytokine receptors (e.g., anti-IL 6R antibody molecules), or anti-TNF α antibody molecules;

(ii) an enzyme, such as ADAMTS13 or a functional variant thereof;

(iii) hormones (e.g., peptide hormones, such as atrial peptides or functional variants thereof);

(iv) cytokines (e.g., IL2 or TNF- α or functional variants thereof);

(v) complement inhibitors (e.g., C3 inhibitors, such as compstatin or pan complement inhibitors, such as PgtE); and/or

(vi) Growth factor inhibitors, such as angiogenin binding peptides, e.g. the A11 angiogenin inhibitor peptide.

21. The finger ring or method of any one of the preceding embodiments, wherein the effector is configured to down-regulate VEGF signaling (e.g., wherein the effector comprises an anti-VEGFR antibody molecule or an anti-VEGF antibody molecule, e.g., bevacizumab or a functional variant thereof, e.g., scFv; or an Fn3 inhibitor), IL-6 signaling (e.g., wherein the effector comprises an anti-IL 6 antibody molecule, e.g., ologozumab or a functional variant thereof, e.g., scFv; an anti-IL 6R antibody molecule, e.g., tollizumab or a functional variant thereof, e.g., scFv; TNF-a signaling (e.g., wherein the effector comprises an anti-TNF a antibody molecule, e.g., adalimumab or a functional variant thereof, e.g., scFv), complement signaling (e.g., an antibody molecule that binds a complement protein (e.g., complement protein 5 or complement protein 3)) Complement 5 signaling (e.g., wherein the effector comprises an anti-complement protein C5 antibody molecule, e.g., eculizumab or a functional variant thereof), complement 3 signaling (e.g., wherein the effector comprises a C3 binding polypeptide, such as compstatin or a functional variant thereof, complement C1 signaling (e.g., wherein the effector comprises a C1 inhibitor, such as pan complement signaling (e.g., wherein the effector comprises a complement inhibitor, e.g., SERPING1 or a functional variant thereof, PgtE or a functional variant thereof), interferon-gamma signaling, DPP-IV signaling (e.g., a peptide inhibitor of DPP-IV, e.g., peptide inhibitors 3-8 amino acids in length), or angiogenin signaling (e.g., wherein the effector comprises an angiogenesis inhibitor, such as an a11 peptide or a functional variant thereof).

22. The ring or method of any of the preceding embodiments, wherein the effector is configured to upregulate atrial peptide signaling (e.g., wherein the effector comprises an atrial peptide or a functional variant thereof), interferon-gamma signaling (e.g., wherein the effector comprises interferon-gamma or a functional variant thereof), erythropoietin signaling (e.g., EPO or a functional variant thereof), GLP-1 signaling (e.g., GLP-1 or a functional variant thereof), growth factor signaling (e.g., wherein the effector comprises a growth factor, e.g., a growth factor of table C or a functional variant thereof), STING/cGAS signaling (e.g., wherein the effector comprises a peptide activator of the STING/cGAS pathway), or cytokine signaling, e.g., IL-2 signaling (e.g., wherein the effector comprises IL-2 or a functional variant thereof).

23. The finger ring body or the method of any one of the preceding embodiments, wherein the effector is configured to modulate a oncology target (e.g., wherein the effector comprises an anti-VEGFR antibody molecule or an anti-VEGF antibody molecule, interferon- γ or a functional variant thereof, IL2 or a functional variant thereof, or an activator of STING/cGAS signaling), or an autoimmune or fibrotic disease target (e.g., wherein the effector comprises an ADAMTS13 or a functional variant thereof, an anti-TNF antibody molecule, an anti-IL 6 antibody molecule, an anti-IL 6R antibody molecule, compstatin or a functional variant thereof, an anti-complement protein C5 antibody molecule, or a PgtE or a functional variant thereof.

24. The finger ring or method of any one of the preceding embodiments, wherein the effector comprises a protease inhibitor, e.g., alpha-1 antitrypsin or a functional variant thereof.

25. The finger ring or method of any one of the preceding embodiments, wherein the effector comprises a secreted enzyme, e.g., ADAMTS13 or a functional variant thereof.

26. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of a ring body composition or an isolated nucleic acid molecule (e.g., an expression vector),

wherein the finger ring composition or isolated nucleic acid molecule comprises a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector); and is

Wherein:

(i) the disease or disorder includes cancer and the effector comprises an anti-VEGF antibody molecule, an anti-VEGFR antibody molecule), IFN- γ or a functional variant thereof (e.g., wherein the disease or disorder is leukemia or lymphoma), IL2 or a functional variant thereof, a peptide activator of the STING/cGAS pathway, or an a11 angiopoietin inhibitory peptide;

(ii) the disease or disorder is a cardiovascular disease or metabolic disease (e.g., type 2 diabetes) and the effector comprises or binds GLP 1;

(iii) The disease or disorder is an autoimmune disease or fibrotic disease, and the effector comprises ADAMTS13 (e.g., wherein the disease or disorder is thrombotic thrombocytopenic purpura), an anti-TNF- α antibody molecule, an anti-IL 6- α antibody molecule, an anti-IL-6R (e.g., anti-soluble IL-6R) antibody molecule, or an inhibitor of STING/cGAS signaling (e.g., an anti-STING antibody molecule or inhibitory peptide);

(iv) the disease or disorder is alpha 1-antitrypsin deficiency and the effector comprises alpha-1 antitrypsin or a functional variant thereof;

(v) the disease or disorder is retinopathy and the effector comprises an a11 angiogenin inhibitory peptide;

(vi) the disease or disorder is age-related macular degeneration (e.g., wet AMD or dry AMD), and the effector comprises a complement inhibitor, e.g., a C3 inhibitor, e.g., compstatin or a functional variant thereof;

(vii) the disease or disorder is hereditary angioedema and the effector comprises a C1 inhibitor, for example SERPING1 or a functional variant thereof;

(viii) the disease or disorder is a viral disease, e.g., hepatitis b or c, and the effector comprises IFN- γ or a functional variant thereof; or

(ix) The disease or disorder is an inflammatory disease, such as rheumatoid arthritis, and the effector comprises an anti-TNF α antibody molecule, such as adalimumab or a functional variant thereof, such as scFv; or an anti-IL 6R antibody molecule (e.g. anti-soluble IL6R), e.g. tollizumab or a functional variant thereof, e.g. scFv;

thereby treating the disease or disorder in the subject.

27. A method of delivering an effector to a subject having a disease or disorder, the method comprising administering to the subject an effective amount of a finger ring composition or an isolated nucleic acid molecule (e.g., an expression vector),

(i) The disease or disorder is or includes cancer and the effector comprises an anti-VEGF antibody molecule, an anti-VEGFR antibody molecule), IFN- γ or a functional variant thereof (e.g., wherein the disease or disorder is leukemia or lymphoma), IL2 or a functional variant thereof, a peptide activator of the STING/cGAS pathway, or an a11 angiogenin-inhibiting peptide;

(ii) The disease or disorder is a cardiovascular disease or metabolic disease (e.g., type 2 diabetes) and the effector comprises or binds GLP 1;

(iii) the disease or disorder is an autoimmune disease or fibrotic disease, and the effector comprises ADAMTS13 (e.g., wherein the disease or disorder is thrombotic thrombocytopenic purpura), an anti-TNF- α antibody molecule, an anti-IL 6- α antibody molecule, an anti-IL-6R (e.g., anti-soluble IL-6R) antibody molecule, or an inhibitor of STING/cGAS signaling (e.g., an anti-STING antibody molecule or inhibitory peptide);

(iv) the disease or disorder is alpha 1-antitrypsin deficiency and the effector comprises alpha-1 antitrypsin or a functional variant thereof;

(v) the disease or disorder is retinopathy and the effector comprises an a11 angiogenin inhibitory peptide;

(vii) the disease or disorder is hereditary angioedema and the effector comprises a C1 inhibitor, for example SERPING1 or a functional variant thereof;

(viii) The disease or disorder is a viral disease, e.g., hepatitis b or c, and the effector comprises IFN- γ or a functional variant thereof; or

thereby delivering the effector to the subject.

28. A method of making a finger ring body composition, the method comprising:

a) providing a host cell comprising one or more nucleic acid molecules encoding a component of the finger ring as described in any of the preceding embodiments;

b) maintaining (e.g., culturing) the host cell under conditions that allow the cell to produce one or more finger rings, thereby producing a finger ring; and

c) the finger ring is formulated, for example, as a pharmaceutical composition suitable for administration to a subject.

29. A method of making a finger ring body composition, the method comprising:

a) providing a plurality of finger rings as described in any of the preceding embodiments;

b) Optionally evaluating the plurality of finger rings of any one of the preceding embodiments for one or more of: contaminants, optical density measurements (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio) as described herein; and is

c) For example, if one or more parameters of (b) meet a specified threshold, then the plurality of finger ring bodies are, for example, formulated as a pharmaceutical composition suitable for administration to a subject.

30. The method of embodiment 28, wherein the ring body composition comprises at least 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵A ring body.

31. The method of embodiment 28 or 30, wherein the finger ring body composition comprises at least 10ml, 20ml, 50ml, 100ml, 200ml, 500ml, 1L, 2L, 5L, 10L, 20L, or 50L.

32. The finger ring or method of any one of the preceding embodiments, wherein the genetic element comprises at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) relative to a wild-type finger ring viral genomic sequence (e.g., as described herein), e.g., an insertion, substitution, enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region).

33. The finger ring or method of any one of the preceding embodiments, wherein the genetic element comprises a region comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the following nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

34. The finger ring or method of any one of the preceding embodiments, wherein the genetic element comprises a sequence comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

35. The finger ring or method of embodiment 34, wherein the genetic element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 80%.

36. The finger ring or method of embodiment 34, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

37. The finger ring or method of embodiment 34, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 80%.

38. The finger ring or method of any one of the preceding embodiments, wherein the effector comprises a signal sequence, e.g., a signal sequence endogenous to the effector, or a heterologous signal sequence.

1000. A polypeptide, such as an ORF1 molecule, comprising one or more of:

(a) a first region comprising an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence described herein (e.g., MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO:216) or MAWGWWKRRRRWWFRKRWTRGRLRRRWPRSARRRPRRRRVRRRRRWRRGRRKTRTYRRRRRFRRRGRK (SEQ ID NO:186), or as listed in any of tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a sequence of at least about 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof),

(b) A second region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity or a sequence comprising at least 6 (e.g., at least 6, 7, 8, 9, 10, 11, or 12) beta strands to a jelly roll region sequence described herein (e.g., PTYTTIPLKQWQPPYKRTCYIKGQDCLIYYSNLRLGMNSTMYEKSIVPVHWPGGGSFSVSMLTLDALYDIHKLCRNWWTSTNQDLPLVRYKGCKITFYQSTFTDYIVRIHTELPANSNKLTYPNTHPLMMMMSKYKHIIPSRQTRRKKKPYTKIFVKPPPQFENKWYFATDLYKIPLLQIHCTACNLQNPFVKPDKLSNNVTLWSLNT (SEQ ID NO:217), or as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10);

(c) a third region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an N22 domain sequence described herein (e.g., TMALTPFNEPIFTQIQYNPDRDTGEDTQLYLLSNATGTGWDPPGIPELILEGFPLWLIYWGFADFQKNLKKVTNIDTNYMLVAKTKFTQKPGTFYLVILNDTFVEGNSPYEKQPLPEDNIKWYPQVQYQLEAQNKLLQTGPFTPNIQGQLSDNISMFYKFYFK (SEQ ID NO:219), or as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10); and

(d) A fourth region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a C-terminal domain (CTD) sequence of an ORF1 of the dactylovirus described herein (e.g., WGGSPPKAINVENPAHQIQYPIPRNEHETTSLQSPGEAPESILYSFDYRHGNYTTTALSRISQDWALKDTVSKITEPDRQQLLKQALECLQISEETQEKKEKEVQQLISNLRQQQQLYRERIISLLKDQ (SEQ ID NO:220), or as listed in any of tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10);

wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, relative to a wild-type ORF1 protein (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, a jelly roll domain, an HVR, N22, or a CTD, e.g., as described herein).

The polypeptide of example 1000, wherein the amino acid sequences of the regions of (a), (b), (c), and (d) have at least 90% sequence identity to their respective references.

1001. The polypeptide of embodiment 1000, wherein the polypeptide comprises:

(i) the first region and the second region;

(ii) the first region and the third region;

(iii) the first region and the fourth region;

(iv) the second region and the third region;

(v) the second region and the fourth region;

(vi) the third region and the fourth region;

(vii) the first region, the second region, and the third region;

(viii) the first region, the second region, and the fourth region;

(ix) the first region, the third region, and the fourth region; or

(x) The second region, the third region, and the fourth region.

1002. A polypeptide, such as an ORF1 molecule, comprising:

(b) A second region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a jelly roll region sequence described herein (e.g., PTYTTIPLKQWQPPYKRTCYIKGQDCLIYYSNLRLGMNSTMYEKSIVPVHWPGGGSFSVSMLTLDALYDIHKLCRNWWTSTNQDLPLVRYKGCKITFYQSTFTDYIVRIHTELPANSNKLTYPNTHPLMMMMSKYKHIIPSRQTRRKKKPYTKIFVKPPPQFENKWYFATDLYKIPLLQIHCTACNLQNPFVKPDKLSNNVTLWSLNT (SEQ ID NO:217), or as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10) or a sequence containing at least 6 beta strands;

1002a. the polypeptide of example 1002, wherein the amino acid sequences of regions (a), (b), (c), and (d) have at least 90% sequence identity to their respective references.

1003. The polypeptide of any one of the preceding embodiments, wherein:

the first region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acids 1-38 of the ORF1 sequence set forth in table 16;

the second region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acids 39-246 of the ORF1 sequence set forth in table 16;

the second region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid 375-537 of the ORF1 sequence set forth in Table 16; and/or

The fourth region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid 538-666 of the ORF1 sequence set forth in Table 16.

1003a. the polypeptide of embodiment 1003, wherein the amino acid sequences of the first, second, third and fourth regions have at least 90% sequence identity to their respective references.

1004. The polypeptide of any one of the preceding embodiments, wherein:

the first region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10;

the second region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a jelly roll region sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10;

the third region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an N22 domain sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10; and/or

The fourth region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a CTD sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1004a. the polypeptide of example 1004, wherein the amino acid sequences of said first, second, third, and fourth regions have at least 90% sequence identity to their respective references.

1005. The polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises, in N-terminal to C-terminal order, the first region, the second region, the third region, and the fourth region.

1006. The polypeptide of any one of the preceding embodiments, wherein the at least one difference comprises at least one difference in the first region relative to an arginine-rich region of a wild-type ORF1 protein.

1007. The polypeptide of any one of the preceding embodiments, wherein the first region comprises an arginine-rich region from the ORF1 protein of an Ring virus other than a wild-type Ring virus having maximum sequence identity to the polypeptide or a portion thereof not including the first region.

1008. The polypeptide of any one of the preceding embodiments, wherein the first region comprises an amino acid sequence having at least 70% sequence identity to an arginine-rich region from a finger ring virus other than a wild-type finger ring virus with which the polypeptide has the greatest sequence identity.

1009. The polypeptide of any one of the preceding embodiments, wherein the first region comprises a polypeptide having less than 15% (e.g., less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) sequence identity to a wild-type finger ring virus genome (e.g., as described herein) or a portion thereof having the same amino acid length as the first region.

1010. A polypeptide according to any one of the preceding embodiments wherein said first region has DNA binding activity and/or nuclear localisation activity.

1011. A polypeptide according to any one of the preceding embodiments wherein the first region comprises a DNA binding region and/or a nuclear localization sequence.

1012. The polypeptide of any one of the preceding embodiments, wherein the at least one difference comprises at least one difference in the second region relative to a jellyroll region of a wild-type ORF1 protein.

1013. The polypeptide of any one of the preceding embodiments, wherein the second region comprises a jellyroll region from the ORF1 protein of an Ring virus other than a wild-type Ring virus having the greatest sequence identity to the polypeptide or a portion thereof not comprising the second region.

1014. The polypeptide of any one of the preceding embodiments, wherein the second region comprises an amino acid sequence having at least 70% sequence identity to a jellyroll region from a finger ring virus other than a wild-type finger ring virus with which the polypeptide has the greatest sequence identity.

1015. The polypeptide of any one of the preceding embodiments, wherein the second region comprises a polypeptide having less than 15% (e.g., less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) sequence identity to a wild-type finger ring virus genome (e.g., as described herein) or a portion thereof having the same amino acid length as the second region.

1016. The polypeptide of any one of the preceding embodiments, wherein the at least one difference comprises at least one difference in the third region relative to the N22 domain of wild-type ORF1 protein.

1017. The polypeptide of any one of the preceding embodiments, wherein the third region comprises the N22 domain from the ORF1 protein of an Ring virus other than a wild-type ring virus having maximum sequence identity to the polypeptide or a portion thereof not including the third region.

1018. A polypeptide according to any one of the preceding embodiments wherein the third region comprises an amino acid sequence which has at least 70% sequence identity to the region N22 from an finger ring virus other than the wild type finger ring virus with which the polypeptide has the greatest sequence identity.

1019. The polypeptide of any one of the preceding embodiments, wherein the third region comprises a polypeptide having less than 15% (e.g., less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) sequence identity to a wild-type finger ring virus genome (e.g., as described herein) or a portion thereof having the same amino acid length as the third region.

1020. The polypeptide of any one of the preceding embodiments, wherein the at least one difference comprises at least one difference in the CTD domain of ORF1 protein relative to wild-type in the fourth region.

1021. The polypeptide of any one of the preceding embodiments, wherein the fourth region comprises a CTD domain from the ORF1 protein of an Ring virus other than a wild-type Ring virus having maximum sequence identity to the polypeptide or a portion thereof not comprising the fourth region.

1022. The polypeptide of any one of the preceding embodiments, wherein the fourth region comprises an amino acid sequence having at least 70% sequence identity to a CTD region from a ring virus other than a wild-type ring virus to which the polypeptide has the greatest sequence identity.

1023. The polypeptide of any one of the preceding embodiments, wherein the fourth region comprises a polypeptide having less than 15% (e.g., less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) sequence identity to a wild-type finger ring virus genome (e.g., as described herein) or a portion thereof having the same amino acid length as the fourth region.

1024. The polypeptide of any one of the preceding embodiments, further comprising an amino acid sequence, e.g., a hypervariable region (HVR) sequence (e.g., an HVR sequence of an ORF1 molecule of a finger virus as described herein), wherein the amino acid sequence comprises at least about 55 (e.g., at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (e.g., about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55-140, or 60-140 amino acids).

1025. The polypeptide of embodiment 1024, wherein the HVR sequence is located between the second region and the third region.

1026. The polypeptide of embodiment 1024 or 1025, wherein the HVR sequence comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an HVR from a wild-type finger ring virus other than the ORF1 protein that has the greatest sequence identity.

1027. The polypeptide of any one of embodiments 1024-1026, wherein the HVR sequence is heterologous with respect to one or more of the first, second, third and/or fourth regions.

1028. The polypeptide of any one of embodiments 1024-1027, wherein the at least one difference comprises at least one difference in the HVR sequence relative to the wild-type ORF1 protein (e.g., from a wild-type ring virus genome, e.g., as described herein).

1029. The polypeptide of any one of embodiments 1024-1028, wherein the HVR sequence comprises an HVR from the ORF1 protein from an finger virus other than a wild-type finger virus having the greatest sequence identity to the polypeptide or a portion thereof not including the HVR type.

1030. The polypeptide of any one of embodiments 1024-1029, wherein the HVR sequence comprises an amino acid sequence having at least about 70% sequence identity to an HVR from an finger ring virus other than the wild-type finger ring virus having the greatest sequence identity to the polypeptide.

1031. The polypeptide of any one of examples 1024-1030, wherein the HVR comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an HVR sequence as set forth in any one of tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1032. The polypeptide of any one of embodiments 1024-1031, wherein the HVR sequence comprises at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acid 247-374 of the ORF1 sequence set forth in Table 16.

1033. The polypeptide of any one of the preceding embodiments, further comprising a heterologous polypeptide, e.g., a polypeptide that is heterologous with respect to one or more of the first, second, third, and/or fourth regions, and/or exogenous with respect to the ring comprising the polypeptide.

1034. The polypeptide of embodiment 1033, wherein the polypeptide lacks a finger ring virus HVR sequence.

1035. The polypeptide of embodiment 1033, wherein the heterologous polypeptide is present on the exterior of the finger loop body.

1036. The polypeptide of embodiment 1033, wherein the heterologous polypeptide is present on the interior of the finger loop body.

1037. The polypeptide of any one of embodiments 1033-1036, wherein the heterologous polypeptide has a functionality that is foreign to the finger ring or the wild-type finger ring virus.

1038. The polypeptide of any one of embodiments 1033-1037, wherein the heterologous polypeptide consists of about 140 or fewer amino acids (e.g., 100, 110, 120, 125, 130, 135, 136, 137, 138, 139, 140, 145, 150, 155, or 160 or fewer amino acids).

1039. The polypeptide of any one of embodiments 1033-1038, wherein the heterologous polypeptide has a size from 50% to 150% relative to, for example, the wild-type HVR region of a finger ring virus as described herein.

1039A. the polypeptide of any one of embodiments 1033 and 1039, wherein the heterologous polypeptide is located between the second region and the third region.

1040. The polypeptide of any one of the preceding embodiments, further comprising one or more amino acids between the first region and the second region, one or more amino acids between the second region and the third region, and/or one or more amino acids between the third region and the fourth region.

1041. The polypeptide of any one of the preceding embodiments, further comprising one or more amino acids at the N-terminus relative to the first region.

1042. The polypeptide of any one of the preceding embodiments, further comprising one or more amino acids C-terminal to the fourth region.

1043. The polypeptide of any one of the preceding embodiments, comprising a plurality of subsequences having at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) contiguous amino acids that have 100% sequence identity to a corresponding subsequence of wild-type finger ring virus ORF1 amino acid sequence, e.g., as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1044. The polypeptide of any one of the preceding embodiments, comprising a plurality of subsequences having at least ten (e.g., 10, 15, 20, 25, 30, 40, or 50) contiguous amino acids that have 80% sequence identity to a corresponding subsequence of the wild-type finger ring virus ORF1 amino acid sequence, e.g., as set forth in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1045. The polypeptide of any one of the preceding embodiments, comprising a plurality of subsequences having at least twenty (e.g., 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100) contiguous amino acids having 60% sequence identity to a corresponding subsequence of the wild-type finger ring virus ORF1 amino acid sequence, e.g., as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1046. The polypeptide according to any one of embodiments 1043-1045, wherein said plurality of subsequences is located in said first region, second region, third region and/or fourth region.

1047. The polypeptide of any one of the preceding embodiments, wherein the first region comprises at least about 40 amino acids (e.g., at least about 50, 60, 70, 80, 90, or 100 amino acids, e.g., about 40-100, 40-90, 40-80, 40-70, 50-100, 50-70, 60-100, 60-90, 60-80, or 60-70 amino acids).

1048. The polypeptide of any one of the preceding embodiments, wherein the first region comprises at least about 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%) basic residues (e.g., arginine, lysine, or a combination thereof).

1049. The polypeptide of any one of the preceding embodiments, wherein the first region comprises at least about 70% (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%) arginine residues.

1050. The polypeptide of any one of the preceding embodiments, wherein the polypeptide forms a homomultimer with additional copies of the polypeptide.

1051. The polypeptide of embodiment 1050, wherein said first region binds to a corresponding first region on additional copies of said polypeptide.

1052. The polypeptide of embodiment 1050, wherein the homopolymer forms a capsid, e.g., encapsulates a nucleic acid, e.g., a genetic element or a ring virus genome or portion thereof.

1053. The polypeptide of any one of the preceding embodiments, wherein the polypeptide is a capsid protein or can form a portion of a capsid.

1054. The polypeptide of any one of the preceding embodiments, wherein the polypeptide has replicase activity.

1055. The polypeptide of any one of the preceding embodiments, wherein the polypeptide binds a nucleic acid (e.g., DNA).

1056. A composite, comprising:

(a) a polypeptide as described in any one of the preceding embodiments, and

1057. A composite, comprising:

(a) ORF1 molecule, and

wherein the ORF1 molecule is bound (e.g., non-covalently bound) to the genetic element,

wherein the ORF1 molecule, genetic element, or both the ORF1 molecule and genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, relative to wild-type ORF1 protein, wild-type dactylovirus genome, or both wild-type ORF1 protein and wild-type dactylovirus genome (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, a jelly roll domain, an HVR, N22, or a CTD, e.g., as described herein) or a genomic region (e.g., one or more of a TATA box, a cap site, transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region, e.g., as described herein).

1058. The complex of embodiment 1056 or 1057, wherein said complex is in vitro, e.g., wherein said complex is in a substantially acellular composition.

1059. The complex of any one of embodiments 1056 and 1058, wherein the complex is in a cell, e.g., a host cell, e.g., a helper cell, e.g., in a nucleus.

1060. The complex of any one of embodiments 1056 and 1059, wherein the ORF1 molecule is part of the exterior of a protein.

1061. The complex of any one of embodiments 1056-1060, wherein the genetic element is undergoing replication.

1062. The complex of any one of embodiments 1056-1061, wherein the complex is in a finger ring body.

1063. The complex of any one of embodiments 1056-1062, wherein the genetic element further comprises a nucleic acid sequence encoding the polypeptide.

1064. The complex of any one of embodiments 1056-1063, wherein the genetic element does not comprise a nucleic acid sequence encoding the polypeptide.

1065. The complex of any one of embodiments 1056-1064, wherein the genetic element comprises a GC-rich region, e.g., as described herein.

1066. The complex of embodiment 1065, wherein the GC-rich region comprises at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the nucleic acid sequence of any one of:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1067. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a polypeptide or complex as described in any one of the preceding embodiments;

(c) a genetic element comprising a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., as described herein); and is

Wherein the genetic element is enclosed within the exterior of the protein.

1068. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising:

(i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., as described herein), and

(ii) a nucleic acid encoding a polypeptide according to any one of the preceding embodiments; and is

Wherein the genetic element is enclosed within the exterior of the protein.

1069. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) an ORF1 molecule or a nucleic acid encoding said ORF1 molecule;

(c) a genetic element comprising a promoter element operably linked to a heterologous nucleic acid sequence (e.g., a DNA sequence) encoding an effector; and is

Wherein the genetic element is enclosed within the exterior of the protein.

1070. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) an ORF1 molecule or a nucleic acid encoding said ORF1 molecule;

(c) a genetic element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a region of at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides comprising the nucleic acid sequence of:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

Wherein the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region);

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1071. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) an ORF1 molecule or a nucleic acid encoding said ORF1 molecule;

(c) A genetic element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a sequence comprising at least 20 (e.g., at least 20, 25, 30, 31, 32, 33, 34, 35, or 36) contiguous nucleotides having a GC content of at least 70% (e.g., at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%);

wherein the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region);

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) Does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1072. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) an ORF1 molecule or a nucleic acid encoding said ORF1 molecule;

wherein:

(i) at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more) of the amino acids of the ORF1 molecule are part of the β -strand;

(ii) the secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β -strands;

(iii) the secondary structure of the ORF1 molecule comprises a ratio of beta-strands to alpha-helices of at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1; and is

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1073. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) an ORF1 molecule or a nucleic acid encoding said ORF1 molecule;

(c) A genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1074. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a region of at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides comprising the nucleic acid sequence of:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

Wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1075. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a sequence comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%; and

Wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring body is configured to deliver the genetic element into a eukaryotic cell; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1076. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector),

wherein the genetic element comprises a region (e.g., a packaging region, e.g., positioned 3' with respect to a nucleic acid sequence encoding the effector) having:

at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a nucleic acid sequence that is: CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGC CATGC (SEQ ID NO: 160);

Wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring is configured to deliver a genetic element into a eukaryotic cell.

1076a. a finger ring, the finger ring comprising:

(i) a genetic element comprising a promoter element and a nucleic acid sequence encoding a therapeutic exogenous effector, wherein the genetic element comprises a sequence having at least 95% sequence identity to a 5' UTR nucleotide sequence from a finger ring virus as described herein (e.g., as listed in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17); and/or

(ii) An proteinaceous outer portion comprising a polypeptide having at least 95% sequence identity to a polypeptide encoded by the ORF1 gene of a ring virus described herein (e.g., as set forth in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17);

wherein the genetic element is enclosed within the protein exterior, and

optionally, wherein the finger ring body is capable of delivering the genetic element into a mammalian cell.

1076b. a ring body, the ring body comprising:

(I) a genetic element comprising: (a) a promoter element, and (b) a nucleic acid sequence encoding an exogenous effector (e.g., an exogenous effector as described herein), wherein the nucleic acid sequence is operably linked to the promoter element; and (c) a 5' UTR domain comprising one of:

(c) (ii) (i) the nucleic acid sequence of nucleotide 323-393 of SEQ ID NO:54, or a nucleic acid sequence which is at least 85% identical thereto;

(c) (ii) the nucleic acid sequence of SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119 or a nucleic acid sequence at least 85% identical thereto; or

(c) (iii) the nucleic acid sequence of nucleotides 117-187 of SEQ ID NO:61, or a nucleic acid sequence which is at least 85% identical thereto;

(II) a proteinaceous outer portion comprising an ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the synthetic finger ring body is capable of delivering the genetic element into a cell of a mammal, such as a human.

1077. The finger ring body of any one of the preceding embodiments, wherein the protein comprises externally an ORF1 molecule.

1078. The finger ring body of any one of the preceding embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98, 99% or 100%) of the protein in the outer portion comprises an ORF1 molecule.

1079. The finger ring body of any one of the preceding embodiments, wherein no more than 1% (e.g., no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%) of the protein in the outer portion comprises an ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 molecule.

1080. The finger loops of any one of the preceding embodiments, wherein the ORF1 molecule comprises an amino acid sequence that is at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to an ORF1 protein listed in or encoded by any one of tables a1-a12, B1-B5, C1-C5, 1-18, 20-37, or D1-D10.

1081. The finger ring body of any one of the preceding embodiments, wherein the ORF1 molecule comprises the polypeptide of any one of the preceding embodiments.

1082. The finger ring body of any one of the preceding embodiments, wherein the genetic element further comprises a nucleic acid sequence encoding the ORF1 molecule.

1083. The finger ring body of any one of the preceding embodiments, wherein the genetic element does not comprise a nucleic acid sequence encoding the ORF1 molecule.

1084. The finger ring body of any one of the preceding embodiments, wherein the genetic element comprises at least 20, 25, 30, 31, 32, 33, 34, 35 or 36 contiguous nucleotides having a GC content of at least 80%.

1085. The finger loop body of any one of the preceding embodiments, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

1086. The finger ring body of any one of the preceding embodiments, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 80%.

1087. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules) comprising a nucleic acid encoding a polypeptide as described in any of the preceding embodiments;

optionally, wherein the isolated nucleic acid composition further comprises at least one difference (e.g., mutation, chemical modification, or epigenetic alteration) relative to a wild-type ring virus genomic sequence (e.g., as described herein), e.g., an insertion, substitution, enzymatic modification, and/or deletion, e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region); and is

Optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1088. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules), wherein the isolated nucleic acid composition comprises a genetic element encoding an ORF1 molecule;

wherein:

(i) at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more) of the amino acids of the ORF1 molecule are part of a β -sheet;

(ii) the secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β -sheets;

(iii) the secondary structure of the ORF1 molecule comprises a ratio of β -sheet to α -helix of at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1; and is

Wherein the genetic element comprises a promoter element, a nucleic acid sequence encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence;

Optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1089. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules) comprising:

(a) a genetic element encoding an ORF1 molecule;

(b) at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

(c) At least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type, refers to a genomic sequence of a retrovirus (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region);

Optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1090. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules), wherein the isolated nucleic acid composition comprises:

(a) a genetic element encoding an ORF1 molecule;

(b) at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%; and is

Wherein the isolated nucleic acid composition comprises at least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type dactylovirus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region); and is

Optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1090a, an isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules), wherein the isolated nucleic acid composition comprises a genetic element comprising a 5' UTR nucleotide sequence from a ring virus as described herein (e.g., as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

1091. The isolated nucleic acid composition of any one of examples 1089-1090, wherein (a) and (b) are portions of the same nucleic acid.

1092. The isolated nucleic acid composition of any one of examples 1089-1091, wherein (a) and (b) are portions of different nucleic acids.

1093. The isolated nucleic acid composition of any of the preceding embodiments, wherein the genetic element further comprises one or more of: a TATA box, an initiation element, a cap site, a transcription initiation site, A5' UTR conserved domain, an ORF1 coding sequence, an ORF1/1 coding sequence, an ORF1/2 coding sequence, an ORF2 coding sequence, an ORF2/2 coding sequence, an ORF2/3 coding sequence, an ORF2/3t coding sequence, an open reading frame region, a poly (a) signal and/or a GC-rich region from a finger ring virus as described herein (e.g., as listed in any of tables a1, A3, A5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1094. The isolated nucleic acid composition of any of the preceding embodiments, wherein the genetic element further comprises a finger ring virus genomic sequence (e.g., as described herein, e.g., as set forth in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1095. The isolated nucleic acid composition of example 1094, further comprising at least one additional copy of a genomic sequence of a ring virus or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto (e.g., 1, 2, 3, 4, 5 or 6 copies in total).

1096. The isolated nucleic acid composition of any of the preceding embodiments, further comprising at least one additional copy (e.g., 1, 2, 3, 4, 5, or 6 copies total) of the genetic element.

1097. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules) comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of a nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

At least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type, refers to a genomic sequence of a retrovirus (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region);

optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1098. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules), wherein the isolated nucleic acid composition comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%; and is

Optionally, wherein the nucleic acid molecule does not comprise:

(i) a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) A deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1099. The isolated nucleic acid composition of any one of the preceding embodiments, wherein the ORF1 molecule comprises the polypeptide of any one of the preceding embodiments.

1100. The isolated nucleic acid composition of any of the preceding embodiments, comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 80%.

1101. The isolated nucleic acid composition of any of the preceding embodiments, comprising at least 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

1102. The isolated nucleic acid composition of any of the preceding embodiments, comprising at least 36 contiguous nucleotides having a GC content of at least 80%.

1103. The isolated nucleic acid composition of any of the preceding embodiments, further comprising one or more of: a promoter element, a nucleic acid sequence encoding an effector (e.g., an exogenous effector or an endogenous effector), and/or a protein binding sequence (e.g., an external protein binding sequence).

1104. The isolated nucleic acid composition of any of the preceding embodiments, comprising at least about 100, 150, 200, 250, 300, 350, 400, 450, or 500 contiguous nucleotides of a wild-type finger ring virus genomic sequence or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1105. An isolated nucleic acid molecule (e.g., an expression vector) comprising a nucleic acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)。

1106. The isolated nucleic acid composition of any of the preceding embodiments, wherein the isolated nucleic acid molecule is circular.

1107. An isolated cell comprising:

(a) a nucleic acid encoding a polypeptide according to any one of the preceding embodiments, wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into the chromosome of a cell, and

(b) a genetic element, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence, wherein optionally the genetic element does not encode an ORF1 polypeptide (e.g., an ORF1 protein).

1108. An isolated cell, such as a host cell, comprising:

(a) a nucleic acid encoding an ORF1 molecule, wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into the chromosome of a cell, and

(b) A genetic element, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence.

1109. An isolated cell, such as a host cell, comprising:

(a) a nucleic acid encoding an ORF1 molecule (e.g., wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into the chromosome of a cell), and

(b) a genetic element that does not encode an ORF1 molecule, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence.

1109a. an isolated cell, e.g., a host cell, comprising:

(i) a nucleic acid molecule (e.g., a first nucleic acid molecule) comprising a nucleic acid sequence that refers to a genetic element of a loop as described herein (e.g., a genetic element that does not encode an ORF1 molecule), and

(ii) optionally, a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more of: an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 (e.g., as set forth in any one of table 16), or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto.

1110. The isolated cell of any of the preceding embodiments, wherein the genetic element not encoding an ORF1 molecule encodes a fragment of an ORF1 molecule, e.g., a fragment that does not form a capsid, e.g., a fragment of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 20, or 10 nucleotides.

1111. An isolated cell, e.g., a host cell, comprising a nucleic acid encoding an ORF1 molecule (e.g., wherein the nucleic acid is a plasmid, is a viral nucleic acid, or is integrated into the chromosome of the cell), wherein the isolated cell does not comprise one or more of an ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 molecule.

1112. An isolated cell, e.g., a host cell, comprising a nucleic acid composition as described in any of the preceding embodiments.

1113. An helper nucleic acid (e.g., a plasmid or viral nucleic acid) encoding an ORF1 molecule, wherein the isolated cell does not comprise one or more of an ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 and/or ORF3 molecule.

1114. A composition, comprising:

(a) an isolated cell as described herein, and

(b) Referred to herein are ring bodies.

1115. A composition, comprising:

(a) a cell that encodes a nucleic acid of the ORF1 molecule (e.g., wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into the chromosome of the cell), and

(b) a genetic element (e.g., within a cell or outside a cell, such as in cell culture media) that does not encode an ORF1 molecule, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) that encodes an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence.

1116. A pharmaceutical composition comprising a polypeptide, complex, finger ring body or isolated nucleic acid as described in any of the preceding embodiments and a pharmaceutically acceptable carrier and/or excipient.

1117. A method of making an ORF1 molecule, the method comprising:

(a) providing a host cell (e.g., a host cell described herein) comprising a nucleic acid encoding a polypeptide as described in any of the preceding embodiments, and

(b) maintaining said host cell under conditions which allow the cell to produce said polypeptide;

thereby producing the ORF1 molecule.

1118. A method of making an ORF1 molecule, the method comprising:

(a) Providing a host cell (e.g., a host cell as described herein) comprising a nucleic acid composition as described in any one of the preceding embodiments, and

(b) maintaining said host cell under conditions which allow the cell to produce said polypeptide;

thereby producing the ORF1 molecule.

1119. The method of embodiment 1117 or 1118, wherein the host cell is a helper cell.

1120. The method of example 1119, wherein the helper cell comprises one or more additional wild-type finger ring viruses encoding one or more additional ORFs (e.g., ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3), e.g., as described herein.

1121. The method of any one of embodiments 1117-1120, wherein the nucleic acid is integrated into the genome of the host cell.

1122. The method of any one of examples 1117-.

1123. The method of any one of embodiments 1117-1122, wherein the host cell produces at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 10,000, or 100,000 copies (e.g., at least about 60 copies) of the polypeptide per finger loop produced by the host cell.

1124. The method of any one of examples 1117-1123, wherein the method comprises providing a plurality of host cells and maintaining the host cells under conditions that allow for the production of at least 1000 copies of the polypeptide/cell.

1125. The method of embodiment 1124, wherein the plurality of host cells produces at least about 1x10⁵、1x10⁶、1x10⁷、1x10⁸、9x10⁸、1x10⁹、1x10¹⁰、1x10¹¹Or 1x10¹²Copies of said polypeptide.

1126. A method of making a finger ring body composition, the method comprising:

(a) providing a helper cell, such as a helper cell described herein;

(b) introducing a genetic element into the helper cell under conditions that allow the cell to produce the finger ring, and

(c) the ring body is formulated, for example, as a pharmaceutical composition suitable for administration to a subject, thereby preparing the ring body composition.

1127. A method of making a finger ring body composition, the method comprising:

(a) providing a host cell;

(b) introducing a helper nucleic acid into the host cell;

(c) introducing a genetic element into the host cell under conditions that allow the cell to produce the finger loop (e.g., before, after, or simultaneously with (b)); and is

(d) Formulating the ring body, e.g., as a pharmaceutical composition suitable for administration to a subject;

Thereby preparing the finger ring composition.

1128. A method of making a finger ring body composition, the method comprising:

(a) providing a helper cell comprising a nucleic acid encoding an ORF1 molecule (e.g., wherein the nucleic acid is a plasmid, viral nucleic acid, or integrated into the helper cell chromosome);

(b) introducing a genetic element into the helper cell under conditions that allow the cell to produce finger loops, wherein the genetic element does not encode an ORF1 molecule, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence; and is

thereby preparing the finger ring composition.

1129. A method of making a finger ring body composition, the method comprising:

(a) providing a host cell;

(b) introducing into said host cell a helper nucleic acid (e.g., wherein said nucleic acid is a plasmid or viral nucleic acid) encoding an ORF1 molecule; and is

(c) Introducing a genetic element into the host cell (e.g., before, after, or simultaneously with (b)) under conditions that allow the cell to produce the finger loop, wherein the genetic element does not encode an ORF1 molecule, wherein the genetic element comprises a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence,

Thereby preparing the ring body.

1130. The method of any one of the preceding embodiments, further comprising separating the finger ring body from the helper or host cell.

1131. The method of any one of the preceding embodiments, wherein providing a helper cell comprises introducing a helper nucleic acid into the host cell, e.g., wherein the helper nucleic acid encodes an ORF1 molecule (e.g., wherein the nucleic acid is a plasmid or viral nucleic acid).

1132. The method of any one of the preceding embodiments, wherein the helper cell comprises the ORF1 molecule.

1133. The method of any one of the preceding embodiments, wherein the nucleic acid comprises one or more of: a TATA box, an initiation element, a cap site, a transcription initiation site, A5' UTR conserved domain, an ORF1 coding sequence, an ORF1/1 coding sequence, an ORF1/2 coding sequence, an ORF2 coding sequence, an ORF2/2 coding sequence, an ORF2/3 coding sequence, an ORF2/3t coding sequence, an open reading frame region, a poly (a) signal and/or a GC-rich region from a finger ring virus as described herein (e.g., as listed in any of tables a1, A3, A5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1134. The method of any one of the preceding embodiments, wherein the nucleic acid comprises a ring virus genomic sequence (e.g., as described herein, e.g., as listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1135. The method of any one of the preceding embodiments, wherein the nucleic acid comprises at least one additional copy of a genomic sequence of a finger ring virus or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto (e.g., 1, 2, 3, 4, 5, or 6 copies in total).

1136. The method of any one of the preceding embodiments, wherein the host cell or helper cell comprises at least one additional copy of the nucleic acid (e.g., 1, 2, 3, 4, 5, or 6 copies total).

1137. The method of any one of the preceding embodiments, wherein the nucleic acid is circular.

1137A. a method for preparing a finger ring body, e.g., a synthetic finger ring body, the method comprising:

a) Providing a host cell comprising:

(i) a nucleic acid molecule, e.g., a first nucleic acid molecule, comprising a nucleic acid sequence of a genetic element of a finger loop, e.g., a synthetic finger loop, as described herein, and

(ii) a nucleic acid molecule, e.g., a second nucleic acid molecule, which encodes one or more of: an amino acid sequence selected from ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 (e.g., as set forth in any one of table 16), or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto; and is

b) Culturing the host cell under conditions suitable for the preparation of the finger ring.

The method of example 1137A, further comprising, prior to step (a), introducing the first nucleic acid molecule and/or the second nucleic acid molecule into the host cell.

The method of embodiment 1137A or 1137B, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

The method of example 1137d, wherein the second nucleic acid molecule is integrated into the genome of the host cell.

The method of example 1137C, wherein the second nucleic acid molecule is a helper (e.g., a helper plasmid or the genome of a helper virus).

The method of any one of embodiments 1137A-1137E, wherein the first nucleic acid comprises one or more of: a TATA box, an initiation element, a cap site, a transcription initiation site, a 5' UTR conserved domain, and/or a GC-rich region from a finger loop virus as described herein (e.g., as listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

1138. A method of delivering an effector to a subject, the method comprising administering to the subject a finger ring body comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

(b) a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding the effector (e.g., an exogenous effector or an endogenous effector), and a region of at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides comprising the nucleic acid sequence of:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

Wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the subject.

1139. A method of delivering an effector to a subject, the method comprising administering to the subject a finger ring body comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

(b) a genetic element comprising a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding the effector (e.g., an exogenous effector or an endogenous effector), and at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a sequence having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%;

Wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the subject.

1140. A method of delivering an effector to a subject, the method comprising administering to the subject a finger ring body comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) Does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the subject.

1141. A method of delivering an effector to a target cell, the method comprising contacting the target cell with a finger ring comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; and is

Wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the target cell.

1142. A method of delivering an effector to a target cell, the method comprising contacting the target cell with a finger ring comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

Wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the target cell.

1143. A method of delivering an effector to a target cell, the method comprising contacting the target cell with a finger ring comprising:

(a) a proteinaceous outer portion comprising an ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) Does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the target cell.

1143a. a method of delivering an effector to a target cell, the method comprising contacting the target cell with a finger ring comprising:

Wherein the genetic element is enclosed within the protein exterior; and is

Optionally, wherein the genetic element:

(i) does not comprise a deletion of nucleotides 3436 to 3607 relative to the wild-type TTV-tth8 genomic sequence, e.g., as described herein;

(ii) does not comprise a deletion of nucleotides 1432 to 2210 relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein; and/or

(iii) Does not comprise a deletion of at least 101 nucleotides relative to the wild-type TTMV-LY2 genomic sequence, e.g., as described herein,

thereby delivering the effector to the target cell.

1144. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element does not encode the amino acid sequence of NCBI accession No. A7XCE8.1.

1145. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the ORF1 molecule comprises an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an ORF1 sequence set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1146. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more) of the amino acids of the ORF1 molecule are part of a β -sheet.

1147. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β -sheets.

1148. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the secondary structure of the ORF1 molecule comprises a ratio of β -sheet to α -helix of at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10: 1.

1149. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the ORF1 molecule comprises an arginine-rich region (e.g., at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an arginine-rich region sequence set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10).

1150. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of example 1149, wherein the arginine-rich region comprises at least 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 consecutive nucleotides comprising at least 40% (e.g., at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, or 95%) arginine residues.

1151. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition or method of example 1149 or 1150, wherein said arginine-rich region is located at the N-terminus or C-terminus of said ORF1 molecule.

1152. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition or method as described in any one of 1149-1151, wherein the arginine-rich region has at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acid sequence TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO:808), RRRYARPYRRRHIRRYRRRRRHFRRRR (SEQ ID NO:809), MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO:216) or MAWGWWKRRRRWWFRKRWTRGRLRRRWPRSARRRPRRRRVRRRRRWRRGRRKTRTYRRRRRFRRRGRK (SEQ ID NO: 186).

1153. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition or method as defined in any one of 1149-1152, wherein the arginine-rich region is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an arginine-rich region sequence set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37 or D1-D10.

1154. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the ORF1 molecule comprises a jellyroll domain, e.g., having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of the jellyroll domain of the ORF1 molecule described herein, e.g., a jellyroll domain having amino acid sequence PTYTTIPLKQWQPPYKRTCYIKGQDCLIYYSNLRLGMNSTMYEKSIVPVHWPGGGSFSVSMLTLDALYDIHKLCRNWWTSTNQDLPLVRYKGCKITFYQSTFTDYIVRIHTELPANSNKLTYPNTHPLMMMMSKYKHIIPSRQTRRKKKPYTKIFVKPPPQFENKWYFATDLYKIPLLQIHCTACNLQNPFVKPDKLSNNVTLWSLNT (SEQ ID NO:217), or a jellyroll domain sequence set forth in any one of table a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1155. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the ORF1 molecule comprises an N22 domain, e.g., has 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of the N22 domain of the ORF1 molecule described herein, e.g., an N22 domain having the amino acid sequence TMALTPFNEPIFTQIQYNPDRDTGEDTQLYLLSNATGTGWDPPGIPELILEGFPLWLIYWGFADFQKNLKKVTNIDTNYMLVAKTKFTQKPGTFYLVILNDTFVEGNSPYEKQPLPEDNIKWYPQVQYQLEAQNKLLQTGPFTPNIQGQLSDNISMFYKFYFK (SEQ ID NO:219), or an N22 domain sequence listed in any one of table a2, a4, A6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10.

1156. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the ORF1 molecule is localized to the nucleus of the cell.

1157. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity relative to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of a wild-type finger loop virus genomic sequence, e.g., as described herein.

1158. A polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method as described in any of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to about 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3450, 3460, 3470, 3480, 3490, 3500, 3510, 3520, 3530, 3540, 3550, 3560, 3570, or 3580 consecutive nucleotides of a genomic sequence of, for example, a wild-type nail-type torque virus (a-type torque virus) (e.g., an evolved branch 1, 2, or 3 of a-type torque virus), as described herein.

1159. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity relative to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of a wild type torque viruse (Betatorquevirus) genomic sequence, e.g., as described herein.

1160. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to about 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3120, 3130, 3140, 3141, or 3142 consecutive nucleotides of a wild-type c-type torque virus (gammatorquirus) genomic sequence, e.g., as described herein.

1161. A polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method as described in any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to at least about 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3450, 3460, 3470, 3480, 3490, 3500, 3510, 3520, 3530, 3540, 3550, 3560, 3570 or 3580 consecutive nucleotides of the genomic sequence of a wild-type torque ringvirus (e.g., the evolved branch 1, 2 or 3 of a torque virus) as described herein (e.g., about 500-.

1162. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least about 500, 1000, 1100, 1200, 1210 or 1219 consecutive nucleotides (e.g., about 500-1000, 500-1100, 500-1200, 500-1219, 1000-1100, 1000-1200 or 1000-1219 consecutive nucleotides) of the wild-type torque virus genomic sequence, e.g., as described herein.

1163. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with respect to at least about 500, 1000, 1500, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3120, 3130, 3140, 3141 or 3142 consecutive nucleotides (e.g., about 500-.

1164. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to about 500, 1000, 1100, 1200, 1210, or 1219 consecutive nucleotides of a wild-type TTMV-LY2 genomic sequence, e.g., as described herein.

1165. A polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises no more than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity relative to about 500, 1000, 1500, 2000,2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3550, 3560, 3570, 3580, or 3581 consecutive nucleotides of a wild-type TTV-tth8 genomic sequence, e.g., as described herein.

1166. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method of any of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises a deletion of at least 1578, 1579, 1580, 1590, 1600, 1650, 1700, 1750 or 2000 nucleotides relative to a wild-type ring virus genomic sequence, e.g., as described herein.

1167. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises a deletion of 1 to 99, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 10 to 99, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 20 to 99, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 30 to 99, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 40 to 99, 40 to 90, 40 to 80, 40 to 70, 40 to 60, or 40 to 50 nucleotides relative to a wild-type ring virus genomic sequence, e.g., as described herein.

1168. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule does not have a 100 nucleotide deletion, a 172 nucleotide deletion, or 1577 nucleotides relative to a wild-type finger ring virus genomic sequence, e.g., as described herein.

1169. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises three or more deletions relative to, for example, a wild-type finger ring virus genomic sequence as described herein.

1170. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a region having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)。

1171. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a region having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a nucleic acid sequence of seq id no:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)。

1172. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a region having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to nucleic acid sequence CCGCCATCTTAAGTAGTTGAGGCGGACGGTGGCGTGAGTTCAAAGGTCACCATCAGCCACACCTACTCAAAATGGTGG (SEQ ID NO: 161).

1173. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a region having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to nucleic acid sequence CTTAAGTAGTTGAGGCGGACGGTGGCGTGAGTTCAAAGGTCACCATCAGCCACACCTACTCAAAATGGTGGACAATTTCTTCCGGGTCAAAGGTTACAGCCGCCATGTTAAAACACGTGACGTATGACGTCACGGCCGCCATTTTGTGACACAAGATGGCCGACTTCCTTCC (SEQ ID NO: 162).

1174. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises at least 20, 25, 30, 31, 32, 33, 34, 35 or 36 contiguous nucleotides having a GC content of at least 80%.

1175. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises at least 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% or 80.6%.

1176. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises at least 36 contiguous nucleotides having a GC content of at least 80%.

1177. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, further comprising a nucleic acid sequence encoding, e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 and/or ORF3 of a finger loop virus, e.g., a wild-type finger loop virus, as described herein.

1178. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the promoter element, nucleic acid sequence encoding the effector, or protein binding sequence has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a promoter element, a nucleic acid sequence encoding an effector, or a protein binding sequence of a finger loop virus in any one of tables a1-a12, B1-B5, C1-C5, or 1-18, respectively.

1179. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a packaging region located 3' relative to a nucleic acid sequence encoding the effector.

1180. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises a packaging region located 5' relative to a nucleic acid sequence encoding the effector.

1181. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises a nucleic acid sequence encoding an finger loop viral protein having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence of ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 of a finger loop virus described herein.

1182. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule comprises single-stranded DNA.

1183. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the genetic element or isolated nucleic acid molecule is circular and/or integrates into the genome of a eukaryotic cell at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element that enters the cell.

1184. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid has at least 75% (e.g., 75%) of a portion of wild-type ring virus sequences (e.g., wild-type Torque Teno Virus (TTV), torque teno parvovirus (TTMV), or TTMDV sequences, e.g., wild-type ring virus sequences, e.g., listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17), or is comprised of about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1600, 1500, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 consecutive nucleotides, at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity.

1185. A polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method as described in any preceding embodiment, wherein the protein binds to a consensus 5' UTR sequence set forth in sequence table 20 having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity.

1186. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any preceding embodiment, wherein the protein binding sequence has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a consensus GC-rich sequence set forth in table 21.

1187. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the protein binding sequence has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a 5' UTR sequence set forth in table 38 and a GC-rich sequence set forth in table 39.

1188. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises a sequence having at least 85% sequence identity to the conserved domain of the 5' UTR of a finger ring virus of a nucleic acid sequence in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

1189. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element or isolated nucleic acid molecule comprises a sequence having at least 85% sequence identity to a GC-rich region of a finger ring virus of a nucleic acid sequence in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

1190. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the promoter element comprises an RNA polymerase II-dependent promoter, an RNA polymerase III-dependent promoter, a PGK promoter, a CMV promoter, an EF-1 a promoter, an SV40 promoter, a CAGG promoter or UBC promoter, a TTV viral promoter, tissue-specific U6 (pollliii), a minimal CMV promoter with an upstream DNA binding site for an activator protein (TetR-VP16, Gal4-VP16, dCas9-VP16, or the like).

1191. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the effector encodes a therapeutic agent, e.g., a therapeutic peptide or polypeptide or a therapeutic nucleic acid.

1192. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the effector comprises a regulatory nucleic acid, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or labels, antigens, peptides, synthetic or analog peptides of naturally bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degrading or self-destroying peptides, small molecules, immune effectors (e.g., affecting sensitivity to immune response/signal), death proteins (e.g., inducers of apoptosis or necrosis), non-lytic inhibitors of tumors (e.g., oncoprotein inhibitors), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein modifying enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors of enzymes, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands, antibodies, receptors, or CRISPR systems or components.

1193. The polypeptide, complex, finger ring body, isolated nucleic acid, cell, composition or method of any of the preceding embodiments, wherein the finger ring body is capable of autonomous replication.

1194. The isolated nucleic acid molecule of any one of the preceding embodiments, wherein the expression vector is selected from the group consisting of: plasmids, cosmids, artificial chromosomes, bacteriophages and viruses.

1195. An isolated cell comprising the isolated nucleic acid or finger ring of any of the preceding embodiments.

1196. The isolated cell of example 195, further comprising, e.g., ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 and/or ORF3 of a wild-type dactylvirus, e.g., as described herein.

1197. A method of delivering an effector to a subject, the method comprising administering to the subject a polypeptide, complex, finger ring body, isolated nucleic acid, isolated cell, or composition of any of the preceding embodiments; wherein the genetic element or isolated nucleic acid molecule encodes an effector, and wherein the effector is expressed in the subject.

1198. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a polypeptide, complex, finger ring, isolated nucleic acid, isolated cell, or composition of any of the preceding embodiments; wherein the genetic element or isolated nucleic acid molecule encodes a therapeutic agent, and wherein the therapeutic agent is expressed in the subject.

1199. A method of delivering an effector to an ex vivo cell or cell population (e.g., a cell or cell population obtained from a subject), the method comprising introducing into the cell or cell population a polypeptide, complex, finger loop, isolated nucleic acid, isolated cell, or composition of any one of the preceding embodiments; wherein the genetic element or isolated nucleic acid molecule encodes an effector, and wherein the effector is expressed in the cell or population of cells.

1200. The finger ring body of any one of the preceding embodiments, wherein the genetic element is single-stranded DNA and has one or both of the following properties: is circular and/or integrates into the genome of a eukaryotic cell at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell.

1201. The finger loop body of any one of the preceding embodiments, wherein the genetic element has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a wild-type finger loop viral sequence (e.g., a wild-type torque loop virus (TTV), parvovirus (TTMV), or TTMDV sequence, e.g., a wild-type finger loop viral sequence, e.g., as listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

1202. The finger loops of any of the preceding embodiments, wherein the protein binding sequence has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a consensus 5 'UTR sequence shown in table 38, or to a consensus GC-rich sequence shown in table 39, or to a consensus 5' UTR sequence shown in table 38 and a consensus GC-rich sequence shown in table 39.

1203. The finger ring of any one of the preceding embodiments, wherein the promoter element comprises an RNA polymerase II-dependent promoter, an RNA polymerase III-dependent promoter, a PGK promoter, a CMV promoter, an EF-1 α promoter, an SV40 promoter, a CAGG promoter or a UBC promoter, a TTV viral promoter, tissue-specific U6 (polllii), a minimal CMV promoter with an upstream DNA binding site for an activator protein (TetR-VP16, Gal4-VP16, dCas9-VP16, etc.).

1204. The finger ring body of any one of the preceding embodiments, wherein the promoter element comprises a TATA box.

1205. The finger loop body of any one of the preceding embodiments, wherein the promoter element is endogenous to a wild-type finger loop virus, e.g., a wild-type finger loop virus sequence listed in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17.

1206. The finger loop body of any one of the preceding embodiments, wherein the promoter element is exogenous to a wild-type finger loop virus, e.g., a wild-type finger loop virus sequence listed in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17.

1207. The finger ring body of any one of the preceding embodiments, wherein the effector encodes a therapeutic agent, e.g., a therapeutic peptide or polypeptide or a therapeutic nucleic acid.

1208. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a regulatory nucleic acid, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or labels, antigens, peptides, synthetic or analog peptides of naturally bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degrading or self-destroying peptides, small molecules, immune effectors (e.g., affecting sensitivity to immune response/signal), death proteins (e.g., inducers of apoptosis or necrosis), non-lytic inhibitors of tumors (e.g., oncoprotein inhibitors), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein modifying enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors of enzymes, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands, antibodies, receptors, or CRISPR systems or components.

1209. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a miRNA.

1210. The finger ring body of any one of the preceding embodiments, wherein the effector, e.g., miRNA, is targeted to a host gene, e.g., modulates expression of a gene, e.g., increases or decreases expression of a gene.

1211. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a miRNA and reduces expression of a host gene.

1212. The finger ring body of any one of the preceding embodiments, wherein the effector comprises a nucleic acid sequence of about 20-200, 30-180, 40-160, 50-140, or 60-120 nucleotides in length.

1213. The finger ring body of any one of the preceding embodiments, wherein the nucleic acid sequence encoding the effector is about 20-200, 30-180, 40-160, 50-140, or 60-120 nucleotides in length.

1214. The finger ring body of any one of the preceding embodiments, wherein the sequence encoding the effector is at least about 100 nucleotides in size.

1215. The finger ring body of any one of the preceding embodiments, wherein the sequence encoding the effector is from about 100 to about 5000 nucleotides in size.

1216. The finger loop of any one of the preceding embodiments, wherein the size of the sequence encoding the effector is about 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500 or 1500-2000 nucleotides.

1217. The finger loop body of any one of the preceding embodiments, wherein the sequence encoding the effector is located at, within, or near (e.g., 5' or 3 ') one or more of the ORF1 locus (e.g., the C-terminus of the ORF1 locus), the miRNA locus, the 5' noncoding region upstream of the TATA box, the 5' UTR, the 3' noncoding region downstream of the polya region, or the noncoding region upstream of the GC-rich region of the genetic element.

1218. The finger loop of example 1217, wherein the sequence encoding the effector is located between the poly a region and the GC-rich region of the genetic element.

1219. The finger loops of any of the preceding embodiments, wherein the protein binding sequence comprises a nucleic acid sequence having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a 5' UTR conserved domain or a GC-rich domain of a wild-type finger loop virus (e.g., a wild-type finger loop virus sequence set forth in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 6, 9, 11, 13, 15, or 17).

1220. The finger loops of any of the preceding embodiments, wherein the genetic element (e.g., the protein binding sequence of the genetic element) is at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to:

(i) A consensus 5' UTR nucleic acid sequence set forth in table 38;

(ii) an exemplary TTV 5' UTR nucleic acid sequence set forth in table 38;

(iii) a TTV-CT30F 5' UTR nucleic acid sequence as set forth in Table 38;

(iv) TTV-HD23a 5' UTR nucleic acid sequences shown in Table 38;

(v) a TTV-JA 205' UTR nucleic acid sequence set forth in Table 38;

(vi) a TTV-TJN 025' UTR nucleic acid sequence set forth in Table 38;

(vii) a TTV-tth 85' UTR nucleic acid sequence as set forth in Table 38;

(viii) a consensus GC enrichment region as shown in table 39;

(ix) exemplary TTV GC rich regions shown in table 39;

(x) TTV-CT30F GC-rich region shown in Table 39;

(xi) TTV-JA20 GC-rich region shown in Table 39;

(xii) TTV-TJN02 GC-rich region shown in Table 39;

(xiii) TTV-HD23a GC rich region shown in Table 39; or

(xiv) TTV-tth8 GC-rich region shown in Table 39.

1221. The finger ring body of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises an outer protein capable of specifically binding to a protein binding sequence.

1222. The finger ring body of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises one or more of: one or more glycosylated proteins, a hydrophilic DNA binding region, a arginine-rich region, a glutamine-rich region, an N-terminal poly-arginine sequence, a variable region, a C-terminal poly-glutamine/glutamine sequence, and one or more disulfide bonds.

1223. The finger ring body of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises one or more of the following characteristics: icosahedral symmetry, recognition and/or binding of molecules that interact with one or more host cell molecules to mediate entry into the host cell, lack of lipid molecules, lack of carbohydrates, pH and temperature stability, resistance to detergents, and being substantially non-immunogenic or substantially non-pathogenic in the host.

1224. The finger ring body of any of the preceding embodiments, wherein the protein comprises externally at least one functional domain providing one or more functions such as species and/or tissue and/or cell selectivity, genetic element binding and/or packaging, immune escape (substantially non-immunogenic and/or tolerogenic), pharmacokinetics, endocytosis and/or cell attachment, nuclear entry, intracellular regulation and localization, exocytosis regulation, reproduction, and nucleic acid protection.

1225. The finger loop of any one of the preceding embodiments, wherein the combined size of the portion of the genetic element other than the effector is about 2.5kb-5kb (e.g., about 2.8kb-4kb, about 2.8kb-3.2kb, about 3.6kb-3.9kb, or about 2.8kb-2.9kb), less than about 5kb (e.g., less than about 2.9kb, 3.2kb, 3.6kb, 3.9kb, or 4kb), or at least 100 nucleotides (e.g., at least 1 kb).

1226. The finger ring body of any one of the preceding embodiments, wherein the genetic element is single-stranded.

1227. The finger ring body of any one of the preceding embodiments, wherein the genetic element is circular.

1228. The finger ring body of any one of the preceding embodiments, wherein the genetic element is DNA.

1229. The finger ring body of any one of the preceding embodiments, wherein the genetic element is negative strand DNA.

1230. The finger ring body of any one of the preceding embodiments, wherein the genetic element comprises an episome.

1231. The finger ring body of any one of the preceding embodiments, wherein the finger ring body has a lipid content of less than 10%, 5%, 2% or 1% by weight, e.g., does not comprise a lipid bilayer.

1232. The finger ring body of any of the preceding embodiments, wherein the finger ring body is resistant to degradation by a detergent (e.g., a mild detergent, e.g., a bile salt, e.g., sodium deoxycholate) relative to a viral particle, e.g., a retrovirus, comprising an outer lipid bilayer.

1233. The finger ring bodies of embodiment 1232, wherein at least about 50% (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%) of the finger ring bodies are not degraded after incubation with a detergent (e.g., 0.5% by weight detergent) at 37 ℃ for 30 minutes.

1234. The finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a deletion of at least one element relative to a wild-type finger loop viral sequence, e.g., a wild-type TTV sequence or a wild-type TTMV sequence, e.g., an element listed in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

1235. The finger loop body of embodiment 1234, wherein the genetic element comprises a deletion comprising a nucleic acid sequence corresponding to:

(i) nucleotide 3436-3607 of the TTV-tth8 sequence, e.g., the nucleic acid sequences shown in Table 5;

(ii) nucleotides 574-;

(iii) nucleotide 1372-1431 of the TTMV-LY2 sequence, such as the nucleic acid sequence shown in Table 15; or

(iv) Nucleotide 2610-2809 of the TTMV-LY2 sequence, for example the nucleic acid sequence shown in Table 15.

1236. The finger loop body of any one of the preceding embodiments, wherein the genetic element comprises at least 72 nucleotides (e.g., at least 73, 74, 75, etc. nucleotides, optionally less than the full length of the genome) of a wild-type ring virus sequence (e.g., a wild-type Torque Teno Virus (TTV), parvovirus (TTMV), or TTMDV sequence, e.g., a sequence listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

1237. The finger ring body of any one of the preceding embodiments, wherein the genetic element further comprises one or more of the following sequences: sequences encoding one or more mirnas, sequences encoding one or more replication proteins, sequences encoding exogenous genes, sequences encoding therapeutic agent sequences, regulatory sequences (e.g., promoters, enhancers), sequences encoding one or more regulatory sequences (siRNA, lncRNA, shRNA) targeting endogenous genes, sequences encoding therapeutic mRNA or proteins, and sequences encoding cytolytic/cytotoxic RNA or proteins.

1238. The finger loop body of any one of the preceding embodiments, wherein the finger loop body further comprises a second genetic element, for example a second genetic element enclosed within the exterior of the protein.

1239. The finger loop body of embodiment 1238, wherein the second genetic element comprises a protein binding sequence, e.g., an external protein binding sequence, e.g., a packaging signal, e.g., a 5' UTR conserved domain or a GC-rich region, e.g., as described herein.

1240. The finger ring body of any one of the preceding embodiments, wherein the finger ring body does not detectably infect bacterial cells, e.g., infects less than 1%, 0.5%, 0.1%, or 0.01% of bacterial cells.

1241. The ring body of any one of the preceding embodiments, wherein the ring body is capable of infecting a mammalian cell, e.g., a human cell, e.g., an immune cell, a liver cell, an epithelial cell, e.g., in vitro.

1242. The finger ring body of any one of the preceding embodiments, wherein the genetic elements are integrated at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of the ring body entering the cell, e.g., wherein the finger ring body is non-integrated.

1243. The finger ring body of any of the preceding embodiments, wherein the genetic element is capable of replication (e.g., by rolling circle replication), e.g., capable of producing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10, per cell²、2x10²、5x10²、10³、2x10³、5x10³Or 10⁴The genetic elements of a genome equivalent are determined, for example, by quantitative PCR assays.

1244. The finger ring body of any one of the preceding embodiments, wherein the genetic element is capable of replication (e.g., by rolling circle replication), e.g., prior to delivery of the genetic element into a cellThe finger ring body is capable of producing at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10 more in said cell than in the amount present in said ring body ²、2x10²、5x10²、10³、2x10³、5x10³Or 10⁴The genomic equivalents of the individual genetic elements are determined, for example, by quantitative PCR assays.

1244a. the finger loop body of embodiment 1243 or 1244, wherein the protein exterior is provided in cis and/or in trans relative to the genetic element.

1244B. the finger loop body according to any one of examples 1243-1244A, wherein a helper nucleic acid (e.g.a helper virus) in the cell encodes the protein exterior or a part thereof (e.g.the ORF1 molecule).

1244℃ the finger loop body of any one of examples 1243-1244B, wherein one or more replication factors (e.g.replicase) are provided in cis and/or trans with respect to the genetic element.

1244d. the finger ring body of example 1244C, wherein a helper nucleic acid (e.g., a helper virus) in the cell encodes the one or more replication factors.

1245. The finger ring body of any one of the preceding embodiments, wherein the genetic element is incapable of replication, e.g., wherein the genetic element is altered at the origin of replication or lacks the origin of replication.

1246. The finger ring body of any one of the preceding embodiments, wherein the genetic element is incapable of self-replication, e.g., replication without integration into the host cell genome.

1247. The ring body of any one of the preceding embodiments, wherein the ring body is substantially non-pathogenic, e.g., does not induce detectable deleterious symptoms in a subject (e.g., increased cell death or toxicity relative to a subject not exposed to the ring body).

1248. The ring body of any one of the preceding embodiments, wherein the ring body is substantially non-immunogenic, e.g., does not induce a detectable and/or unwanted immune response, e.g., detected according to the method described in example 4.

1249. The finger ring body of embodiment 1248, wherein the substantially non-immunogenic finger ring body has a potency in a subject that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the potency in a reference subject that lacks an immune response.

1250. The finger loop body of embodiment 1248 or 1249, wherein the immune response comprises one or more antibodies specific for the finger loop body or a portion thereof, or a product encoded by a nucleic acid thereof; a cellular response (e.g., an immune effector cell (e.g., T cell or NK cell) response) against the finger ring body or cells comprising the finger ring body; or phagocytosis of the finger ring or a cell comprising the finger ring by macrophages.

1251. The finger ring body of any of the preceding embodiments, wherein the finger ring body is less immunogenic than an AAV, e.g., elicits an immune response that is less than that detected by an equivalent amount of an AAV, induces an antibody prevalence of less than 70% (e.g., less than about 60%, 50%, 40%, 30%, 20%, or 10% antibody prevalence), as determined by an assay described herein, or is substantially non-immunogenic.

1252. The finger ring of any of the preceding embodiments, wherein a population having at least 1000 of the finger rings is capable of delivering at least about 100 copies (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies) of the genetic element into one or more of the eukaryotic cells.

1253. The finger ring of any one of the preceding embodiments, wherein the population of finger rings (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of delivering the genetic element into at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the population of eukaryotic cells, e.g., wherein the eukaryotic cells are HEK293T cells, as described in example 22.

1254. The finger ring body of any one of the preceding embodiments, wherein the population of the finger ring body (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of separating at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 8,000, 1x10 per cell⁴、1x10⁵、1x10⁶、1x10⁷Or more copies of the genetic element into the population of eukaryotic cells, e.g., wherein the eukaryotic cell is a HEK293T cell, as described in example 22.

1255. The finger ring of any one of the preceding embodiments, wherein the population of the finger ring (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of the genetic element per cell) is capable of associating 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-10, 10-20, 20-50, 50-100, 100-1000, 1000-10, 10-20, 20-50, 100-and 1000-10 genome equivalents of each cell⁴、1x10⁴-1x10⁵、1x10⁴-1x10⁶、1x10⁴-1x10⁷、1x10⁵-1x10⁶、1x10⁵-1x10⁷Or 1x10⁶-1x10⁷The multiple copies of the genetic element are delivered into a population of the eukaryotic cell, e.g., wherein the eukaryotic cell is a HEK293T cell, as described in example 22.

1256. The finger ring body of any one of the preceding embodiments, wherein the finger ring body is present after at least two passages.

1257. The finger ring body of any one of the preceding embodiments, wherein the finger ring body is produced by a method comprising at least two passages.

1258. The finger ring body of any one of the preceding embodiments, wherein the finger ring body selectively delivers or is present at higher levels (e.g., preferably accumulates) in a desired cell type, tissue or organ (e.g., bone marrow, blood, heart, GI, skin, photoreceptors in the retina, epithelial lining, or pancreas).

1259. The finger ring body of any one of the preceding embodiments, wherein the eukaryotic cell is a mammalian cell, e.g., a human cell.

1260. The finger ring body of any one of the preceding embodiments, wherein the finger ring body or a copy thereof is detectable in the cell 24 hours (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 30 days, or 1 month) after delivery into the cell.

1261. The finger ring body of any of the preceding embodiments, wherein the finger ring body is present in the cell pellet and supernatant at a level of at least about 10, e.g., relative to the amount of finger ring body used to infect the cells, after 3-4 days post infection, e.g., using an infectivity assay, e.g., an assay according to example 7 ⁸Multiple (e.g., about 10)⁵10 times of⁶10 times of⁷10 times of⁸10 times of⁹Multiple or 10 times¹⁰Fold) genome equivalents/mL.

1262. A composition comprising the finger ring of any of the preceding embodiments.

1263. A pharmaceutical composition comprising the finger ring of any one of the preceding embodiments, and a pharmaceutically acceptable carrier or excipient.

1264. The composition or pharmaceutical composition of embodiment 1262 or 1263, which comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more ring bodies, e.g., synthetic ring bodies.

1265. The composition or pharmaceutical composition of any one of embodiments 1262-1264, comprising at least 10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹A synthetic ring finger.

1266. The composition or pharmaceutical composition of any one of embodiments 1262-1265, which has one or more of the following characteristics:

a) the pharmaceutical composition meets drug or Good Manufacturing Practice (GMP) standards;

b) the pharmaceutical composition is made according to Good Manufacturing Practice (GMP);

c) the pharmaceutical composition has a level of the pathogen below a predetermined reference value, e.g., is substantially free of the pathogen;

d) the pharmaceutical composition has a level of contaminants below a predetermined reference value, e.g., is substantially free of contaminants;

e) The pharmaceutical composition has a predetermined level of non-infectious particles or a predetermined ratio of particles to infectious units (e.g., <300:1, <200:1, <100:1, or <50:1), or

f) The pharmaceutical compositions are of low immunogenicity or are substantially non-immunogenic, e.g., as described herein.

1267. The composition or pharmaceutical composition of any of embodiments 1262-1266, wherein the pharmaceutical composition has a level of contaminants below a predetermined reference value, e.g., is substantially free of contaminants.

1268. The composition or pharmaceutical composition of embodiment 1267, wherein said contaminant is selected from the group consisting of: mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and/or host cell RNA), process impurities of animal origin (e.g., serum albumin or trypsin), replication-competent factors (RCA), such as replication-competent viruses or unwanted finger rings (e.g., finger rings other than the desired finger ring, e.g., a synthetic finger ring as described herein), free viral capsid proteins, exogenous factors, and aggregates.

1269. The composition or pharmaceutical composition of embodiment 1268, wherein said contaminant is host cell DNA and the threshold amount is about 10ng host cell DNA per said dose of pharmaceutical composition.

1270. The composition or pharmaceutical composition of any one of embodiments 1262-1269, wherein the pharmaceutical composition comprises less than 10% (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) by weight of contaminants.

1271. The use of a ring body, composition, or pharmaceutical composition of any of the preceding embodiments for treating a disease or disorder (e.g., as described herein) in a subject.

1272. The finger ring body, composition, or pharmaceutical composition of any of the preceding embodiments for use in treating a disease or disorder (e.g., as described herein) in a subject.

1273. A method of treating a disease or disorder (e.g., as described herein) in a subject, the method comprising administering to the subject a finger ring (e.g., a synthetic finger ring body) or a pharmaceutical composition described in any of the preceding embodiments.

1274. A method of modulating, e.g., enhancing or inhibiting a biological function (e.g., as described herein) in a subject, the method comprising administering to the subject a finger ring (e.g., a synthetic finger ring) or a pharmaceutical composition as described in any of the preceding embodiments.

1275. The method of any one of embodiments 1273-1274, wherein the ring does not comprise an exogenous effector.

1276. The method of any one of embodiments 1273-1275, wherein the finger ring comprises a wild-type finger ring virus, e.g., as described herein.

1277. The method of any one of embodiments 1273-1276, wherein administering the finger ring body, e.g., a synthetic finger ring body, results in delivery of the genetic element to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the target cell population in the subject.

1278. The method of any one of embodiments 1273-1277, wherein administering the finger ring body, e.g., a synthetic finger ring body, results in delivery of the effector to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the target cell population in the subject.

1279. The method of embodiment 1277 or 1278, wherein said target cells comprise mammalian cells, e.g., human cells, e.g., blood cells, skin cells, muscle cells, nerve cells, adipocytes, endothelial cells, immune cells, hepatocytes, lung epithelial cells, e.g., in vitro.

1280. The method of any one of embodiments 1277-1279, wherein the target cell is present in the liver or lung.

1281. The method of any one of embodiments 1277-1280, wherein the target cells into which the genetic element is delivered each receive at least 10, 50, 100, 500, 1000, 10,000, 50,000, 100,000 or more copies of the genetic element.

1282. The method of any one of embodiments 1273-1281, wherein the effector comprises a miRNA, and wherein the miRNA reduces the level of, e.g., at least 10%, 20%, 30%, 40% or 50% of a target protein or RNA in a cell or population of cells (e.g., into which the ring is delivered).

1283. A method of delivering a finger ring body (e.g., a synthetic finger ring body) to a cell, the method comprising contacting the finger ring body of any of the preceding embodiments with a cell (e.g., a eukaryotic cell, e.g., a mammalian cell).

1284. The method of embodiment 1283, further comprising contacting a helper virus with the cell, wherein the helper virus comprises a polynucleotide, e.g., a polynucleotide encoding an external protein, e.g., an external protein capable of binding the external protein binding sequence, and optionally a lipid envelope.

1285. The method of embodiment 1284, wherein the helper virus is contacted with the cell before, simultaneously with, or after the finger ring is contacted with the cell.

1286. The method of embodiment 1283, further comprising contacting a helper polynucleotide with the cell.

1287. The method of embodiment 1286, wherein the helper polynucleotide comprises a sequence polynucleotide encoding an external protein, e.g., an external protein capable of binding the external protein binding sequence and a lipid envelope.

1288. The method of embodiment 1286, wherein the helper polynucleotide is RNA (e.g., mRNA), DNA, plasmid, viral polynucleotide, or any combination thereof.

1289. The method of any one of embodiments 1286-1288, wherein the helper polynucleotide is contacted with the cell prior to, simultaneously with, or after the finger loop body is contacted with the cell.

1290. The method of any one of embodiments 1283-1289, further comprising contacting an accessory protein (e.g., a growth factor) with the cell.

1291. The method of embodiment 1290, wherein the accessory protein comprises a viral replication protein or a capsid protein.

1292. A host cell comprising the finger ring of any of the preceding embodiments.

1293. A nucleic acid molecule comprising a promoter element, a sequence encoding an effector (e.g., a payload), and an external protein binding sequence,

Wherein the nucleic acid molecule is single-stranded DNA, and wherein the nucleic acid molecule is circular and/or integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of a nucleic acid molecule entering a cell;

wherein the effector is not derived from TTV and is not SV 40-miR-S1;

wherein the nucleic acid molecule does not comprise a polynucleotide sequence of TTMV-LY;

wherein the promoter element is capable of directing expression of an effector in a eukaryotic cell.

1294. A genetic element comprising:

(i) a promoter element and a sequence encoding an effector, e.g., a payload, optionally wherein the effector is exogenous relative to a wild-type ring virus sequence;

(ii) at least 72 contiguous nucleotides having at least 75% sequence identity to a wild-type finger ring viral sequence (e.g., at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, or 150 nucleotides); or at least 100 contiguous nucleotides having at least 72% (e.g., at least 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99, or 100%) sequence identity to a wild-type finger ring virus sequence; and

(iii) Protein binding sequences, e.g. external protein binding sequences, and

wherein the nucleic acid construct is a single-stranded DNA; and

wherein the nucleic acid construct is circular and/or integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell.

1295. A method of manufacturing a ring body composition, the method comprising:

a) providing a host cell comprising one or more nucleic acid molecules encoding components of a finger loop, such as a synthetic finger loop described herein, e.g., wherein the finger loop comprises a protein external and a genetic element, such as a genetic element comprising a promoter element, an effector-encoding sequence (e.g., an endogenous or exogenous effector), and a protein-binding sequence (e.g., an external protein-binding sequence, such as a packaging signal);

b) producing a finger ring body from the host cell, thereby producing a finger ring body; and is

c) The finger ring is formulated, for example, as a pharmaceutical composition suitable for administration to a subject.

1296. A method of making a synthetic ring body composition, the method comprising:

a) providing a plurality of ring bodies, compositions or pharmaceutical compositions as described in any of the preceding embodiments;

b) Optionally evaluating the plurality of finger ring bodies, compositions or pharmaceutical compositions of any of the preceding embodiments for one or more of: contaminants, optical density measurements (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio, e.g., as determined by fluorescence and/or ELISA) as described herein; and is

1297. The method of embodiment 1296, wherein the ring body composition comprises at least 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵A ring body, or wherein the ring body composition comprises at least 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵Each ring genome/mL.

1298. The method of embodiment 1296 or 1297, wherein the finger ring body composition comprises at least 10ml, 20ml, 50ml, 100ml, 200ml, 500ml, 1L, 2L, 5L, 10L, 20L, or 50L.

1299. A reaction mixture comprising a finger ring body according to any preceding embodiment and a helper virus, wherein the helper virus comprises a polynucleotide, for example a polynucleotide encoding an external protein, for example an external protein capable of binding to the external protein binding sequence, and optionally a lipid envelope.

1300. A reaction mixture comprising a finger ring body as described in any one of the preceding embodiments and a second nucleic acid sequence encoding one or more of the amino acid sequences selected from any one of ORF2, ORF2/2, ORF2/3, ORF2t/3, ORF1, ORF1/1, or ORF1/2, or an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto, selected from table a2, a4, A6, a8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18, 20-37, or D1-D10.

1301. The reaction mixture of embodiment 1300, wherein the second nucleic acid sequence is a portion of the genetic element.

1302. The reaction mixture of example 1301, wherein the second nucleic acid sequence is not part of the genetic element, e.g., the second nucleic acid sequence is contained in a helper cell or helper virus.

1303. A synthetic finger ring comprising:

a genetic element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds the genetic element to the non-pathogenic external protein, and (iii) a sequence encoding an effector, e.g., a regulatory nucleic acid; and

A protein associated with (e.g., encapsulating or blocking) the genetic element.

1304. A pharmaceutical composition comprising:

a) a ring body, comprising:

a protein associated with (e.g., encapsulating or blocking) the genetic element; and

b) a pharmaceutical excipient.

1305. A pharmaceutical composition comprising:

a) at least 10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹A finger ring body (e.g., a synthetic finger ring body described herein) comprising:

a protein associated with (e.g., encapsulating or blocking) the genetic element;

b) a pharmaceutical excipient, and optionally,

c) less than a predetermined amount: mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and/or host cell RNA), process impurities of animal origin (e.g., serum albumin or trypsin), replication competent factors (RCA), such as replication competent viruses or unwanted finger rings, free viral capsid proteins, exogenous factors, endogenous factors, and/or aggregates.

1306. The ring body or composition of any of the preceding embodiments, further comprising at least one of the following features: the genetic element is a single-stranded DNA; the genetic element is circular; the ring body is non-integrated; the finger ring body has a sequence, structure and/or function based on an Ring virus or other non-pathogenic virus, and the finger ring body is non-pathogenic.

1307. The finger ring body or composition of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises a non-pathogenic outer protein.

1308. The finger ring body or composition of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises one or more of: one or more glycosylated proteins, a hydrophilic DNA binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal poly-arginine sequence, a variable region, a C-terminal poly-glutamine/glutamine sequence, and one or more disulfide bonds.

1309. The finger ring body or composition of any one of the preceding embodiments, wherein the proteinaceous outer portion comprises one or more of the following characteristics: icosahedral symmetry, recognition and/or binding of molecules that interact with one or more host cell molecules to mediate entry into the host cell, lack of lipid molecules, lack of carbohydrates, contain one or more desired carbohydrates (e.g., glycosylation), pH and temperature stability, detergent resistance, and are non-immunogenic or non-pathogenic in the host.

1310. The finger ring body or composition of any one of the preceding embodiments, wherein the sequence encoding the non-pathogenic external protein comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to one or more of the sequences listed in table 19, or a fragment thereof.

1311. The finger ring body or composition of any of the preceding embodiments, wherein the non-pathogenic external protein comprises at least one functional domain that provides one or more functions such as species and/or tissue and/or cell tropism, viral genome binding and/or packaging, immune escape (non-immunogenic and/or tolerogenic), pharmacokinetics, endocytosis and/or cell attachment, nuclear entry, intracellular regulation and localization, exocytosis regulation, reproduction, and nucleic acid protection.

1312. The finger ring body or composition of any one of the preceding embodiments, wherein the effector comprises a regulatory nucleic acid, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; therapeutic agents, such as fluorescent tags or labels, antigens, peptide therapeutics, synthetic or analog peptides of naturally occurring bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degraded or self-destructing peptides, and various degraded or self-destructing peptides, small molecules, immune effectors (e.g., affecting sensitivity to immune response/signal), death proteins (e.g., inducers of apoptosis or necrosis), non-lytic inhibitors of tumors (e.g., oncoprotein inhibitors), epigenetic modifiers, epigenetic enzymes, transcription factors, DNA or protein modifying enzymes, DNA intercalators, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors of enzymes, protein synthesis effectors or inhibitors, nucleases, protein fragments or domains, ligands or receptors, antibodies, or receptors, and the like, And CRISPR systems or components.

The finger ring body or composition of any one of the preceding embodiments, wherein the effector comprises an antibody molecule that binds VEGF or VEGFR.

1313. The finger ring body or composition of any one of the preceding embodiments, wherein the effector comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more miRNA sequences described herein.

1314. The finger ring body or composition of the preceding embodiments, wherein the effector, e.g., miRNA, is targeted to a host gene, e.g., modulates expression of the gene.

1315. The finger loops or compositions of the preceding embodiments, wherein the miRNA comprises a sequence at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more miRNA sequences described herein.

1316. The finger ring body or composition of any one of the preceding embodiments, wherein the genetic element further comprises one or more of the following sequences: sequences encoding one or more mirnas, sequences encoding one or more replication proteins, sequences encoding exogenous genes, sequences encoding therapeutic agent sequences, regulatory sequences (e.g., promoters, enhancers), sequences encoding one or more regulatory sequences (siRNA, lncRNA, shRNA) targeting endogenous genes, sequences encoding therapeutic mRNA or proteins, and sequences encoding cytolytic/cytotoxic RNA or proteins.

1317. The finger ring body or composition of any one of the preceding embodiments, wherein the genetic element has one or more of the following characteristics: non-integrated with the genome of the host cell, episomal nucleic acid, single-stranded DNA, about 1kb to 10kb, present in the nucleus, capable of binding to endogenous proteins, and producing micrornas that target host genes.

1318. The finger loop body or composition of any one of the preceding embodiments, wherein the genetic element comprises at least one viral sequence or is at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to one or more of the sequences listed in table 23 or fragments thereof (e.g., fragments encoding ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 molecules, and/or fragments comprising one or more of a TATA box, cap site, transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region).

1319. The finger ring body or composition of the previous embodiments, wherein the viral sequence is from at least one of: single-stranded DNA viruses (e.g., dactylovirus, binavirus, circovirus, geminivirus, kenovirus, filovirus, parvovirus, and sipara virus), double-stranded DNA viruses (e.g., adenovirus, pitchvirus, vesiculovirus, african swine fever virus, baculovirus, forskovirus, orbivirus, titre virus, adenovirus, herpesvirus, iridovirus, lipoviridae, linear virus, and poxvirus), RNA viruses (e.g., alphavirus, fungating baculovirus, hepatitis virus, barley virus, tobacco mosaic virus, tobacco rattle virus, trigonovirus, rubella virus, birnavirus, sacoviruses, split virus, and reovirus).

1320. The finger loops or compositions of the preceding embodiments, wherein the viral sequence is from one or more non-finger loop viruses, such as adenovirus, herpesvirus, pox virus, vaccinia virus, SV40, papilloma virus, RNA virus (e.g., retrovirus, e.g., lentivirus), single stranded RNA virus (e.g., hepatitis virus), or double stranded RNA virus (e.g., rotavirus).

1321. The finger loop body or composition of any one of the preceding embodiments, wherein the protein binding sequence interacts with an arginine-rich region external to the protein.

1322. The finger ring body or the composition of any one of the preceding embodiments, wherein the finger ring body is capable of replicating in a mammalian cell, such as a human cell.

1323. The finger ring body or composition of the preceding embodiments, wherein the finger ring body is non-pathogenic and/or non-integrating in a host cell.

1324. The finger ring body or composition of any one of the preceding embodiments, wherein the finger ring body is non-immunogenic in a host.

1325. The finger ring body or composition of any one of the preceding embodiments, wherein the finger ring body inhibits/enhances one or more viral properties, e.g., selectivity, e.g., infectivity, e.g., immunosuppression/activation, in a host or host cell.

1326. The finger loops or compositions of the preceding embodiments, wherein the amount of the finger loops is sufficient to modulate (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of phenotype, viral level, gene expression, competition with other viruses, disease state, etc.).

1327. The composition of any one of the preceding embodiments, further comprising at least one virus or vector comprising the genome of the virus, e.g., a variant of the finger loop, e.g., a common/native virus.

1328. The composition of any one of the preceding embodiments, further comprising a heterologous moiety, at least one small molecule, an antibody, a polypeptide, a nucleic acid, a targeting agent, an imaging agent, a nanoparticle, and combinations thereof.

1329. A vector comprising a genetic element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds the genetic element to the non-pathogenic external protein, and (iii) a sequence encoding an effector, such as a regulatory nucleic acid.

1330. The vector of the preceding embodiment, wherein the genetic element is incapable of integrating with the genome of the host cell.

1331. The vector of any one of the preceding embodiments, wherein the genetic element is capable of replication in a mammalian cell, e.g., a human cell.

1332. The vector of any one of the preceding embodiments, further comprising an exogenous nucleic acid sequence, e.g., selected to modulate the expression of a gene, e.g., a human gene.

1333. A pharmaceutical composition comprising a carrier as described in any one of the preceding embodiments and a pharmaceutical excipient.

1334. The composition of the preceding embodiment, wherein the vector is non-pathogenic and/or non-integrating in the host cell.

1335. The composition of any one of the preceding embodiments, wherein the vector is non-immunogenic in the host.

1336. The composition of the preceding embodiments, wherein the amount of the vector is sufficient to modulate (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of phenotype, viral level, gene expression, competition with other viruses, disease state, etc.).

1337. The composition of any one of the preceding embodiments, further comprising at least one virus or vector comprising the genome of the virus, e.g., a variant of the finger ring body, a common/native virus, a helper virus, a non-finger ring virus.

1338. The composition of any one of the preceding embodiments, further comprising a heterologous moiety, at least one small molecule, an antibody, a polypeptide, a nucleic acid, a targeting agent, an imaging agent, a nanoparticle, and combinations thereof.

1339. A method of producing, propagating and harvesting a finger ring body as described in any of the preceding embodiments.

1340. A method of designing and manufacturing a carrier as claimed in any one of the preceding embodiments.

1341. A method of administering to a subject an effective amount of a composition as described in any one of the preceding embodiments.

1342. A method of delivering a nucleic acid or protein payload to a target cell, tissue or subject, the method comprising contacting the target cell, tissue or subject with a nucleic acid composition comprising (a) a first DNA sequence derived from a virus, wherein the first DNA sequence is sufficient to produce particles capable of infecting the target cell, tissue or subject, and (a) a second DNA sequence encoding the nucleic acid or protein payload, the improvement comprising:

the first DNA sequence comprises at least 500 (at least 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000) nucleotides with at least 80% (at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity to a corresponding sequence set forth in any one of tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, or

The first DNA sequence encodes a sequence having at least 80% (at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity to an ORF listed in Table A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10, or

The first DNA sequence comprises a sequence having at least 90% (at least 95%, 97%, 99%, 100%) sequence identity to a consensus sequence listed in table 19.

1343. A method of delivering a nucleic acid or protein effector to a target cell, tissue or subject, the method comprising contacting the target cell, tissue or subject with a finger loop or a nucleic acid composition of any one of the preceding embodiments, the nucleic acid composition comprising (a) a first DNA sequence derived from a virus, wherein the first DNA sequence is sufficient to produce a finger loop of any one of the preceding embodiments that is capable of infecting the target cell, tissue or subject, and (a) a second DNA sequence encoding the nucleic acid or protein effector.

1344. A codon-optimized nucleic acid molecule encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type dactylovirus ORF1, ORF2 or ORF3 amino acid sequence.

1345. The codon-optimized nucleic acid molecule of example 1344 encoding an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type ring virus ORF1 amino acid sequence, e.g., as set forth in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37 or D1-D10.

1346. A pharmaceutical composition comprising:

(a) a ring body, such as a ring body as described in any of the preceding embodiments, and

(b) a carrier selected from a vesicle, a Lipid Nanoparticle (LNP), an erythrocyte, an exosome (e.g., a mammalian or plant exosome), or a fusion.

2001. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

(b) genetic elements comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector) and a protein binding sequence (e.g., an external protein binding sequence),

wherein the genetic element:

(i) has at least 72.2% (e.g., at least 72.2%, 72.3%, 72.4%, 72.5%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 1;

(ii) At least 68.4% (e.g., at least 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 3;

(iii) has at least 81.7% (e.g., at least 81.7%, 81.8%, 81.9%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 5;

(iv) at least 92.6% (e.g., at least 92.6%, 92.7%, 92.8%, 92.9%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 7;

(v) has at least 65% (e.g., at least 65%, 66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 9; or

(vi) Has at least 65% (e.g., at least 65%, 66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 11;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring is configured to deliver a genetic element into a eukaryotic cell.

2002. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

Wherein the genetic element comprises no more than about:

(i)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 1;

(ii)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 11160, 1170, 1171, 1172, 1173, or 1174 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 3;

(iii)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 5;

(iv)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, or 280 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 7;

(v)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 9; or

(vi)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to the ring virus sequences as set forth in table a 11;

Wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring is configured to deliver a genetic element into a eukaryotic cell.

2002. A finger ring body, the finger ring body comprising:

(a) a proteinaceous outer portion;

wherein the genetic element comprises no more than about:

(iii)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to a finger ring virus sequence as set forth in table B3;

Optionally, wherein the genetic element comprises at least one difference (e.g., mutation, chemical modification, or epigenetic change), e.g., insertion, substitution, enzymatic modification, and/or deletion, relative to a wild-type ring virus genomic sequence (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription start site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or GC-rich region);

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the ring is configured to deliver a genetic element into a eukaryotic cell.

2003. The finger loop body of any of the preceding embodiments, wherein the genetic element is not a naturally occurring sequence (e.g., comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic change), e.g., an insertion, a substitution, an enzymatic modification, and/or a deletion, e.g., of a domain (e.g., one or more of a TATA box, a cap site, a transcription initiation site, 5' UTR, Open Reading Frame (ORF), poly (a) signal, or a GC-rich region) relative to a wild-type finger loop virus sequence (e.g., a wild-type torque loop virus (TTV), torque loop parvovirus (TTMV), or TTMDV sequence, e.g., a wild-type finger loop virus sequence, e.g., as listed in any of tables B1-B5, a1, A3, a5, a7, a9, a11, 1, 3, 5, 7, 9, 11, or 13)).

2004. The finger ring of any one of the preceding embodiments, comprising a polypeptide comprising an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence of a finger ring virus ORF1 molecule (e.g., the finger ring virus ORF1 sequence listed in any of tables C1-C5, a2, a4, a6, A8, a10, or a 12).

2005. The finger ring body of embodiment 2004, wherein the protein externally comprises the polypeptide.

2006. The finger loop body of embodiment 2005, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) of the protein in the outer portion of the protein comprises the polypeptide.

2007. The finger ring body of any one of the preceding embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98, 99% or 100%) of the protein in the outer portion comprises an ORF1 molecule.

2008. The finger loop body of any one of the preceding embodiments, comprising a nucleic acid molecule (e.g., in a genetic element) encoding an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an amino acid sequence of a finger loop virus ORF1 molecule (e.g., a finger loop virus ORF1 sequence set forth in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12).

2009. The finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a region comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the following nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCc (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

2010. The finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a 5' UTR region and/or a GC-rich region as described herein (e.g., listed in tables 38 or 39, respectively).

2011. An isolated nucleic acid molecule (e.g., an expression vector) comprising a genetic element that:

(iv) at least 92.6% (e.g., at least 92.6%, 92.7%, 92.8%, 92.9%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a finger ring virus sequence as set forth in table a 7;

2012. An isolated nucleic acid molecule (e.g., an expression vector) comprising a genetic element comprising no more than about:

2012a. an isolated nucleic acid molecule (e.g., an expression vector) comprising a genetic element comprising no more than about:

(iii)1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 610, 620, 630, 640, 650, 660, 670, 671, or 672 nucleotide differences, e.g., substitutions, insertions, or deletions, relative to a finger ring virus sequence as set forth in table B3;

2013. The isolated nucleic acid molecule of any of the preceding embodiments, wherein the genetic element is not a naturally occurring sequence (e.g., comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), such as an insertion, substitution, enzymatic modification, and/or deletion, for example, a deletion of a domain (e.g., one or more of a TATA box, cap site, transcription initiation site, 5' Open Reading Frame (ORF), poly (utra) signal, or GC-rich region) relative to a wild-type dactylovirus sequence (e.g., a wild-type dactylovirus (TTV), minicirus finesse (TTMV), or TTMDV sequence, e.g., a wild-type dactylovirus sequence, e.g., as listed in any of tables B1-B5, a1, A3, a5, a7, a9, a11, 1, 3, 5, 7, 9, 11, or 13)).

2014. The isolated nucleic acid molecule of any of the preceding embodiments, wherein the isolated nucleic acid molecule comprises a nucleic acid encoding an ORF1 molecule (e.g., an ORF1 molecule listed in any one of tables C1-C5, a2, a4, a6, A8, a10, or a12, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto);

Wherein:

(i) at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more) of the amino acids of the ORF1 molecule are part of a β -sheet;

(ii) the secondary structure of the ORF1 molecule comprises at least three (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) β -sheets;

(iii) the secondary structure of the ORF1 molecule comprises a ratio of β -sheet to α -helix of at least 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1; and is

2015. The isolated nucleic acid molecule of any of the preceding embodiments, comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the following nucleic acid sequence:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

2016. The isolated nucleic acid molecule of any of the preceding embodiments comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

2017. The isolated nucleic acid molecule of any one of the preceding embodiments, wherein the genetic element further comprises one or more of: a TATA box, a start element, a cap site, a transcription start site, a 5' UTR conserved domain, an ORF1 coding sequence, an ORF1/1 coding sequence, an ORF1/2 coding sequence, an ORF2 coding sequence, an ORF2/2 coding sequence, an ORF2/3 coding sequence, an ORF2/3t coding sequence, an open reading frame region, a poly (a) signal, and/or a GC-rich region from an finger ring virus described herein (e.g., as listed in any of tables B1-B5, a1, A3, a5, a7, a9, or a 35 11), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

2018. The isolated nucleic acid molecule of any of the preceding embodiments, wherein the genetic element further comprises at least one or two copies (e.g., 1, 2, 3, 4, 5, or 6 copies) of, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, to a ring virus genomic sequence (e.g., as described herein, e.g., as listed in any of tables B1-B5, a1, A3, a5, a7, a9, a11, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

2019. The isolated nucleic acid molecule of any of the preceding embodiments, further comprising at least one additional copy (e.g., 1, 2, 3, 4, 5, or 6 copies total) of the genetic element.

2020. The isolated nucleic acid molecule of any one of the preceding embodiments, wherein the isolated nucleic acid molecule is circular.

2021. An isolated nucleic acid composition (e.g., comprising one, two, or more nucleic acid molecules) comprising a nucleic acid encoding the isolated nucleic acid of any of the preceding embodiments.

2022. The isolated nucleic acid of any of the preceding embodiments, wherein the genetic element further comprises a promoter element, a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an endogenous effector or an exogenous effector), and/or a protein binding sequence (e.g., an external protein binding sequence).

2022a. the isolated nucleic acid molecule of any of the preceding embodiments, wherein said genetic element comprises an insertion or substitution in the hypervariable domain (HVD) of said ORF 1.

2023. The finger ring body or isolated nucleic acid molecule of any one of the preceding embodiments, wherein the genetic element comprises one or more of: a TATA box, a start site, a 5' UTR conserved domain, ORF1, ORF2, sequences downstream of ORF2, ORF2, ORF3, and/or a GC-rich region, or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, e.g., as set forth in any one of tables B1-B5, a1, A3, a5, a7, a9, or a 11.

2024. The finger loop body or isolated nucleic acid of any one of the preceding embodiments, comprising (e.g., in the protein exterior) or encoding one or more polypeptides comprising: an amino acid sequence of ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 and/or ORF3 selected from any one of tables C1-C5, a2, a4, a6, A8, a10 or a12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

2025. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element comprises a sequence comprising at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

2026. The finger loop body or the isolated nucleic acid of embodiment 2025, wherein the genetic element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides having a GC content of at least 80%.

2027. The finger loop body or the isolated nucleic acid of embodiment 2025, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, or 80.6%.

2028. The finger loop body or the isolated nucleic acid of embodiment 2025, wherein the genetic element comprises at least 36 contiguous nucleotides having a GC content of at least 80%.

2029. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element comprises a region (e.g., a packaging region) comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 consecutive nucleotides of the following nucleic acid sequences:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)；

Or a nucleic acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

2030. The finger loop body or the isolated nucleic acid of embodiment 2029, wherein the packaging region is located 3' relative to the nucleic acid sequence encoding the effector.

2031. A polypeptide comprising one or more of:

(a) a first region comprising an amino acid sequence having at least 70% (e.g., at least about 70, 80, 90, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence of an ring virus ORF1 molecule described herein (e.g., the ring virus ORF1 sequence listed in any of tables C1-C5, a2, a4, a6, A8, a10, or a 12);

(b) A second region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a jellyroll region sequence of an ORF1 molecule described herein (e.g., the sequence of an ORF1 of an dactylovirus listed in any of tables C1-C5, a2, a4, a6, A8, a10, or a 12);

(c) a third region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an N22 domain sequence of an ORF1 molecule described herein (e.g., an ORF1 sequence of a circovirus listed in any of tables C1-C5, a2, a4, a6, A8, a10, or a 12); and/or

(d) A fourth region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an annular virus ORF 1C-terminal domain (CTD) sequence of an annular virus ORF1 molecule described herein (e.g., an annular virus ORF1 sequence listed in any of tables C1-C5, a2, a4, A6, a8, a10, or a 12);

Wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change), e.g., an insertion, substitution, chemical or enzymatic modification, and/or deletion, relative to a wild-type ORF1 protein (e.g., as described herein), e.g., a deletion of a domain (e.g., one or more of an arginine-rich region, a jelly roll domain, an HVR, N22, or a CTD, e.g., as described herein).

2031a. the polypeptide of example 2031, comprising one or more of:

(a) a first region comprising an amino acid sequence having at least 90% sequence identity to an arginine-rich region sequence of an ring virus ORF1 molecule described herein (e.g., the ring virus ORF1 sequence set forth in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12);

(b) a second region comprising an amino acid sequence having at least 90% sequence identity to a jellyroll region sequence of an ring virus ORF1 molecule described herein (e.g., the ring virus ORF1 sequence set forth in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12);

(c) a third region comprising an amino acid sequence having at least 90% sequence identity to an N22 domain sequence of an ORF1 molecule described herein (e.g., an ORF1 sequence of a finger listed in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12); and/or

(d) A fourth region comprising an amino acid sequence having at least 90% sequence identity to a ring virus ORF 1C-terminal domain (CTD) sequence of the ring virus ORF1 molecules described herein (e.g., the ring virus ORF1 sequences listed in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12);

2032. The polypeptide of embodiment 2031, wherein the polypeptide comprises:

(i) the first region and the second region;

(ii) the first region and the third region;

(iii) the first region and the fourth region;

(iv) the second region and the third region;

(v) the second region and the fourth region;

(vi) the third region and the fourth region;

(vii) the first region, the second region, and the third region;

(viii) The first region, the second region, and the fourth region;

(ix) the first region, the third region, and the fourth region; or

(x) The second region, the third region, and the fourth region.

2033. The polypeptide of embodiment 2031 or 2032, wherein said polypeptide comprises, in order from N-terminus to C-terminus, said first region, said second region, said third region and said fourth region.

2034. The polypeptide of any one of the preceding embodiments, further comprising an amino acid sequence, e.g., a hypervariable region (HVR) sequence (e.g., an HVR sequence of an ORF1 molecule of a finger virus as described herein), wherein the amino acid sequence comprises at least about 55 (e.g., at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (e.g., about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55-140, or 60-140 amino acids).

2035. The polypeptide of example 2034, wherein the HVR comprises an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an annular virus ORF1 HVR sequence of an annular virus ORF1 molecule described herein (e.g., an annular virus ORF1 sequence listed in any one of tables C1-C5, a2, a4, a6, A8, a10, or a 12).

2036. The polypeptide of embodiment 2034 or 2035, wherein the HVR sequence is located between the second region and the third region.

2037. The polypeptide of any one of embodiments 2034-2036, wherein the HVR comprises one or more characteristics of an HVR as described herein.

2038. A polypeptide comprising the amino acid sequence of, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to, ORF 4/1, ORF 4/2, ORF 4/3, ORF2 4/3 and/or ORF 4 in any one of tables C1-C5, a2, a4 or a4, and wherein said polypeptide further comprises a sequence (e.g., as described herein, e.g., as in any one of tables C4-C4, a4, or a 4), relative to, or at least one of the addition of a mutation, e.g., a chemical difference, e.g., a conjugate, in any one of tables C8672-C4-C, a 4/1, ORF 4/2, ORF 4, and/or at least one of its sequence identity thereto, and wherein said polypeptide is further comprises a4, e.g., a4, and wherein said polypeptide is a4, and said polypeptide is not a, Insertions, substitutions and/or deletions, for example deletions of a domain.

2039. A polypeptide comprising the amino acid sequence of ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 in any one of tables C1-C5, a2, a4, a6, A8, a10, or a12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

2040. A polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97% or 98% but not more than 99% sequence identity to an amino acid sequence of ORF1, ORF2, ORF2 or ORF3 selected from any one of tables C1-C5, a2, a4, a6, A8, a10 or a 12.

2041. A polypeptide having at least 1 but no more than 2, 5, 10, 20, 50 or 100 amino acid differences, e.g., substitutions, insertions or deletions, relative to an amino acid sequence of ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 and/or ORF3 selected from any one of tables C1-C5, a2, a4, a6, A8, a10 or a 12.

2042. The polypeptide of any one of the preceding embodiments, wherein the polypeptide is an isolated polypeptide.

2043. A composite, comprising:

(a) a polypeptide as described in any one of the preceding embodiments, and

2044. The composite of embodiment 2043, wherein the composite comprises one or more features of a composite as described herein.

2045. A fusion protein comprising a first amino acid sequence selected from the group consisting of ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 molecules in any one of tables C1-C5, a2, a4, a6, A8, a10, or a12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and a heterologous portion.

2046. A fusion protein comprising a first amino acid sequence selected from the ORF1 molecules in any one of tables C1-C5, a2, a4, a6, A8, a10, or a12, or having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, and a heterologous portion.

2047. The fusion protein of any one of the preceding embodiments, wherein the heterologous moiety comprises a targeting moiety.

2048. The fusion protein of any one of the preceding embodiments, wherein the first amino acid sequence comprises at least one difference (e.g., a mutation or chemical modification), e.g., a conjugation, addition, insertion, substitution, and/or deletion, e.g., a deletion of a domain, relative to wild-type dactylovirus ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 sequences (e.g., as described herein, e.g., as listed in any of tables C1-C5, a2, a4, A6, a8, a10, or a 12).

2049. A host cell comprising a finger ring body, an isolated nucleic acid, a fusion protein, or a polypeptide as described in any one of the preceding embodiments.

2050. A reaction mixture comprising a finger ring body and a helper virus as described in any preceding embodiment, wherein the helper virus comprises a polynucleotide, for example a polynucleotide encoding an external protein, for example an external protein that binds to the external protein binding sequence, and optionally a lipid envelope.

2051. A method of treating a disease or disorder in a subject, the method comprising administering to the subject a finger ring body, an isolated nucleic acid molecule, a fusion protein or polypeptide of any of the preceding embodiments, or a pharmaceutical composition of any of the preceding embodiments.

2052. The method of example 2051, wherein the disease or disorder is selected from the group consisting of an immune disorder, an infectious disease, an inflammatory disorder, an autoimmune disease, a cancer (e.g., a solid tumor), and a gastrointestinal disorder.

2053. The finger ring body, isolated nucleic acid, fusion protein, or polypeptide of any of the preceding embodiments for use in treating a disease or disorder in a subject.

2054. The use of embodiment 2053, wherein the disease or disorder is selected from an immune disorder, an infectious disease, an inflammatory disorder, an autoimmune disease, a cancer (e.g., a solid tumor, e.g., lung cancer), and a gastrointestinal disorder.

2055. The finger ring body, the isolated nucleic acid, the composition or the pharmaceutical composition of any one of the preceding embodiments, for use in treating a disease or disorder in a subject.

2055a. a ring body, an isolated nucleic acid, a composition or a pharmaceutical composition as described in any of the preceding embodiments for use as a medicament.

2056. A method of modulating, e.g., inhibiting or enhancing a biological function in a subject, the method comprising administering to the subject a finger ring body, an isolated nucleic acid, a fusion protein or polypeptide as described in any one of the preceding embodiments, or a pharmaceutical composition as described in any one of the preceding embodiments.

2057. A method of delivering a finger ring body to a cell, the method comprising contacting the finger ring body, isolated nucleic acid, fusion protein or polypeptide of any of the preceding embodiments with a cell, e.g., a eukaryotic cell, e.g., a mammalian cell.

2058. The method of embodiment 2057, further comprising contacting a helper virus with the cell, wherein the helper virus comprises a polynucleotide, e.g., a polynucleotide that encodes an external protein, e.g., an external protein that binds to the external protein binding sequence, and optionally a lipid envelope.

2059. The method of embodiment 2058, wherein the helper virus is contacted with the cell before, simultaneously with, or after the finger ring body is contacted with the cell.

2060. The method of embodiment 2057, further comprising contacting an helper polynucleotide with the cell.

2061. The method of embodiment 2060, wherein the helper polynucleotide comprises a sequence polynucleotide that encodes an external protein, e.g., an external protein that binds to the external protein binding sequence and a lipid envelope.

2062. The method of embodiment 2060, wherein the helper polynucleotide is an RNA (e.g., mRNA), a DNA, a plasmid, a viral polynucleotide, or any combination thereof.

2063. The method of any one of embodiments 2060-2062, wherein the helper polynucleotide is contacted with the cell prior to, simultaneously with, or after the finger ring body is contacted with the cell.

2064. The method of any one of embodiments 2057-2063, further comprising contacting an accessory protein with the cell.

2065. The method of embodiment 2064, wherein the helper protein comprises a viral replication protein or a capsid protein.

2066. A method of delivering a nucleic acid or protein effector to a target cell, tissue or subject, the method comprising contacting the target cell, tissue or subject with a nucleic acid composition comprising (a) a first DNA sequence derived from a virus, wherein the first DNA sequence is sufficient to produce particles that infect the target cell, tissue or subject, and (a) a second DNA sequence encoding the nucleic acid or protein effector, the improvement comprising:

The first DNA sequence comprises at least 500 (at least 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1600, 1800, 2000) nucleotides having at least 80% (at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity to a corresponding sequence set forth in any one of tables B1-B5, A1, A3, A5, A7, A9, or A11, or

The first DNA sequence encodes a sequence having at least 80% (at least 85%, 90%, 95%, 97%, 99%, 100%) sequence identity to an Ring virus ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, and/or ORF3 molecule (e.g., listed in any one of tables C1-C5, a2, a4, A6, A8, A10, or A12).

2067. A method of manufacturing a ring body composition, the method comprising:

a) providing a host cell comprising one or more nucleic acid molecules encoding a component of a finger loop body as described in any of the preceding embodiments, e.g., a synthetic finger loop body as described herein, e.g., wherein the finger loop body comprises a protein external and a genetic element, e.g., a genetic element comprising a promoter element, an effector-encoding sequence (e.g., an endogenous effector or an exogenous effector), and a protein-binding sequence (e.g., an external protein-binding sequence, e.g., a packaging signal);

b) Producing a finger ring body from the host cell, thereby producing a finger ring body; and is

c) Formulating the ring body, e.g., as a pharmaceutical composition suitable for administration to a subject;

optionally, wherein the one or more nucleic acid molecules encode an accessory protein.

2068. A method of manufacturing a ring body composition, the method comprising:

a) providing a plurality of finger rings as described in any of the preceding embodiments;

b) optionally evaluating the plurality of finger rings of any one of the preceding embodiments for one or more of: contaminants, optical density measurements (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio) as described herein; and is

2069. The method of embodiment 2068, wherein the ring body composition comprises at least 10⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵A ring body.

2070. The method of embodiment 2068 or 2069, wherein the ring body composition comprises at least 10ml, 20ml, 50ml, 100ml, 200ml, 500ml, 1L, 2L, 5L, 10L, 20L, or 50L.

2071. The finger ring body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element is configured to replicate in a mammalian cell, e.g., a human cell.

2072. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element further comprises an exogenous nucleic acid sequence, e.g., selected for modulating the expression of a gene, e.g., a human gene.

2073. The finger ring body or the isolated nucleic acid of any of the preceding embodiments, wherein at least 60% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the protein binding sequence consists of G or C.

2074. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element comprises a sequence of at least 80, 90, 100, 110, 120, 130, or 140 nucleotides in length, at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) or about 70-100%, 75-95%, 80-95%, 85-95%, or 85-90% of the positions of the sequence consisting of a G or a C.

2075. The finger ring body or the isolated nucleic acid of any of the preceding embodiments, wherein the protein binding sequence binds to an arginine-rich region outside of the protein.

2076. The finger ring body or the isolated nucleic acid of any of the preceding embodiments, wherein the proteinaceous outer comprises an outer protein that specifically binds to the protein binding sequence.

2077. The finger loop or the isolated nucleic acid of any of the preceding embodiments, wherein the combined size of the portion of the genetic element other than the effector is about 2.5kb-5kb (e.g., about 2.8kb-4kb, about 2.8kb-3.2kb, about 3.6kb-3.9kb, or about 2.8kb-2.9kb), less than about 5kb (e.g., less than about 2.9kb, 3.2kb, 3.6kb, 3.9kb, or 4kb), or at least 100 nucleotides (e.g., at least 1 kb).

2078. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element is single-stranded.

2079. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element is circular.

2080. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element is DNA.

2081. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element is a negative strand DNA.

2082. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the genetic element comprises an episome.

2083. The finger loop body or the isolated nucleic acid of any of the preceding embodiments, wherein the finger loop body is present at a higher level (e.g., preferentially accumulates in) in a desired organ or tissue relative to other organs or tissues.

2084. The finger ring body or the isolated nucleic acid of any of the preceding embodiments, wherein the eukaryotic cell is a mammalian cell, e.g., a human cell.

2085. A composition comprising a finger ring body or an isolated nucleic acid as described in any one of the preceding embodiments.

2086. A pharmaceutical composition comprising a finger ring body or an isolated nucleic acid and a pharmaceutically acceptable carrier or excipient.

2087. A pharmaceutical composition comprising:

a) at least 10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹A finger ring body as described in any of the preceding embodiments;

b) a pharmaceutical excipient, and optionally,

2088. The composition or pharmaceutical composition of embodiment 2085 or 2086, comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more, finger rings, e.g., synthetic finger rings.

2089. The composition or pharmaceutical composition of any one of embodiments 2085-2088, comprising at least 10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹A ring body.

2090. A pharmaceutical composition comprising:

a) at least 10³、10⁴、10⁵、10⁶、10⁷、10⁸Or 10⁹As in any of the previous embodimentsThe ring body of one finger ring;

b) a pharmaceutical excipient, and optionally,

2091. The composition or pharmaceutical composition of any one of embodiments 2085-2090, having one or more of the following characteristics:

a) the pharmaceutical composition meets drug or Good Manufacturing Practice (GMP) standards;

b) the pharmaceutical composition is made according to Good Manufacturing Practice (GMP);

c) The pharmaceutical composition has a level of the pathogen below a predetermined reference value, e.g., is substantially free of the pathogen;

d) the pharmaceutical composition has a level of contaminants below a predetermined reference value, e.g., is substantially free of contaminants;

e) the pharmaceutical composition has a predetermined level of non-infectious particles or a predetermined ratio of particles to infectious units (e.g., <300:1, < 200:1, < 100:1, or <50:1), or

f) The pharmaceutical compositions are of low immunogenicity or are substantially non-immunogenic, e.g., as described herein.

2092. The composition or pharmaceutical composition of any one of embodiments 2085-2091, wherein the pharmaceutical composition has a level of contaminants below a predetermined reference value, e.g., is substantially free of contaminants.

2093. The composition or pharmaceutical composition of embodiment 92, wherein the contaminant is selected from the group consisting of: mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and/or host cell RNA), process impurities of animal origin (e.g., serum albumin or trypsin), replication-competent factors (RCA), such as replication-competent viruses or unwanted finger rings (e.g., finger rings other than the desired finger ring, e.g., a synthetic finger ring as described herein), free viral capsid proteins, exogenous factors, and aggregates.

2094. The composition or pharmaceutical composition of embodiment 2093, wherein the contaminant is host cell DNA and the threshold amount is about 500ng host cell DNA per the dose pharmaceutical composition.

2095. The composition or pharmaceutical composition of any one of embodiments 2085-2094, wherein the pharmaceutical composition comprises less than 10% (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) by weight of contaminants.

2096. The method of any one of the preceding embodiments, wherein the finger ring body does not comprise an exogenous effector.

2097. The method of any one of the preceding embodiments, wherein administration of the ring body, e.g., a synthetic ring body, results in delivery of the genetic element to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the target cell population in the subject.

2098. The method of any one of the preceding embodiments, wherein administration of the ring body, e.g., a synthetic ring body, results in delivery of the exogenous effector to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of the target cell population in the subject.

2099. The method of embodiment 2097 or 2098, wherein the target cell comprises, e.g., an in vitro mammalian cell, e.g., a human cell, e.g., an immune cell, a liver cell, a lung epithelial cell.

2100. The method of any one of embodiments 2097-2099, wherein the target cell is present in the liver or lung.

2101. The method of any one of embodiments 2097-2100, wherein the target cells into which the genetic element is delivered each receive at least 10, 50, 100, 500, 1000, 10,000, 50,000, 100,000 or more copies of the genetic element.

2102. The method of any one of the preceding embodiments, wherein the effector comprises a miRNA, and optionally wherein the miRNA reduces the level of, e.g., at least 10%, 20%, 30%, 40% or 50% of a target protein or RNA in a cell or population of cells (e.g., into which the finger ring is delivered).

2103. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the genetic element (e.g., the 5' UTR of the genetic element) is physically associated (e.g., bound) to the protein exterior (e.g., an ORF1 molecule external to the protein).

2104. A polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method according to any one of the preceding embodiments, wherein the genetic element enclosed outside the protein is resistant to endonuclease digestion, e.g. as determined according to the method described in Martin et al (2013, hum. gene ther. methods [ human gene therapy and methods ]24(4):253-269, incorporated herein in its entirety by reference); optionally, the amount of DNase used therein is about 60U/ml or about 300U.

2105. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element comprises a sequence of at least 100 nucleotides in length, the sequence consisting of a G or a C in at least 80% of the positions.

2106. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element is a circular single-stranded DNA.

2107. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element does not comprise one or more bacterial plasmid elements (e.g., a bacterial origin of replication or a selectable marker, e.g., a bacterial resistance gene).

2108. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element is integrated at a frequency of less than 1% of the finger loop entering a mammalian cell.

2109. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the promoter element is exogenous or endogenous to a wild-type finger loop virus.

2110. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the exogenous effector is a therapeutic exogenous effector, e.g., a therapeutic peptide, a therapeutic polypeptide, or a therapeutic nucleic acid (e.g., miRNA).

2111. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein a population having at least 1000 (e.g., at least 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 50,000, 75,000, 100,000, 200,000, 500,000, 1,000,000 or more) of the finger rings delivers at least 100 (e.g., at least 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 50,000, 100,000 or more) copies of the genetic element into one or more mammalian cells.

2112. The polypeptide, complex, finger ring body, isolated nucleic acid, cell, composition or method of any one of the preceding embodiments, wherein the finger ring body comprises one or more polypeptides comprising one or more of: an amino acid sequence selected from the group consisting of ORF2, ORF2/2, ORF2/3, ORF1, ORF1/1 or ORF1/2 (e.g., as described herein) of a finger virus or an amino acid sequence having at least 95% sequence identity thereto.

2113. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element comprises a nucleic acid sequence encoding: an amino acid sequence selected from the group consisting of ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1 or ORF1/2 (e.g., as described herein), or an amino acid sequence having at least 95% sequence identity thereto.

2114. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the finger loop does not comprise a polynucleotide encoding one or both of a replication factor and a capsid protein, or wherein the finger loop is replication defective.

2115. The polypeptide, complex, finger ring, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the finger ring is contacted with the cell in vitro or in vivo.

2116. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition, or method of any one of the preceding embodiments, wherein the finger loop does not comprise a polypeptide having at least 95% sequence identity to finger ring virus ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 (e.g., as described herein).

2117. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition or method of any of the preceding embodiments, wherein the genetic element is capable of being amplified by rolling circle replication (e.g., in a cell, e.g., a host cell, e.g., a mammalian cell, e.g., a human cell, e.g., HEK293T or a549 cell), e.g., producing at least 2, 4, 8, 16, 32, 64, 128, 256, 518, or 1024 copies.

2118. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element is produced from a double stranded circular DNA molecule.

2119. The polypeptide, complex, finger loop body, isolated nucleic acid, cell, composition or method of embodiment 2118, wherein the double stranded circular DNA molecule is produced by in vitro circularization.

2118. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element is produced from a DNA molecule comprising two copies of the nucleic acid sequence of the genetic element.

2119. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein two copies of the nucleic acid sequence of the genetic element are arranged in tandem in the DNA molecule.

2120. A nucleic acid molecule comprising two copies of a nucleic acid sequence comprising a 5' UTR referring to a genomic genetic element (e.g., a genetic element as described in any of the preceding embodiments).

2121. A nucleic acid molecule comprising a promoter element; a nucleic acid sequence encoding an exogenous effector; a 5' UTR sequence as set forth in any one of tables B1-B5, or a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto; and the GC-rich region set forth in any one of tables B1-B5, or a nucleic acid sequence having at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity thereto.

2122. The nucleic acid molecule of embodiment 2121, wherein the nucleic acid molecule is single-stranded or double-stranded.

2123. The nucleic acid molecule of embodiment 2121, wherein the nucleic acid molecule is circular.

2124. The polypeptide, complex, finger loop, isolated nucleic acid, cell, composition or method of any preceding embodiment, wherein the genetic element comprises a 5' UTR comprising the nucleic acid sequence:

CGGGAGCCX₁CGAGGTGAGTGAAACCACCGAGGTCTAGGGGCAATTCGGGCTAGGGCAGTCTAGCGGAACGGG, wherein X₁Is C or absent, or a nucleic acid sequence at least 95% identical thereto.

3001. A synthetic finger ring comprising:

(I) a genetic element comprising:

(a) a promoter element which is capable of expressing a promoter sequence,

(b) a nucleic acid sequence encoding an exogenous effector, wherein the nucleic acid sequence is operably linked to the promoter element,

wherein the exogenous effector is a secreted therapeutic agent selected from the group consisting of:

(i) antibody molecules that bind a growth factor (e.g., VEGF) or a growth factor receptor (e.g., VEGF receptor), or a cytokine or cytokine receptor;

(ii) an enzyme, such as ADAMTS13 or a functional variant thereof;

(iii) a hormone, such as a hormone of table B or a functional variant thereof;

(iv) a cytokine, such as a cytokine from table a (e.g., IL2 or TNF-a), or a functional variant thereof);

(v) complement inhibitors, such as C3 inhibitors (e.g., compstatin) or pan complement inhibitors (e.g., PgtE);

(vi) Growth factors, such as those of Table C,

(vi) growth factor inhibitors, such as those of Table C,

(vii) coagulation factors, e.g. of Table D, or

(viii) Modulators of STING/cGAS signaling;

(i) 54, or a nucleic acid sequence which is at least 85% identical thereto;

(ii) 113, 114, 115, 116, 117, 118, 119 or a nucleic acid sequence at least 85% identical thereto; or

(iii) 61, or a nucleic acid sequence which is at least 85% identical thereto;

(II) a proteinaceous outer portion comprising an ORF1 molecule;

wherein the genetic element is enclosed within the protein exterior; and is

Wherein the synthetic finger loop body is capable of delivering the genetic element into a human cell.

3002. The synthetic finger ring body of embodiment 3001, wherein the exogenous effector is an antibody molecule that binds a growth factor or a growth factor receptor, such as VEGF or VEGFR.

3003. The synthetic finger ring body of embodiment 3001, wherein the secreted therapeutic agent is a secreted polypeptide.

3004. The synthetic finger ring body of embodiment 3001, wherein said ORF1 molecule comprises the amino acid sequence of SEQ ID NO:217, or an amino acid sequence at least 90% identical thereto.

3005. The synthetic finger loop body of any one of the preceding embodiments, wherein the ORF1 molecule is encoded by nucleotides 612-2612 of SEQ ID NO 54.

3006. The synthetic finger loop of any one of the preceding embodiments, wherein the genetic element comprises the nucleic acid sequence of nucleotides 2868-2929 of SEQ ID NO:54, or a nucleic acid sequence having at least 85% sequence identity thereto.

3007. The synthesis of any one of the preceding embodiments is a loop, wherein the ORF1 molecule comprises an amino acid sequence comprising one or more of: an amino acid sequence of an arginine-rich region, a jelly roll domain, a hypervariable domain, an N22 domain, and/or a C-terminal domain as set forth in table 16, or an amino acid sequence having at least 85% identity thereto.

3008. The synthetic finger ring body of any one of the preceding embodiments, wherein the ORF1 molecule comprises the amino acid sequence of SEQ ID NO:58, or a nucleic acid sequence having at least 85% sequence identity thereto.

3009. The synthetic finger ring of any of the preceding embodiments, further comprising a polypeptide comprising the amino acid sequence of ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16, or an amino acid sequence at least 85% identical thereto.

3010. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element encodes an amino acid sequence of, or an amino acid sequence having at least 85% identity to, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16.

3011. The synthetic finger loop body of any one of the preceding embodiments, wherein the synthetic finger loop body does not comprise a polypeptide comprising the amino acid sequence of ORF2, ORF2/2, ORF2/3, TAIP, ORF1/1, or ORF1/2, or an amino acid sequence at least 85% identical thereto, as set forth in table 16.

3012. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element does not encode an amino acid sequence of, or has at least 85% identity to, ORF1, ORF2, ORF2/2, ORF2/3, ORF1/1, or ORF1/2 as set forth in table 16.

3013. The synthetic finger ring body of any one of the preceding embodiments, wherein the ORF1 molecule comprises the amino acid sequence YNPX ²DXGX²N (SEQ ID NO:829), wherein XⁿEach independently is a contiguous sequence of any n amino acids.

3014. The synthetic finger ring body of embodiment 3013, wherein the ORF1 molecule further comprises a nucleotide sequence located in the amino acid sequence YNPX²DXGX²N (SEQ ID NO:829) flanking a first beta-strand and a second beta-strand, e.g., wherein the first beta-strand comprises the amino acid sequence YNPX²DXGX²N (SEQ ID NO:829) and/or wherein the second beta-chain comprises the amino acid sequence YNPX²DXGX²The second asparagine (N) residue (from N to C) of N (SEQ ID NO: 829).

3015. The synthetic finger ring body of any one of the preceding embodiments, wherein the ORF1 molecule comprises, in order in the N-terminal to C-terminal direction, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and a ninth beta strand.

3016. The synthetic finger loop body of any of the preceding embodiments, wherein the genetic element is capable of being amplified in a host cell by rolling circle replication, e.g., producing at least 8 copies.

3017. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element is single-stranded.

3018. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element is circular.

3019. The synthetic finger ring body of any one of the preceding embodiments, wherein the genetic element is DNA.

3020. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element is negative strand DNA.

3021. The synthetic finger ring body of any one of the preceding embodiments, wherein the genetic element is integrated at a frequency of less than 10%, 8%, 6%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1% of the finger ring body entering the cell, e.g., wherein the synthetic finger ring body is non-integrated.

3022. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a sequence of a consensus 5' UTR nucleic acid sequence set forth in table 16-1.

3023. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a sequence of a consensus GC-rich region shown in table 16-2.

3024. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element comprises a sequence of at least 100 nucleotides in length, at least 70% (e.g., about 70% -100%, 75% -95%, 80% -95%, 85% -95%, or 85% -90%) of the sequence consisting of a G or a C at a position.

3025. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element comprises the nucleic acid sequence of SEQ ID NO: 120.

3026. The synthetic finger ring body of any one of the preceding embodiments, wherein the promoter element is exogenous to a wild-type finger ring virus.

3027. The synthetic finger ring body of any one of the preceding embodiments, wherein the promoter element is endogenous to a wild-type finger ring virus.

3028. The synthetic finger ring body of any of the preceding embodiments, wherein the exogenous effector comprises a peptide, a synthetic or analogous peptide of a naturally occurring biologically active peptide, an agonist peptide or antagonist peptide, a competitive inhibitor of an enzyme, a ligand, or an antibody.

3029. The synthetic finger loop of any of the preceding embodiments, wherein the nucleic acid sequence encoding the exogenous effector is about 20-200, 30-180, 40-160, 50-140, 60-120, 200-2000, 200-500, 500-1000, 1000-1500, or 1500-2000 nucleotides in length.

3030. The synthetic finger loop body of any one of the preceding embodiments, wherein the genetic element is about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0kb in length.

3031. The synthetic finger ring body of any of the preceding embodiments, wherein the synthetic finger ring body is capable of infecting a human cell, e.g., a blood cell, a skin cell, a muscle cell, a nerve cell, an adipocyte, an endothelial cell, an immune cell, a liver cell, a lung epithelial cell, e.g., in vitro.

3032. The synthetic finger ring of any of the preceding embodiments, which is substantially non-immunogenic, e.g., does not induce a detectable and/or unwanted immune response, e.g., detected according to the method described in example 4.

3033. The synthetic finger ring body of embodiment 3032, wherein the substantially non-immunogenic finger ring body has a potency of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the potency in a reference subject that lacks an immune response in the subject.

3034. The synthetic finger ring of any one of the preceding embodiments, wherein a population having at least 1000 of the finger rings is capable of delivering at least about 100 copies (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies) of the genetic element into one or more human cells.

3035. A pharmaceutical composition comprising the synthetic finger ring of any one of the preceding embodiments, and a pharmaceutically acceptable carrier or excipient.

3036. A pharmaceutical composition as described in embodiment 3035 comprising at least 10³、10⁴、105、10⁶、10⁷、10⁸Or 10⁹A synthetic ring finger.

3037. A pharmaceutical composition as described in example 3035 or 3036, wherein the pharmaceutical composition has a predetermined ratio of particles to infectious units (e.g., <300:1, <200:1, <100:1, or <50: 1).

3038. A reaction mixture, comprising:

(i) a first nucleic acid (e.g., a double-stranded or single-stranded circular DNA) comprising the sequence of the genetic element of the synthetic finger loop described in any of the preceding embodiments, and

3039. A reaction mixture as described in example 3038, wherein said first nucleic acid and said second nucleic acid are in the same nucleic acid molecule.

3040. A reaction mixture as described in example 3038, wherein said first nucleic acid and said second nucleic acid are different nucleic acid molecules.

3041. The reaction mixture of embodiment 3038, wherein said first nucleic acid and said second nucleic acid are different nucleic acid molecules and wherein said second nucleic acid is provided as a double-stranded circular DNA.

3042. The reaction mixture of embodiment 3038, wherein said first nucleic acid and said second nucleic acid are different nucleic acid molecules and wherein said first nucleic acid and said second nucleic acid are provided as double-stranded circular DNA.

3043. The reaction mixture of embodiment 3040, wherein the second nucleic acid sequence is contained in a helper cell or helper virus.

3044. A method of making a synthetic finger ring, the method comprising:

a) providing a host cell comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence of the genetic element of the synthetic finger loop body as described in any one of the preceding embodiments, and

b) Incubating the host cell under conditions suitable for the preparation of a synthetic finger ring body;

Thereby preparing the synthetic finger ring.

3045. The method of embodiment 3044, further comprising, prior to step (a), introducing the first nucleic acid molecule and/or the second nucleic acid molecule into the cell.

3046. The method of example 3045, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

3047. The method of any one of embodiments 3044 or 3045, wherein the second nucleic acid molecule is integrated into the genome of the host cell.

3048. The method of any one of embodiments 3044 and 3047, wherein the second nucleic acid molecule is a helper (e.g., a helper plasmid, or a helper virus genome).

3049. The method as described in example 3044 and 3047, wherein the second nucleic acid molecule encodes a polypeptide comprising the amino acid sequence [ W/F ]]X⁷HX³CX¹CX⁵ORF2 molecule of H (SEQ ID NO:949), wherein XⁿIs a contiguous sequence of any n amino acids.

3050. A method of manufacturing a synthetic finger ring formulation, the method comprising:

a) providing a plurality of synthetic finger ring bodies as described in example 3001-3034, a pharmaceutical composition as described in any one of examples 3035-3037, or a reaction mixture as described in any one of examples 3038-3043;

b) Optionally evaluating the plurality of synthetic ring bodies as described in example 3001-3034 for one or more of: contaminants, optical density measurements (e.g., OD 260), particle number (e.g., by HPLC), infectivity (e.g., particle: infectious unit ratio) as described herein; and is

3051. A host cell, comprising:

(i) a first nucleic acid molecule comprising a nucleic acid sequence of the genetic element of the synthetic finger loop body as described in any one of the preceding embodiments, and

3052. A method of delivering an exogenous effector (e.g., a therapeutic exogenous effector) to a mammalian cell, the method comprising:

(a) providing a synthetic ring body according to any one of the preceding embodiments; and is

(b) Contacting a mammalian cell with the synthetic finger ring;

wherein the synthetic finger ring body is capable of delivering the genetic element into the mammalian cell; and

thereby delivering the therapeutic exogenous effector to the mammalian cell.

3053. Use of a synthetic finger ring body as described in any one of examples 3001-3034 or a pharmaceutical composition as described in any one of examples 3035-3037 for delivering the genetic element to a host cell.

3054. Use of a synthetic finger ring body as described in any one of examples 3001-3034 or a pharmaceutical composition as described in any one of examples 3035-3037 for treating a disease or disorder in a subject.

3055. The use of example 3054, wherein said disease or disorder is cancer, e.g., a solid tumor.

3056. A synthetic finger ring body as described in any one of examples 3001-3034 or a pharmaceutical composition as described in any one of examples 3035-3037 for use in treating a disease or disorder in a subject.

3057. A method of treating a disease or disorder in a subject, the method comprising administering to the subject a synthetic finger ring body as described in any one of examples 3001-3034 or a pharmaceutical composition as described in any one of examples 3035-3037, wherein the disease or disorder is a cancer, for example a solid tumor.

3058. Use of a synthetic finger ring body as described in any one of examples 3001-3034 or a pharmaceutical composition as described in any one of examples 3035-3037 in the manufacture of a medicament for treating a disease or disorder in a subject, optionally wherein the disease or disorder is cancer, for example a solid tumor.

3059. The method, composition or use of any one of examples 3052-3058, wherein said disease or disorder is associated with altered VEGF levels or activity relative to healthy tissue, e.g., wherein said altered level or activity is an increased level or activity.

3060. The method, composition for use, or use of any one of examples 3052-3058, wherein said disease or disorder is associated with increased angiogenesis.

3061. The method, use composition or use of any one of examples 3052-3058, wherein said exogenous effector comprises a VEGF inhibitor, e.g., an antibody molecule directed against VEGF or a VEGF receptor.

Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Drawings

The embodiments of the invention described in detail below will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings exemplary embodiments of the invention. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Figure 1A is a diagram showing percent sequence similarity of the amino acid regions of the capsid protein sequences.

Figure 1B is a graph showing percent sequence similarity of capsid protein sequences.

Fig. 2 is a diagram illustrating one embodiment of a ring body.

Figure 3 depicts a schematic of the kanamycin vector encoding LY1 strain of TTMiniV ("finger ring 1").

Figure 4 depicts a schematic of the kanamycin vector encoding LY2 strain of TTMiniV ("finger ring 2").

FIG. 5 depicts the transfection efficiency of finger rings synthesized in 293T and A549 cells.

FIGS. 6A and 6B depict quantitative PCR results demonstrating successful infection of 293T cells with synthetic finger rings.

FIGS. 7A and 7B depict quantitative PCR results demonstrating successful infection of A549 cells by synthetic finger ring.

FIGS. 8A and 8B depict quantitative PCR results demonstrating successful infection of Raji cells with synthetic finger ring.

FIGS. 9A and 9B depict quantitative PCR results demonstrating successful infection of Jurkat cells by synthetic finger ring.

FIGS. 10A and 10B depict quantitative PCR results demonstrating successful infection of Chang cells by synthetic ring bodies.

FIGS. 11A-11B are a series of graphs showing luciferase expression in nLuc transfected or infected cells with TTMV-LY2 Δ 574-1371, Δ 1432-2210, 2610. Luminescence was observed in infected cells, indicating successful replication and packaging.

FIG. 11C is a diagram depicting a phylogenetic tree of type A torque teno virus (torque teno virus; TTV), with the evolutionary branches highlighted. At least 100 finger-ring virus strains are represented. Exemplary sequences from several clades are provided herein, for example in tables A1-A12, B1-B5, C1-C5, and 1-18.

Fig. 12 is a schematic diagram illustrating an exemplary workflow for generating finger loops (e.g., replication-competent or replication-defective finger loops described herein).

Fig. 13 is a graph showing primer specificity for primer sets designed to quantify TTV and TTMV genome equivalents. Quantitative PCR based on SYBR green chemistry showed a distinct peak for each amplification product using either TTMV or TTV specific primer sets (as shown) on plasmids encoding the respective genomes.

Figure 14 is a series of graphs showing PCR efficiency for quantifying TTV genome equivalents by qPCR. Increasing concentrations of primers and fixed concentrations of hydrolysis probe (250nM) were used with two different commercial qPCR premixes. The 90% -110% efficiency yields the lowest error propagation in the quantification process.

Fig. 15 is a graph showing an exemplary amplification plot for linear amplification of TTMV (target 1) or TTV (target 2) using 7 genome equivalent concentrations log 10. Quantification of genome equivalents with high PCR efficiency and linearity using 7 10-fold dilutions (R) ²TTMV：0.996；R² TTV：0.997)。

FIGS. 16A-16B are a series of graphs showing quantification of TTMV genomic equivalents in finger ring stocks. (A) Amplification profiles of two stocks, each diluted 1:10 and run in duplicate. (B) Two samples identical to panel A, here in the linear rangeThe case (2) is displayed. Shown are the upper and lower limits of two representative samples. PCR efficiency: 99.58%, R²：0988。

FIG. 17 is a graph showing the fold change in miR-625 expression in HEK293T cells transfected with the indicated plasmids.

Figure 18 is a graph showing aligned pairwise identities of representative sequences from each of the torque teno virus clades. The DNA sequences of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02 and TTV-HD16d were aligned. The pairwise percent identity in a 50bp sliding window is shown along the length of the alignment. The brackets above indicate the non-coding and coding regions of pairwise identity, where pairwise identity is indicated. The following parenthesis indicates regions of high or low sequence conservation.

FIG. 19 is a graph showing pairwise identity of amino acid alignments of putative proteins in seven evolved branches of A-type torque virus. The amino acid sequences of the putative proteins from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02 and TTV-HD16d were aligned. The pairwise percent identity in a 15aa sliding window is shown along the length of each alignment. Pairwise identity of open reading frame DNA sequences and protein amino acid sequences is indicated. (. about) the putative ORF2t/3 amino acid sequences were aligned against TTV-CT30F, TTV-tth8, TTV-16 and TTV-TJN 02.

FIG. 20 is a graph showing the high conservation of the 5' UTR internal domain in the evolved branches of seven A-ringviruses (SEQ ID NO 810-. The conserved domain sequences of the 71-bp 5' UTR of each representative type A torque teno virus were aligned. The sequence has 95.2% pairwise identity between seven evolutionary branches.

Figure 21 is a graph showing an alignment of GC-rich domains from seven evolved branches of a type a torque teno virus. Each of the dactyloviruses has a region downstream of the ORF with a GC content of greater than 70%. Shown are alignments of GC-rich regions from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16 d. The regions vary in length, but when aligned they show 75.4% pairwise identity.

FIG. 22 is a diagram showing the mitochondrial infection of Raji B cells with a miRNA encoding an n-myc interacting protein (NMI). Shown is the quantification of the genome equivalents of finger rings detected after infection of Raji B cells (arrows) or control cells with the ring gene encoded by NMI miRNA.

FIG. 23 is a diagram showing the mitochondrial infection of Raji B cells with a miRNA encoding an n-myc interacting protein (NMI). Western blot shows that finger loops encoding mirnas against NMI reduced the expression of NMI protein in Raji B cells, whereas Raji B cells infected with finger loops lacking the mirnas showed comparable NMI protein expression to controls.

Figure 24 is a series of graphs showing quantification of finger ring particles produced in host cells following infection with a finger ring comprising the coding sequence for an endogenous miRNA and a corresponding finger ring in which the coding sequence for an endogenous miRNA has been deleted.

FIGS. 25A-25C are a series of graphs showing the intracellular localization of the ORF from TTMV-LY2 fused to a nanoluciferase. (A) In Vero cells, ORF2 (top row) appears to localize to the cytoplasm, while ORF1/1 (bottom row) appears to localize to the nucleus. (B) In HEK293 cells, ORF2 (top row) appeared to be localized to the cytoplasm, while ORF1/1 (bottom row) appeared to be localized to the nucleus. (C) Localization patterns of ORF1/2 and ORF2/2 in cells.

FIG. 26 is a series of graphs showing sequential deletion controls in the 3' non-coding region (NCR) of TTV-tth 8. The top row shows the structure of the wild-type TTV-tth8 finger ring virus. The second row shows TTV-tth8, which is missing 36 nucleotides in the GC-rich region of the 3' NCR (Δ 36nt (GC)). The third row shows TTV-tth8 with a deletion of 36 nucleotides and an additional deletion of the miRNA sequence, resulting in a total deletion of 78 nucleotides (Δ 36nt (gc) Δ miR). The fourth row shows TTV-tth8 deleted 171 nucleotides from 3 'NCR, which includes a 36 nucleotide deletion region and a miRNA sequence (Δ 3' NCR).

FIGS. 27A-27D are a series of graphs showing that a sequential deletion in the 3' NCR of TTV-tth8 has a significant effect on the level of ORF transcripts from the dactylovirus. Shown are the expression of ORF1 and ORF2(A) at day 2, ORF1/1 and ORF2/2(B) at day 2, ORF1/2 and ORF2/3(C) at day 2 and ORF2t3(D) at day 2.

FIGS. 28A-28B are a series of graphs showing constructs for generating finger loops expressing nanoluciferases (A) and a series of finger loop/plasmid combinations for transfecting cells (B)

FIGS. 29A-29C are a series of graphs showing the expression of nanoluciferases in finger ring-injected mice. (A) Nano-luciferase expression in mice from day 0-9 post injection. (B) Nano-luciferase expression in mice injected with various finger ring/plasmid construct combinations, as shown. (C) Quantification of the luciferase luminescence detected in vivo in mice after injection. Group A received TTMV-LY2 vector + -nano-luciferase. Group B received the nano-luciferase protein and TTMV-LY2 ORF.

FIG. 29D is a schematic representation of the genomic organization of a representative finger ring from seven different evolutionary branches of a type A torque teno virus. The sequences of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02 and TTV-HD16d are aligned, with the key regions labeled. Putative Open Reading Frames (ORFs) are shown in light gray, TATA boxes in dark gray, and key putative regulatory regions in medium gray, including the initiation element, 5' UTR conserved domain, and GC-rich region (e.g., as shown).

Figure 30 is a schematic diagram showing an exemplary workflow for determining endogenous targets for pre-dactylovirus mirnas.

FIGS. 31A-31B are a series of graphs showing that a concatenated dactylovirus plasmid can increase production of dactylovirus or dactylovirus. (A) Exemplary tandem refers to a plasmid map of a viral plasmid. (B) Transfection of HEK293T cells with a tandem finger virus plasmid produced four times the number of viral genomes as a plasmid carrying a single copy.

FIG. 31C is a gel electrophoresis image showing circularization of TTMV-LY2 plasmids pVL46-063 and pVL 46-240.

Fig. 31D is a chromatogram showing copy numbers of linear and circular TTMV-LY2 constructs, as determined by Size Exclusion Chromatography (SEC).

FIG. 32 is a graph showing an alignment of the 36 nucleotide GC-rich region from the nine finger ring virus genome sequences and the consensus sequence based thereon (SEQ ID NO 818 and 827, respectively, in appearance order).

FIG. 33 is a series of charts showing the structure of ORF1 from Ring virus strains LY2 and CBD 203. The putative domain is labeled: arginine-rich region (arg-rich), core region, including the jelly roll domain, hypervariable region (HVR), N22 region, and C-terminal domain (CTD), as shown.

FIG. 34 is a diagram showing the structure of ORF1 from the P.sub.B strain CBS 203. Residues showing high similarity between a group of 110 torque viruses are indicated. Indicated are residues 60% -79.9% similar, residues 80% -99.9% similar and residues 100% similar in all strains evaluated.

Fig. 35 is a graph showing the following: an aligned consensus sequence of 258 sequences from torque teno virus A (SEQ ID NO:828) with residues with high similarity scores highlighted in dark grey (100%), medium grey (80-99.9%), light grey (60% -80%). The putative domains are indicated in boxes. The block diagram below the consensus sequence also indicates the percent identity, where the middle gray box indicates 100% identity, the light gray box indicates 30% -99% identity, and the dark gray box indicates less than 30% identity.

FIG. 36 is a schematic diagram showing the domains of an ORF1 molecule from a dactylovirus and the hypervariable regions to be replaced by hypervariable domains from a different dactylovirus.

FIG. 37 is a schematic drawing showing the domain of ORF1 and the hypervariable region to be replaced by a protein or peptide of interest (POI) from a non-ring virus source.

FIG. 38 is a series of graphs showing the design of exemplary finger ring genetic elements based on finger ring virus genomes. The protein coding region was deleted from the genome of the dactylovirus (left), leaving the non-coding region of the dactylovirus (NCR), including the viral promoter, 5' UTR conserved domain (5CD) and GC-rich region. The payload DNA is inserted into a non-coding region of the protein-coding locus (right). The resulting finger ring contains payload DNA (including open reading frames, genes, non-coding RNA, etc.) and the necessary elements for cis replication and packaging of the finger ring virus, but lacks the necessary protein elements for replication and packaging.

FIG. 39 is a bar graph showing the successful transduction of the human lung-derived cell line EKVX by finger ring comprising a genetic element encoding exogenous human immunoadhesin.

FIG. 40 is a graph showing the following: finger rings based on tth8 or LY2 designed to contain sequences encoding human erythropoietin (hEpo) can deliver a functional transgene to mammalian cells.

Fig. 41A and 41B are a series of graphs showing engineered finger ring bodies that were detectably administered to mice seven days after intravenous injection.

FIG. 42 is a graph showing the following: the engineered finger ring encoding hGH was administered intravenously seven days after which hGH mRNA was detected in the cell fraction of whole blood.

FIGS. 43A-43D are a series of graphs illustrating a highly conserved motif in ORF2 of the finger virus. FIG. 43 discloses SEQ ID NO 949.

FIGS. 44A and 44B are a series of graphs showing evidence of full-length ORF1 mRNA expression in human tissues.

FIG. 45 is a graph showing the following: the ability of the In Vitro Circularized (IVC) TTV-tth8 genome (IVC TTV-tth8) to produce copies of the TTV-tth8 genome at the expected density in HEK293T cells compared to the TTV-tth8 genome in the plasmid.

Fig. 46 is a series of graphs showing the ability of In Vitro Circularized (IVC) LY2 genome (WT LY2 IVC) and wild type LY2 genome in plasmid (WT LY2 plasmid) to produce copies of LY2 genome at expected densities in Jurkat cells.

FIG. 47 is a graph showing the following: alignment of the secondary structures of the jelly-roll domains of the ORF1 proteins from viruses A, B and C (SEQ ID NO: 950-975). These secondary structural elements are highly conserved.

FIG. 48 is a graph showing the following: conserved sequence and secondary structure of the ORF1 motif located in the domain of N22 (SEQ ID NO 976-1000 and 851, respectively, in order of appearance). The conserved YNPXDXGXXN (SEQ ID NO:829) motif of human TTV ORF1 has a conserved secondary structure. In particular, tyrosine in the motif breaks the beta chain, with the second beta chain starting from the terminal asparagine of the motif.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

Definition of

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Unless otherwise indicated, the terms set forth below are generally to be understood in their consensus.

When the term "comprising" is used in the present description and claims, other elements are not excluded. For the purposes of the present invention, the term "consisting of … …" is considered to be a preferred embodiment of the term "comprising". If in the following a group is defined comprising at least a certain number of embodiments, this is to be understood as preferably also disclosing a group consisting of only these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a", "an", "the", this includes a plural of that noun unless something else is specifically stated.

The word "compound, composition, product, etc., for treatment, modulation, etc." is to be understood to mean a compound, composition, product, etc., which is itself suitable for the indicated purpose of treatment, modulation, etc. The word "compound, composition, product, etc., for treatment, modulation, etc." additionally discloses as an example that such compound, composition, product, etc., is for treatment, modulation, etc.

As used herein, "cytokine molecule" includes wild-type cytokines or variants thereof that bind to the same receptor as the wild-type cytokine. In some embodiments, the cytokine molecule comprises a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a wild-type cytokine. In some embodiments, a variant comprises a fusion polypeptide having a first region at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type cytokine and a second region heterologous to the wild-type cytokine. In some embodiments, the cytokine molecule comprises or consists of a fragment of a wild-type cytokine.

The words "a compound, composition, product, etc. for …", "use of a compound, composition, product, etc. for the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for …" or "a compound, composition, product, etc. for use as a medicament … …" mean that these compounds, compositions, products, etc. will be used in a method of treatment that can be practiced on a human or animal body. They are considered to be the equivalent disclosures of embodiments and claims relating to methods of treatment and the like. If the examples or claims thus refer to "a compound for use in the treatment of a human or animal suspected of having a disease", this is also considered to disclose "the use of the compound in the manufacture of a medicament for the treatment of a human or animal suspected of having a disease" or "a method of treatment by administering the compound to a human or animal suspected of having a disease". The word "compound, composition, product, etc., for treatment, modulation, etc." is to be understood to mean a compound, composition, product, etc., which is itself suitable for the indicated purpose of treatment, modulation, etc.

If examples of terms, values, quantities, etc. are provided below in parentheses, this is to be understood as meaning that the examples mentioned in the parentheses may constitute embodiments. For example, if it is stated that "in an embodiment, a nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence encoding ORF1 of Table 1 (e.g., nucleotide 571-2613 of a nucleic acid sequence of Table 1)," some embodiments relate to a nucleic acid molecule comprising a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to nucleotide 571-2613 of a nucleic acid sequence of Table 1.

As used herein, the term "refers to a loop body" refers to a vector comprising a genetic element, e.g., an episome, e.g., circular DNA, enclosed in the exterior of a protein. As used herein, "synthetic finger ring" generally refers to a finger ring that does not occur naturally, e.g., has a different sequence relative to a wild-type virus (e.g., a wild-type finger ring virus as described herein). In some embodiments, the synthetic finger ring is engineered or recombined, e.g., comprises a genetic element comprising a difference or modification relative to a wild-type viral genome (e.g., a wild-type finger ring viral genome as described herein). In some embodiments, blocking in the protein exterior comprises 100% coverage by the protein exterior, and less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50%, or less. For example, there may be gaps or discontinuities outside the protein (e.g., such that the protein exterior is permeable to water, ions, peptides, or small molecules) as long as the genetic element remains in the protein exterior, e.g., prior to entering the host cell. In some embodiments, the ring body is purified, e.g., isolated from its original source and/or substantially free (> 50%, > 60%, > 70%, > 80%, > 90%) of other components.

As used herein, the term "annular vector" refers to a vector that comprises sufficient nucleic acid sequence derived from or highly similar to (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) an annular viral genome sequence or a contiguous portion thereof to allow packaging into the exterior of a protein (e.g., a capsid) and further comprises a heterologous sequence. In some embodiments, the ring vector is a viral vector or a naked nucleic acid. In some embodiments, the ring vector comprises a sequence at least about 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 3500 consecutive nucleotides of a native ring virus sequence to which it or highly similar (at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical). In some embodiments, the finger ring vector further comprises one or more of finger ring virus ORF1, ORF2, or ORF 3. In some embodiments, the heterologous sequence comprises a multiple cloning site, comprises a heterologous promoter, comprises a coding region for a therapeutic protein, or encodes a therapeutic nucleic acid. In some embodiments, the capsid is a wild-type dactylovirus capsid. In embodiments, the ring vector comprises a genetic element described herein, e.g., comprises a genetic element comprising a promoter, a sequence encoding a therapeutic effector, and a capsid-binding sequence.

As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes full-length antibodies and antibody fragments (e.g., scFv). In some embodiments, the antibody molecule is a multispecific antibody molecule, e.g., the antibody molecule comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules are generally characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope.

As used herein, a nucleic acid that "encodes" refers to a nucleic acid sequence that encodes an amino acid sequence or a functional polynucleotide (e.g., a non-coding RNA, such as an siRNA or miRNA).

As used herein, an "exogenous" agent (e.g., effector, nucleic acid (e.g., RNA), gene, payload, protein) refers to an agent that is not included in or encoded by a corresponding wild-type virus (e.g., a ring virus as described herein). In some embodiments, the exogenous factor is not naturally occurring, e.g., a protein or nucleic acid having a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein or nucleic acid. In some embodiments, the exogenous factor is not naturally present in the host cell. In some embodiments, the exogenous agent is naturally present in the host cell, but is exogenous to the virus. In some embodiments, the exogenous factor is naturally present in the host cell, but is not present at a desired level or at a desired time.

A "heterologous" agent or element (e.g., effector, nucleic acid sequence, amino acid sequence), as used herein with respect to another agent or element (e.g., effector, nucleic acid sequence, amino acid sequence), refers to an agent or element that is not naturally found together, e.g., in a wild-type virus, e.g., a ring virus. In some embodiments, a heterologous nucleic acid sequence can be present in the same nucleic acid as a naturally occurring nucleic acid sequence (e.g., a sequence naturally present in a finger ring virus). In some embodiments, the heterologous agent or element is exogenous with respect to the ring virus upon which other (e.g., remaining) elements of the ring body are based.

As used herein, the term "genetic element" refers to a nucleic acid sequence, typically in a loop. It is understood that the genetic element may be produced as naked DNA and optionally further assembled into the exterior of the protein. It is also understood that the ring may have its genetic element inserted into the cell, resulting in the genetic element being present in the cell, while the protein does not necessarily enter the cell externally.

As used herein, the term "ORF 1 molecule" refers to a polypeptide having the activity and/or structural characteristics of a ring virus ORF1 protein (e.g., a ring virus ORF1 protein as described herein, e.g., as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a functional fragment thereof. In some cases, the ORF1 molecule can comprise one or more (e.g., 1, 2, 3, or 4) of: a first region comprising at least 60% basic residues (e.g., at least 60% arginine residues), a second region comprising at least about six beta strands (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands), a third region comprising the structure or activity of a domain from a ring virus N22 (e.g., as described herein, e.g., from the N22 domain of the ring virus ORF1 protein as described herein), and/or a fourth region comprising the structure or activity of a ring virus C-terminal domain (CTD) (e.g., as described herein, e.g., from the CTD of the ring virus ORF1 protein as described herein). In some cases, the ORF1 molecule comprises the first, second, third, and fourth regions in N-terminal to C-terminal order. In some cases, the finger ring comprises an ORF1 molecule comprising, in N-terminal to C-terminal order, the first, second, third, and fourth regions. In some cases, the ORF1 molecule can comprise a polypeptide encoded by an ring virus ORF1 nucleic acid (e.g., as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17). In some cases, the ORF1 molecule can further comprise a heterologous sequence, e.g., a hypervariable region (HVR), e.g., an HVR from, e.g., the finger ring virus ORF1 protein described herein. As used herein, "dactylovirus ORF1 protein" refers to an ORF1 protein encoded by a dactylovirus genome (e.g., a wild-type dactylovirus genome, e.g., as described herein), e.g., an ORF1 protein having an amino acid sequence as set forth in any one of tables a2, a4, A6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10, or an ORF1 protein encoded by an ORF1 gene as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

As used herein, the term "ORF 2 molecule" refers to a polypeptide having the activity and/or structural characteristics of a ring virus ORF2 protein (e.g., a ring virus ORF2 protein as described herein, e.g., as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a functional fragment thereof. As used herein, "dactylovirus ORF2 protein" refers to an ORF2 protein encoded by a dactylovirus genome (e.g., a wild-type dactylovirus genome, e.g., as described herein), e.g., an ORF2 protein having an amino acid sequence as set forth in any one of tables a2, a4, A6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10, or an ORF2 protein encoded by an ORF2 gene as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17.

The term "proteinaceous external" as used herein refers to an external component that is predominantly (e.g., > 50%, > 60%, > 70%, > 80%, > 90%) proteinaceous.

As used herein, the term "regulatory nucleic acid" refers to a nucleic acid sequence that modifies the expression, e.g., transcription and/or translation, of a DNA sequence encoding an expression product. In embodiments, the expression product comprises RNA or protein.

As used herein, the term "regulatory sequence" refers to a nucleic acid sequence that modifies transcription of a target gene product. In some embodiments, the regulatory sequence is a promoter or an enhancer.

As used herein, the term "replication protein" refers to a protein, e.g., a viral protein, used during infection, viral genome replication/expression, viral protein synthesis, and/or viral component assembly.

As used herein, an "essentially non-pathogenic" organism, particle, or component refers to an organism, particle (e.g., virus or ring, e.g., as described herein), or component thereof that does not cause or induce a detectable disease or pathogenic condition, e.g., in a host organism, e.g., a mammal, e.g., a human. In some embodiments, administration of the finger ring to a subject may result in a mild reaction or side effect, which is acceptable as part of the standard of care.

As used herein, the term "non-pathogenic" refers to an organism or component thereof that does not cause or induce a detectable disease or pathogenic condition, e.g., in a host organism, e.g., a mammal, e.g., a human.

As used herein, a "substantially non-integrated" genetic element refers to a genetic element, e.g., a virus or to a genetic element in a loop, e.g., as described herein, wherein less than about 0.01%, 0.05%, 0.1%, 0.5%, or 1% of the genetic elements entering a host cell (e.g., a eukaryotic cell) or organism (e.g., a mammal, e.g., a human) are integrated into the genome. In some embodiments, the genetic element is not detectably integrated into, for example, the genome of the host cell. In some embodiments, integration of a genetic element into a genome can be detected using techniques described herein, such as nucleic acid sequencing, PCR detection, and/or nucleic acid hybridization.

As used herein, an "essentially non-immunogenic" organism, particle, or component refers to an organism, particle (e.g., a virus or a ring, e.g., as described herein), or a component thereof that does not elicit or induce an undesirable or untargeted immune response, e.g., in a host tissue or organism (e.g., a mammal, e.g., a human). In embodiments, the substantially non-immunogenic organism, particle, or component does not produce a detectable immune response. In an embodiment, the substantially non-immunogenic finger ring does not produce a detectable immune response to a protein comprising an amino acid sequence as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, or encoded by a nucleic acid sequence as set forth in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17. In embodiments, immune responses (e.g., undesired or untargeted immune responses) are detected by determining the presence or level of an antibody (e.g., the presence or level of an anti-finger ring antibody, e.g., antibodies directed against a finger ring as described herein) in a subject, e.g., according to the anti-TTV antibody detection methods described in Tsuda et al (1999; J.Virol. methods [ J.Virol methods ]77: 199-189; incorporated herein by reference) and/or the methods described in Kakkola et al (2008; Virology [ Virology ]382: 182-189; incorporated herein by reference) for determining anti-TTV IgG levels. Antibodies to a ring virus or ring body based thereon can also be detected by methods known in the art for detecting anti-viral antibodies, such as methods for detecting anti-AAV antibodies, e.g., Calcedo et al (2013; front.

As used herein, "subsequence" refers to a nucleic acid sequence or amino acid sequence contained within a larger nucleic acid sequence or amino acid sequence, respectively. In some cases, a subsequence may comprise a domain or functional fragment of a larger sequence. In some cases, a subsequence may comprise a fragment of a larger sequence that, when separated from the larger sequence, is capable of forming a secondary and/or tertiary structure similar to that formed when present with the remainder of the larger sequence. In some cases, a subsequence may be replaced with another sequence (e.g., a subsequence comprising an exogenous sequence or a sequence heterologous to the remainder of the larger sequence, e.g., a corresponding subsequence from a different finger ring virus).

As used herein, "treatment" and "treating" and their cognates refer to the medical management of a subject with the intent to ameliorate, improve, stabilize, prevent or cure a disease, pathological condition or disorder. The term includes active treatment (treatment intended to ameliorate a disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to alleviate symptoms), prophylactic treatment (treatment intended to prevent, minimize, or partially or completely inhibit the progression of the associated disease, pathological condition, or disorder); and supportive therapy (therapy to supplement another therapy).

As used herein, the term "virome" refers to a virus in a particular environment, e.g., a part of the body, e.g., in an organism, e.g., in a cell, e.g., in a tissue.

The present invention relates generally to finger ring bodies, such as synthetic finger ring bodies, and uses thereof. The present invention provides finger ring bodies, compositions comprising finger ring bodies, and methods of making or using finger ring bodies. The ring body is typically used as a delivery vehicle, e.g., for delivering therapeutic agents to eukaryotic cells. Typically, the finger loop will include a genetic element comprising a nucleic acid sequence enclosed within the exterior of the protein (e.g., encoding an effector, such as an exogenous effector or an endogenous effector). The ring body can include a deletion of one or more sequences (e.g., regions or domains as described herein) relative to the ring virus sequences (e.g., as described herein). The finger ring may be used as a substantially non-immunogenic vehicle for delivering a genetic element or effector encoded therein (e.g., a polypeptide or nucleic acid effector, e.g., as described herein) into a eukaryotic cell, e.g., to treat a disease or disorder in a subject comprising the cell.

Content listing

I. Finger ring body

A. Finger ring virus

ORF1 molecules

ORF2 molecules

D. Genetic elements

E. Protein binding sequences

F.5' UTR region

GC enrichment region

H. Effector

I. Protein exterior

II. vector

Composition III

IV. host cells

Method of use

VI. production method

Administration/delivery

I. Finger ring body

In some aspects, the invention described herein includes compositions and methods of using and making finger ring bodies, and therapeutic compositions. In some embodiments, the finger ring has a sequence, structure, and/or function based on a finger ring virus (e.g., a finger ring virus described herein, e.g., a finger ring virus comprising a nucleic acid or polypeptide comprising a sequence set forth in any of tables a1-a12, B1-B5, C1-C5, 1-18, 20-37, or D1-D10), or a fragment thereof, or other substantially non-pathogenic virus (e.g., a symbiotic virus, a common virus, a natural virus). In some embodiments, a finger ring based on a finger ring virus comprises at least one element that is foreign to the finger ring virus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector within a genetic element of the finger ring. In some embodiments, a finger ring based on a finger ring virus comprises at least one element that is heterologous to another element from the finger ring virus, e.g., an effector-encoding nucleic acid sequence, e.g., a promoter element, that is heterologous to another linked nucleic acid sequence. In some embodiments, the finger ring comprises a genetic element (e.g., circular DNA, e.g., single-stranded DNA) comprising at least one element that is heterologous with respect to the remainder of the genetic element and/or the proteinaceous exterior (e.g., an exogenous element encoding an effector, e.g., as described herein). The finger ring body can be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) for entering a payload into a host, such as a human. In some embodiments, the ring is capable of replicating in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the finger ring body is substantially non-pathogenic and/or substantially non-integrating in mammalian (e.g., human) cells. In some embodiments, the finger ring is substantially non-immunogenic in a mammal, such as a human. In some embodiments, the ring is replication defective. In some embodiments, the ring body has replication capability.

In some embodiments, the finger ring comprises the finger ring body or a component thereof (e.g., a genetic element, e.g., comprising a sequence encoding an effector and/or a proteinaceous outer portion), e.g., as described in PCT application No. PCT/US2018/037379, which is incorporated herein by reference in its entirety.

In one aspect, the invention includes a ring body comprising (i) a genetic element comprising a promoter element, a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a payload), and a protein binding sequence (e.g., an external protein binding sequence, e.g., a packaging signal), wherein the genetic element is single-stranded DNA and has one or both of the following properties: is circular and/or integrates into the genome of a eukaryotic cell at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell; and (ii) a proteinaceous outer portion; wherein the genetic element is enclosed within the protein exterior; and wherein the finger ring body is capable of delivering the genetic element into the eukaryotic cell.

In some embodiments of the finger rings described herein, the genetic element is integrated at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell. In some embodiments, less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the genetic elements of the plurality of finger loops administered to the subject will integrate into the genome of one or more host cells of the subject. In some embodiments, for example, the genetic elements of the population of finger loops described herein integrate into the genome of the host cell at a frequency that is less than the frequency of a comparable AAV viral population, e.g., at a frequency that is about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or less than the frequency of a comparable AAV viral population.

In one aspect, the invention includes a ring body comprising: (i) a genetic element comprising a promoter element and a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, such as a payload), and a protein binding sequence (e.g., an external protein binding sequence), wherein the genetic element has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a wild-type finger ring virus sequence (e.g., a wild-type torque ring virus (TTV), a parvovirus (TTMV), or a TTMDV sequence, e.g., a wild-type finger ring virus sequence listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17); and (ii) a proteinaceous outer portion; wherein the genetic element is enclosed within the protein exterior; and wherein the finger ring body is capable of delivering the genetic element into the eukaryotic cell.

In one aspect, the invention includes a ring body comprising:

a) a genetic element comprising (i) a sequence encoding an external protein (e.g., a non-pathogenic external protein), (ii) an external protein binding sequence that binds the genetic element to the non-pathogenic external protein, and (iii) a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector); and

b) A protein associated with (e.g., encapsulating or blocking) the genetic element.

In some embodiments, the finger ring comprises a sequence or expression product from (or has > 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology to) a non-enveloped, circular, single-stranded DNA virus. An animal circular single-stranded DNA virus generally refers to a subgroup of single-stranded DNA (ssdna) viruses that infect eukaryotic non-plant hosts and have a circular genome. Thus, animal circular ssDNA viruses can be distinguished from those infecting prokaryotes (i.e., the family of picornaviridae and filamentous bacterioviridae) and those infecting plants (i.e., the family of geminiviridae and the family of dwarfing viruses). They can also be distinguished from linear ssDNA viruses (i.e., parvoviridae) that infect non-plant eukaryotes.

In some embodiments, the finger ring body modulates host cell function, e.g., transiently or chronically. In certain embodiments, cell function is stably altered, e.g., modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or more, or any time therebetween. In certain embodiments, cell function is transiently altered, e.g., modulation persists for no more than about 30 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time therebetween.

In some embodiments, the genetic element comprises a promoter element. In an embodiment, the promoter element is selected from the group consisting of an RNA polymerase II dependent promoter, an RNA polymerase III dependent promoter, a PGK promoter, a CMV promoter, an EF-1 alpha promoter, an SV40 promoter, a CAGG promoter or UBC promoter, a TTV viral promoter, tissue specific U6(pollIII), a minimal CMV promoter with an upstream DNA binding site for an activator protein (TetR-VP16, Gal4-VP16, dCas9-VP16, etc.). In embodiments, the promoter element comprises a TATA box. In embodiments, the promoter element is endogenous to a wild-type finger ring virus (e.g., as described herein).

In some embodiments, the genetic element comprises one or more of the following characteristics: single-stranded, circular, negative-stranded, and/or DNA. In embodiments, the genetic element comprises an episome. In some embodiments, the combined size of the portion of the genetic element other than the effector is about 2.5kb-5kb (e.g., about 2.8kb-4kb, about 2.8kb-3.2kb, about 3.6kb-3.9kb, or about 2.8kb-2.9kb), less than about 5kb (e.g., less than about 2.9kb, 3.2kb, 3.6kb, 3.9kb, or 4kb), or at least 100 nucleotides (e.g., at least 1 kb).

In some cases, the finger rings, compositions comprising finger rings, methods of using the finger rings, and the like described herein are based in part on examples that illustrate how different effectors (e.g., mirnas (e.g., for IFN or miR-625), shrnas, and the like) and protein binding sequences (e.g., DNA sequences that bind to capsid proteins such as Q99153) combine with the exterior of proteins (e.g., capsids disclosed in Arch Virol [ virology ] (2007)152: 1961-. In embodiments, the effector can silence the expression of a factor such as interferon. The examples further describe how to prepare finger ring bodies by inserting effectors into sequences derived from, for example, a ring virus. Based on these examples, the following description considers variations of the specific findings and combinations considered in the examples. For example, the skilled person will appreciate from the examples that a particular miRNA serves only as an example of an effector, while other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the particular capsids used in the examples can be replaced with substantially non-pathogenic proteins as described below. The particular dactylovirus sequences described in the examples may also be replaced with the dactylovirus sequences described below. These considerations apply analogously to protein binding sequences, regulatory sequences such as promoters, etc. Independently thereof, those skilled in the art will specifically consider these embodiments in close relation to the examples.

In some embodiments, the finger ring body or genetic elements contained in the finger ring body are introduced into a cell (e.g., a human cell). In some embodiments, for example, an effector (e.g., an RNA, such as a miRNA) encoded by a genetic element of a finger loop is expressed in a cell (e.g., a human cell), e.g., once the finger loop or genetic element is introduced into the cell. In embodiments, the finger loops or genetic elements contained therein are introduced into a cell to modulate (e.g., increase or decrease) the level of a target molecule (e.g., a target nucleic acid, e.g., RNA, or a target polypeptide) in the cell, e.g., by altering the expression level of the target molecule in the cell. In embodiments, the introduction of the finger loop body or the genetic element comprised therein reduces the level of interferon produced by the cell. In embodiments, the finger loop or genetic element contained therein is introduced into a cell to modulate (e.g., increase or decrease) the function of the cell. In embodiments, introduction of the finger loop or genetic element contained therein into a cell modulates (e.g., increases or decreases) the viability of the cell. In embodiments, introduction of the finger loops or genetic elements contained therein into a cell reduces the viability of the cell (e.g., cancer cell).

In some embodiments, a finger ring (e.g., a synthetic finger ring) described herein induces an antibody prevalence of less than 70% (e.g., less than about 60%, 50%, 40%, 30%, 20%, or 10% antibody prevalence). In the examples, antibody prevalence is determined according to methods known in the art. In embodiments, antibody prevalence is determined by detecting antibodies in a biological sample against a dactylovirus (e.g., as described herein) or a dactylosome based thereon, e.g., according to the anti-TTV antibody detection method described in Tsuda et al (1999; J. Virol. methods [ J. Virol. methods ]77: 199-206; incorporated herein by reference) and/or the method of determining anti-TTV IgG seropositivity as described in Kakkola et al (2008; Virology [ Virology ]382: 182-189; incorporated herein by reference). Antibodies to a ring virus or ring body based thereon can also be detected by methods known in the art for detecting anti-viral antibodies, such as methods for detecting anti-AAV antibodies, e.g., Calcedo et al (2013; front.

In some embodiments, a genetic element that is under-replicated, replication-defective, or replication-incompetent does not encode all of the necessary machinery or components required for replication of the genetic element. In some embodiments, the replication-defective genetic element does not encode a replication factor. In some embodiments, the replication-defective genetic element does not encode one or more ORFs (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, and/or ORF2t/3, e.g., as described herein). In some embodiments, a machine or component not encoded by a genetic element may be provided in trans (e.g., using a helper virus, such as a helper virus or helper plasmid, or encoded in a nucleic acid comprised by the host cell, e.g., integrated into the genome of the host cell), e.g., such that the genetic element can replicate in the presence of the machine or component provided in trans.

In some embodiments, the genetic element that is under-packaged, defective-packaged, or incapacitated-packaged cannot be packaged into the exterior of the protein (e.g., wherein the exterior of the protein comprises the capsid or portion thereof, e.g., comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., as described herein). In some embodiments, the packaging-deficient genetic element is packaged into the protein exterior with an efficiency of less than 10% (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) as compared to a wild-type finger ring virus (e.g., as described herein). In some embodiments, a packaging-defective genetic element cannot be packaged into the exterior of a protein even in the presence of a factor (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3) that allows packaging of a genetic element of a wild-type finger ring virus (e.g., as described herein). In some embodiments, a packaging-deficient genetic element is packaged into the protein exterior with an efficiency of less than 10% (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) as compared to a wild-type finger ring virus (e.g., as described herein), even in the presence of a factor (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3) that allows packaging of the genetic element of the wild-type finger ring virus (e.g., as described herein).

In some embodiments, the packaging competent genetic element can be packaged into the protein exterior (e.g., wherein the protein exterior comprises a capsid or portion thereof, e.g., comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., as described herein). In some embodiments, the packaging competent genetic element is packaged into the protein exterior with an efficiency of at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more) as compared to a wild-type finger ring virus (e.g., as described herein). In some embodiments, the packaging competent genetic element can be packaged into the protein exterior in the presence of a factor (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3) that allows packaging of the genetic element of a wild-type finger ring virus (e.g., as described herein). In some embodiments, in the presence of a factor (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3) that allows packaging of a genetic element of a wild-type finger ring virus (e.g., as described herein), the packaging competent genetic element is packaged into the protein exterior with an efficiency of at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more) as compared to the wild-type finger ring virus (e.g., as described herein).

Finger ring virus

In some embodiments, for example, the finger ring described herein includes sequences or expression products derived from a finger ring virus. In some embodiments, the finger ring includes one or more sequences or expression products that are exogenous to the finger ring virus. In some embodiments, the finger ring includes one or more sequences or expression products that are endogenous to the finger ring virus. In some embodiments, the finger ring body includes one or more sequences or expression products that are heterologous with respect to one or more other sequences or expression products in the finger ring body. A ring virus typically has a single-stranded circular DNA genome with negative polarity. The ring virus is generally not associated with any human disease. However, the high incidence of asymptomatic dactyloviridae in one or more control cohort groups, significant genomic diversity within the dactyloviridae, inability to transmit the agent in vitro with a history of disease, and the lack of one or more animal models of dactyloviridae disease have hampered attempts to correlate dactyloviridae infection with human disease (Yzebe et al, Panminerva Med. [ Parnieva medicine ] (2002)44: 167-.

Ring viruses are commonly transmitted by oronasal or fecal infection, maternal-fetal transmission, and/or intrauterine transmission (Gerner et al, ped. infection. Dis. J. [ J. pediatric infectious diseases ] (2000)19: 1074-. In some cases, the infected person may be characterized by prolonged (months to years) dacycloviral viremia. Humans can co-infect more than one gene group or strain (Saback, et al, Scad.J.Infect.Dis. [ Scandinavian J.Infect ] (2001)33: 121-. It has been suggested that these gene groups can be recombined in infected individuals (Rey et al, infection. [ infection ] (2003)31: 226-233). Double-stranded isoform (replicative) intermediates are found in several tissues such as liver, peripheral blood mononuclear cells and bone marrow (Kikuchi et al, J.Med.Virol. [ J.J.J. [ J.Med.Virol ] (2000)61: 165-.

In some embodiments, the genetic element comprises a nucleotide sequence encoding: an amino acid sequence, or a functional fragment thereof, or a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences described herein (e.g., to a finger ring virus amino acid sequence).

In some embodiments, the finger ring described herein comprises one or more nucleic acid molecules (e.g., genetic elements described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring viral sequence, or fragment thereof, e.g., described herein. In embodiments, the finger ring comprises a nucleic acid sequence selected from the group consisting of: a sequence shown in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In embodiments, the finger ring comprises a polypeptide comprising a sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

In some embodiments, the finger ring described herein comprises one or more nucleic acid molecules (e.g., a genetic element as described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of any of the finger ring viruses described herein (e.g., finger ring viral sequences annotated or encoded by any of tables a1-a12, B1-B5, C1-C5, or 1-18) a TATA box, a cap site, an initiation element, a transcription initiation site, a 5' UTR conserved domain, ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3, an open reading frame region, a poly (a) signal, a GC-rich region, or any combination thereof. In some embodiments, the nucleic acid molecule comprises a capsid protein encoding sequence, such as an ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, ORF2t/3 sequence of any of the finger viruses described herein (e.g., finger ring virus sequences encoded by the sequences annotated or listed in any of tables A1-A12 or 1-18). In embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an ORF1 or ORF2 protein (e.g., an ORF1 or ORF2 amino acid sequence shown in any of tables a2, a4, A6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37 or D1-D10, or an ORF1 or ORF2 amino acid sequence encoded by a nucleic acid sequence shown in any of tables a1, A3, A5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15 or 17). In embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Ring Virus ORF1 protein (e.g., an ORF1 amino acid sequence set forth in any of tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37 or D1-D10 or an ORF1 amino acid sequence encoded by a nucleic acid sequence set forth in any of tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15 or 17).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A1 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table A1 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A1 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table A1 (e.g., nucleotide 335. sub.703 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table A1 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table A1 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table A1 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table a1 (e.g., nucleotides 77-81 of a nucleic acid sequence of table a 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table a1 (e.g., nucleotides 95-110 of a nucleic acid sequence of table a 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table a1 (e.g., nucleotide 105 of a nucleic acid sequence of table a 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a1 (e.g., nucleotide 165-235 of the nucleic acid sequence of table a 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of the open reading frame region of a finger loop virus of table a1 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table a 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A1 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table A1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table A1 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table A1).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A3 (e.g., nucleotide 599-2887 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table A3 (e.g., nucleotides 599-724 and/or 2414-2887 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A3 (e.g., nucleotides 599-724 and/or 2643-2849 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger ring virus ORF2 of Table A3 (e.g., nucleotide 342-728 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table A3 (e.g., nucleotides 342-724 and/or 2414-2849 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table A3 (e.g., nucleotides 342 and/or 2643 and 3057 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table A3 (e.g., nucleotides 87-91 of a nucleic acid sequence of table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of an annular ring initiation element of Table A3 (e.g., nucleotide 105-120 of a nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table A3 (e.g., nucleotide 115 of a nucleic acid sequence of table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table A3 (e.g., nucleotide 175-245 of the nucleic acid sequence of table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of the open reading frame region of an finger ring virus of Table A3 (e.g., nucleotide 2626-2846 of the nucleic acid sequence of Table A3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A3 (e.g., nucleotides 3052-3058 of the nucleic acid sequence of Table A3).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A5 (e.g., nucleotide 556-2904 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table A5 (e.g., nucleotides 556-687 and/or 2422-2904 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A5 (e.g., nucleotides 556-687 and/or 2564-2878 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table A5 (e.g., nucleotide 305-691 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/2 of Table A5 (e.g., nucleotides 305-687 and/or 2422-2878 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table A5 (e.g., nucleotides 305-687 and/or 2564-3317 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of Table A5 (e.g., nucleotides 305-360 and/or 2564-3317 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table a5 (e.g., nucleotides 50-55 of a nucleic acid sequence of table a 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table a5 (e.g., nucleotides 68-83 of the nucleic acid sequence of table a 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table a5 (e.g., nucleotide 78 of a nucleic acid sequence of table a 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a5 (e.g., nucleotide 138-208 of the nucleic acid sequence of table a 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of the open reading frame region of an finger ring virus of Table A5 (e.g., nucleotide 2626-2846 of the nucleic acid sequence of Table A5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A5, e.g., nucleotide 3316-3319 of the nucleic acid sequence of Table A5.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A7 (e.g., nucleotide 589-2889 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table A7 (e.g., nucleotides 589 and/or 2362 and 2889 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A7 (e.g., nucleotides 589-711 and/or 2555-2863 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table A7 (e.g., nucleotide 353-715 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table A7 (e.g., nucleotides 353-711 and/or 2362-2863 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table A7 (e.g., nucleotides 353-711 and/or 2555-3065 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table A7 (e.g., nucleotides 353-432 and/or 2555-3065 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table a7 (e.g., nucleotides 86-90 of a nucleic acid sequence of table a 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of an annular ring initiation element of Table A7 (e.g., nucleotide 104-119 of a nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table a7 (e.g., nucleotide 114 of a nucleic acid sequence of table a 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a7 (e.g., nucleotide 174-244 of the nucleic acid sequence of table a 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the open reading frame region of an finger ring virus of Table A7 (e.g., nucleotides 2555-2863 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A7 (e.g., nucleotide 3062-plus 3066 of the nucleic acid sequence of Table A7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger loop virus of Table A7 (e.g., nucleotide 3720-3742 of a nucleic acid sequence of Table A7).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A9 (e.g., nucleotide 511-2793 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table A9 (e.g., nucleotides 511-711 and/or 2326-2793 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A9 (e.g., nucleotides 511-711 and/or 2525-2767 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table A9 (e.g., nucleotide 272-637 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table A9 (e.g., nucleotide 272-27633 and/or 2326-2767 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table A9 (e.g., nucleotide 272-633 and/or 2525-2984 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of Table A9 (e.g., nucleotide 272-633 and/or 2525-2984 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table a9 (e.g., nucleotides 12-17 of a nucleic acid sequence of table a 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table a9 (e.g., nucleotides 30-45 of a nucleic acid sequence of table a 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table a9 (e.g., nucleotide 40 of a nucleic acid sequence of table a 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a9 (e.g., nucleotide 100-171 of the nucleic acid sequence of table a 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the open reading frame region of a finger loop virus of Table A9 (e.g., nucleotides 2525-2767 of the nucleic acid sequence of Table A9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A9 (e.g., nucleotide 2981-2985 of the nucleic acid sequence of Table A9).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table A11 (e.g., nucleotide 704-3001 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/1 of Table A11 (e.g., nucleotides 704-826 and/or 2534-3001 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table A11 (e.g., nucleotides 704-826 and/or 2721-2975 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table A11 (e.g., nucleotides 465-830 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/2 of Table A11 (e.g., nucleotides 465 and/or 2534 and 2975 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table A11 (e.g., nucleotides 465-826 and/or 2721-3192 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table A11 (e.g., nucleotides 465-595 and/or 2721-3192 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a TATA box nucleotide sequence of an finger ring virus of table a11 (e.g., nucleotide 206-210 of a nucleic acid sequence of table a 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of an annular ring viral initiation element of Table A11 (e.g., nucleotide 224-239 of a nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table a11 (e.g., nucleotide 234 of a nucleic acid sequence of table a 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a11 (e.g., nucleotide 294-364 of the nucleic acid sequence of table a 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of an open reading frame region of an finger ring virus of Table A11 (e.g., nucleotides 2721-2975 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table A11 (e.g., nucleotides 3189 and 3193 of the nucleic acid sequence of Table A11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table a11 (e.g., nucleotide 3844-3895 of the nucleic acid sequence of table a 11).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table B1 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table B1 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table B1 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table B1 (e.g., nucleotide 335-703 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table B1 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table B1 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table B1 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table B1 (e.g., nucleotides 77-81 of a nucleic acid sequence of table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table B1 (e.g., nucleotides 95-110 of a nucleic acid sequence of table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table B1 (e.g., nucleotide 105 of a nucleic acid sequence of table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B1 (e.g., nucleotide 165-235 of the nucleic acid sequence of table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger loop virus of table B1 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table B1 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table B1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table B1 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table B1).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table B2 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table B2 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table B2 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table B2 (e.g., nucleotide 335-703 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table B2 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table B2 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table B2 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table B2 (e.g., nucleotides 77-81 of a nucleic acid sequence of table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table B2 (e.g., nucleotides 95-110 of a nucleic acid sequence of table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table B2 (e.g., nucleotide 105 of a nucleic acid sequence of table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B2 (e.g., nucleotide 165-235 of the nucleic acid sequence of table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger loop virus of table B2 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table B2 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table B2). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table B2 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table B2).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table B3 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table B3 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table B3 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table B3 (e.g., nucleotide 335-703 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table B3 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table B3 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table B3 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table B3 (e.g., nucleotides 77-81 of a nucleic acid sequence of table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table B3 (e.g., nucleotides 95-110 of a nucleic acid sequence of table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table B3 (e.g., nucleotide 105 of a nucleic acid sequence of table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B3 (e.g., nucleotide 165-235 of the nucleic acid sequence of table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger loop virus of table B3 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table B3 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table B3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table B3 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table B3).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table B4 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table B4 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table B4 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table B4 (e.g., nucleotide 335-703 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table B4 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table B4 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table B4 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table B4 (e.g., nucleotides 77-81 of a nucleic acid sequence of table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table B4 (e.g., nucleotides 95-110 of a nucleic acid sequence of table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table B4 (e.g., nucleotide 105 of a nucleic acid sequence of table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B4 (e.g., nucleotide 165-235 of the nucleic acid sequence of table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger loop virus of table B4 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table B4 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table B4). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table B4 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table B4).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1 of Table B5 (e.g., nucleotide 574-2775 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table B5 (e.g., nucleotides 574-699 and/or 2326-2775 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table B5 (e.g., nucleotides 574 and/or 2552-2759 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2 of Table B5 (e.g., nucleotide 335-703 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table B5 (e.g., nucleotides 335-699 and/or 2326-2759 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table B5 (e.g., nucleotides 335-699 and/or 2552-2957 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of the finger ring virus of Table B5 (e.g., nucleotides 335 and/or 2552 and 2957 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table B5 (e.g., nucleotides 77-81 of a nucleic acid sequence of table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table B5 (e.g., nucleotides 95-110 of a nucleic acid sequence of table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus transcription start site nucleotide sequence of table B5 (e.g., nucleotide 105 of a nucleic acid sequence of table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B5 (e.g., nucleotide 165-235 of the nucleic acid sequence of table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger loop virus of table B5 (e.g., nucleotide 2535-2746 of the nucleic acid sequence of table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a poly (A) signal nucleotide sequence of Table B5 (e.g., nucleotide 2953-2958 of the nucleic acid sequence of Table B5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of an finger ring virus of Table B5 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table B5).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 1 (e.g., nucleotide 571-2613 of a nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/1 of Table 1 (e.g., nucleotides 571-587 and/or 2137-2613 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/2 of Table 1 (e.g., nucleotides 571-687 and/or 2339-2659 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2 of Table 1 (e.g., nucleotide 299-691 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF2/2 of Table 1 (e.g., nucleotides 299-687 and/or 2137-2659 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table 1 (e.g., nucleotides 299-687 and/or 2339-2831 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the ORF2t/3 nucleotide sequence of Table 1 (e.g., nucleotides 299-348 and/or 2339-2831 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring virus TATA box of table 1 (e.g., nucleotides 84-90 of a nucleic acid sequence of table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a cap site of a finger loop virus of Table 1 (e.g., nucleotide 107-114 of a nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 1 (e.g., nucleotide 114 of a nucleic acid sequence of table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 1 (e.g., nucleotides 177-247 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a three open reading frame region of a finger ring virus of Table 1 (e.g., nucleotide 2325-2610 of a nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 1 (e.g., nucleotides 2813-2818 of the nucleic acid sequence of Table 1). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of a finger loop virus of Table 1 (e.g., nucleotide 3415-3570 of the nucleic acid sequence of Table 1).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 3 (e.g., nucleotide 729-2972 of a nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/1 of Table 3 (e.g., nucleotides 729-908 and/or 2490-2972 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/2 of Table 3 (e.g., nucleotides 729-908 and/or 2725-3039 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the finger loop virus ORF2 of Table 3 (e.g., nucleotide 412 and 912 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/2 of Table 3 (e.g., nucleotides 412-908 and/or 2490-3039 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF2/3 of Table 3 (e.g., nucleotides 412-908 and/or 2725-3208 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a TATA box nucleotide sequence of a finger ring virus of table 3 (e.g., nucleotide 112-119 of the nucleic acid sequence of table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of an annular ring initiation element of Table 3 (e.g., nucleotide 128-148 of a nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 3 (e.g., nucleotide 148 of a nucleic acid sequence of table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 3 (e.g., nucleotide 204-273 of the nucleic acid sequence of table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of a finger ring virus of Table 3 (e.g., nucleotides 2699-2969 of the nucleic acid sequence of Table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ringed viral poly (a) signal nucleotide sequence of table 3 (e.g., nucleotides 3220-3225 of the nucleic acid sequence of table 3). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 3 (e.g., nucleotide 3302-3541 of the nucleic acid sequence of table 3).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 5 (e.g., nucleotide 599-2830 of a nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 5 (e.g., nucleotides 599-715 and/or 2363-2830 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the Ring Virus ORF1/2 of Table 5 (e.g., nucleotides 599-715 and/or 2565-2789 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2 of Table 5 (e.g., nucleotide 336-719 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 5 (e.g., nucleotides 336-715 and/or 2363-2789 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF2/3 of Table 5 (e.g., nucleotides 336-715 and/or 2565-3015 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of Table 5 (e.g., nucleotides 336-388 and/or 2565-3015 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring virus TATA box of table 5 (e.g., nucleotides 83-88 of a nucleic acid sequence of table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a cap site of a finger loop virus of table 5 (e.g., nucleotide 104-111 of a nucleic acid sequence of table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 5 (e.g., nucleotide 111 of a nucleic acid sequence of table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 5 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of a finger ring virus of table 5 (e.g., nucleotides 2551-2786 of a nucleic acid sequence of table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 5 (e.g., nucleotides 3011-3016 of the nucleic acid sequence of Table 5). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a GC-rich nucleotide sequence of a finger loop virus of Table 5 (e.g., nucleotides 3632-3753 of a nucleic acid sequence of Table 5).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 7 (e.g., nucleotide 586-2928 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 7 (e.g., nucleotides 586-717 and/or 2446-2928 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF1/2 of Table 7 (e.g., nucleotides 586-717 and/or 2675-2902 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of ORF2 of Table 7 (e.g., nucleotide 335-721 of a nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 7 (e.g., nucleotides 335. sub.717 and/or 2446. sub.2902 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF2/3 of Table 7 (e.g., nucleotides 335. sub.717 and/or 2675. sub.3109 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table 7 (e.g., nucleotides 82-87 of a nucleic acid sequence of table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus initiation element nucleotide sequence of table 7 (e.g., nucleotides 95-115 of a nucleic acid sequence of table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 7 (e.g., nucleotide 115 of a nucleic acid sequence of table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 7 (e.g., nucleotide 170-238 of the nucleic acid sequence of table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the open reading frame region of a finger ring virus of table 7 (e.g., nucleotide 2640-2899 of the nucleic acid sequence of table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 7 (e.g., nucleotides 3106-3114 of the nucleic acid sequence of Table 7). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 7 (e.g., nucleotides 3768-3878 of the nucleic acid sequence of Table 7).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 9 (e.g., nucleotide 588-2873 of a nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 9 (e.g., nucleotides 588-722 and/or 2412-2873 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF1/2 of Table 9 (e.g., nucleotides 588-722 and/or 2638-2847 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2 of Table 9 (e.g., nucleotide 331-726 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 9 (e.g., nucleotides 331-722 and/or 2412-2847 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF2/3 of Table 9 (e.g., nucleotides 331-722 and/or 2638-3058 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of Table 9 (e.g., nucleotides 331-380 and/or 2638-3058 of the nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table 9 (e.g., nucleotides 82-86 of a nucleic acid sequence of table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger loop viral initiation element of Table 9 (e.g., nucleotide 100-115 of a nucleic acid sequence of Table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 9 (e.g., nucleotide 115 of a nucleic acid sequence of table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 9 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an apheresis virus of table 9 (e.g., nucleotide 2699-2969 of a nucleic acid sequence of table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an ringed viral poly (a) signal nucleotide sequence of table 9 (e.g., nucleotides 3220-3225 of the nucleic acid sequence of table 9). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 9 (e.g., nucleotide 3302-3541 of the nucleic acid sequence of table 9).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 11 (e.g., nucleotide 599-2839 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 11 (e.g., nucleotides 599-727 and/or 2381-2839 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/2 of Table 11 (e.g., nucleotides 599-727 and/or 2619-2813 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of ORF2 of Table 11 (e.g., nucleotide 357-731 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 11 (e.g., nucleotides 357-727 and/or 2381-2813 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table 11 (e.g., nucleotides 357-727 and/or 2619-3021 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2t/3 of Table 11 (e.g., nucleotides 357 and 406 and/or 2619 and 3021 of the nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring virus TATA box of table 11 (e.g., nucleotides 89-90 of a nucleic acid sequence of table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a cap site of a finger loop virus of table 11 (e.g., nucleotide 107-114 of a nucleic acid sequence of table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 11 (e.g., nucleotide 114 of a nucleic acid sequence of table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 11 (e.g., nucleotide 174-244 of the nucleic acid sequence of table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of an open reading frame region of an finger loop virus of table 11 (e.g., nucleotide 2596-2810 of a nucleic acid sequence of table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 11 (e.g., nucleotide 3017-3022 of a nucleic acid sequence of Table 11). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a GC-rich nucleotide sequence of Table 11 (e.g., nucleotides 3691-3794 of a nucleic acid sequence of Table 11).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of ORF1 of Table 13 (e.g., nucleotide 599-2896 of a nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 13 (e.g., nucleotides 599-724 and/or 2411-2896 of the nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the ORF1/2 of Table 13 (e.g., nucleotides 599-724 and/or 2646-2870 of the nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of an ORF2 of Table 13 (e.g., nucleotide 357-728 of a nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 13 (e.g., nucleotides 357 and/or 2411 and 2870 of the nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table 13 (e.g., nucleotides 357 and/or 2646 and 3081 of the nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table 13 (e.g., nucleotides 82-86 of a nucleic acid sequence of table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus starting element nucleotide sequence of table 13 (e.g., nucleotides 94-115 of a nucleic acid sequence of table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 13 (e.g., nucleotide 115 of a nucleic acid sequence of table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 13 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of a finger ring virus of Table 13 (e.g., nucleotides 2629-2867 of a nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 13 (e.g., nucleotides 3076-3086 of a nucleic acid sequence of Table 13). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 13 (e.g., nucleotides 3759-3866 of a nucleic acid sequence of Table 13).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1 of Table 15 (e.g., nucleotide 612-2612 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/1 of Table 15 (e.g., nucleotides 612-719 and/or 2274-2612 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/2 of Table 15 (e.g., nucleotides 612-719 and/or 2449-2589 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of ORF2 of Table 15 (e.g., nucleotide 424-723 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 15 (e.g., nucleotides 424-719 and/or 2274-2589 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table 15 (e.g., nucleotides 424-719 and/or 2449-2812 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a TATA box nucleotide sequence of a finger ring virus of table 15 (e.g., nucleotides 237-. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a cap site of a finger loop virus of table 15 (e.g., nucleotide 260-267 of a nucleic acid sequence of table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 15 (e.g., nucleotide 267 of a nucleic acid sequence of table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 15 (e.g., nucleotide 323-393 of the nucleic acid sequence of table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of the open reading frame region of an Ring Virus of Table 15 (e.g., nucleotides 2441-2586 of a nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 15 (e.g., nucleotide 2808-2813 of the nucleic acid sequence of Table 15). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 15 (e.g., nucleotide 2868-2929 of the nucleic acid sequence of Table 15).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1 of Table 17 (e.g., nucleotide 432-2453 of the nucleic acid sequence of Table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of the finger loop virus ORF1/1 of Table 17 (e.g., nucleotides 432-584 and/or 1977-2453 of the nucleic acid sequence of Table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF1/2 of Table 17 (e.g., nucleotides 432-584 and/or 2197-2388 of the nucleic acid sequence of Table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of ORF2 of Table 17 (e.g., nucleotides 283-. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/2 of Table 17 (e.g., nucleotides 283-584 and/or 1977-2388 of the nucleic acid sequence of Table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of ORF2/3 of Table 17 (e.g., nucleotides 283-. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus TATA box nucleotide sequence of table 17 (e.g., nucleotides 21-25 of a nucleic acid sequence of table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring viral cap site nucleotide sequence of table 17 (e.g., nucleotides 42-49 of a nucleic acid sequence of table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a finger ring viral transcription start site of table 17 (e.g., nucleotide 49 of a nucleic acid sequence of table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 17 (e.g., nucleotide 117-187 of the nucleic acid sequence of table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of the open reading frame region of an finger ring virus of table 17 (e.g., nucleotides 2186-2385 of the nucleic acid sequence of table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a poly (A) signal nucleotide sequence of Table 17 (e.g., nucleotides 2676-2681 of a nucleic acid sequence of Table 17). In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 17 (e.g., nucleotide 3054-3172 of the nucleic acid sequence of Table 17).

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 2.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 4.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 6.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table A8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table A8.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 10.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 12.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C1. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of a finger ring virus TAIP of table C1.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of a finger ring virus TAIP of table C2.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C3. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of a finger ring virus TAIP of table C3.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of a finger ring virus TAIP of table C4.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C5. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of a finger ring virus TAIP of table C5.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF1/1 of a finger ring virus of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2/3 of a finger ring virus of table 2. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2t/3 of a finger ring virus of table 2.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF1/2 of a finger ring virus of table 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2/2 of a finger ring virus of table 4. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2/3 of a finger ring virus of table 4.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/3 of table 6. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2t/3 of a finger ring virus of table 6.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF1/1 of table 8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF1/2 of a finger ring virus of table 8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2/2 of a finger ring virus of table 8. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2/3 of a finger ring virus of table 8.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF1 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF2 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/3 of table 10. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2t/3 of a finger ring virus of table 10.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF1 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF1/2 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF2 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF2/2 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF2/3 of table 12. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF2t/3 of a finger ring virus of table 12.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF1 of table 14. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 14. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 14. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ring virus ORF2 of table 14. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 14. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/3 of table 14.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 16. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 16. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 16. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 16. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 16. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/3 of table 16.

In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 18. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/1 of table 18. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1/2 of table 18. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 18. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/2 of table 18. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF2/3 of table 18.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 2. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 574-2775 of the nucleic acid sequence of Table A1. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a2 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 4. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 599-2887 of the nucleic acid sequence of Table A3. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a4 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 6. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 6. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop virus ORF1 nucleic acid sequence of nucleotide 556-2904 of the nucleic acid sequence of Table A5. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a6 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table A8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table A8. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 589-2889 of the nucleic acid sequence of Table A7. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a8 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 10. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 511-2793 of the nucleic acid sequence of Table A9. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a10 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table a 12. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table a 12. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 704-3001 of the nucleic acid sequence of Table A11. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table a12 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the finger ring virus ORF1/1 amino acid sequence of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table C1. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the TAIP amino acid sequence of the finger ring virus of table C1. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 512-2545 of the nucleic acid sequence of Table B1. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table C1 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the finger ring virus ORF1/1 amino acid sequence of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table C2. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the TAIP amino acid sequence of the finger ring virus of table C2. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 501 of the nucleic acid sequence of Table B2 and the finger loop ORF1 nucleic acid sequence of 2489. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table C2 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the finger ring virus ORF1/1 amino acid sequence of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table C3. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the TAIP amino acid sequence of the finger ring virus of table C3. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 572-2758 of the nucleic acid sequence of Table B3. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table C3 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the finger ring virus ORF1/1 amino acid sequence of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table C4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the TAIP amino acid sequence of the finger ring virus of table C4. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 581-2884 finger ring virus ORF1 nucleic acid sequence of the nucleic acid sequence of Table B4. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table C4 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the finger ring virus ORF1/1 amino acid sequence of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table C5. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the TAIP amino acid sequence of the finger ring virus of table C5. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotide 614-2911 of the nucleic acid sequence of Table B5. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger loop virus ORF1 protein of table C5 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, finger bodies described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 2. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1/1 of table 2. In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1/2 of table 2. In embodiments, finger bodies described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 2. In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2/2 of table 2. In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2/3 of table 2. In embodiments, finger bodies described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger virus of table 2. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop virus ORF1 nucleic acid sequence of nucleotides 571-2613 of the nucleic acid sequence of Table 1. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 2 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table 4. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table 4. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 729-2972 of a nucleic acid sequence of Table 3, referring to the finger virus ORF1 nucleic acid sequence. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 4 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, finger bodies described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 6. In embodiments, finger bodies described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 6. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1/2 of table 6. In embodiments, finger bodies described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 6. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2/2 of table 6. In embodiments, finger bodies described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table 6. In embodiments, finger bodies described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of a finger virus of table 6. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 599-2830 finger loop ORF1 nucleic acid sequence of a nucleic acid sequence of Table 5. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 6 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF1/1 of finger ring virus of table 8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF1/2 of finger ring virus of table 8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2/2 of finger ring virus of table 8. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2/3 of finger ring virus of table 8. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 586-. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 8 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2/3 of finger ring virus of table 10. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of finger ring virus of table 10. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop viral ORF1 nucleic acid sequence of nucleotides 588-2873 of the nucleic acid sequence of Table 9. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 10 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1 of table 12. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1/1 of table 12. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1/2 of table 12. In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2 of table 12. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2/2 of table 12. In embodiments, finger loops described herein comprise proteins having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2/3 of table 12. In embodiments, finger bodies described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2t/3 of a finger virus of table 12. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 599-2839 finger loop ORF1 nucleic acid sequence of a nucleic acid sequence of Table 11. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop) comprises the finger ring virus ORF1 protein of table 12 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 14. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 14. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table 14. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 14. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table 14. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2/3 of finger ring virus of table 14. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the nucleotide 599-2896 finger loop ORF1 nucleic acid sequence of a nucleic acid sequence of Table 13. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 14 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF1 of table 16. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 16. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table 16. In embodiments, finger loops described herein comprise proteins having an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger loop virus ORF2 of table 16. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table 16. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of ORF2/3 of a finger ring virus of table 16. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 612-. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop) comprises the finger ring virus ORF1 protein of table 16 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 18. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/1 of table 18. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1/2 of table 18. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2 of table 18. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/2 of table 18. In embodiments, the finger ring described herein comprises a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF2/3 of table 18. In some embodiments, the ORF1 molecule (e.g., contained within a finger loop) comprises a polypeptide encoded by the finger loop ORF1 nucleic acid sequence of nucleotides 432-2453 of the nucleic acid sequence of Table 17. In some embodiments, the ORF1 molecule (e.g., contained in a finger loop body) comprises the finger ring virus ORF1 protein of table 18 or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of ORF1 of a finger virus described herein. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 2. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 4. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1 of table 6. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 8. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1 of table 10. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1 of table 12. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 14. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence of finger ring virus ORF1 of table 16. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table 18. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 2. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 4. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 6. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table A8. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 10. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table a 12. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C1. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C2. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C3. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C4. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of finger ring virus ORF1 of table C5.

In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the ORF1 molecule encoded by an ring virus ORF1 nucleic acid described herein. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table 1. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table 3. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid listed in table 5. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid listed in table 7. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table 9. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid listed in table 11. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid listed in table 13. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table 15. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table 17. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 1. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 3. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 5. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 7. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 9. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ring virus ORF1 nucleic acid set forth in table a 11. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table B1. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table B2. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table B3. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table B4. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an ORF1 nucleic acid of a finger ring virus listed in table B5.

In some embodiments, the polypeptide comprises an amino acid sequence as set forth in any one of tables 2, 4, 6, 8, 10, 12, 14, 16, or 18 (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3 sequence), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

TABLE A1. novel Ring Virus nucleic acid sequences (type A ringlet viruses)

Name TTV-RTx1

Genus/evolved branch A-type ringworm virus, evolved branch 6

Accession number SRR2167793

The complete sequence is as follows: 3648bp

Note that:

TABLE A2 novel Ring Virus amino acid sequences (Levolavirus type A, clade 6)

TABLE A3 novel Ring Virus nucleic acid sequences (type A ringlet viruses)

Name TTV-RTx2

Genus/evolved branch A-type ringworm virus, evolved branch 6

Accession number SRR3479021

The complete sequence is as follows: 3704bp

Note that:

TABLE A4 novel Ring Virus amino acid sequences (Cycloviron A, clade 6)

TABLE A5 novel Ring Virus nucleic acid sequences (type A ringlet viruses)

Name TTV-RTx3

Genus/clade A-type ringworm virus, clade 4

Accession number SRR3479781

The complete sequence is as follows: 3653bp

Note that:

TABLE A6 novel Ring Virus amino acid sequences (Levolavirus type A, clade 4)

TABLE A7. novel finger ring virus nucleic acid sequences (ringlet A virus)

Name TTV-RTx4

Genus/clade A-type ringworm virus, clade 4

Accession number SRR3481579

The complete sequence is as follows: 3742bp

Note that:

TABLE A8. novel finger ring virus amino acid sequence (Levolavirus type A, clade 4)

TABLE A9. novel finger ring virus nucleic acid sequences (ringlet A virus)

The name TTV-RTx5b

Genus/clade A-type ringworm virus, clade 5

Accession number SRR3481639

The complete sequence is as follows: 3553bp

Note that:

note: modifications to maintain reading frame:

- "C" insertion ORF 2430

- "N" insertion ORF 11842

TABLE A10 novel Ring Virus amino acid sequences (Cycloviron A, clade 5)

TABLE A11 novel Ring Virus nucleic acid sequences (type A ringlet viruses)

Name TTV-RTx6

Genus/clade A-type ringworm virus, clade 5

Accession number SRR3438066

The complete sequence is as follows: 3896bp

Note that:

TABLE A12 novel Ring Virus amino acid sequence (Levolavirus type A, clade 5)

TABLE 1 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 1)

Name TTV-CT30F

Genus/clade A-type ringworm virus, clade 1

Accession number AB064597.1

The complete sequence is as follows: 3570bp

Note that:

TABLE 2 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 1)

TABLE 3 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 2)

Name TTV-P13-1

Genus/evolved branch A-type ringworm virus, evolved branch 2

Accession number KT163896.1

The complete sequence is as follows: 3451bp

Note that:

TABLE 4 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 2)

TABLE 5 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 3)

The name TTV-tth8

Genus/clade A-type ringworm virus, clade 3

Accession number AJ620231.1

The complete sequence is as follows: 3753bp

Note that:

TABLE 6 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 3)

TABLE 7 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 4)

Name TTV-HD20a

Genus/clade A-type ringworm virus, clade 4

Accession number FR751492.1

The complete sequence is as follows: 3878bp

Note that:

TABLE 8 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 4)

TABLE 9 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 5)

Name TTV-16(TUS01)

Genus/clade A-type ringworm virus, clade 5

Accession number AB017613.1

The complete sequence is as follows: 3818bp

Note that:

TABLE 10 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 5)

TABLE 11 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 6)

Name TTV-TJN02

Genus/evolved branch A-type ringworm virus, evolved branch 6

Accession number AB028669.1

The complete sequence is as follows: 3794bp

Note that:

TABLE 12 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 6)

TABLE 13 exemplary Ring Virus nucleic acid sequences (Levolavirus type A, clade 7)

Name TTV-HD16d

Genus/clade A-type ringworm virus, clade 7

Accession number FR751479.1

The complete sequence is as follows: 3866bp

Note that:

TABLE 14 exemplary Ring Virus amino acid sequences (Levolavirus type A, clade 7)

TABLE 15 exemplary Ring Virus nucleic acid sequences (type B ringlet Virus)

Name TTMV-LY2

Genus/evolution of branched b-ringworm virus

Accession number JX134045.1

The complete sequence is as follows: 2797bp

Note that:

TABLE 16 exemplary Ring Virus amino acid sequences (type B ringlet Virus)

TABLE 17 exemplary Ring Virus nucleic acid sequences (C-type ringlet Virus)

The name TTMDV-MD1-073

Genus/evolution of branched C-type ringworm virus

Accession number AB290918.1

The complete sequence is as follows: 3242bp

Note that:

TABLE 18 exemplary Ring Virus amino acid sequences (C-type ringlet Virus)

TABLE B1 exemplary Ring Virus nucleic acid sequences (C-type ringlet Virus)

Name ring 3.1

Genus/evolution of branched C-type ringworm virus

The complete sequence is as follows: 3264bp

Note that:

TABLE C1 exemplary Ring Virus amino acid sequence (C-type ringlet Virus)

TABLE B2 exemplary Ring Virus nucleic acid sequences (C-type ringlet Virus)

Name ring 4.0

Genus/evolution of branched C-type ringworm virus

The complete sequence is as follows: 3176bp of

Note that:

TABLE C2. exemplary finger Ring Virus amino acid sequence (C type ringlet Virus)

Table B3. exemplary Ring Virus nucleic acid sequences (Cyclovirome A) -clade 1

Name ring 5.2

Genus/clade a-type ringvirus clade 1

The complete sequence is as follows: 3696bp

Note that:

table C3. exemplary Ring Virus amino acid sequence (Cycloviron A) evolved branch 1

Table B4. exemplary finger Ring Virus nucleic acid sequence (Cycloviron A) -clade 3

Name ring 6.0

Genus/clade A-type ringworm virus-clade 3

The complete sequence is as follows: 3828bp

Note that:

TABLE C4 exemplary Ring Virus amino acid sequence (Cycloviron A) evolutionary Branch 3

Table B5. exemplary finger Ring Virus nucleic acid sequence (Cycloviron A) -clade 7

Name ring 7

Genus/clade A-type ringworm virus-clade 7

The complete sequence is as follows: 3815bp

Note that:

table C5. exemplary Ring Virus amino acid sequence (Cycloviron A) -clade 7

In some embodiments, the finger ring comprises a nucleic acid comprising a sequence set forth in PCT application No. PCT/US2018/037379 (incorporated herein by reference in its entirety). In some embodiments, the finger ring comprises a polypeptide comprising a sequence set forth in PCT application number PCT/US2018/037379 (incorporated herein by reference in its entirety).

In some embodiments, the finger ring comprises a finger virus genome, e.g., identified according to the method described in example 9. In some embodiments, the finger ring comprises a finger virus sequence or a portion thereof, as described in example 13.

In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF1 motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF1/1 motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF1/2 motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF2/2 motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF2/3 motif, e.g., as shown in table 19. In some embodiments, the finger ring comprises a genetic element comprising a common finger ring virus ORF2t/3 motif, e.g., as shown in table 19. In some embodiments, as shown in table 19, X represents any amino acid. In some embodiments, as shown in table 19, Z represents glutamic acid or glutamine. In some embodiments, as shown in table 19, B represents aspartic acid or asparagine. In some embodiments, as shown in table 19, J represents leucine or isoleucine.

TABLE 19 consensus motifs in the Open Reading Frame (ORF) of finger-like viruses

ORF1 molecule

In some embodiments, the finger ring comprises an ORF1 molecule and/or a nucleic acid encoding an ORF1 molecule. Typically, the ORF1 molecule comprises a polypeptide having the structural features and/or activity of a ring virus ORF1 protein (e.g., a ring virus ORF1 protein as described herein, e.g., as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10), or a functional fragment thereof. In some embodiments, the ORF1 molecule comprises a truncation relative to an ring virus ORF1 protein (e.g., a ring virus ORF1 protein as described herein, e.g., as listed in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10). In some embodiments, the ORF1 molecule is truncated to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids of the ring virus ORF1 protein. In some embodiments, the ORF1 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger virus ORF1 protein sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10. In some embodiments, the ORF1 molecule comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to, for example, a leptovirus a, b, or c ORF1 protein as described herein. The ORF1 molecule can generally bind to a nucleic acid molecule, such as DNA (e.g., a genetic element, e.g., as described herein). In some embodiments, the ORF1 molecule is localized to the nucleus. In certain embodiments, the ORF1 molecule is localized to the nucleolus of the cell.

Without wishing to be bound by theory, ORF1 molecules may be capable of binding to other ORF1 molecules, e.g., forming a proteinaceous exterior (e.g., as described herein). Such ORF1 molecules can be described as having the ability to form capsids. In some embodiments, the protein may coat the nucleic acid molecule (e.g., a genetic element as described herein) externally. In some embodiments, multiple ORF1 molecules can form multimers, e.g., to create a protein exterior. In some embodiments, the multimer can be a homomultimer. In other embodiments, the multimer can be a heteromultimer (e.g., comprising a plurality of different ORF1 molecules). It is also contemplated that the ORF1 molecule may have replicase activity.

In some embodiments, the ORF1 molecule can comprise one or more of the following: a first region comprising an arginine-rich region, e.g., a region having at least 60% basic residues (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% basic residues; e.g., 60% -90%, 60% -80%, 70% -90%, or 70-80% basic residues), and a second region comprising a jellyroll domain, e.g., at least six beta strands (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands).

Arginine-rich region

The arginine-rich region has at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence described herein, or a sequence of at least about 40 amino acids comprising at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof).

Jelly roll domains

The jelly roll domain or region comprises (e.g., consists of): a polypeptide (e.g., a domain or region comprised in a larger polypeptide) comprising one or more (e.g., 1, 2, or 3) of the following features:

(i) at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or more) of the amino acids of the jelly roll domain are part of one or more β -sheets;

(ii) the secondary structure of the jellyroll domain comprises at least four (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12) β -strands; and/or

(iii) The tertiary structure of the jellyroll domain comprises at least two (e.g., at least 2, 3, or 4) β -sheets; and/or

(iv) The jellyroll domain comprises a ratio of beta-sheet to alpha-helix of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1.

In certain embodiments, the jellyroll domain comprises two β -sheets.

In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) β -sheets comprise about eight (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12) β -strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) β -sheets comprise eight β -strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta-sheets comprise seven beta-strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) β -sheets comprise six β -strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) β -sheets comprise five β -strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) β -sheets comprise four β -strands.

In some embodiments, the jellyroll domain comprises a first β -sheet oriented antiparallel to a second β -sheet. In certain embodiments, the first β -sheet comprises about four (e.g., 3, 4, 5, or 6) β -strands. In certain embodiments, the second β -sheet comprises about four (e.g., 3, 4, 5, or 6) β -strands. In embodiments, the first and second β -sheets comprise a total of about eight (e.g., 6, 7, 8, 9, 10, 11, or 12) β -strands.

In certain embodiments, the jellyroll domain is a component of a capsid protein (e.g., an ORF1 molecule as described herein). In certain embodiments, the jellyroll domain has self-assembly activity. In some embodiments, the polypeptide comprising the jelly roll domain binds to another copy of the polypeptide comprising the jelly roll domain. In some embodiments, the jellyroll domain of the first polypeptide binds to a jellyroll domain of the second copy of the polypeptide.

The ORF1 molecule can also include a third region comprising structure or activity directed to a N22 domain of a ring virus (e.g., as described herein, e.g., from the N22 domain of the ring virus ORF1 protein as described herein), and/or a fourth region comprising structure or activity directed to a C-terminal domain (CTD) of a ring virus (e.g., as described herein, e.g., from the CTD of the ring virus ORF1 protein as described herein). In some embodiments, the ORF1 molecule comprises the first, second, third, and fourth regions in N-terminal to C-terminal order.

In some embodiments, the ORF1 molecule can further comprise a hypervariable region (HVR), such as an HVR from, for example, the finger ring virus ORF1 protein described herein. In some embodiments, an HVR is located between the second region and the third region. In some embodiments, an HVR comprises at least about 55 (e.g., at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (e.g., about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55-140, or 60-140 amino acids).

In some embodiments, the first region can bind a nucleic acid molecule (e.g., DNA). In some embodiments, the basic residue is selected from arginine, histidine, or lysine, or a combination thereof. In some embodiments, the first region comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% arginine residues (e.g., 60% -90%, 60% -80%, 70% -90%, or 70-80% arginine residues). In some embodiments, the first region comprises about 30-120 amino acids (e.g., about 40-120, 40-100, 40-90, 40-80, 40-70, 50-100, 50-90, 50-80, 50-70, 60-100, 60-90, or 60-80 amino acids). In some embodiments, the first region comprises the structure or activity of an arginine-rich region of viral ORF1 (e.g., an arginine-rich region from, e.g., the ring virus ORF1 protein described herein). In some embodiments, the first region comprises a nuclear localization signal.

In some embodiments, the second region comprises a jelly roll domain, e.g., the structure or activity of a viral ORF1 jelly roll domain (e.g., a jelly roll domain from, e.g., a ring virus ORF1 protein as described herein). In some embodiments, the second region is capable of binding to a second region of another ORF1 molecule, e.g., forming the outer portion of a protein (e.g., the capsid) or a portion thereof.

In some embodiments, the fourth region is exposed on the surface of the exterior of the protein (e.g., the exterior of the protein comprising a multimer of, for example, an ORF1 molecule as described herein).

In some embodiments, the first region, the second region, the third region, the fourth region, and/or the HVRs each comprise less than four (e.g., 0, 1, 2, or 3) beta-sheets.

In some embodiments, one or more of the first region, the second region, the third region, the fourth region, and/or the HVRs may be replaced with a heterologous amino acid sequence (e.g., the corresponding region from a heterologous ORF1 molecule). In some embodiments, the heterologous amino acid sequence has a desired functionality, e.g., as described herein.

In some embodiments, the ORF1 molecule comprises a plurality of conserved motifs (e.g., motifs comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids) (e.g., as shown in figure 34). In some embodiments, a conserved motif can exhibit 60%, 70%, 80%, 85%, 90%, 95%, or 100% sequence identity to one or more of the wild-type ringworm virus's ORF1 proteins (e.g., ringworm A virus, clade 1; ringworm A virus, clade 2; ringworm A virus, clade 3; ringworm A virus, clade 4; ringworm A virus, clade 5; ringworm A virus, clade 6; ringworm A virus, clade 7; ringworm B virus; and/or terworm C virus). In embodiments, the conserved motifs each have a length of 1-1000 (e.g., between 5-10, 5-15, 5-20, 10-15, 10-20, 15-20, 5-50, 5-100, 10-50, 10-100, 10-1000, 50-100, 50-1000, or 100-1000) amino acids. In certain embodiments, the conserved motifs consist of about 2% -4% (e.g., about 1% -8%, 1% -6%, 1% -5%, 1% -4%, 2% -8%, 2% -6%, 2% -5%, or 2% -4%) of the sequence of the ORF1 molecule and each displays 100% sequence identity to the corresponding motif in the wild-type, claded ORF1 protein of the dactylovirus. In certain embodiments, the conserved motifs consist of about 5% -10% (e.g., about 1% -20%, 1% -10%, 5% -20%, or 5% -10%) of the sequence of ORF1 molecule, and each displays 80% sequence identity to the corresponding motif in the wild-type, clade ORF1 protein. In certain embodiments, the conserved motifs consist of about 10% -50% (e.g., about 10% -20%, 10% -30%, 10% -40%, 10% -50%, 20% -40%, 20% -50%, or 30% -50%) of the sequence of ORF1 molecule and each displays 60% sequence identity to the corresponding motif in the wild-type, claded ORF1 protein. In some embodiments, the conserved motifs comprise one or more amino acid sequences as set forth in table 19.

In some embodiments, the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change) relative to, for example, a wild-type ORF1 protein as described herein (e.g., as set forth in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10).

Conserved ORF1 motif in the N22 domain

In some embodiments, a polypeptide described herein (e.g., an ORF1 molecule) comprises an amino acid sequence of YNPX²DXGX²N (SEQ ID NO:829), wherein XⁿIs a contiguous sequence of any n amino acids. For example, X²Representing a contiguous sequence of any two amino acids. In some embodiments, YNPX²DXGX²N (SEQ ID NO:829) is contained within the N22 domain of the ORF1 molecule, for example as described herein. In some embodiments, the genetic element described herein comprises a coding amino acid sequence YNPX²DXGX²N (SEQ ID NO:829) (e.g., a nucleic acid sequence encoding, for example, an ORF1 molecule as described herein), wherein XⁿIs a contiguous sequence of any n amino acids.

In some embodiments, the polypeptide (e.g., ORF1 molecule) comprises a conserved secondary structure, e.g., flanked by and/or comprises YNPX²DXGX²The N motif (SEQ ID NO:829), for example in the domain of N22. In some embodiments, the conserved secondary structure comprises the first beta strand and/or the second beta strand. In some embodiments, the first beta strand is about 5-6 (e.g., 3, 4, 5, 6, 7, or 8) amino acids in length. In some embodiments, the first beta chain comprises a position in YNPX ²DXGX²The tyrosine (Y) residue at the N-terminus of the N (SEQ ID NO:829) motif. In some embodiments, YNPX²DXGX²The N (SEQ ID NO:829) motif comprises a random coil (e.g., a random coil of about 8-9 amino acids). In some embodiments, the second beta strand is about 7-8 (e.g., 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second beta chain comprises a position in YNPX²DXGX²The asparagine (N) residue at the C-terminus of the N (SEQ ID NO:829) motif.

Exemplary YNPX²DXGX²The secondary structure flanking the N (SEQ ID NO:829) motif is described in example 47 and FIG. 48. In some embodiments, the ORF1 molecule comprises a region comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structural elements (e.g., the beta strands) shown in figure 48. In some embodiments, the ORF1 molecule comprises a region comprising the flanking YNPX shown in FIG. 48²DXGX²One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structural elements (e.g., beta strands) of the N (SEQ ID NO:829) motif (e.g., as described herein).

Conserved secondary structural motifs in ORF1 jelly roll domains

In some embodiments, a polypeptide described herein (e.g., an ORF1 molecule) comprises one or more secondary structural elements comprised by an ORF1 protein of a finger virus (e.g., as described herein). In some embodiments, the ORF1 molecule comprises one or more secondary structural elements comprised by the jellyroll domain of the ORF1 protein of an Ring virus (e.g., as described herein). Typically, the ORF1 jelly roll domain comprises a secondary structure comprising, in order in the direction from N-terminus to C-terminus, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and a ninth beta strand. In some embodiments, the ORF1 molecule comprises a secondary structure comprising, in order from the N-terminus to the C-terminus, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and/or a ninth beta strand.

In some embodiments, a pair of conserved secondary structural elements (i.e., beta strands and/or alpha helices) are separated by a gap amino acid sequence, e.g., comprising a random coil sequence, a beta strand or an alpha helix, or a combination thereof. The gap amino acid sequence between conserved secondary structural elements may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. In some embodiments, the ORF1 molecule can further comprise one or more additional beta strands and/or alpha helices (e.g., in a jellyroll). In some embodiments, continuous beta strands or continuous alpha helices may be combined. In some embodiments, the first beta strand and the second beta strand are comprised in a larger beta strand. In some embodiments, the third beta strand and the fourth beta strand are comprised in a larger beta strand. In some embodiments, the fourth beta strand and the fifth beta strand are comprised in a larger beta strand. In some embodiments, the sixth beta strand and the seventh beta strand are comprised in a larger beta strand. In some embodiments, the seventh beta strand and the eighth beta strand are comprised in a larger beta strand. In some embodiments, the eighth beta strand and the ninth beta strand are comprised in a larger beta strand.

In some embodiments, the first beta strand is about 5-7 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second beta strand is about 15-16 (e.g., 13, 14, 15, 16, 17, 18, or 19) amino acids in length. In some embodiments, the first alpha helix is about 15-17 (e.g., 13, 14, 15, 16, 17, 18, 19, or 20) amino acids in length. In some embodiments, the third beta strand is about 3-4 (e.g., 1, 2, 3, 4, 5, or 6) amino acids in length. In some embodiments, the fourth beta strand is about 10-11 (e.g., 8, 9, 10, 11, 12, or 13) amino acids in length. In some embodiments, the fifth beta strand is about 6-7 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second alpha helix is about 8-14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) amino acids in length. In some embodiments, the second alpha helix can be broken down into two smaller alpha helices (e.g., separated by a random coil sequence). In some embodiments, each of the two smaller alpha helices is about 4-6 (e.g., 2, 3, 4, 5, 6, 7, or 8) amino acids in length. In some embodiments, the sixth beta strand is about 4-5 (e.g., 2, 3, 4, 5, 6, or 7) amino acids in length. In some embodiments, the seventh beta strand is about 5-6 (e.g., 3, 4, 5, 6, 7, 8, or 9) amino acids in length. In some embodiments, the eighth beta strand is about 7-9 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids in length. In some embodiments, the ninth beta strand is about 5-7 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length.

Exemplary jelly roll domain secondary structures are described in example 47 and fig. 47. In some embodiments, the ORF1 molecule comprises a region comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structural elements (e.g., the beta strand and/or alpha helix) of any of the jelly roll domain secondary structures shown in figure 47.

Exemplary ORF1 sequences

In some embodiments, a polypeptide described herein (e.g., an ORF1 molecule) comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of the finger ring virus ORF1 subsequences, e.g., as described in any of tables 20-37 or D1-D10. In some embodiments, the finger ring described herein comprises an ORF1 molecule, the ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more finger ring virus ORF1 subsequences, e.g., as described in any of tables 20-37 or D1-D10. In some embodiments, the finger ring described herein comprises a nucleic acid molecule (e.g., a genetic element) encoding an ORF1 molecule, which ORF1 molecule comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more finger ring viral ORF1 subsequences, e.g., as described in any of tables 20-37 or D1-D10.

In some embodiments, one or more of the finger ring virus ORF1 subsequences comprise one or more of an arginine (Arg) -rich domain, a jelly roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD) (e.g., as set forth in any of tables 20-37 or D1-D10), or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the ORF1 molecule comprises multiple subsequences from different finger ring viruses (e.g., any combination of ORF1 subsequences selected from the group consisting of the group of clade 1-7 subsequences of the type a torque tenoviruses listed in tables 20-37 or D1-D10). In embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly roll domain, an N22 domain, and a CTD from one dactylovirus, and an HVR from another. In embodiments, the ORF1 molecule comprises one or more of the jelly roll domain, HVR, N22 domain, and CTD from one finger ring virus, and an Arg-rich domain from another. In embodiments, the ORF1 molecule comprises one or more of the Arg-rich domain, HVR, N22 domain, and CTD from one finger ring virus, and a jelly roll domain from another. In embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly roll domain, an HVR, and a CTD from one finger ring virus, and an N22 domain from another. In embodiments, the ORF1 molecule comprises one or more of the Arg-rich domain, the jelly roll domain, the HVR, and the N22 domain from one finger ring virus, and the CTD from another.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 20 (e.g., amino acids 1-66 of table 20). In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the Arg-rich region of table 21. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 20 (e.g., amino acids 67-277 of table 20). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 21. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 20 (e.g., amino acids 278 and 347 of Table 20). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 21. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table 20 (e.g., amino acid 348-513 of Table 20). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 21. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 20 (e.g., amino acids 513 and 680 of Table 20). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 21.

In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 22 (e.g., amino acids 1-69 of table 22). In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 23. In embodiments, one or more of the sub-sequences of ORF1 of a finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 22 (e.g., amino acids 70-279 of table 22). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 23. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 22 (e.g., amino acid 280-411 of Table 22). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 23. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table 22 (e.g., amino acid 412-578 of Table 22). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 23. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 22 (e.g., amino acid 579-747 of Table 22). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 23.

In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 24 (e.g., amino acids 1-68 of table 24). In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 25. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 24 (e.g., amino acids 69-280 of table 24). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 25. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 24 (e.g., amino acids 281-413 of Table 24). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 25. In embodiments, one or more of the sub-sequences of ORF1 of the dactylovirus comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table 24 (e.g., amino acid 414 and 479 of Table 24). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 25. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 24 (e.g., amino acid 580-743 of Table 24). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 25.

In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 26 (e.g., amino acids 1-74 of table 26). In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 27. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 26 (e.g., amino acids 75-284 of table 26). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 27. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 26 (e.g., amino acids 285-445 of Table 26). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 27. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table 26 (e.g., amino acid 446-611 of Table 26). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 27. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 26 (e.g., amino acids 612-780 of Table 26). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 27.

In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 28 (e.g., amino acids 1-75 of table 28). In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 29. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 28 (e.g., amino acids 75-284 of table 28). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 29. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 28 (e.g., amino acids 285-432 of Table 28). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 29. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table 28 (e.g., amino acid 433-599 of Table 28). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 29. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 28 (e.g., amino acids 600-780 of Table 28). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 29.

In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 30 (e.g., amino acids 1-77 of table 30). In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the Arg-rich region of table 31. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 30 (e.g., amino acids 78-286 of table 30). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 31. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 30 (e.g., amino acid 287-416 of Table 30). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 31. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table 30 (e.g., amino acids 417 and 585 of Table 30). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 31. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 30 (e.g., amino acids 586 and 746 of Table 30). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 31.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 32 (e.g., amino acids 1-74 of table 32). In embodiments, one or more of the finger loop virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 33. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 32 (e.g., amino acids 75-286 of table 32). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 33. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 32 (e.g., amino acids 287-428 of Table 32). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 33. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table 32 (e.g., amino acids 429-595 of Table 32). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 33. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 32 (e.g., amino acids 596-765 of Table 32). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 33.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table 34 (e.g., amino acids 1-38 of table 34). In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the Arg-rich region of table 35. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table 34 (e.g., amino acids 39-246 of table 34). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 35. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 34 (e.g., amino acids 247 and 374 of Table 34). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 35. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table 34 (e.g., amino acids 375 and 537 of Table 34). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 35. In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 34 (e.g., amino acid 538-666 of Table 34). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 35.

In embodiments, one or more of the sub-sequences of ORF1 of the finger ring virus comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the Arg-rich region of table 36 (e.g., amino acids 1-57 of table 36). In embodiments, one or more of the sub-sequences of ORF1 of a finger ring virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the Arg-rich region of table 37. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 36 (e.g., amino acids 58-259 of table 36). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jellyroll amino acid sequence of table 37. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table 36 (e.g., amino acids 260 and 351 of Table 36). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table 37. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table 36 (e.g., amino acid 352-510 of Table 36). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an N22 domain amino acid sequence of table 37. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table 36 (e.g., amino acids 511-673 of Table 36). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table 37.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table D1 (e.g., amino acids 1-66 of table D1). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Arg-rich region amino acid sequence of table D2. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D1 (e.g., amino acids 67-277 of table D1). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D2. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the HVR amino acid sequence of Table D1 (e.g., amino acids 278 and 347 of Table D1). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table D2. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table D1 (e.g., amino acids 348 and 513 of Table D1). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the N22 domain amino acid sequence of table D2. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table D1 (e.g., amino acids 513-680 of Table D1). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table D2.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table D3 (e.g., amino acids 1-66 of table D3). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Arg-rich region amino acid sequence of table D4. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D3 (e.g., amino acids 67-277 of table D3). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D4. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the HVR amino acid sequence of Table D3 (e.g., amino acids 278 and 347 of Table D3). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table D4. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table D3 (e.g., amino acids 348 and 513 of Table D3). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the N22 domain amino acid sequence of table D4. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table D3 (e.g., amino acids 513-680 of Table D3). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table D4.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table D5 (e.g., amino acids 1-66 of table D5). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Arg-rich region amino acid sequence of table D6. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D5 (e.g., amino acids 67-277 of table D5). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D6. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the HVR amino acid sequence of Table D5 (e.g., amino acids 278 and 347 of Table D5). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table D6. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the N22 domain amino acid sequence of Table D5 (e.g., amino acids 348 and 513 of Table D5). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the N22 domain amino acid sequence of table D6. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table D5 (e.g., amino acids 513-680 of Table D5). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table D6.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table D7 (e.g., amino acids 1-57 of table D7). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Arg-rich region amino acid sequence of table D8. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D7 (e.g., amino acids 58-259 of table D7). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D8. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table D7 (e.g., amino acid 260-351 of Table D7). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table D8. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table D7 (e.g., amino acids 352-510 of Table D7). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the N22 domain amino acid sequence of table D8. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table D7 (e.g., amino acid 511-673 of Table D7). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table D8.

In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Arg-rich region amino acid sequence of table D9 (e.g., amino acids 1-57 of table D9). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Arg-rich region amino acid sequence of table D10. In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D9 (e.g., amino acids 58-259 of table D9). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a jelly roll amino acid sequence of table D10. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HVR amino acid sequence of Table D9 (e.g., amino acid 260-351 of Table D9). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an HVR amino acid sequence of table D10. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the N22 domain of Table D9 (e.g., amino acids 352-510 of Table D9). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the N22 domain amino acid sequence of table D10. In embodiments, one or more of the sub-sequences of ORF1 of the finger virus comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the CTD amino acid sequence of Table D9 (e.g., amino acid 511-673 of Table D9). In embodiments, one or more of the finger ring virus ORF1 subsequences comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CTD region amino acid sequence of table D10.

TABLE 20 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 1)

The complete sequence is as follows: 680 AA

Note that:

TABLE 21 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 1)

TABLE 22 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 2)

The complete sequence is as follows: 747 AA

Note that:

TABLE 23 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 2)

TABLE 24 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 3)

The complete sequence is as follows: 743 AA

Note that:

TABLE 25 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 3)

TABLE 26 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 4)

The complete sequence is as follows: 780 AA

Note that:

TABLE 27 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 4)

TABLE 28 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 5)

The complete sequence is as follows: 761 AA

Note that:

TABLE 29 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 5)

TABLE 30 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 6)

The complete sequence is as follows: 746 AA

Note that:

TABLE 31 exemplary Ring Virus ORF1 amino acid subsequence (ringlet A virus, clade 6)

TABLE 32 exemplary Ring virus ORF1 amino acid subsequence (ringlet A virus, clade 7)

The complete sequence is as follows: 765 AA

Note that:

TABLE 33 exemplary Ring Virus ORF1 amino acid subsequence (Levolavirus type A, clade 7)

TABLE 34 exemplary Ring Virus ORF1 amino acid subsequence (torque B virus)

The complete sequence is as follows: 666 AA

Note that:

TABLE 35 exemplary Ring Virus ORF1 amino acid subsequence (torque B virus)

TABLE 36 exemplary Ring Virus ORF1 amino acid subsequence (C type torque virus)

The complete sequence is as follows: 673 AA

Note that:

TABLE 37 exemplary Ring Virus ORF1 amino acid subsequence (C type torque virus)

TABLE D1 exemplary Ring Virus ORF1 amino acid subsequence (C type torque virus)

The complete sequence is as follows: 677 AA

Note that:

TABLE D2. exemplary Ring Virus ORF1 amino acid subsequence (third type torque teno virus)

TABLE D3 exemplary Ring Virus ORF1 amino acid subsequence (C type torque virus)

The complete sequence is as follows: 662 AA

Note that:

TABLE D4. exemplary Ring Virus ORF1 amino acid subsequence (third type torque teno virus)

Table D5. exemplary finger Virus ORF1 amino acid subsequence (Lennovirus A) clade 1

The complete sequence is as follows: 728 AA

Note that:

table D6. exemplary finger Virus ORF1 amino acid subsequence (Lennovirus A) clade 1

Table D7. exemplary finger Ring Virus ORF1 amino acid subsequence (ringvirus A) -clade 3

The complete sequence is as follows: 767 AA

Note that:

table D8. exemplary finger Ring Virus ORF1 amino acid subsequence (ringvirus A) -clade 3

Table D9. exemplary finger Ring Virus ORF1 amino acid subsequence (ringvirus A) -clade 7

The complete sequence is as follows: 766 AA

Note that:

TABLE D10 exemplary Ring Virus ORF1 amino acid subsequence (ringworm virus A) -clade 7

Consensus ORF1 domain sequence

In some embodiments, the ORF1 molecule, e.g., as described herein, comprises one or more of a jelly roll domain, an N22 domain, and/or a C-terminal domain (CTD). In some embodiments, the jelly roll domain comprises an amino acid sequence having a jelly roll domain consensus sequence as described herein (e.g., as set forth in any one of tables 37A-37C). In some embodiments, the N22 domain comprises an amino acid sequence having an N22 domain consensus sequence as described herein (e.g., as set forth in any one of tables 37A-37C). In some embodiments, the CTD domain comprises an amino acid sequence having a CTD domain consensus sequence as described herein (e.g., as listed in any one of tables 37A-37C). In some embodiments, "(X) _a-b) "the amino acids listed in any one of the formats in tables 37A-37C comprise a contiguous series of amino acids, wherein the series comprises at least a and at most b amino acids. In certain embodiments, all amino acids in the series are the same. In other embodiments, the series includes at least two (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) different amino acids.

TABLE 37A. miniloop virus type A ORF1 domain consensus sequence

TABLE 37B Leptospira B ORF1 Domain consensus sequences

TABLE 37C. miniringvirus type C ORF1 domain consensus sequence

In some embodiments, the jellyroll domain comprises the jellyroll domain amino acid as set forth in any one of tables 21, 23, 25, 27, 29, 31, 33, 35, D2, D4, D6, D8, D10, or 37A-37C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the N22 domain comprises N22 domain amino acid as set forth in any one of tables 21, 23, 25, 27, 29, 31, 33, 35, D2, D4, D6, D8, D10, or 37A-37C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the CTD domain comprises CTD domain amino acid as set forth in any one of tables 21, 23, 25, 27, 29, 31, 33, 35, D2, D4, D6, D8, D10, or 37A-37C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

ORF2 molecule

In some embodiments, the finger ring comprises an ORF2 molecule and/or a nucleic acid encoding an ORF2 molecule. Typically, the ORF2 molecule comprises a polypeptide having the structural features and/or activity of a ring virus ORF2 protein (e.g., a ring virus ORF2 protein as described herein, e.g., as set forth in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18), or a functional fragment thereof. In some embodiments, the ORF2 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger virus ORF2 protein sequence as set forth in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18.

In some embodiments, the ORF2 molecule comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the a, b, or c leptovirus ORF2 protein. In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the A torque teno virus ORF2 protein) is 250 or fewer amino acids in length (e.g., about 150-200 amino acids). In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the b-type torque virus ORF2 protein) is about 50-150 amino acids in length. In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the C-type torque virus ORF2 protein) is about 100-200 amino acids (e.g., about 100-150 amino acids) in length. In some embodiments, the ORF2 molecule comprises a helix-turn-helix motif (e.g., a helix-turn-helix motif comprising two alpha helices flanking a turn region). In some embodiments, the ORF2 molecule does not comprise the amino acid sequence of ORF2 protein of TTV isolate TA278 or of TTV isolate SANBAN. In some embodiments, the ORF2 molecule has protein phosphatase activity. In some embodiments, the ORF2 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic change) relative to, for example, a wild-type ORF2 protein as described herein (e.g., as shown in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18).

Conserved ORF2 motif

In some embodiments, a polypeptide described herein (e.g., an ORF2 molecule) comprises the amino acid sequence [ W/F]X⁷HX³CX¹CX⁵H (SEQ ID NO:949), wherein XⁿIs a contiguous sequence of any n amino acids. In the examples, X⁷Representing a contiguous sequence of any seven amino acids. In the examples, X³Representing a contiguous sequence of any three amino acids. In the examples, X¹Represents any single amino acid. In the examples, X⁵Representing a contiguous sequence of any five amino acids. In some embodiments, [ W/F ]]Can be tryptophan or phenylalanine. In some embodiments, [ W/F ]]X⁷HX³CX¹CX⁵H (SEQ ID NO:949) is contained within the N22 domain of, for example, the ORF2 molecule as described herein. In some embodiments, the genetic elements described herein comprise a coding amino acid sequence [ W/F [ ]]X⁷HX³CX¹CX⁵H (SEQ ID NO:949) (e.g., a nucleic acid sequence encoding, for example, an ORF2 molecule as described herein), wherein XⁿIs a contiguous sequence of any n amino acids.

Genetic elements

In some embodiments, the ring body comprises a genetic element. In some embodiments, the genetic element has one or more of the following characteristics: is essentially non-integrated with the genome of the host cell, is an episomal nucleic acid, is a single-stranded DNA, is circular, of about 1 to 10kb, exists within the nucleus, and can bind to endogenous proteins to produce effectors, such as polypeptides or nucleic acids (e.g., RNA, iRNA, microrna) that target genes, activities, or functions of the host or target cell. In one embodiment, the genetic element is substantially non-integrated DNA. In some embodiments, the genetic element comprises a packaging signal, such as a sequence that binds a capsid protein. In some embodiments, the genetic element has less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to a wild-type dactylovirus nucleic acid sequence, in addition to the packaging or capsid binding sequence, e.g., less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to a dactylovirus nucleic acid sequence, e.g., as described herein. In some embodiments, the genetic element has less than 500, 450, 400, 350, 300, 250, 200, 150, or 100 contiguous nucleotides that are at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a dactylovirus nucleic acid sequence, in addition to the packaging or capsid binding sequence. In certain embodiments, the genetic element is a circular single-stranded DNA comprising a promoter sequence, a sequence encoding a therapeutic effector, and a capsid-binding protein.

In one embodiment, the genetic element has at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a ring virus nucleic acid sequence or fragment thereof, e.g., as described herein (e.g., as described in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15 or 17), or encodes an amino acid sequence having at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a ring virus amino acid sequence (e.g., as described in any of tables a2, a4, A6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16 or 18) or fragment thereof, or a fragment thereof. In embodiments, the genetic element comprises a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a payload), e.g., a polypeptide effector (e.g., a protein) or a nucleic acid effector (e.g., a non-coding RNA, e.g., miRNA, siRNA, mRNA, incrna, RNA, DNA, antisense RNA, gRNA).

In some embodiments, the genetic element is less than 20kb in length (e.g., less than about 19kb, 18kb, 17kb, 16kb, 15kb, 14kb, 13kb, 12kb, 11kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb or less). In some embodiments, the genetic element independently or additionally has a length greater than 1000b (e.g., at least about 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb, 4.7kb, 4.8, 4.9, 5kb or greater). In some embodiments, the genetic element is about 2.5kb-4.6kb, 2.8kb-4.0kb, 3.0kb-3.8kb, or 3.2kb-3.7kb in length. In some embodiments, the genetic element has a length of about 1.5-2.0, 1.5-2.5, 1.5-3.0, 1.5-3.5, 1.5-3.8, 1.5-3.9, 1.5-4.0, 1.5-4.5, or 1.5-5.0 kb. In some embodiments, the genetic element has a length of about 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-3.8, 2.0-3.9, 2.0-4.0, 2.0-4.5, or 2.0-5.0 kb. In some embodiments, the genetic element has a length of about 2.5-3.0, 2.5-3.5, 2.5-3.8, 2.5-3.9, 2.5-4.0, 2.5-4.5, or 2.5-5.0 kb. In some embodiments, the genetic element has a length of about 3.0-5.0, 3.5-5.0, 4.0-5.0, or 4.5-5.0 kb. In some embodiments, the genetic element has a length of about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5, or 4.5-5.0 kb.

In some embodiments, the genetic element comprises one or more features described herein, e.g., a sequence encoding a substantially non-pathogenic protein, a protein binding sequence, one or more sequences encoding a regulatory nucleic acid, one or more regulatory sequences, one or more sequences encoding a replication protein, and other sequences. In some embodiments, a substantially non-pathogenic protein comprises an amino acid sequence, or a functional fragment thereof, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences described herein (referring to the ring virus amino acid sequences, e.g., listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18).

In embodiments, the genetic element is produced from double-stranded circular DNA (e.g., produced by in vitro circularization). In some embodiments, the genetic element is produced by rolling circle replication of double stranded circular DNA. In embodiments, rolling circle replication occurs in a cell (e.g., a host cell, e.g., a mammalian cell, e.g., a human cell, e.g., a HEK293T cell, an a549 cell, or a Jurkat cell). In embodiments, the genetic element may be exponentially amplified by rolling circle replication in the cell. In embodiments, the genetic element may be amplified linearly by rolling circle replication in the cell. In embodiments, the double-stranded circular DNA or genetic element is capable of producing at least 2, 4, 8, 16, 32, 64, 128, 256, 518, 1024 or more times the original amount by rolling circle replication in a cell. In embodiments, a double-stranded circular DNA, e.g., as described herein, is introduced into a cell.

In some embodiments, the double-stranded circular DNA and/or genetic element does not comprise one or more bacterial plasmid elements (e.g., a bacterial origin of replication or a selectable marker, such as a bacterial resistance gene). In some embodiments, the double-stranded circular DNA and/or genetic element does not comprise a bacterial plasmid backbone.

In one embodiment, the invention includes a genetic element comprising a nucleic acid sequence (e.g., a DNA sequence) encoding (i) a substantially non-pathogenic external protein, (ii) an external protein binding sequence that binds the genetic element to the substantially non-pathogenic external protein, and (iii) a regulatory nucleic acid. In such embodiments, the genetic element can comprise one or more sequences having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any of the nucleotide sequences of a native viral sequence (e.g., a native dactylovirus sequence, e.g., as described herein).

Protein binding sequences

The strategy employed by many viruses is that the viral capsid proteins recognize specific protein binding sequences in their genomes. For example, in viruses with an unsegmented genome (e.g., yeast L-A virus), there is a secondary structure (stem loop) and specific sequences at the 5' end of the genome, which are used to bind viral capsid proteins. However, viruses with segmented genomes, such as reoviridae, orthomyxoviridae (influenza), bunyaviridae, and arenaviruses, require packaging of each genome segment. Some viruses utilize complementary regions of the fragments to help the virus include one of each genomic molecule. Other viruses have specific binding sites for each different fragment. See, for example, Curr Opin Struct Biol [ latest view of structural biology ] month 2 2010; 20(1) 114-; and Journal of Virology (2003),77(24),13036 and 13041.

In some embodiments, the genetic element encodes a protein binding sequence that binds to a substantially non-pathogenic protein. In some embodiments, the protein binding sequence facilitates packaging of the genetic element into the exterior of the protein. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a substantially non-pathogenic protein. In some embodiments, the genetic element comprises a protein binding sequence as described in example 8. In some embodiments, the genetic element comprises a protein binding sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a 5' UTR conserved domain or a GC-rich domain of an finger ring virus sequence (e.g., as shown in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17).

In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a1 (e.g., nucleotide 165-235 of the nucleic acid sequence of table a 1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an finger ring virus GC-rich nucleotide sequence of Table A1 (e.g., nucleotides 3620 and 3648 of the nucleic acid sequence of Table A1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table A3 (e.g., nucleotide 175-245 of the nucleic acid sequence of table A3). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a5 (e.g., nucleotide 138-208 of the nucleic acid sequence of table a 5). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a7 (e.g., nucleotide 174-244 of the nucleic acid sequence of table a 7). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table A7 (e.g., nucleotide 3720-3742 of the nucleic acid sequence of Table A7). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a9 (e.g., nucleotide 100-171 of the nucleic acid sequence of table a 9). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a11 (e.g., nucleotide 294-364 of the nucleic acid sequence of table a 11). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table a1 (e.g., nucleotide 3844-3895 of the nucleic acid sequence of table a 11).

In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 1 (e.g., nucleotides 177-247 of the nucleic acid sequence of Table 1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 1 (e.g., nucleotide 3415-3570 of the nucleic acid sequence of Table 1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 3 (e.g., nucleotide 204-273 of the nucleic acid sequence of Table 3). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table 3 (e.g., nucleotide 3302-3541 of the nucleic acid sequence of Table 3). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 5 (e.g., nucleotide 170-240 of the nucleic acid sequence of Table 5). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 5 (e.g., nucleotides 3632-3753 of the nucleic acid sequence of Table 5). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 7 (e.g., nucleotide 170-238 of the nucleic acid sequence of Table 7). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 7 (e.g., nucleotides 3768 and 3878 of the nucleic acid sequence of Table 7). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 9 (e.g., nucleotide 170-240 of the nucleic acid sequence of Table 9). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table 9 (e.g., nucleotide 3302-3541 of the nucleic acid sequence of Table 9). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 11 (e.g., nucleotide 174-244 of the nucleic acid sequence of Table 11). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table 11 (e.g., nucleotides 3691-3794 of the nucleic acid sequence of Table 11). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 13 (e.g., nucleotide 170-240 of the nucleic acid sequence of Table 13). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 13 (e.g., nucleotides 3759-3866 of a nucleic acid sequence of Table 13). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 15 (e.g., nucleotide 323-393 of the nucleic acid sequence of Table 15). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table 15 (e.g., nucleotide 2868-2929 of the nucleic acid sequence of Table 15). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 17 (e.g., nucleotide 117-187 of the nucleic acid sequence of Table 17). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table 17 (e.g., nucleotide 3054-3172 of the nucleic acid sequence of Table 17).

In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B1 (e.g., nucleotide 185-255 of the nucleic acid sequence of table B1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an finger ring virus GC-rich nucleotide sequence of Table B1 (e.g., nucleotides 3141-3264 of the nucleic acid sequence of Table B1). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B2 (e.g., nucleotide 185-254 of the nucleic acid sequence of table B2). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an finger ring virus GC-rich nucleotide sequence of table B2 (e.g., nucleotides 3076-3176 of the nucleic acid sequence of table B2). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B3 (e.g., nucleotide 178-248 of the nucleic acid sequence of table B3). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table B3 (e.g., nucleotide 3555-3696 of the nucleic acid sequence of Table B3). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B4 (e.g., nucleotide 176-246 of the nucleic acid sequence of table B4). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of Table B4 (e.g., nucleotide 3720-3828 of the nucleic acid sequence of Table B4). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B5 (e.g., nucleotide 170-240 of the nucleic acid sequence of table B5). In embodiments, the protein binding sequence has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an finger ring virus GC-rich nucleotide sequence of table B5 (e.g., nucleotides 3716-3815 of the nucleic acid sequence of table B5).

5' UTR region

In some embodiments, a genetic element (e.g., a protein-binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a nucleic acid sequence set forth in table 38 and/or figure 20. In some embodiments, the genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence of a consensus 5' UTR sequence shown in table 38, wherein X₁、X₂、X₃、X₄And X₅Each independently is any nucleotide, e.g. wherein X₁G or T, X₂Either C or A, X₃G or A, X₄Is ═ T or C, and X₅A, C or T). In embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a consensus 5' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of a genetic element)) Nucleic acid sequences having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to exemplary TTV 5' UTR sequences set forth in table 38 are included. In embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-CT30F 5' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-HD23a 5' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-JA 205' UTR sequence set forth in table 38. In embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-TJN 025' UTR sequence set forth in table 38. In embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-tth 85' UTR sequence set forth in table 38.

In embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a type a torque virus consensus 5' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an a-type torque teno virus evolved branch 15' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a leptovirus type a clade 25' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an a-type torque teno virus evolved branch 35' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an a-type torque teno virus evolved branch 45' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a leptovirus type a clade 55' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a leptovirus type a clade 65' UTR sequence set forth in table 38. In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an a-type torque teno virus evolved branch 75' UTR sequence set forth in table 38.

In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a1 (e.g., nucleotide 165-235 of the nucleic acid sequence of table a 1). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table A3 (e.g., nucleotide 175-245 of the nucleic acid sequence of table A3). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a5 (e.g., nucleotide 138-208 of the nucleic acid sequence of table a 5). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a7 (e.g., nucleotide 174-244 of the nucleic acid sequence of table a 7). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a9 (e.g., nucleotide 100-171 of the nucleic acid sequence of table a 9). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table a11 (e.g., nucleotide 294-364 of the nucleic acid sequence of table a 11).

In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of Table 1 (e.g., nucleotides 177-247 of the nucleic acid sequence of Table 1). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 3 (e.g., nucleotide 204-273 of the nucleic acid sequence of table 3). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 5 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 5). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 7 (e.g., nucleotide 170-238 of the nucleic acid sequence of table 7). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 9 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 9). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 11 (e.g., nucleotide 174-244 of the nucleic acid sequence of table 11). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 13 (e.g., nucleotide 170-240 of the nucleic acid sequence of table 13). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 15 (e.g., nucleotide 323-393 of the nucleic acid sequence of table 15). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table 17 (e.g., nucleotide 117-187 of the nucleic acid sequence of table 17).

In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B1 (e.g., nucleotide 185-255 of the nucleic acid sequence of table B1). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B2 (e.g., nucleotide 185-254 of the nucleic acid sequence of table B2). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B3 (e.g., nucleotide 178-248 of the nucleic acid sequence of table B3). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B4 (e.g., nucleotide 176-246 of the nucleic acid sequence of table B4). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence of a conserved domain of the finger loop virus 5' UTR of table B5 (e.g., nucleotide 170-240 of the nucleic acid sequence of table B5).

TABLE 38 exemplary 5' UTR sequences from finger-Ring viruses

GC enrichment region

In some embodiments, a genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a nucleic acid sequence set forth in table 39 and/or any of figures 20 and 32. In embodiments, the genetic element (e.g., a protein binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a GC-rich sequence shown in table 39.

In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a 36 nucleotide GC-rich sequence shown in table 39 (e.g., 36 nucleotide consensus GC-rich sequence 1, 36 nucleotide consensus GC-rich sequence 2, 136 nucleotide region of TTV evolution branch, 336 nucleotide region of TTV evolution branch, 136 nucleotide region of TTV evolution 3 isolate GH, 3sle 193236 nucleotides region of TTV evolution branch, 4ctdc 00236 nucleotide region of TTV evolution branch, 536 nucleotide region of TTV evolution branch, 636 nucleotide region of TTV evolution branch, or 736 nucleotide region of TTV evolution branch). In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence comprising at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a 36-nucleotide GC-rich sequence shown in table 39 (e.g., 36-nucleotide consensus GC-rich sequence 1, 36-nucleotide consensus GC-rich sequence 2, 136-nucleotide region of TTV evolution branch, 336-nucleotide region of TTV evolution branch, 136-nucleotide region of TTV evolution branch 3 isolate GH, 3sle 193236-nucleotide region of TTV evolution branch, 4ctdc 00236-nucleotide region of TTV evolution branch, 536-nucleotide region of TTV evolution branch, 636-nucleotide region of TTV evolution branch, or 736-nucleotide region of TTV evolution branch).

In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence that is at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a parvovirus type a GC-rich region sequence (e.g., selected from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, or TTV-HD16d, e.g., as listed in table 39). In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 104, 105, 108, 110, 111, 115, 120, 122, 130, 140, 145, 150, 155, or 156 consecutive nucleotides of a type a torque teno virus GC-rich region sequence (e.g., selected from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, or TTV-HD16d, e.g., as listed in table 39).

In embodiments, the 36 nucleotide GC-rich sequence is selected from:

(i)CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC(SEQ ID NO:160)，

(ii)GCGCTX₁CGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO:164), wherein X₁Selected from T, G or A;

(iii)GCGCTTCGCGCGCCGCCCACTAGGGGGCGTTGCGCG(SEQ ID NO:165)；

(iv)GCGCTGCGCGCGCCGCCCAGTAGGGGGCGCAATGCG(SEQ ID NO:166)；

(v)GCGCTGCGCGCGCGGCCCCCGGGGGAGGCATTGCCT(SEQ ID NO:167)；

(vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC(SEQ ID NO:168)；

(vii)GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC(SEQ ID NO:169)；

(viii)GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC(SEQ ID NO:170)；

(ix) GCGCTACGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or

(x)GCGCTACGCGCGCGCGCCGGGGGGCTCTGCCCCCCC(SEQ ID NO:172)。

In an embodiment, the genetic element (e.g., a protein binding sequence of a genetic element) comprises nucleic acid sequence CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (SEQ ID NO: 160).

In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence of a consensus GC-rich sequence shown in table 39, where X is₁、X₄、X₅、X₆、X₇、X₁₂、X₁₃、X₁₄、X₁₅、X₂₀、X₂₁、X₂₂、X₂₆、X₂₉、X₃₀And X₃₃Each independently is any nucleotide, and wherein X₂、X₃、X₈、X₉、X₁₀、X₁₁、X₁₆、X₁₇、X₁₈、X₁₉、X₂₃、X₂₄、X₂₅、X₂₇、X₂₈、X₃₁、X₃₂And X₃₄Each independently is absent or any nucleotide. In some embodiments, X₁To X₃₄Is each independently a nucleotide (or is absent) specified in table 39. In embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an exemplary TTV GC-enriched sequence (e.g., full sequence, fragment 1, fragment 2, fragment 3, or any combination thereof, e.g., fragments 1-3 in sequence) shown in table 39. In embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-CT30F GC-rich sequence shown in table 39 (e.g., full sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1-7 in sequence). In embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a sequence that is homologous to the TTV-HD23a GC enrichment sequence shown in table 39 A nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity (e.g., full sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, or any combination thereof, e.g., fragments 1-6 in sequence). In embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-JA20 GC-enriched sequence (e.g., the full sequence, fragment 1, fragment 2, or any combination thereof, such as fragments 1 and 2 in sequence) shown in table 39. In embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-TJN02 GC-rich sequence shown in table 39 (e.g., full sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1-8 in sequence). In embodiments, the genetic element (e.g., a protein-binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-tth8 GC-rich sequence shown in table 39 (e.g., full sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, fragment 9, or any combination thereof, e.g., fragments 1-6 in sequence). In embodiments, a genetic element (e.g., a protein-binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 7 shown in table 39. In embodiments, a genetic element (e.g., a protein-binding sequence of a genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 8 shown in table 39. In embodiments, the genetic element (e.g., heritage) Protein binding sequences of the elements) include nucleic acid sequences having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 9 shown in table 39.

TABLE 39 exemplary GC enrichment sequences from dactyloviruses

In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 1 (e.g., nucleotide 3415-3570 of the nucleic acid sequence of table 1). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 3 (e.g., nucleotide 3302-3541 of the nucleic acid sequence of table 3). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 5 (e.g., nucleotides 3632-3753 of the nucleic acid sequence of table 5). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 7 (e.g., nucleotides 3768 and 3878 of the nucleic acid sequence of Table 7). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 9 (e.g., nucleotide 3302-3541 of a nucleic acid sequence of table 9). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 11 (e.g., nucleotides 3691-3794 of the nucleic acid sequence of table 11). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 13 (e.g., nucleotides 3759-3866 of a nucleic acid sequence of table 13). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of Table 15 (e.g., nucleotide 2868-2929 of a nucleic acid sequence of Table 15). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a finger loop virus GC-rich nucleotide sequence of table 17 (e.g., nucleotide 3054-3172 of the nucleic acid sequence of table 17).

In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table B1 (e.g., nucleotides 3141-3264 of the nucleic acid sequence of table B1). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table B2 (e.g., nucleotides 3076-3176 of the nucleic acid sequence of table B2). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table B3 (e.g., nucleotide 3555-3696 of the nucleic acid sequence of table B3). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table B4 (e.g., nucleotide 3720-3828 of the nucleic acid sequence of table B4). In embodiments, the genetic element comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a finger ring virus GC-rich nucleotide sequence of table B5 (e.g., nucleotides 3716-3815 of the nucleic acid sequence of table B5).

Effector

In some embodiments, the genetic element may comprise one or more sequences encoding a functional effector, such as an endogenous effector or an exogenous effector, such as a therapeutic polypeptide or nucleic acid, such as a cytotoxic or cytolytic RNA or protein. In some embodiments, the functional nucleic acid is a non-coding RNA. In some embodiments, the functional nucleic acid is an encoding RNA. Effectors may modulate biological activity, such as increasing or decreasing enzyme activity, gene expression, cell signaling, and cell or organ function. Effector activity may also include binding to regulatory proteins to modulate the activity of a modulator, such as transcription or translation. Effector activity may also include activator or inhibitor functions. For example, effectors may induce enzymatic activity by triggering an increase in substrate affinity in the enzyme, e.g., fructose 2, 6-diphosphate activates phosphofructokinase 1 and increases the rate of glycolysis in response to insulin. In another example, effectors may inhibit binding of a substrate to a receptor and inhibit its activation, e.g., naltrexone and naloxone bind opioid receptors without activating them and block the ability of the receptors to bind opioids. Effector activity may also include modulating the stability/degradation of proteins and/or the stability/degradation of transcripts. For example, proteins can be targeted for degradation by the polypeptide cofactor ubiquitin on the proteins to label them for degradation. In another example, the effector inhibits enzyme activity by blocking the active site of the enzyme, e.g., methotrexate is a structural analog of tetrahydrofolate, a coenzyme of dihydrofolate reductase, that binds 1000-fold more to dihydrofolate reductase than to natural substrates and inhibits nucleotide base synthesis.

In some embodiments, the sequence encoding the effector is part of a genetic element, e.g., it may be inserted at an insertion site as described in examples 10, 12, or 22. In embodiments, effector-encoding sequences are inserted into the genetic element at the noncoding region, e.g., the noncoding region located 3' of the open reading frame and 5' of the GC-rich region of the genetic element, in the 5' noncoding region upstream of the TATA box, in the 5' UTR, in the 3' noncoding region downstream of the polya signal or upstream of the GC-rich region. In embodiments, the effector-encoding sequence is inserted into the genetic element at about nucleotide 3588 of a TTV-tth8 plasmid, e.g., as described herein, or at about nucleotide 2843 of a TTMV-LY2 plasmid, e.g., as described herein. In embodiments, the effector-encoding sequence is inserted into the genetic element at or within nucleotide 336-3015 of, for example, the TTV-tth8 plasmid described herein or at or within nucleotide 242-2812 of, for example, the TTV-LY2 plasmid described herein. In some embodiments, the sequence encoding the effector replaces a portion or all of an open reading frame (e.g., an ORF described herein, e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, and/or ORF2t/3 shown in any one of tables a1-a12, B1-B5, C1-C5, or 1-18).

In some embodiments, the sequence encoding the effector comprises 100-2000, 100-1000, 100-500, 100-200, 200-2000, 200-1000, 200-500, 500-1000, 500-2000 or 1000-2000 nucleotides. In some embodiments, the effector is a nucleic acid or protein payload, e.g., as described in example 11.

Regulatory nucleic acids

In some embodiments, the effector is a regulatory nucleic acid. The regulatory nucleic acid modifies the expression of the endogenous gene and/or the exogenous gene. In one embodiment, the regulatory nucleic acid targets a host gene. Regulatory nucleic acids can include, but are not limited to, nucleic acids that hybridize to an endogenous gene (e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA, as described elsewhere herein), nucleic acids that hybridize to an exogenous nucleic acid (e.g., viral DNA or RNA), nucleic acids that hybridize to RNA, nucleic acids that interfere with gene transcription, nucleic acids that interfere with RNA translation, nucleic acids that stabilize RNA or destabilize RNA (e.g., by targeted degradation), and nucleic acids that modulate DNA or RNA binding factors. In embodiments, the regulatory nucleic acid encodes a miRNA.

In some embodiments, the regulatory nucleic acid comprises an RNA or RNA-like structure (depending on the particular RNA structure, e.g., miRNA 5-30bp, lncRNA 200-500bp) that typically comprises 5-500 base pairs and may have a nucleobase sequence that is identical (or complementary) or nearly identical (or substantially complementary) to a coding sequence in or encoding a target gene expressed in a cell.

In some embodiments, the regulatory nucleic acid comprises a nucleic acid sequence, e.g., a guide rna (grna). In some embodiments, the DNA targeting moiety comprises a guide RNA or a nucleic acid encoding a guide RNA. Short synthetic RNAs of grnas can consist of a "scaffold" sequence necessary for binding to an incomplete effector moiety and a user-defined targeting sequence of about 20 nucleotides for genomic targets. In practice, the guide RNA sequence is typically designed to have a length of 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and is complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for designing effective guide RNAs. Gene editing is also achieved using chimeric "single guide RNAs" ("sgrnas"), an engineered (synthetic) single RNA molecule that mimics the naturally occurring crRNA-tracrRNA complex and comprises a tracrRNA (for binding a nuclease) and at least one crRNA (to direct the nuclease to edit a target sequence). Chemically modified sgrnas have also been demonstrated to be effective in genome editing; see, for example, Hendel et al (2015) Nature Biotechnol. [ Nature Biotechnology ], 985-.

A regulatory nucleic acid comprises a gRNA that recognizes a particular DNA sequence (e.g., a sequence adjacent to or within a promoter, enhancer, silencer, or repressor of a gene).

Certain regulatory nucleic acids can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules comprise RNA or RNA-like structures that typically comprise 15-50 base pairs (e.g., about 18-25 base pairs) and have a nucleobase sequence that is identical (complementary) or nearly identical (substantially complementary) to a coding sequence in a target gene expressed in a cell. RNAi molecules include, but are not limited to: short interfering rnas (sirnas), double-stranded rnas (dsrnas), micrornas (mirnas), short hairpin rnas (shrnas), partial duplexes, and dicer substrates (U.S. patent nos. 8,084,599, 8,349,809, and 8,513,207).

Long non-coding rna (lncrna) is defined as a non-protein coding transcript longer than 100 nucleotides. This somewhat arbitrary restriction distinguishes lncRNA from small regulatory RNAs (e.g., microRNA (miRNA), short interfering RNA (siRNA), and other short RNAs). Typically, most (about 78%) lncrnas are characterized as tissue-specific. Divergent lncrnas (accounting for a large proportion of about 20% of the total lncrnas in a mammalian genome) that are transcribed in the opposite direction to nearby protein-encoding genes may regulate transcription of nearby genes.

The genetic element may encode a regulatory nucleic acid having a sequence that is substantially complementary or fully complementary to all or a fragment of an endogenous gene or gene product (e.g., mRNA). The regulatory nucleic acid may be complementary to sequences at the boundary between an intron and an exon, thereby preventing the newly generated nuclear RNA transcript of the specific gene from maturing into mRNA for transcription. Regulatory nucleic acids complementary to a particular gene can hybridize to the mRNA of that gene and prevent its translation. The antisense regulatory nucleic acid may be DNA, RNA or derivatives or hybrids thereof.

The length of the regulatory nucleic acid that hybridizes to a transcript of interest can be between 5 and 30 nucleotides, between about 10 and 30 nucleotides, or about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. The degree of identity of the regulatory nucleic acid to the target transcript should be at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

The genetic element may encode a regulatory nucleic acid, such as a microrna (mirna) molecule that is identical to about 5 to about 25 contiguous nucleotides of the target gene. In some embodiments, the miRNA sequence targets an mRNA and begins with a dinucleotide AA, has a GC content of about 30% -70% (about 30% -60%, about 40% -60%, or about 45% -55%), and does not have a high percentage of identity to any nucleotide sequence other than the target in the mammalian genome into which it is to be introduced, e.g., as determined by a standard BLAST search.

In some embodiments, the regulatory nucleic acid is at least one miRNA, e.g., 2, 3, 4, 5, 6, or more. In some embodiments, the genetic element comprises a sequence encoding a miRNA having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to any of the nucleotide sequences described herein or a sequence complementary to the sequence.

siRNA and shRNA are analogous to intermediates in the processing pathway of the endogenous microRNA (miRNA) gene (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNA may be used as miRNA and vice versa (Zeng et al, Mol Cell [ molecular cytology ]9: 1327-. Like siRNA, micrornas down-regulate target genes using RISC, but unlike siRNA, most animal mirnas do not cleave mRNA. In contrast, miRNAs reduce protein export through translational inhibition or poly A removal and mRNA degradation (Wu et al, Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ]103: 4034-. The known miRNA binding site is located within the mRNA 3' UTR; miRNAs appear to target sites that are almost completely complementary to 2-8 nucleotides at the 5' end of the miRNA (Rajewsky, Nat Genet [ Nature genetics ]38 supplement: S8-13,2006; Lim et al, Nature [ Nature ]433: 769-. This area is called the seed area. Since siRNA and miRNA are interchangeable, exogenous siRNA down-regulates mRNA that is seed complementary to siRNA (Birmingham et al, Nat Methods [ Nature Methods ]3:199-204, 2006). Multiple target sites within the 3' UTR lead to stronger downregulation (Doench et al, Genes Dev [ Gene and development ]17:438-442, 2003).

The list of known miRNA sequences can be found in databases maintained by research organizations such as the Wellcome Trust Sanger Institute (Wellcome true Sanger Institute), the Pennsylvania Bioinformatics Center (Penn Center for Bioinformatics), the Schlumbering Cancer Center (Central Sloan Kettering Cancer Center), and the European molecular Biology Laboratory (European molecular Biology Laboratory). Known effective siRNA sequences and homologous binding sites are also well represented in the relevant literature. RNAi molecules are readily designed and produced by techniques known in the art. Furthermore, computational tools exist that increase the chance of finding effective and specific motifs (Lagana et al, Methods mol. Bio. [ molecular biology Methods ],2015,1269: 393-412).

The regulatory nucleic acid can regulate the expression of an RNA encoded by the gene. Because multiple genes may share some degree of sequence homology with each other, in some embodiments, regulatory nucleic acids may be designed to target a class of genes with sufficient sequence homology. In some embodiments, a regulatory nucleic acid may comprise a sequence that is complementary to a sequence shared between different gene targets or that is unique to a particular gene target. In some embodiments, the regulatory nucleic acid can be designed to target a conserved region of RNA sequences with homology between several genes, thereby targeting several genes in a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In some embodiments, the regulatory nucleic acid can be designed to target sequences unique to a particular RNA sequence of a single gene.

In some embodiments, the genetic element may comprise one or more sequences encoding regulatory nucleic acids that regulate the expression of one or more genes.

In one embodiment, grnas described elsewhere herein are used as part of a CRISPR system for gene editing. For gene editing purposes, the finger ring can be designed to include one or more guide RNA sequences corresponding to a desired target DNA sequence; see, e.g., Cong et al (2013) Science, 339: 819. 823; ran et al (2013) Nature Protocols [ Nature laboratory Manual ],8: 2281-2308. At least about 16 or 17 nucleotides of the gRNA sequence typically allow Cas 9-mediated DNA cleavage to occur; for Cpf1, at least about 16 nucleotides of the gRNA sequence are required to achieve detectable DNA cleavage.

Therapeutic peptides or polypeptides

In some embodiments, the genetic element comprises a sequence encoding a therapeutic peptide or polypeptide, e.g., a secreted polypeptide, e.g., an antibody molecule, an enzyme, a hormone, a cytokine, a complement inhibitor, a growth factor, or a growth factor inhibitor, e.g., as described herein. Such therapeutic agents include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acid analogs. Such therapeutic agents typically have a molecular weight of less than about 5,000 grams/mole, a molecular weight of less than about 2,000 grams/mole, a molecular weight of less than about 1,000 grams/mole, a molecular weight of less than about 500 grams/mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. Such therapeutic agents may include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viral or microbial particles, synthetic molecules, and agonists or antagonists thereof.

In some embodiments, the genetic element comprises a sequence encoding a peptide, e.g., a therapeutic peptide. The peptide may be linear or branched. The peptide is about 5 to about 500 amino acids, about 15 to about 400 amino acids, about 20 to about 325 amino acids, about 25 to about 250 amino acids, about 50 to about 150 amino acids, or any range therebetween in length.

Exemplary secreted therapeutic agents are described herein, e.g., in the table below.

Table a. exemplary cytokines and cytokine receptors

In some embodiments, the effectors described herein comprise a cytokine of table a or a functional variant thereof, e.g., a homolog (e.g., an ortholog or paralog) or fragment. In some embodiments, a functional variant binds to a corresponding cytokine receptor with a Kd that is no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type cytokine for the same receptor under the same conditions. In some embodiments, the effector comprises a fusion protein comprising a first region (e.g., a cytokine polypeptide of table a) and a second heterologous region. In some embodiments, the first region is a first cytokine polypeptide of table a. In some embodiments, the second region is a second cytokine polypeptide of table a, wherein the first and second cytokine polypeptides form cytokine heterodimers with each other in wild-type cells. In some embodiments, the polypeptide of table a, or a functional variant thereof, comprises a signal sequence, e.g., an effector endogenous signal sequence, or a heterologous signal sequence. In some embodiments, the finger ring encoding a cytokine or functional variant thereof of table a is used to treat a disease or disorder described herein.

In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind to a cytokine of table a. In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind to a cytokine receptor of table a. In some embodiments, the antibody molecule comprises a signal sequence.

Exemplary cytokines and cytokine Receptors are described, for example, in Akdis et al, "nterleukins (from IL-1 to IL-38), interferons, transforming growth factor β, and TNF- α: Receptors, functions, and roles in diseases [ interleukins (from IL-1 to IL-38), interferons, transforming growth factor β, and TNF- α: receptors, function and role in disease ] ", 2016 month 10, vol 138, phase 4, p 984-1010, which is incorporated herein by reference in its entirety, including table I therein.

Table b. exemplary polypeptide hormones and receptors

In some embodiments, the effectors described herein comprise a hormone of table B or a functional variant thereof, such as a homolog (e.g., an ortholog or paralog) or fragment. In some embodiments, a functional variant binds to a corresponding receptor with a Kd that is no more than 10%, 20%, 30%, 40% or 50% higher than the Kd of the corresponding wild-type hormone for the same receptor under the same conditions. In some embodiments, the polypeptide of table B, or a functional variant thereof, comprises a signal sequence, e.g., an effector endogenous signal sequence, or a heterologous signal sequence. In some embodiments, the finger ring encoding a hormone of table B or a functional variant thereof is used to treat a disease or disorder described herein.

In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind the hormones of table B. In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind the hormone receptors of table B. In some embodiments, the antibody molecule comprises a signal sequence.

Table c. exemplary growth factors

In some embodiments, the effectors described herein comprise a growth factor of table C or a functional variant thereof, such as a homolog (e.g., an ortholog or paralog) or fragment. In some embodiments, a functional variant binds to a corresponding receptor with a Kd that is no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type growth factor for the same receptor under the same conditions. In some embodiments, the polypeptide of table C, or a functional variant thereof, comprises a signal sequence, e.g., an effector endogenous signal sequence, or a heterologous signal sequence. In some embodiments, the finger ring encoding a growth factor of table C or a functional variant thereof is used to treat a disease or disorder described herein.

In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind a growth factor of table C. In some embodiments, the effectors described herein comprise antibody molecules (e.g., scFv) that bind to a growth factor receptor of table C. In some embodiments, the antibody molecule comprises a signal sequence.

Exemplary Growth Factors and Growth factor Receptors are described, for example, in "Classification of Growth Factors and Their Receptors ]" Holland-Frei Cancer Medicine [ Holland-Frei Cancer Medicine ] 6 th edition by Bafico et al, which is incorporated herein by reference in its entirety.

TABLE D coagulation-related factors

Effector	Indications of
		Factor I (fibrinogen)	Fibrinogenemia
Factor II	Deficiency of factor II
		Factor IX	Hemophilia B
Factor V	European disease (Owren's disease)
		Factor VIII	Hemophilia A
Factor X	Stuart-Prower factor deficiency
		Factor XI	Hemophilia C
Factor XIII	Deficiency of fibrin stabilizing factor
		vWF	Von Willebrand disease

In some embodiments, the effector described herein comprises a polypeptide of table D or a functional variant thereof, such as a homolog (e.g., an ortholog or paralog) or fragment. In some embodiments, a functional variant catalyzes the same reaction as a corresponding wild-type protein, e.g., at a rate that is not less than 10%, 20%, 30%, 40%, or 50% less than the wild-type protein. In some embodiments, the polypeptide of table D, or a functional variant thereof, comprises a signal sequence, e.g., an effector endogenous signal sequence, or a heterologous signal sequence. In some embodiments, the finger ring encoding a polypeptide of table D or a functional variant thereof is used to treat a disease or disorder of table D.

In some embodiments, a functional variant of a wild-type protein comprises a protein having one or more activities of the wild-type protein, e.g., the functional variant catalyzes the same reaction as a corresponding wild-type protein, e.g., catalyzes at a rate that is not less than 10%, 20%, 30%, 40%, or 50% less than the wild-type protein. In some embodiments, a functional variant binds to the same binding partner that the wild-type protein binds to, e.g., has a Kd no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd for the same binding partner of the corresponding wild-type protein under the same conditions. In some embodiments, the functional variant has a polypeptide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of the wild-type polypeptide. In some embodiments, a functional variant comprises a homolog (e.g., an ortholog or paralog) of the corresponding wild-type protein. In some embodiments, the functional variant is a fusion protein. In some embodiments, the fusion comprises a first region having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the corresponding wild-type protein, and a second heterologous region. In some embodiments, the functional variant comprises or consists of a fragment of the corresponding wild-type protein.

Effector of STING modulators

In some embodiments, the secreted effectors described herein modulate STING/cGAS signaling. In some embodiments, the STING modulator is a polypeptide, e.g., a viral polypeptide or a functional variant thereof. For example, the effector may be contained in a "Message in a bottle" from free anti-inflammatory of STING signalling reducing RNA virus infection by Maringer et al [ information in: an experimental training learned from antagonism of STING signaling during RNA virus infection "Cytokine & Growth Factor Reviews" volume 25, phase 6, month 12 2014, page 669-679 (which is incorporated herein by reference in its entirety). Other STING modulators (e.g., activators) are described, for example, in: wang et al, "STING activator c-di-GMP enzymes of peptide vaccines in melanomas-bearing mice [ anti-tumor effect of STING activator c-di-GMP-enhancing peptide vaccine on melanoma-bearing mice ]," Cancer Immunol immunotherapy [ Cancer immunology and immunotherapy ].2015 8 months; 64(8) 1057-66.doi:10.1007/s00262-015 and 1713-5.2015 year 5-19 days electronic publication; bose "cGAS/STING Pathway in Cancer: Jekkyl and Hyde store of Cancer Immune Response [ cGAS/STING Pathway in Cancer: jeklyl and Hyde stories of cancer immune response "Int J Mol Sci. [ journal of international molecular science ]2017 for 11 months; 18(11) 2456; and Fu et al "STING aginst formulated vaccines cancer resistance to PD-1 blockade [ STING agonists formulated cancer vaccines can cure tumors resistant to PD-1 blockade ]" Sci Transl Med. [ scientific transformation medicine ]2015 year 4, month 15; 283ra52, each of which is incorporated by reference herein in its entirety.

Some examples of peptides include, but are not limited to, fluorescent tags or labels, antigens, therapeutic peptides, synthetic or analog peptides of naturally bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, targeting or cytotoxic peptides, degraded or self-destructing peptides, and various degraded or self-destructing peptides. Peptides described herein that can be used in the present invention also include antigen binding peptides, such as antigen binding antibodies or antibody-like fragments, such as single chain antibodies, Nanobodies (see, e.g., Steeland et al 2016.Nanobodies as therapeutics: big opportunities for Nanobodies as therapeutics: small antibodies ] Drug discovery 21 (7: 1076-. Such antigen binding peptides may bind to cytoplasmic, nuclear or intracellular antigens.

In some embodiments, the genetic element comprises a sequence encoding a protein, such as a therapeutic protein. Some examples of therapeutic proteins may include, but are not limited to, hormones, cytokines, enzymes, antibodies, transcription factors, receptors (e.g., membrane receptors), ligands, membrane transporters, secreted proteins, peptides, carrier proteins, structural proteins, nucleases, or components thereof.

In some embodiments, the compositions or finger rings described herein comprise a polypeptide linked to a ligand capable of targeting a specific location, tissue, or cell.

Regulatory sequences

In some embodiments, the genetic element comprises a regulatory sequence, such as a promoter or enhancer, operably linked to a sequence encoding an effector.

In some embodiments, the promoter comprises a DNA sequence adjacent to a DNA sequence encoding the expression product. The promoter may be operably linked to an adjacent DNA sequence. A promoter generally increases the amount of a product expressed by a DNA sequence compared to the amount of the product expressed in the absence of the promoter. Promoters from one organism may be used to enhance the expression of products from DNA sequences from another organism. For example, vertebrate promoters can be used to express jellyfish GFP in vertebrates. In addition, one promoter element may increase the amount of product expressed from multiple DNA sequences linked in series. Thus, a promoter element may enhance the expression of one or more products. Various promoter elements are well known to those of ordinary skill in the art.

In one embodiment, high levels of constitutive expression are desired. Examples of such promoters include, but are not limited to, the retroviral Rous Sarcoma Virus (RSV) Long Terminal Repeat (LTR) promoter/enhancer, the Cytomegalovirus (CMV) immediate early promoter/enhancer (see, e.g., Boshart et al, Cell [ Cell ],41: 521-19 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β -actin promoter, and the phosphoglycerate kinase (PGK) promoter.

In another embodiment, an inducible promoter may be desired. Inducible promoters are those regulated in cis or trans by exogenously supplied compounds, including but not limited to the zinc-induced sheep Metallothionein (MT) promoter; dexamethasone (Dex) inducible Mouse Mammary Tumor Virus (MMTV) promoter; the T7 polymerase promoter system (WO 98/10088); the tetracycline repression system (Gossen et al, Proc. Natl. Acad. Sci. USA, Proc. Sci. Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline inducible system (Gossen et al, Science [ Science ],268: 1766-; RU486 inducible system (Wang et al, nat. Biotech. [ Nature Biotechnology ],15: 239-; and rapamycin inducible systems (Magari et al, J.Clin.Invest. [ J.Clin.J. ] [ J.C. ],100: 2865-. Other types of inducible promoters that can be used herein are those that are regulated by a particular physiological state, such as temperature, acute phase, or only in replicating cells.

In some embodiments, a native promoter of the gene or nucleic acid sequence of interest is used. Where expression of a desired gene or nucleic acid sequence should mimic natural expression, a natural promoter may be used. Where expression of a gene or other nucleic acid sequence must be regulated temporally or developmentally, either in a tissue-specific manner or in response to a particular transcriptional stimulus, a native promoter may be used. In another embodiment, other natural expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic natural expression.

In some embodiments, the genetic element comprises a gene operably linked to a tissue-specific promoter. For example, if expression in skeletal muscle is desired, a promoter active in muscle may be used. These include promoters from the synthetic muscle promoters encoding skeletal muscle alpha-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, and having higher activity than the natural promoters. See Li et al, nat. Biotech. [ Nature Biotechnology ],17: 241-. Examples of tissue-specific promoters are known for: hepatic albumin, Miyatake et al J.Virol [ J.Virol ],71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al, Gene Ther [ Gene therapy ]3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthrot et al, hum. Gene Ther. [ human Gene therapy ],7:1503-14(1996), bone (osteocalcin, Stein et al, mol. biol. Rep. [ molecular biology report ],24:185-96(1997), bone sialoprotein, Chen et al, J.bone Miner. Res. [ J.Osseikagaku et al ]11:654-64(1996)), lymphocytes [ lymphocytes ] (CD2, Hansal et al, J.Immunol. [ J.Immunol ],161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor A chain), neurons (neuron-specific enolase (NSE) promoter, Andersen et al cell. mol. Neurobiol. [ cell and molecular neurobiology (503), 13: 15-15 (1993), neurofilament gene, Piccsil et al, Pimcial et al, Nature et al, Procclic acid et al, USA: vgf, 19811: 5632, neuron (15: 373-84 (1995)); and the like.

Genetic elements may include enhancers, such as DNA sequences adjacent to the DNA sequence encoding the gene. Enhancer elements are typically located upstream of a promoter element, or may be located downstream of or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into one or more products). Thus, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of the DNA sequence encoding the product. Enhancer elements can increase the amount of recombinant product expressed by the DNA sequence beyond the increased expression provided by the promoter element. Multiple enhancer elements are readily available to one of ordinary skill in the art.

In some embodiments, the genetic element comprises one or more Inverted Terminal Repeat (ITRs) flanking a sequence encoding an expression product described herein. In some embodiments, the genetic element comprises one or more Long Terminal Repeats (LTRs) flanking a sequence encoding an expression product described herein. Examples of promoter sequences that may be used include, but are not limited to, the simian virus 40(SV40) early promoter, the Mouse Mammary Tumor Virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, and the Rous sarcoma virus promoter.

Replication proteins

In some embodiments, the genetic elements of a finger ring, e.g., a synthetic finger ring, can include sequences encoding one or more replication proteins. In some embodiments, the ring can be replicated by rolling circle replication, e.g., the synthesis of the leader and the lag is uncoupled. In such an embodiment, the ring body contains three additional elements: i) a gene encoding an initiator protein, ii) a double-stranded origin, and iii) a single-stranded origin. A Rolling Circle Replication (RCR) protein complex comprising a replication protein binds to the leader chain and destabilizes the origin of replication. The RCR complex cleaves the genome to generate a free 3' OH terminus. The cellular DNA polymerase initiates viral DNA replication from the free 3' OH terminus. After replication of the genome, the RCR complex covalently closes the loop. This results in the release of a positive circular single stranded parent DNA molecule and a circular double stranded DNA molecule consisting of a negative parent strand and a newly synthesized positive strand. Single-stranded DNA molecules may be encapsidated or involved in a second round of replication. See, e.g., Virology Journal 2009,6:60 doi 10.1186/1743-422X-6-60.

The genetic element may comprise a sequence encoding a polymerase, for example an RNA polymerase or a DNA polymerase.

Other sequences

In some embodiments, the genetic element further comprises a nucleic acid encoding a product (e.g., a ribozyme, a therapeutic mRNA encoding a protein, an exogenous gene).

In some embodiments, the genetic element comprises one or more sequences that affect the species and/or tissue and/or cell tropism (e.g., capsid protein sequences), infectivity (e.g., capsid protein sequences), immunosuppression/activation (e.g., regulation of nucleic acids), viral genome binding and/or packaging, immune escape (non-immunogenic and/or tolerance), pharmacokinetics, endocytosis and/or cell attachment, nuclear entry, intracellular regulation and localization, exocytosis regulation, reproduction, and nucleic acid protection of the ring in the host or host cell.

In some embodiments, the genetic element may comprise other sequences, including DNA, RNA, or artificial nucleic acids. Other sequences may include, but are not limited to, genomic DNA, cDNA, or sequences encoding tRNA, mRNA, rRNA, miRNA, gRNA, siRNA, or other RNAi molecules. In one embodiment, the genetic element comprises a sequence encoding an siRNA to target a different locus of the same gene expression product as the regulatory nucleic acid. In one embodiment, the genetic element comprises a sequence encoding an siRNA to target a gene expression product different from the regulatory nucleic acid.

In some embodiments, the genetic element further comprises one or more of the following sequences: sequences encoding one or more mirnas, sequences encoding one or more replication proteins, sequences encoding exogenous genes, sequences encoding therapeutic agents, regulatory sequences (e.g., promoters, enhancers), sequences encoding one or more regulatory sequences targeting endogenous genes (siRNA, lncRNA, shRNA), and sequences encoding therapeutic mRNA or proteins.

The length of the additional sequence may be about 2nt to about 5000nt, about 10nt to about 100nt, about 50nt to about 150nt, about 100nt to about 200nt, about 150nt to about 250nt, about 200 to about 300nt, about 250nt to about 350nt, about 300nt to about 500nt, about 10nt to about 1000nt, about 50nt to about 1000nt, about 100nt to about 1000nt, about 1000nt to about 2000nt, about 2000nt to about 3000nt, about 3000nt to about 4000nt, about 4000nt to about 5000nt, or any range therebetween.

Encoded gene

For example, a genetic element can include a gene associated with a signaling biochemical pathway, such as a gene or polynucleotide associated with a signaling biochemical pathway. Examples include genes or polynucleotides associated with diseases. A "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that produces a transcription or translation product at an abnormal level or in an abnormal form in cells derived from diseased tissue as compared to non-disease control tissues or cells. It may be a gene that is expressed at abnormally high levels; it may be a gene that is expressed at abnormally low levels, where changes in expression are associated with the onset and/or progression of disease. A gene associated with a disease also refers to a gene having one or more mutations or genetic variations that directly cause the cause or are in linkage disequilibrium with one or more genes that cause the cause.

Examples of disease-associated genes and polynucleotides are available from the McKurick-Narses Institute of Genetic Medicine, Inc. of Hopkins University (Barlmo, McKumock-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)) and the National Center for Biotechnology Information, National Library of Medicine (Besseda, Md.) (National Center for Biotechnology Information, National Library of Medicine, Bethesda, Md.). Examples of disease-associated genes and polynucleotides are listed in U.S. patent nos.: 8,697,359, which is incorporated herein by reference in its entirety. Specific disease Information is available from the McKurick-Narses Institute of Genetic Medicine, Johns Hopkins University (Barbarmo, McKusick-National offices of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)) and the National Center for Biotechnology Information, National Library of Medicine (Besserda, Md.) (National Center for Biotechnology Information, National Library of Medicine). Examples of genes and polynucleotides associated with biochemical pathways of signaling are listed in U.S. patent nos.: 8,697,359, which is incorporated herein by reference in its entirety.

Furthermore, as described elsewhere herein, the genetic element may encode a targeting moiety. This can be achieved, for example, by inserting polynucleotides encoding sugars, glycolipids or proteins, such as antibodies. Other methods for generating targeting moieties are known to those skilled in the art.

Viral sequences

In some embodiments, the genetic element comprises at least one viral sequence. In some embodiments, the sequence has homology or identity to one or more sequences from a single-stranded DNA virus, such as a dactylovirus, a bunavirus, a circovirus, a geminivirus, a kenovirus, a filovirus, a parvovirus, a tripavirus, and a sipara virus. In some embodiments, the sequence has homology or identity to one or more sequences from a double-stranded DNA virus, such as adenovirus, bottled virus, vesicular virus, african swine fever virus, baculovirus, forskovirus, orbivirus, trichoviridae, adenitis virus, herpes virus, iridovirus, lipomavirus, nima virus, and poxvirus. In some embodiments, the sequence has homology or identity to one or more sequences from an RNA virus, e.g., an alphavirus, a fungal baculovirus, a hepatitis virus, a barley virus, a tobacco mosaic virus, a tobacco rattle virus, a triangle virus, a rubella virus, a birnavirus, a capsovirus, a split virus, and a reovirus.

In some embodiments, the genetic element may comprise one or more sequences from a non-pathogenic virus, such as a symbiotic virus (symbian virus), such as a commensal virus (commensal virus), such as a natural virus, e.g., a ring virus. Recent changes in nomenclature have classified three dactyloviruses capable of infecting human cells into the genera alpha-type torque teno virus (TT), beta-type torque teno virus (TTM), and c-type torque teno virus (TTMD) of the dactyloviridae family of viruses. To date, the ring virus has not been associated with any human disease. In some embodiments, the genetic element may comprise a sequence having homology or identity to a torque teno virus (TT), a non-enveloped single-stranded DNA virus having a circular negative-sense genome. In some embodiments, the genetic element may comprise a sequence having homology or identity to SEN virus, sentinel virus, TTV-like parvovirus, and TT virus. Different types of TT viruses have been described, including TT virus genotype 6, the TT virus population, the TTV-like virus DXL1 and the TTV-like virus DXL 2. In some embodiments, the genetic element may comprise a sequence having homology or identity to a smaller virus, a ringlet-like parvovirus (TTM), or a third virus with a genome size between TTV and TTMV, referred to as a ringlet-like mesovirus (TTMD). In some embodiments, the genetic element can comprise one or more sequences or fragments of sequences from a non-pathogenic virus having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any of the nucleotide sequences described herein.

In some embodiments, the genetic element can comprise one or more sequences or fragments of sequences from a substantially non-pathogenic virus having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any of the nucleotide sequences described herein, e.g., in table 41.

Table 41: examples of finger viruses and their sequences. Accession numbers and related sequence information are available at www.ncbi.nlm.nih.gov/genbank/for reference 12/11/2018.

In some embodiments, the genetic element comprises one or more sequences having homology or identity to one or more sequences from one or more non-finger ring viruses, such as adenovirus, herpes virus, pox virus, vaccinia virus, SV40, papilloma virus, RNA virus (e.g., retrovirus, such as lentivirus), single-stranded RNA virus (e.g., hepatitis virus), or double-stranded RNA virus (e.g., rotavirus). Since in some embodiments, the recombinant retrovirus is defective, assistance may be provided to produce infectious particles. This assistance can be provided, for example, by using a helper cell line that contains plasmids encoding all the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable cell lines for replicating the finger rings described herein include cell lines known in the art, such as a549 cells, which can be modified as described herein. The genetic element may additionally comprise a gene encoding a selectable marker, so that the desired genetic element can be identified.

In some embodiments, the genetic element comprises a non-silent mutation, e.g., a base substitution, deletion, or addition that results in an amino acid difference in the encoded polypeptide, so long as the sequence retains at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the polypeptide encoded by the first nucleotide sequence or is otherwise useful in the practice of the present invention. In this regard, certain conservative amino acid substitutions may be made which are generally considered not to inactivate the overall function of the protein: for example, for positively charged amino acids (and vice versa), lysine, arginine and histidine; for negatively charged amino acids (and vice versa), aspartic acid and glutamic acid; for certain groups of electrically neutral amino acids (in all cases vice versa), (1) alanine and serine, (2) asparagine, glutamine and histidine, (3) cysteine and serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6) methionine, leucine and isoleucine, (7) phenylalanine, methionine, leucine and tyrosine, (8) serine and threonine, (9) tryptophan and tyrosine, (10) and for example tyrosine, tryptophan and phenylalanine. Amino acids can be classified according to physical properties and contributions to secondary and tertiary protein structure. Conservative substitutions are considered in the art as the replacement of one amino acid by another with similar properties.

The identity of two or more nucleic acid or polypeptide sequences having the same or a specified percentage of identical nucleotide or amino acid residues (e.g., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity within a particular region when compared and aligned for maximum correspondence over a comparison window or designated region) can be measured using a BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (e.g., see NCBI website www.ncbi.nlm.nih.gov/BLAST/etc.). Identity may also refer to or be used in addition to a test sequence. Identity also includes sequences with deletions and/or additions as well as sequences with substitutions. As described herein, the algorithm takes into account gaps, etc. Identity may exist in regions of at least about 10 amino acids or nucleotides in length, about 15 amino acids or nucleotides in length, about 20 amino acids or nucleotides in length, about 25 amino acids or nucleotides in length, about 30 amino acids or nucleotides in length, about 35 amino acids or nucleotides in length, about 40 amino acids or nucleotides in length, about 45 amino acids or nucleotides in length, about 50 amino acids or nucleotides in length, or more.

In some embodiments, the genetic element comprises a nucleotide sequence having at least about 75% nucleotide sequence identity, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any of the nucleotide sequences set forth herein, e.g., as in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, 17 or 41. Since the genetic code is degenerate, a homologous nucleotide sequence may include any number of "silent" base changes, i.e., nucleotide substitutions that still encode the same amino acid.

Gene editing components

The genetic elements of the finger ring may include one or more genes encoding components of a gene editing system. Exemplary gene editing systems include clustered regulatory short palindromic repeats (CRISPR) systems, Zinc Finger Nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). Methods based on ZFN, TALEN and CRISPR have been described, for example, in Gaj et al Trends Biotechnol. [ biotechnological Trends ]31.7(2013): 397-; CRISPR gene editing methods are described, for example, in Guan et al, Application of CRISPR-Cas system in gene therapy: Pre-clinical development in animal model, [ Application of CRISPR-Cas system in gene therapy: preclinical progression in animal models ] DNA Repair [ DNA Repair ]2016 for 10 months; 46:1-8.doi: 10.1016/J.dnarep.2016.07.004; zheng et al, Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells [ Precise gene deletion and replacement in human cells using the CRISPR/Cas9 system ] BioTechniques [ BioTechniques ], Vol.57, No. 3, 9/2014, p.115-124.

CRISPR systems are adaptive defense systems originally found in bacteria and archaea. CRISPR systems use RNA-guided nucleases (e.g., Cas9 or Cpf1), referred to as CRISPR-associated or "Cas" endonucleases, to cleave exogenous DNA. In a typical CRISPR/Cas system, endonucleases are directed to a target nucleotide sequence (e.g., a site in the genome to be sequence edited) by targeting a sequence-specific non-coding "guide RNA" of a single-or double-stranded DNA sequence. Three classes (I-III) CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes class II Cas endonucleases, such as Cas9, CRISPR RNA ("crRNA") and trans-activating crRNA ("tracrRNA"). crRNA comprises a "guide RNA," i.e., an RNA sequence of about 20 nucleotides that generally corresponds to a target DNA sequence. The crRNA also contains a region to which the tracrRNA binds to form a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid. The crRNA/tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence must be generally adjacent to an "protospacer adjacent motif" ("PAM") that is specific for a given Cas endonuclease; however, PAM sequences appear to be spread throughout a given genome.

In some embodiments, the finger ring comprises a gene of a CRISPR endonuclease. For example, some CRISPR endonucleases identified from different prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5' -NNAGAA (Streptococcus thermophilus) CRISPR1, 5 '-NGGNG (Streptococcus thermophilus CRISPR3), and 5' -NNNGATT (Neisseria meningitidis). Some endonucleases, such as Cas9 endonuclease, are associated with a G-rich PAM site, such as 5'-NGG, and blunt-end cleave the target DNA 3 nucleotides upstream (5') from the PAM site. Another class II CRISPR system comprises the V-endonuclease Cpf1 smaller than Cas 9; examples include AsCpf1 (from an aminoacetococcus species (Acylaminococcus sp.)) and LbCpf1 (from a Trichospiraceae species (Lachnospiraceae sp.)). Cpf1 endonuclease was associated with a T-rich PAM site such as 5' -TTN. Cpf1 also recognized the 5' -CTA PAM motif. Cpf1 cleaves target DNA by introducing misplaced or staggered double-stranded breaks with 5 'overhangs of 4 or 5 nucleotides, for example, by cleaving target DNA in which the 5 nucleotide misplaced or staggered cleavage is located 18 nucleotides downstream (3') from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand; the 5 nucleotide overhang created by this mis-cut allows more precise genome editing of a DNA insertion by homologous recombination than a DNA insertion cut at a blunt end. See, e.g., Zetsche et al (2015) Cell [ cells ],163: 759-771.

A plurality of CRISPR-associated (Cas) genes may be included in the finger loop body. Specific examples of genes are those encoding Cas proteins from class II systems (including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C 3). In some embodiments, the finger ring includes a gene encoding a Cas protein, e.g., a Cas9 protein, which may be from any of a variety of prokaryotic species. In some embodiments, the ring body includes a gene encoding a particular Cas protein, e.g., a particular Cas9 protein, selected to recognize a particular Protospacer Adjacent Motif (PAM) sequence. In some embodiments, the finger ring comprises nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, which can be introduced into a cell, zygote, embryo or animal, e.g., to allow for recognition and modification of sites comprising the same, similar, or different PAM motifs. In some embodiments, the finger ring includes a gene encoding a modified Cas protein with an inactivated nuclease (e.g., nuclease-deficient Cas 9).

The wild-type Cas9 protein produces Double Strand Breaks (DSBs) on specific DNA sequences targeted by grnas, while a number of CRISPR endonucleases with modified functions are known, for example: the "nickase" version of Cas9 produces only single strand breaks; cas9 ("dCas 9") which has no catalytic activity does not cleave the target DNA. A gene encoding dCas9 can be fused to a gene encoding an effector domain to inhibit (CRISPRi) or activate (CRISPRa) expression of the target gene. For example, the gene may encode a fusion of Cas9 with a transcriptional silencer (e.g., KRAB domain) or transcriptional activator (e.g., dCas9-VP64 fusion). A gene encoding catalytically inactive Cas9(dCas9) fused to fokl nuclease ("dCas 9-fokl") can be included to produce DSBs at target sequences homologous to both grnas. See, for example, many CRISPR/Cas9 plasmids are disclosed in and publicly available from the alder gene plasmid library (addge repository) (addge, west dney street No. 75 (Sidney St.), unit 550A, xigeshire, ma 02139; addge. Ran et al (2013) Cell [ Cell ],154:1380-1389 describe a "double nickase" Cas9 that introduces two independent double-stranded breaks, each of which is directed by an independent guide RNA, to achieve more accurate genome editing.

CRISPR techniques for editing genes of eukaryotes are disclosed in U.S. patent application publications 2016/0138008 a1 and US 2015/0344912 a1 and U.S. patent 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965 and 8,906,616. Cpf1 endonuclease and corresponding guide RNA and PAM sites are disclosed in U.S. patent application publication 2016/0208243 a 1.

In some embodiments, the finger ring comprises a gene encoding a polypeptide described herein (e.g., a targeted nuclease, e.g., Cas9, e.g., wild-type Cas9, nickase Cas9 (e.g., Cas 9D 10A), inactivated Cas9(dCas9), eSpCas9, Cpf1, C2C1, or C2C3), and a gRNA. The selection of the gene encoding the nuclease and one or more grnas depends on whether the targeted mutation is a deletion, substitution, or addition of nucleotides, such as a nucleotide deletion, substitution, or addition of a target sequence. Genes encoding catalytically inactive endonucleases (e.g., inactivating Cas9(dCas9, e.g., D10A; H840A)) linked to all or part (e.g., a biologically active portion) of the effector domain(s) (e.g., VP64) produce chimeric proteins that can modulate the activity and/or expression of one or more target nucleic acid sequences.

As used herein, a "biologically active portion of an effector domain" is a portion that maintains the function (e.g., complete, partial, or minimal function) of an effector domain (e.g., a "minimal" or "core" domain). In some embodiments, the finger ring includes a gene encoding a fusion of dCas9 with all or a portion of one or more effector domains to produce a chimeric protein useful in the methods described herein. Thus, in some embodiments, the finger ring comprises a gene encoding a dCas 9-methylase fusion. In other embodiments, the finger ring includes a gene encoding a fusion of dCas9 enzyme with a site-specific gRNA to target an endogenous gene.

In other aspects, the finger ring comprises a gene encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector domains (all or a biologically active portion) fused to dCas 9.

Protein exterior

In some embodiments, the ring, e.g., a synthetic ring, comprises a proteinaceous outer portion enclosing a genetic element. The proteinaceous outer portion may comprise a substantially non-pathogenic outer protein incapable of eliciting an unwanted mammalian immune response. The protein exterior of the finger ring body typically comprises a substantially non-pathogenic protein that can self-assemble into an icosahedral structure that makes up the protein exterior.

In some embodiments, the protein outer protein is encoded by a sequence of a genetic element of the finger loop (e.g., in cis with the genetic element). In other embodiments, the proteinaceous outer protein is encoded by a nucleic acid that is separated from the genetic elements of the finger loop (e.g., in trans with the genetic elements).

In some embodiments, the protein, e.g., a substantially non-pathogenic protein and/or a proteinaceous outer protein, comprises one or more glycosylated amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In some embodiments, a protein, e.g., a substantially non-pathogenic protein and/or a proteinaceous outer protein, comprises at least one hydrophilic DNA binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal poly-arginine sequence, a variable region, a C-terminal poly-glutamine/glutamine sequence, and one or more disulfide bonds.

In some embodiments, the protein is a capsid protein, e.g., a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a protein encoded by any of the nucleotide sequences encoding capsid proteins described herein (e.g., the ring virus ORF1 sequence or capsid protein sequence set forth in any of tables 1-18, a1-a12, B1-B5, C1-C5, D1-D10, or 20-37). In some embodiments, the protein or functional fragment of a capsid protein is encoded by a nucleotide sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the nucleotide sequences described herein, e.g., a finger ring viral capsid sequence or capsid protein sequence listed in any of tables a1-a12, B1-B5, C1-C5, D1-D10, or 20-37. In some embodiments, the protein comprises a capsid protein or a functional fragment of a capsid protein encoded by a capsid nucleotide sequence or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any of the nucleotide sequences described herein, e.g., the ring virus capsid sequences or capsid protein sequences listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15 or 17.

In some embodiments, the ring comprises a nucleotide sequence encoding: a capsid protein or a functional fragment of a capsid protein or a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the amino acid sequences described herein (e.g., the dactylovirus capsid sequence or capsid protein sequence listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16 or 18). In some embodiments, the ring comprises a nucleotide sequence encoding: a capsid protein or a functional fragment of a capsid protein or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the amino acid sequences described herein (e.g., the dactylovirus capsid sequence or capsid protein sequence listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16 or 18).

In some embodiments, the finger ring comprises a nucleotide sequence encoding about position 1 to about position 150 (e.g., or any subset of amino acids within each range, e.g., about position 20 to about position 35, about position 25 to about position 30, about position 26 to about position 30), about position 150 to about position 390 (e.g., or any subset of amino acids within each range, e.g., about position 200 to about position 380, about position 205 to about position 375, about position 205 to about position 371), about position 390 to about position 525, about position 525 to about position 850 (e.g., or any subset of amino acids within each range, e.g., about position 840 to about position 530) having an amino acid sequence described herein (e.g., as listed in any of tables a2, a4, A6, a8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18 or as shown in figure 1) or a functional fragment thereof, An amino acid sequence of about 545 to about 830, about 550 to about 820), about 850 to about 950 (e.g., or any subset of amino acids within each range, such as about 860 to about 940, about 870 to about 930, about 880 to about 923). In some embodiments, the protein comprises an amino acid sequence, or a functional fragment thereof, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence described herein (referring to a ring virus amino acid sequence, e.g., as listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18 or as shown in fig. 1) from about position 1 to about 150 (e.g., or any subset of amino acids within each range described herein), from about position 150 to about position 390, from about position 390 to about position 525, from about position 525 to about position 850, from about position 850 to about position 950.

In some embodiments, the protein comprises an amino acid sequence, or a functional fragment thereof, or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the amino acid sequences or amino acid ranges described herein (referring to the ring virus amino acid sequence, e.g., listed in any of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18, or as shown in figure 1). In some embodiments, a range of amino acids with less sequence identity may provide for differences in one or more of the properties described herein, as well as cell/tissue/species specificity (e.g., tropism).

In some embodiments, the ring body lacks lipids outside the protein. In some embodiments, the ring lacks a lipid bilayer, e.g., a viral envelope. In some embodiments, the interior of the ring body is completely covered (e.g., 100% covered) by the protein exterior. In some embodiments, the interior of the finger ring is covered by less than 100% of the exterior of the protein, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. In some embodiments, the protein includes gaps or discontinuities on the exterior, e.g., such that it is permeable to water, ions, peptides, or small molecules, so long as the genetic element remains in the finger loop body.

In some embodiments, the protein comprises externally one or more proteins or polypeptides that specifically recognize and/or bind to the host cell, e.g., complement the protein or polypeptide, to mediate entry of the genetic element into the host cell.

In some embodiments, the protein comprises externally one or more of: one or more glycosylated proteins, a hydrophilic DNA binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal poly-arginine sequence, a variable region, a C-terminal poly-glutamine/glutamine sequence, and one or more disulfide bonds. For example, the protein comprises externally a protein encoded by the finger ring virus ORF1 described herein.

In some embodiments, the protein exterior includes one or more of the following characteristics: icosahedral symmetry, recognition and/or binding of molecules that interact with one or more host cell molecules to mediate entry into the host cell, lack of lipid molecules, lack of carbohydrates, pH and temperature stability, resistance to detergents, and be substantially non-immunogenic or non-pathogenic in the host.

II. vector

The genetic elements described herein may be comprised in a vector. Suitable carriers and methods for making and using the same are well known in the art.

In one aspect, the invention includes a vector comprising a genetic element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds the genetic element to the non-pathogenic external protein, and (iii) a sequence encoding a regulatory nucleic acid.

The genetic element or any sequence within the genetic element may be obtained using any suitable method. Various recombinant methods are known in the art, for example, using standard techniques, screening libraries from cells having viral sequences, obtaining the sequences from vectors known to contain the sequences, or isolating the sequences directly from cells and tissues containing the sequences. Alternatively or in combination, part or all of the genetic elements may be produced synthetically, rather than cloned.

In some embodiments, the vector includes regulatory elements, nucleic acid sequences homologous to the target gene, and various reporter constructs for causing expression of the reporter in a living cell and/or when an intracellular molecule is present in the target cell.

The reporter gene is used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some readily detectable property (e.g., enzymatic activity). After the DNA is introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al, 2000FEBSletters [ Proc. Federation of European Biochemical society ]479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, a construct with the smallest 5' flanking region showing the highest expression level of the reporter gene is identified as a promoter. Such promoter regions may be linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.

In some embodiments, the vector is substantially non-pathogenic and/or substantially non-integrating in the host cell, or is substantially non-immunogenic in the host.

In some embodiments, the amount of vector is sufficient to modulate at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of one or more of phenotype, viral level, gene expression, competition with other viruses, disease state, and the like.

Model system for quantifying ring body activity

The ring bodies described herein can be assayed in a number of animal models and in vitro models. For example, a mouse model deficient in A1AT (for A1AT α -1-antitrypsin deficiency) can be used to quantify the activity of finger rings encoding α -1-antitrypsin or a functional variant thereof. In some embodiments, a C1E-INH-deficient mouse model (for hereditary angioedema) can be used to quantify the activity of the ring body encoding a C1 inhibitor or a functional variant thereof. In some embodiments, an in vitro vWF cleavage assay (as representative of thrombotic thrombocytopenic purpura) can be used to quantify the activity of ring bodies that encode ADAMTS13 or functional variants thereof.

Composition III

The ring members or vectors described herein may also be included in a pharmaceutical composition having, for example, a pharmaceutical excipient as described herein. In some embodiments, the pharmaceutical composition comprises at least 10 ⁵、10⁶、10⁷、10⁸、10⁹、10¹⁰、10¹¹、10¹²、10¹³、10¹⁴Or 10¹⁵A ring body. In some embodiments, the pharmaceutical composition comprises about 10⁵-10¹⁵、10⁵-10¹⁰Or 10¹⁰-10¹⁵A ring body. In some embodiments, the pharmaceutical composition comprises about 10⁸(e.g., about 10)⁵、10⁶、10⁷、10⁸、10⁹Or 10¹⁰) One genome equivalent/mL refers to the loop body. In some embodiments, the pharmaceutical composition comprises 10⁵-10¹⁰、10⁶-10¹⁰、10⁷-10¹⁰、10⁸-10¹⁰、10⁹-10¹⁰、10⁵-10⁶、10⁵-10⁷、10⁵-10⁸、10⁵-10⁹、10⁵-10¹¹、10⁵-10¹²、10⁵-10¹³、10⁵-10¹⁴、10⁵-10¹⁵Or 10¹⁰-10¹⁵A finger ring of one genome equivalent per mL, e.g., as determined according to the method of example 18. In some embodiments, the pharmaceutical composition comprises sufficient ring bodies to incorporate at least 1, 2, 5, or 10, 100, 500, 1000, 2000, 5000, 8,000, 1x10 of the genetic elements contained in the ring body⁴、1x10⁵、1x10⁶、1x10⁷A number of copies/cells or greater are delivered into the eukaryotic cell population. In some embodiments, the pharmaceutical composition comprises sufficient ring bodies to contain at least about 1x10 of the genetic element contained in the ring body⁴、1x10⁵、1x10⁶、1x10⁷Or about 1x10⁴-1x10⁵、1x10⁴-1x10⁶、1x10⁴-1x10⁷、1x10⁵-1x10⁶、1x10⁵-1x10⁷Or 1x10⁶-1x10⁷The individual copies/cell are delivered to a population of eukaryotic cells.

In some embodiments, the pharmaceutical composition has one or more of the following characteristics: the pharmaceutical composition meets drug or Good Manufacturing Practice (GMP) standards; the pharmaceutical composition is made according to Good Manufacturing Practice (GMP); the pharmaceutical composition has a level of the pathogen below a predetermined reference value, e.g., is substantially free of the pathogen; the pharmaceutical composition has a level of contaminants below a predetermined reference value, e.g., is substantially free of contaminants; or the pharmaceutical composition has low immunogenicity or is substantially non-immunogenic, e.g., as described herein.

In some embodiments, the pharmaceutical composition comprises less than a threshold amount of one or more contaminants. Exemplary contaminants that are desirably not included or minimally included in the pharmaceutical composition include, but are not limited to, host cell nucleic acids (e.g., host cell DNA and/or host cell RNA), components of animal origin (e.g., serum albumin or trypsin), replication competent viruses, non-infectious particles, free viral capsid proteins, exogenous factors, and aggregates. In embodiments, the contaminant is host cell DNA. In embodiments, the composition comprises less than about 10ng of host cell DNA per dose. In embodiments, the level of host cell DNA in the composition is reduced by filtration and/or enzymatic degradation of the host cell DNA. In embodiments, the pharmaceutical composition consists of less than 10% (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) by weight of contaminants.

In one aspect, the invention described herein includes a pharmaceutical composition comprising:

a) a finger loop comprising a genetic element comprising (i) a sequence encoding a non-pathogenic external protein, (ii) an external protein binding sequence that binds the genetic element to the non-pathogenic external protein, and (iii) a sequence encoding a regulatory nucleic acid; and the exterior of a protein associated with (e.g., encapsulating or blocking) the genetic element; and

b) A pharmaceutical excipient.

Vesicle

In some embodiments, the composition further comprises a carrier component, such as a microparticle, liposome, vesicle, or exosome. In some embodiments, the liposomes comprise a spherical vesicle structure composed of a monolayer or multilayer lipid bilayer surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are generally biocompatible, non-toxic, and can deliver hydrophilic and lipophilic Drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery [ J. Drug Delivery ], Vol.2011, article ID 469679, p.12, 2011.doi: 10.1155/2011/469679).

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Vesicles may include, but are not limited to, DOTMA, DOTAP, DOTIM, DDAB, alone or in combination with cholesterol to produce DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Methods of preparing multilamellar vesicle lipids are known in the art (see, e.g., U.S. patent No. 6,693,086, the teachings of which are incorporated herein by reference for the preparation of multilamellar vesicle lipids). Although vesicle formation is spontaneous when the lipid membrane is mixed with an aqueous solution, vesicle formation can also be accelerated by applying force in the form of shaking using a homogenizer, sonicator or a squeezing device (for review, see, for example, Spuch and Navarro, Journal of Drug Delivery, vol.2011, article ID 469679, page 12, 2011.doi: 10.1155/2011/469679). Extruded lipids can be prepared by extrusion through filters of reduced size, as described in Templeton et al, Nature Biotech [ Nature Biotechnology ],15:647-652,1997, the teachings of which are incorporated herein by reference with respect to the preparation of extruded lipids.

As described herein, additives may be added to the vesicles to alter their structure and/or properties. For example, cholesterol or sphingomyelin may be added to the mixture to help stabilize the structure and prevent leakage of the internal cargo. In addition, vesicles may be prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol and dicetyl phosphate. (for reviews, see, e.g., Spuch and Navarro, Journal of Drug Delivery [ Journal of Drug Delivery ], Vol.2011, article ID 469679, p.12, 2011.doi: 10.1155/2011/469679). Also, the vesicles may be surface modified during or after synthesis to include reactive groups complementary to the reactive groups on the recipient cells. Such reactive groups include, but are not limited to, maleimide groups. For example, vesicles may be synthesized to include maleimide-conjugated phospholipids, such as, but not limited to, DSPE-MaL-PEG 2000.

Vesicular formulations may be composed primarily of natural phospholipids and lipids (e.g., 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, and monosialoganglioside). Formulations consisting of phospholipids alone are less stable in plasma. However, manipulation of the lipid membrane with cholesterol reduces the rapid release of the encapsulated cargo, or 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery [ Journal of Drug Delivery ], vol.2011, article ID 469679, p.12, 2011.doi: 10.1155/2011/469679).

In embodiments, lipids may be used to form lipid microparticles. Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and the co-lipids distearoylphosphatidylcholine, cholesterol and PEG-DMG that can be formulated using spontaneous vesicle formation procedures (see, e.g., novobransteva, Molecular Therapy-Nucleic Acids (2012)1, e 4; doi: 10.1038/mtna.2011.3). The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/distearoylphosphatidylcholine/cholesterol/PEG-DMG). Tekmira has about 95 patent family combinations in the united states and abroad relating to various aspects of lipid particles and lipid particle formulations (see, e.g., U.S. patent 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and european patent numbers 1766035; 1519714; 1781593 and 1664316), all of which may be used and/or adapted for use in the present invention.

In some embodiments, the microparticles comprise one or more cured polymers arranged in a random manner. The microparticles may be biodegradable. Biodegradable microparticles can be synthesized, for example, using methods known in the art, including but not limited to solvent evaporation, hot melt microencapsulation, solvent removal, and spray drying. Exemplary methods for synthesizing microparticles are described in Bershteyn et al, Soft Matter [ Soft materials ]4:1787- & 1787,2008 and US 2008/0014144A 1, the specific teachings of which are incorporated herein by reference with respect to microparticle synthesis.

Exemplary synthetic polymers that can be used to form the biodegradable microparticles include, but are not limited to, aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA), copolymers of lactic and glycolic acid (PLGA), Polycaprolactone (PCL), polyanhydrides, poly (ortho) acid esters, polyurethanes, poly (butyric acid), poly (propionic acid), and poly (lactide-caprolactone), as well as natural polymers such as albumin, alginates, and other polysaccharides, including dextran and cellulose, collagen, chemical derivatives thereof, including substitution, addition of chemical groups such as alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely made by those skilled in the art, albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers, and mixtures thereof. Typically, these materials degrade by enzymatic hydrolysis or exposure to water, by surface or bulk corrosion.

The diameter of the microparticles ranges from 0.1 to 1000 micrometers (μm). In some embodiments, their diameters range in size from 1 μm to 750 μm, or from 50 μm to 500 μm, or from 100 μm to 250 μm. In some embodiments, their diameters range in size from 50 μm to 1000 μm, 50 μm to 750 μm, 50 μm to 500 μm, or 50 μm to 250 μm. In some embodiments, their diameters range in size from.05 μm to 1000 μm, 10 μm to 1000 μm, 100 μm to 1000 μm, or 500 μm to 1000 μm. In some embodiments, they are about 0.5 μm, about 10 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, or about 1000 μm in diameter. As used in the context of particle diameter, the term "about" refers to +/-5% of the absolute value.

In some embodiments, the ligand is conjugated to the microparticle surface through functional chemical groups (carboxylic acids, aldehydes, amines, thiols, and hydroxyls) present on the particle surface and present on the ligand to be attached. Functionality may be introduced into the microparticles by, for example, incorporating a stabilizer having functional chemical groups during emulsion preparation of the microparticles.

Another example of introducing functional groups into microparticles is by directly crosslinking the particles and ligands with a homobifunctional or heterobifunctional crosslinking agent after microparticle preparation. The procedure may use appropriate chemicals and a class of crosslinkers (CDI, EDAC, glutaraldehyde, etc., as discussed in more detail below) or any other crosslinker that couples ligands to the particle surface by chemically modifying the particle surface after preparation. This also includes procedures by which amphipathic molecules (e.g., fatty acids, lipids, or functional stabilizers) can be passively adsorbed and adhered to the particle surface, thereby introducing functional end groups for attachment to ligands.

In some embodiments, the microparticles may be synthesized to include one or more targeting groups on their outer surface to target specific cells or tissue types (e.g., cardiomyocytes). Such targeting groups include, but are not limited to, receptors, ligands, antibodies, and the like. These targeting groups bind their partners to the cell surface. In some embodiments, the microparticles will integrate into the lipid bilayer that makes up the cell surface and deliver mitochondria into the cell.

The microparticles may also comprise a lipid bilayer on their outermost surface. The bilayer may be composed of one or more lipids of the same or different types. Examples include, but are not limited to, phospholipids such as phosphorylcholine and phosphoinositide. Specific examples include, but are not limited to, DMPC, DOPC, DSPC, and various other lipids, such as those described herein for liposomes.

In some embodiments, the carrier comprises nanoparticles, such as described herein.

In some embodiments, the vesicles or microparticles described herein are functionalized with a diagnostic agent. Examples of diagnostic agents include, but are not limited to, commercially available imaging agents for Positron Emission Tomography (PET), Computer Assisted Tomography (CAT), single photon emission computed tomography, X-ray, fluoroscopy, and Magnetic Resonance Imaging (MRI); and a contrast agent. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper and chromium.

Carrier

The compositions (e.g., pharmaceutical compositions) described herein can comprise, be formulated with, and/or be delivered in a carrier. In one aspect, the invention includes a composition, e.g., a pharmaceutical composition, comprising a carrier (e.g., a vesicle, a liposome, a lipid nanoparticle, an exosome, an erythrocyte, an exosome (e.g., a mammalian or plant exosome)), a fusion comprising (e.g., encapsulating) a composition described herein (e.g., a ring body, a ring virus, a ring vector, or a genetic element described herein).

In some embodiments, the compositions and systems described herein can be formulated in liposomes or other similar vesicles. Typically, liposomes are spherical vesicle structures consisting of a monolayer or multilamellar lipid bilayer surrounding an inner aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes typically have one or more (e.g., all) of the following characteristics: biocompatible, non-toxic, can deliver hydrophilic and lipophilic Drug molecules, can protect their cargo from degradation by plasma enzymes, and can transport their cargo across biological membranes and the Blood Brain Barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery [ J. Drug Delivery ], Vol.2011, article ID 469679, p.12, 2011.doi: 10.1155/2011/469679; and Zylberg & Matosevic.2016.Drug Delivery [ Drug Delivery ],23:9, 3319-.

Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods of preparing multilamellar vesicle lipids are known (see, e.g., U.S. patent No. 6,693,086, the teachings of which are incorporated herein by reference for their preparation). Although vesicle formation is spontaneous when the lipid membrane is mixed with an aqueous solution, vesicle formation can also be accelerated by applying force in the form of shaking using a homogenizer, sonicator or a squeezing device (for review, see, for example, Spuch and Navarro, Journal of Drug Delivery, vol.2011, article ID 469679, p.12, 2011.doi: 10.1155/2011/469679). The extruded lipids can be prepared, for example, by extrusion through a filter for size reduction, as described in empleton et al, Nature Biotech [ Nature Biotech ],15:647-652, 1997.

Lipid Nanoparticles (LNPs) are another example of a carrier that provides a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein. See, for example, Gordillo-Galeano et al European Journal of pharmaceuticals and biopharmaceuticals [ European Journal of pharmacy ] Vol. 133, 12 months 2018, p. 285-308. Nanostructured Lipid Carriers (NLCs) are modified Solid Lipid Nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and drug loading, and prevent drug leakage. Polymeric Nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipopolymer Nanoparticles (PLN), a novel carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNP and liposomes. PLN consists of a core-shell structure; the polymer core provides a stable structure and the phospholipid shell provides good biocompatibility. Thus, the two components improve the efficiency of drug encapsulation, facilitate surface modification, and prevent leakage of water-soluble drugs. For reviews, see, e.g., Li et al 2017, Nanomaterials [ Nanomaterials ]7,122; doi:10.3390/nano 7060122.

Exosomes may also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al, 2016, 7 months, Acta pharmaceutical Sinica B. [ Pharmacology paper ] Vol.6, No. 4, pp 287-296; doi.org/10.1016/j.apsb.2016.02.001.

Ex vivo differentiated red blood cells may also be used as carriers for the compositions described herein. See, for example, WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; shi et al 2014, Proc Natl Acad Sci USA [ Proc Natl Acad of sciences USA ]111(28) 10131-; us patent 9,644,180; huang et al 2017 Nature Communications [ Nature Communications ]8: 423; shi et al 2014, Proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ]111(28) 10131-.

The fusion compositions, for example, as described in WO 2018208728, can also be used as a vehicle to deliver the compositions described herein.

Membrane penetrating polypeptides

In some embodiments, the composition further comprises a Membrane Penetrating Polypeptide (MPP) to carry the component into the cell or across a membrane, such as a cell or nuclear membrane. Membrane-penetrating polypeptides capable of facilitating transport of a substance across membranes include, but are not limited to, cell-penetrating peptides (CPPs) (see, e.g., U.S. Pat. No. 8,603,966), fusion peptides for intracellular Delivery in plants (see, e.g., Ng et al, PLoS One,2016,11: e0154081), protein transduction domains, Trojan peptides, and Membrane Translocation Signals (MTS) (see, e.g., Tung et al, Advanced Drug Delivery Reviews [ Advanced Drug Delivery discussion ]55: 281-. Some MPPs are rich in amino acids, such as arginine, with positively charged side chains.

Membrane penetrating polypeptides have the ability to induce membrane penetration of components and can undergo macromolecular translocation within cells of multiple tissues in vivo following systemic administration. A membrane-penetrating polypeptide may also refer to a peptide that passes from the external environment into the intracellular environment (including the cytoplasm, organelles such as mitochondria or the nucleus) in amounts significantly in excess of those achievable by passive diffusion when contacted with a cell under appropriate conditions.

The components transported across the membrane may be reversibly or irreversibly linked to the membrane-penetrating polypeptide. The linker may be a chemical bond, such as one or more covalent bonds or non-covalent bonds. In some embodiments, the linker is a peptide linker. Such linkers may be between 2-30 amino acids, or longer. Joints include flexible, rigid, or cuttable joints.

Combination of

In one aspect, a finger ring body or a composition comprising a finger ring body described herein may further comprise one or more heterologous moieties. In one aspect, a finger loop body or a composition comprising a finger loop body described herein may further comprise one or more heterologous moieties in the fusion. In some embodiments, the heterologous moiety can be linked to a genetic element. In some embodiments, the heterologous moiety can be blocked in the protein exterior as part of the finger loop body. In some embodiments, the heterologous moiety can be administered with the finger ring.

In one aspect, the invention includes a cell or tissue comprising any of the finger loops and heterologous moieties described herein.

In another aspect, the invention includes a pharmaceutical composition comprising a finger ring body and a heterologous moiety as described herein.

In some embodiments, the heterologous moiety can be a virus (e.g., an effector (e.g., a drug, a small molecule), a targeting agent (e.g., a DNA targeting agent, an antibody, a receptor ligand), a tag (e.g., a fluorophore, a photosensitizer, such as KillerRed), or an editing or targeting moiety described herein.

Virus

In some embodiments, the composition may further comprise a virus as a heterologous moiety, such as a single-stranded DNA virus, e.g., a dactylovirus, a bunavirus (Bidnavirus), a circovirus, a geminivirus, a genovirus (genovirus), a filovirus, a parvovirus, and a spiavirus (Spiravirus). In some embodiments, the composition can further comprise a double-stranded DNA virus, such as adenovirus, a bottled virus, a vesicular virus, an african swine fever virus, a baculovirus, a forskovirus (Fusellovirus), a orbivirus, a trichoviridae, an adenitis virus, a herpes virus, an iridovirus, a lipomavirus, a Nimavirus (Nimavirus), and a poxvirus. In some embodiments, the composition may further comprise an RNA virus, such as an alphavirus, a fungal baculovirus, a hepatitis virus, a barley virus, a tobacco mosaic virus, a tobacco rattle virus, a trigonovirus (tricornavir), a rubella virus, a birnavirus, a capsovirus, a split virus, and a reovirus. In some embodiments, the finger ring is administered with a virus as the heterologous moiety.

In some embodiments, the heterologous moiety can comprise a non-pathogenic virus, e.g., a symbiotic virus, a commensal virus, a natural virus. In some embodiments, the non-pathogenic virus is one or more dactyloviruses, e.g., a type a ringlet virus (TT), a type b ringlet virus (TTM), and a type c ringlet virus (TTMD). In some embodiments, the dactylovirus may include a torque teno virus (TT), SEN virus, sentinel virus, TTV-like parvovirus, TT virus genotype 6, TT virus group, TTV-like virus DXL1, TTV-like virus DXL2, torque teno-like parvovirus (TTM), or torque teno-like mesovirus (TTMD). In some embodiments, the non-pathogenic virus comprises one or more sequences having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any of the nucleotide sequences described herein, e.g., as listed in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, 17, or 41.

In some embodiments, the heterologous moiety can comprise one or more viruses identified as deficient in the subject. For example, a composition comprising finger rings and one or more viral components or viruses that are imbalanced in the subject or have a ratio that is different from a reference value, e.g., a healthy subject, can be administered to a subject identified as having a viral deficiency.

In some embodiments, the heterologous moiety can comprise one or more non-ring viruses, such as an adenovirus, a herpesvirus, a pox virus, a vaccinia virus, SV40, a papilloma virus, an RNA virus (e.g., a retrovirus, such as a lentivirus), a single-stranded RNA virus (e.g., a hepatitis virus), or a double-stranded RNA virus (e.g., a rotavirus). In some embodiments, the finger ring or virus is defective or requires assistance to produce infectious particles. Such assistance may be provided, for example, by using a helper cell line comprising a nucleic acid encoding one or more of the replication-defective finger loops or the (e.g. all) structural genes of the virus, e.g. a plasmid or DNA integrated into the genome, under the control of regulatory sequences within the LTRs. Suitable cell lines for replicating the finger rings described herein include cell lines known in the art, such as a549 cells, which can be modified as described herein.

Targeting moieties

In some embodiments, the compositions or finger rings described herein can further comprise a targeting moiety, e.g., a targeting moiety that specifically binds to a molecule of interest present on a target cell. The targeting moiety can modulate a particular function of the molecule or cell of interest, modulate a particular molecule (e.g., an enzyme, protein, or nucleic acid), e.g., a particular molecule downstream of the molecule of interest in the pathway, or specifically bind to the target to locate the finger ring or genetic element. For example, a targeting moiety may include a therapeutic agent that interacts with a particular molecule of interest to increase, decrease, or otherwise modulate its function.

Labelling or monitoring moieties

In some embodiments, the compositions or finger loops described herein can further comprise a label to label or monitor the finger loops or genetic elements described herein. The labeled moiety or the monitoring moiety may be removed by chemical or enzymatic cleavage, such as proteolysis or intein splicing. Affinity tags can be used to purify tagged polypeptides using affinity techniques. Some examples include Chitin Binding Protein (CBP), Maltose Binding Protein (MBP), glutathione-S-transferase (GST), and poly (His) tags. Solubilization tags can be used to help recombinant proteins expressed in chaperone deficient species (e.g., E.coli) to help the protein fold properly and prevent its precipitation. Some examples include Thioredoxin (TRX) and poly (NANP). The labeling moiety or monitoring moiety may comprise a light-sensitive label, such as fluorescence. Fluorescent labels are useful for visualization. GFP and variants thereof are some examples commonly used as fluorescent tags. Protein tags may allow specific enzymatic modifications (e.g., biotinylation by biotin ligase) or chemical modifications (e.g., fluorescence imaging by reaction with FlaSH-EDT 2) to occur. The labeling moieties or monitoring moieties are typically combined to link the protein to a plurality of other components. The labeling moiety or monitoring moiety can also be removed by specific proteolytic or enzymatic cleavage (e.g., by TEV protease, thrombin, factor Xa, or enteropeptidase).

Nanoparticles

In some embodiments, the composition or finger ring described herein may further comprise a nanoparticle. The nanoparticles comprise inorganic materials having a size between about 1 to about 1000 nanometers, between about 1 to about 500 nanometers, between about 1 to about 100nm, between about 50nm to about 300nm, between about 75nm to about 200nm, between about 100nm to about 200nm, and any range therebetween. Nanoparticles generally have a composite structure on the nanometer scale. In some embodiments, the nanoparticles are generally spherical, although different morphologies are possible depending on the composition of the nanoparticles. The portion of the nanoparticle in contact with the environment external to the nanoparticle is typically identified as the surface of the nanoparticle. In the nanoparticles described herein, the size constraints may be limited to two dimensions, such that the nanoparticles comprise a composite structure having a diameter of about 1 to about 1000nm, where the particular diameter depends on the composition of the nanoparticle and the intended use of the nanoparticle according to experimental design. For example, nanoparticles used for therapeutic applications typically have a size of about 200nm or less.

Other desirable properties of the nanoparticles (e.g., surface charge and steric stability) may also vary, given the particular application of interest. In Davis et al, Nature ]2008, volume 7, pages 771-782; duncan, Nature [ Nature ]]2006 volume 6, page 688-701; and Allen, Nature [ Nature]Exemplary properties that may be desirable in clinical applications (e.g., cancer treatment) are described in 2002, Vol 2, pages 750-763, each of which is incorporated herein by reference in its entirety. Other properties can be identified by the skilled person upon reading the present invention. The size and properties of the nanoparticles can be detected by techniques known in the art. Exemplary techniques for detecting particle size include, but are not limited to, Dynamic Light Scattering (DLS) and various microscopic techniques, such as Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). Exemplary techniques for detecting particle morphology include, but are not limited to, TEM and AFM. Exemplary techniques for detecting the surface charge of nanoparticles include, but are not limited to, zeta potential methods. Other techniques suitable for detecting other chemical properties include by¹H、¹¹B. And¹³c and¹⁹f NMR, UV/Vis and infrared/raman spectroscopy and fluorescence spectroscopy (when nanoparticles are used in combination with fluorescent labels) and other techniques that the skilled person can identify.

Small molecules

In some embodiments, the compositions or finger ring described herein may further comprise a small molecule. Small molecule moieties include, but are not limited to, small peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, synthetic polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including heteroorganic and organometallic compounds) typically having a molecular weight of less than about 5,000 g/mole, e.g., organic or inorganic compounds having a molecular weight of less than about 2,000 g/mole, e.g., organic or inorganic compounds having a molecular weight of less than about 1,000 g/mole, e.g., organic or inorganic compounds having a molecular weight of less than about 500 g/mole, as well as salts, esters, and other pharmaceutically acceptable forms of such compounds. Small molecules may include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viral or microbial particles, synthetic molecules, and agonists or antagonists.

Examples of suitable small molecules include those described in: "The Pharmacological Basis of Therapeutics ]," Goodman and Gilman, McGraw-Hill, New York, N.Y. (1996), ninth edition, in The following sections: drugs Acting at Synaptic and Neuroeffector Junctional Sites; drugs action on the Central Nervous System; drug Therapy of infection [ auto-active substance: pharmacological treatment of inflammation ]; water, Salts and Ions; drugs Affecting Renal Function and Electrolyte Metabolism; cardiovacular Drugs; drugs Affecting Gastrointestinal Function; drugs influencing Uterine Motility; chemotherapy of Parasitic Infections; chemical of Microbial Diseases [ Chemotherapy for Microbial Diseases ]; chemotherapy of Neoplastic Diseases [ Chemotherapy for Neoplastic disease ]; drugs Used for Immunosuppression [ Drugs for Immunosuppression ]; drugs Acting on Blood-Forming organs; hormones and Hormone Antagonists; [ hormones and hormone antagonists ]; vitamins, Dermatology; and toxicoly [ Toxicology ], are all incorporated herein by reference. Some examples of small molecules include, but are not limited to, prion drugs such as tacrolimus, ubiquitin ligase or HECT ligase inhibitors such as heclin, histone modifying drugs such as sodium butyrate, enzyme inhibitors such as 5-azacytidine, anthracyclines such as doxorubicin, beta-lactams such as penicillin, antibacterial agents, chemotherapeutic agents, antiviral agents, modulators from other organisms such as VP64, and poorly bioavailable drugs such as poorly pharmacokinetic chemotherapeutic drugs.

In some embodiments, the small molecule is an epigenetic modifier, for example, such as those described in de Groote et al nuc. Exemplary small molecule epigenetic modifiers are described, for example, in Lu et al j.biomolecular Screening [ journal of biomolecular Screening ]17.5(2012):555-71 (e.g., table 1 or 2), which is incorporated herein by reference. In some embodiments, the epigenetic modifier comprises vorinostat or romidepsin. In some embodiments, the epigenetic modifier comprises an inhibitor of a class I, II, III and/or IV Histone Deacetylase (HDAC). In some embodiments, the epigenetic modifier comprises an activator of SirTI. In some embodiments, the epigenetic modifier comprises mangosteen, Lys-CoA, C646, (+) -JQI, I-BET, BICI, MS120, dzneep, UNC0321, EPZ004777, AZ505, AMI-I, pyrazolamide 7b, benzo [ d ] imidazole 17b, acylated dapsone derivatives (e.g., PRMTI), methystat, 4 '-dicarboxyl-2, 2' -bipyridine, SID 85736331, hydroxamate analog 8, tanylcypromie, biguanide and biguanide polyamine analogs, UNC669, vadadazole, decitabine, sodium phenylbutyrate (SDB), Lipoic Acid (LA), quercetin, valproic acid, hydralazine, sulfamethoxine, green tea extracts (e.g., epigallocatechin gallate (EGCG)), curcumin, sulforaphane, and/or allicin/diallyl disulfide. In some embodiments, the epigenetic modifier inhibits DNA methylation, e.g., an inhibitor of DNA methyltransferase (e.g., 5-azacytidine and/or decitabine). In some embodiments, the epigenetic modifier modifies histone modifications, such as histone acetylation, histone methylation, histone ubiquitination, and/or histone phosphorylation. In some embodiments, the epigenetic modifier is an inhibitor of histone deacetylase (e.g., vorinostat and/or trichostatin a).

In some embodiments, the small molecule is a pharmaceutically active agent. In one embodiment, the small molecule is an inhibitor of a metabolic activity or component. Useful classes of pharmaceutically active agents include, but are not limited to, antibiotics, anti-inflammatory agents, angiogenic or vasoactive agents, growth factors, and chemotherapeutic (anti-tumor) agents (e.g., tumor suppressors). One or a combination of molecules from the classes and examples described herein or from (Orme-Johnson 2007, Methods Cell Biol. [ Methods of Cell biology ] 2007; 80:813-26) may be used. In one embodiment, the invention includes a composition comprising an antibiotic, an anti-inflammatory, an angiogenic or vasoactive agent, a growth factor, or a chemotherapeutic agent.

Peptides or proteins

In some embodiments, the compositions or finger rings described herein may further comprise a peptide or protein. Peptide moieties may include, but are not limited to, peptide ligands or antibody fragments (e.g., antibody fragments that bind to a receptor such as an extracellular receptor), neuropeptides, hormone peptides, peptide drugs, toxic peptides, viral or microbial peptides, synthetic peptides, and agonist or antagonist peptides.

The peptide moiety may be linear or branched. The peptide is about 5 to about 200 amino acids, about 15 to about 150 amino acids, about 20 to about 125 amino acids, about 25 to about 100 amino acids, or any range therebetween in length.

Some examples of peptides include, but are not limited to, fluorescent tags or labels, antigens, antibodies, antibody fragments such as single domain antibodies, ligands and receptors such as glucagon-like peptide-1 (GLP-1), GLP-2 receptor 2, cholecystokinin b (cckb), and somatostatin receptors, peptide therapeutics such as those that bind to specific cell surface receptors such as G-protein coupled receptors (GPCRs) or ion channels, synthetic or analog peptides of naturally bioactive peptides, antimicrobial peptides, pore-forming peptides, tumor-targeting or cytotoxic peptides, and degraded or self-destroying peptides such as apoptosis-inducing peptide signaling or photoactive peptides.

Peptides described herein that can be used in the present invention also include small antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single chain antibodies, Nanobodies (see, e.g., Steeland et al 2016.Nanobodies as therapeutics: big chance of Nanobodies as therapeutic: minibody: 21(7): 1076-113). Such small antigen-binding peptides can bind cytoplasmic, nuclear, intracellular antigens.

In some embodiments, the compositions or finger rings described herein comprise a polypeptide linked to a ligand capable of targeting a specific location, tissue, or cell.

Oligonucleotide aptamers

In some embodiments, the compositions or finger ring described herein may further comprise an oligonucleotide aptamer. The aptamer moiety is an oligonucleotide or peptide aptamer. Oligonucleotide aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can bind preselected targets (including proteins and peptides) with high affinity and specificity.

Oligonucleotide aptamers are nucleic acid species that can be engineered to bind to various molecular targets (e.g., small molecules, proteins, nucleic acids, and even cells, tissues, and organisms) by repeated rounds of in vitro selection or equivalently SELEX (systematic evolution of ligands by exponential enrichment). Aptamers provide differentiated molecular recognition and can be generated by chemical synthesis. In addition, aptamers may have desirable storage characteristics and cause little immunogenicity in therapeutic applications.

Both DNA and RNA aptamers can exhibit robust binding affinity to a variety of targets. For example, DNA and RNA aptamers have been selected for lysozyme, thrombin, human immunodeficiency virus trans-response element (HIV TAR) (see en. wikipedia. org/wiki/Aptamer-site _ note-10), hemin, interferon gamma, Vascular Endothelial Growth Factor (VEGF), Prostate Specific Antigen (PSA), dopamine and non-classical oncogenes, heat shock factor 1(HSF 1).

Peptide aptamers

In some embodiments, the compositions or finger ring described herein may further comprise a peptide aptamer. Peptide aptamers have one (or more) short variable peptide domain, including peptides with low molecular weight 12-14 kDa. Peptide aptamers can be designed to specifically bind to and interfere with protein-protein interactions within cells.

Peptide aptamers are artificial proteins that are selected or engineered to bind to a particular target molecule. These proteins include one or more peptide loops of variable sequence. They are usually isolated from combinatorial libraries and often subsequently improved by directed mutagenesis or multiple rounds of variable region mutagenesis and selection. In vivo, peptide aptamers can bind to cellular protein targets and exert biological effects, including interfering with the normal protein interactions of their targeting molecules with other proteins. In particular, variable peptide aptamer loops attached to a transcription factor binding domain are screened against target proteins attached to the transcription factor activation domain. The in vivo binding of the peptide aptamers to their targets by this selection strategy was detected as the expression of downstream yeast marker genes. Such experiments identified specific proteins that bind to the aptamer, as well as aptamer-disrupted protein interactions to elicit a phenotype. In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modifications of their target proteins, or alter the subcellular localization of the target.

Peptide aptamers can also recognize targets in vitro. They have been found to replace antibodies in biosensors and to be useful for detecting active protein isoforms from populations comprising inactive and active protein forms. Derivatives known as tadpoles, in which the peptide aptamer "head" is covalently linked to a unique sequence double-stranded DNA "tail", can quantify the scarce target molecule in the mixture by PCR of its DNA tail (e.g., using the quantitative real-time polymerase chain reaction).

Peptide aptamer selection can be performed using different systems, but the most used today is the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display techniques (e.g., mRNA display, ribosome display, bacterial display, and yeast display). These experimental procedures are also known as biopanning. Among peptides obtained from biopanning, a mimotope can be considered a peptide aptamer. All peptides panned from the combinatorial peptide library have been stored in a special database named MimoDB.

IV. host

The invention further relates to a host or host cell comprising a finger ring as described herein. In some embodiments, the host or host cell is a plant, insect, bacterial, fungal, vertebrate, mammalian (e.g., human), or other organism or cell. In certain embodiments, as demonstrated herein, the provided rings infect a range of different host cells. Target host cells include cells of mesodermal, endodermal or ectodermal origin. Target host cells include, for example, epithelial cells, muscle cells, leukocytes (e.g., lymphocytes), kidney tissue cells, lung tissue cells.

In some embodiments, the finger ring is substantially non-immunogenic in the host. Meaning that the loop or genetic element is unable to produce an undesirable substantial response by the host's immune system. Some immune responses include, but are not limited to, humoral immune responses (e.g., production of antigen-specific antibodies) and cell-mediated immune responses (e.g., lymphocyte proliferation).

In some embodiments, the host or host cell is contacted with the finger ring (e.g., infection). In some embodiments, the host is a mammal, such as a human. The amount of the finger ring body in the host can be measured at any time after administration. In certain embodiments, the time course of ring body growth in culture is determined.

In some embodiments, the finger ring is heritable, such as the finger rings described herein. In some embodiments, the ring is linearly transported from the mother to the child in a fluid and/or cell. In some embodiments, the daughter cells from the original host cell comprise finger rings. In some embodiments, the mother transfers the ring to the child or from the host cell to the daughter cell with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the efficiency of transfer of the mother to the child or from the host cell to the daughter cell is at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the finger ring body in the host cell has a transport efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% during meiosis. In some embodiments, the ring in the host cell has a transport efficiency during mitosis of at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the ring in the cell has a transmission efficiency of about 10% -20%, 20% -30%, 30% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -75%, 75% -80%, 80% -85%, 85% -90%, 90% -95%, 95% -99%, or any percentage therebetween.

In some embodiments, the ring body, e.g., the ring body, replicates within a host cell. In one embodiment, the ring is capable of replicating in a mammalian cell, such as a human cell. In other embodiments, the ring is defective or incapable of replication.

Although in some embodiments, the loop replicates in the host cell, it does not integrate into the genome of the host, e.g., does not integrate with the host chromosome. In some embodiments, the ring has a negligible recombination frequency, e.g., with the host's chromosome. In some embodiments, the frequency of recombination of the ring with, for example, a chromosome of the host is, for example, less than about 1.0cM/Mb, 0.9cM/Mb, 0.8cM/Mb, 0.7cM/Mb, 0.6cM/Mb, 0.5cM/Mb, 0.4cM/Mb, 0.3cM/Mb, 0.2cM/Mb, 0.1cM/Mb, or lower.

Method of use

The finger ring bodies and compositions comprising the finger ring bodies described herein can be used, for example, in methods of treating a disease, disorder, or condition in a subject (e.g., a mammalian subject, e.g., a human subject) in need thereof. Administration of the pharmaceutical compositions described herein can be, for example, by parenteral (including intravenous, intratumoral, intraperitoneal, intramuscular, intracavity, and subcutaneous) administration. The finger ring body can be administered alone or formulated into a pharmaceutical composition.

The finger ring body may be administered in the form of a unit dose composition, for example, a unit dose parenteral composition. Such compositions are typically prepared by mixing, and may be suitable for parenteral administration. Such compositions may be in the form of, for example, injectable and infusible solutions or suspensions or suppositories or aerosols.

In some embodiments, administration of a finger loop, or a composition comprising the same, e.g., as described herein, can result in delivery of the genetic element comprised by the finger loop, e.g., into a target cell of a subject.

The ring bodies or compositions thereof, e.g., comprising an effector (e.g., endogenous or exogenous effector), described herein can be used to deliver the effector to a cell, tissue, or subject. In some embodiments, the ring body or a composition thereof is used to deliver an effector to bone marrow, blood, heart, GI, or skin. Delivery of effectors by administration of the finger ring compositions described herein can modulate (e.g., increase or decrease) the expression level of a non-coding RNA or polypeptide in a cell, tissue, or subject. Modulating expression levels in this manner can result in altered functional activity in the cell into which the effector is delivered. In some embodiments, the modulated functional activity can be enzymatic, structural, or modulated in nature.

In some embodiments, the ring or copy thereof is detectable in the cell 24 hours (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 30 days, or 1 month) after delivery into the cell. In embodiments, the ring body or composition thereof mediates an effect on a target cell and the effect persists for at least 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or, 1, 2, 3, 6, or 12 months. In some embodiments (e.g., wherein the loop or composition thereof comprises a genetic element encoding an exogenous protein), the effect persists for less than 1, 2, 3, 4, 5, 6, or 7 days, 2, 3, or 4 weeks, or 1, 2, 3, 6, or 12 months.

Examples of diseases, disorders, and conditions that can be treated with the ring bodies described herein or compositions comprising the ring bodies include, but are not limited to: immune disorders, interferon diseases (e.g., type I interferon disease), infectious diseases, inflammatory disorders, autoimmune disorders, cancer (e.g., solid tumors, such as lung cancer, non-small cell lung cancer, e.g., tumors expressing a gene responsive to nir-625 (e.g., caspase 3)), and gastrointestinal disorders. In some embodiments, the finger ring modulates (e.g., increases or decreases) activity or function in a cell that is in contact with the finger ring. In some embodiments, the finger ring modulates (e.g., increases or decreases) the level or activity of a molecule (e.g., a nucleic acid or protein) in a cell contacted with the finger ring. In some embodiments, the ring reduces the viability of a cell, e.g., a cancer cell, that is in contact with the ring, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more. In some embodiments, the finger ring comprises an effector, e.g., a miRNA, e.g., miR-625, that reduces the viability of cells, e.g., cancer cells, contacted with the finger ring by, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more. In some embodiments, the finger ring increases apoptosis of a cell, e.g., a cancer cell, contacted with the finger ring, e.g., by increasing caspase-3 activity, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. In some embodiments, the ring comprises an effector, e.g., a miRNA, e.g., miR-625, that increases apoptosis, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, of a cell, e.g., a cancer cell, that is in contact with the ring, e.g., by increasing caspase-3 activity.

VI. production method

Generation of genetic elements

Methods for preparing the genetic elements of the finger ring are described, for example, in Khudyakov & Fields, Artificial DNA: Methods and Applications [ Artificial DNA: methods and applications ], CRC press (2002); zhao, Synthetic Biology Tools and Applications [ Synthetic Biology: tools and applications ], (first edition), Academic Press (2013); and Egli & Herdewijn, Chemistry and Biology of Artificial Nucleic Acids [ Chemistry and Biology of Artificial Nucleic Acids ], (first edition), Wiley-VCH (2012).

In some embodiments, the genetic element may be designed using computer-aided design tools. The ring can be divided into smaller overlapping blocks (e.g., in the range of about 100bp to about 10kb fragments or individual ORFs) that are more easily synthesized. These DNA fragments are synthesized from a set of overlapping single-stranded oligonucleotides. The resulting contigs are then assembled into larger DNA blocks, e.g., finger loops. The fragments or ORFs can be assembled into finger loops, e.g., recombined in vitro or unique restriction sites at the 5 'and 3' ends to achieve ligation.

Alternatively, genetic elements can be synthesized using design algorithms that resolve the ring into oligonucleotide-length fragments, creating optimal design conditions for synthesis given the complexity of the sequence space. Oligonucleotides were then chemically synthesized on semiconductor-based high-density chips, each of which could synthesize over 200,000 individual oligonucleotides. By means such as The assembly technique of (3) assembles oligonucleotides to construct longer DNA fragments from smaller oligonucleotides. This is done in a parallel fashion, so that hundreds or thousands of synthetic DNA fragments can be constructed at a time.

Sequence verification can be performed for each genetic element or fragment of a genetic element. In some embodiments, AnyDot.chips (Genovox, Germany) can be used for high throughput sequencing of RNA or DNA, which allows monitoring of biological processes (e.g., miRNA expression or allelic variability (SNP detection). specifically, AnyDot.chips increase detection of nucleotide fluorescence signals by 10x-50 x.AnyDot.chips and methods of use are described in part in International published application Nos. WO 02088382, WO 03020968, WO 03031947, WO 2005044836, PCTEP 05105657, PCMEP 05105655, and German patent application Nos. DE 10149786, DE 10214395, DE 10356837, DE 102004009704, DE 102004025696, DE 102004025746, DE 102004025694, DE 102004025695, DE 102004025744, DE 102004025745, and DE 102005012301.

Other high throughput sequencing systems are included in Venter, j., et al Science [ Science ]2001, month 2, day 16; adams, M. et al Science [ Science ] 24/3/2000; and M.J, Leven, et al Science 299: 682-; and those disclosed in US published application nos. 20030044781 and 2006/0078937. In general, such systems involve sequencing a target nucleic acid molecule having multiple bases by adding bases in time via a polymerization reaction measured on the nucleic acid molecule, i.e., tracking the activity of a nucleic acid polymerase on a template nucleic acid molecule to be sequenced in real time. The sequence can then be deduced by identifying which base is incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerase at each step of the base addition sequence. A polymerase is provided on the target nucleic acid molecule complex at a position suitable for movement along the target nucleic acid molecule and extending the oligonucleotide primer at the active site. A plurality of labeled types of nucleotide analogs are provided in proximity to the active site, each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. Extending a growing nucleic acid strand by using a polymerase to add a nucleotide analog to the nucleic acid strand at an active site, wherein the added nucleotide analog is complementary to a nucleotide of the target nucleic acid at the active site. Identifying the nucleotide analogs added to the oligonucleotide primers as a result of the polymerizing step. The steps of providing labeled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analogs are repeated, thereby further extending the nucleic acid strand and identifying the sequence of the target nucleic acid.

In some embodiments, shotgun sequencing is performed. In shotgun sequencing, DNA is randomly divided into many small fragments, which are sequenced using the chain termination method to obtain reads. By performing several rounds of such fragmentation and sequencing, multiple overlapping reads of the target DNA can be obtained. The computer program then uses the overlapping ends of the different reads to assemble them into a continuous sequence.

In some embodiments, the factors for replication or packaging may be provided in cis or in trans relative to the genetic element. For example, when provided in cis, the genetic element may comprise one or more genes encoding, for example, an ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3 or ORF2t/3 of a finger ring virus as described herein. In some embodiments, replication and/or packaging signals may be incorporated into the genetic element, e.g., to induce amplification and/or encapsulation. In some embodiments, this is done in the context of a larger region of the genome of the finger ring (e.g., insertion of an effector into a specific site in the genome, or replacement of a viral ORF with an effector).

In another example, when provided in trans, the genetic element may lack a gene encoding one or more of, for example, an ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3, or ORF2t/3 of a finger ring virus as described herein; the one or more proteins may be provided, for example, by another nucleic acid, such as a helper nucleic acid. In some embodiments, minimal cis-signals (e.g., 5' UTR and/or GC-rich regions) are present in the genetic element. In some embodiments, the genetic element does not encode a replication or packaging factor (e.g., replicase and/or capsid protein). In some embodiments, such factors may be provided by one or more helper nucleic acids (e.g., helper viral nucleic acids, helper plasmids, or helper nucleic acids integrated into the genome of the host cell). In some embodiments, the helper nucleic acids express proteins and/or RNAs sufficient to induce amplification and/or packaging, but may lack their own packaging signals. In some embodiments, the genetic element and helper nucleic acid are introduced into the host cell (e.g., simultaneously or separately), resulting in amplification and/or packaging of the genetic element but not the helper nucleic acid.

In vitro cyclization

In some cases, the genetic element to be packaged into the exterior of the protein is a single-stranded circular DNA. In some cases, the genetic element may be introduced into the host cell in a form other than single-stranded circular DNA. For example, the genetic element may be introduced into the host cell as a double-stranded circular DNA. The double-stranded circular DNA can then be converted to single-stranded circular DNA in a host cell (e.g., a host cell comprising a suitable enzyme for rolling circle replication, such as a dactylovirus Rep proteinFor example, Rep68/78, Rep60, RepA、RepB、Pre、MobM、TraX、TrwC、Mob02281、Mob02282、NikB、ORF50240、NikK、TecH. OrfJ or Trai, e.g., as Wawrzyniak et al 2017, front. Microbiol. [ microbiology front line ]]2353, the preparation method is as follows; just list The enzyme producedIncorporated herein by reference). In some embodiments, the double-stranded circular DNA is produced by in vitro circularization, e.g., as described in example 35. Typically, in vitro circularized DNA constructs can be produced by digesting a plasmid containing the genetic element sequence to be packaged such that the genetic element sequence is cleaved into linear DNA molecules. The resulting linear DNA may then be ligated, e.g., using DNA ligase, to form double-stranded circular DNA. In some cases, double-stranded circular DNA resulting from in vitro circularization can undergo rolling circle replication, e.g., as described herein. Without wishing to be bound by theory, in vitro circularization is expected to produce a double stranded DNA construct that can undergo rolling circle replication without further modification, thereby enabling the generation of single stranded circular DNA of suitable size for packaging into a finger ring body, e.g., as described herein. In some embodiments, the double-stranded DNA construct is smaller than a plasmid (e.g., a bacterial plasmid). In some embodiments, the double stranded DNA construct is excised from a plasmid (e.g., a bacterial plasmid) and then circularized, e.g., by in vitro circularization.

Production of finger ring bodies

Genetic elements and vectors comprising genetic elements prepared as described herein can be used in a variety of ways to express finger loops in appropriate host cells. In some embodiments, the genetic element and the vector comprising the genetic element are transfected into an appropriate host cell, and the resulting RNA can direct high level expression of ring gene products such as non-pathogenic proteins and protein binding sequences. Host cell systems that provide high levels of expression include continuous cell lines that provide viral function, such as cell lines that are superinfected with APV or MPV, respectively, cell lines engineered to complement APV or MPV function, and the like.

In some embodiments, the ring body is produced as described in any one of examples 1, 2, 5, 6, or 15-17.

In some embodiments, the finger ring body is cultured in vitro in a continuous animal cell line. According to one embodiment of the invention, the cell line may comprise a porcine cell line. Cell lines contemplated in the context of the present invention include immortalized porcine cell lines such as, but not limited to, porcine kidney epithelial cell lines PK-15 and SK, the single myeloid cell line 3D4/31 and the testicular cell line ST. In addition, other mammalian cell lines are included, such as CHO cells (Chinese hamster ovary), MARC-145, MDBK, RK-13, EEL. Additionally or alternatively, particular embodiments of the methods of the present invention utilize animal cell lines that are epithelial cell lines, i.e., cell lines of cells of the epithelial lineage. Cell lines susceptible to infection by the finger ring include, but are not limited to, cell lines of human or primate origin, such as human or primate renal cancer cell lines.

In some embodiments, the genetic element and the vector comprising the genetic element are transfected into a cell line that expresses the viral polymerase protein to effect expression of the finger loop. For this purpose, transformed cell lines expressing the protein loop polymerase can be used as suitable host cells. Host cells can be similarly engineered to provide other viral functions or additional functions.

To prepare the finger rings disclosed herein, the genetic elements disclosed herein or vectors comprising the genetic elements can be used to transfect cells that provide the finger ring protein and the functions required for replication and production. Alternatively, cells can be transfected with a helper virus before, during, or after transfection with a genetic element or vector comprising a genetic element disclosed herein. In some embodiments, helper virus can be used to supplement the production of incomplete viral particles. Helper viruses may have conditional growth defects, such as host range limitations or temperature sensitivity, allowing for subsequent selection of transfected viruses. In some embodiments, the helper virus can provide one or more replication proteins for use by the host cell to achieve expression of the finger ring. In some embodiments, a host cell can be transfected with a vector encoding a viral protein, such as one or more replication proteins. In some embodiments, the helper virus comprises antiviral susceptibility.

The genetic elements disclosed herein or vectors comprising genetic elements can be replicated and generated into finger ring particles by a variety of techniques known in the art, for example, U.S. Pat. nos. 4,650,764; U.S. patent nos. 5,166,057; U.S. patent nos. 5,854,037; european patent publications EP 0702085a 1; U.S. patent application serial No. 09/152,845; international patent publications PCT WO 97/12032; WO 96/34625; european patent publication EP-A780475; WO 99/02657; WO 98/53078; WO 98/02530; WO 99/15672; WO 98/13501; WO 97/06270; and EPO 78047 SA1, each of which is incorporated by reference herein in its entirety.

The production of cell cultures containing finger rings according to the invention can be carried out on different scales, for example in flasks, roller bottles or bioreactors. The medium used to culture the cells to be infected is known to the skilled person and may typically comprise standard nutrients required for cell viability, but may also comprise other nutrients depending on the cell type. Optionally, the culture medium may be protein-free and/or serum-free. Depending on the cell type, the cells may be cultured in suspension or on a substrate. In some embodiments, different media are used for growth of the host cell and production of the finger ring.

Purification and isolation of the finger ring body can be performed according to Methods known to those skilled in the art of virus production, such as Rinaldi et al, DNA Vaccines: Methods and Protocols (Methods in Molecular Biology) [ DNA vaccine: methods and protocols (methods in molecular biology) ], 3 rd edition 2014, Humana press.

In one aspect, the invention includes a method of in vitro replication and propagation of finger rings as described herein, which may include the steps of: (a) transfecting the linearized genetic element into a cell line sensitive to ring infection; (b) harvesting the cells and isolating cells that show the presence of the genetic element; (c) culturing the cells obtained in step (b) for at least three days, for example for at least one week or more, depending on the experimental conditions and gene expression; and (d) harvesting the cells of step (c).

In some embodiments, the ring body can be introduced into a host cell line that is grown to high cell density. In some embodiments, the finger ring bodies can be harvested and/or purified by separating solutes based on biophysical properties, such as ion exchange chromatography or tangential flow filtration, prior to formulation with pharmaceutical excipients.

Administration/delivery

The compositions (e.g., pharmaceutical compositions comprising the finger rings described herein) can be formulated to include pharmaceutically acceptable excipients. The pharmaceutical composition may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances. The pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical formulations can be found in: for example, Remington The Science and Practice of Pharmacy 21st ed. [ Remington: pharmaceutical science and practice, 21st edition ], Lippincott Williams & Wilkins,2005 (incorporated herein by reference).

Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, such as a non-human animal, e.g., a non-human mammal. The modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well known, and ordinary veterinary pharmacologists can design and/or make such modifications, if at all, by only ordinary experimentation. Subjects contemplated for administration of the pharmaceutical composition include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals, such as cows, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or birds, including commercially relevant birds, such as poultry, chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or later developed in the pharmacological arts. Generally, such a preparation method comprises the following steps: the active ingredient is combined with excipients and/or one or more other auxiliary ingredients, and the product is then separated, shaped and/or packaged if necessary and/or desired.

In one aspect, the invention features a method of delivering a ring body to a subject. The method comprises administering to a subject a pharmaceutical composition comprising a finger ring as described herein. In some embodiments, the administered finger ring replicates in the subject (e.g., becomes part of the subject's virome).

The pharmaceutical composition may comprise wild-type or native viral elements and/or modified viral elements. Finger loops may include one or more sequences (e.g., nucleic acid sequences or nucleic acid sequences encoding amino acid sequences thereof) in any of tables a1-a12, B1-B5, C1-C5, or 1-18, or sequences having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any of these nucleotide sequences, or sequences complementary to sequences in any of tables a1-a12, B1-B5, C1-C5, or 1-18. The finger ring can comprise a nucleic acid molecule comprising a nucleic acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to one or more sequences in any one of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, 17, or 41. The finger ring can comprise a nucleic acid molecule encoding an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to any one of the amino acid sequences in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18. The finger ring may comprise a polypeptide comprising an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to any one of the amino acid sequences in any one of tables a2, a4, a6, A8, a10, a12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, or 18. The finger ring may comprise a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% nucleotide sequence identity to one or more of the sequences in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, 17 or 41, or to any of the nucleotide sequences in any of tables a1, A3, a5, a7, a9, a11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, 17 or 41, or to a sequence complementary thereto.

In some embodiments, the finger ring is sufficient to increase (stimulate) endogenous gene and protein expression, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, as compared to a reference, e.g., a healthy control. In certain embodiments, the finger ring is sufficient to reduce (inhibit) endogenous gene and protein expression, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, as compared to a reference, e.g., a healthy control.

In some embodiments, the finger ring inhibits/enhances one or more viral properties, e.g., tropism, infectivity, immunosuppression/activation, e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, in the host or host cell as compared to a reference, e.g., a healthy control.

In some embodiments, a pharmaceutical composition is administered to a subject, the pharmaceutical composition further comprising one or more viral strains not represented in the viral genetic information.

In some embodiments, a pharmaceutical composition comprising a finger ring as described herein is administered at a dose and for a time sufficient to modulate viral infection. Some non-limiting examples of viral infections include adeno-associated virus, alphavirus, Australian bat rabies virus, BK polyoma virus, Banna virus, Bama forest virus, Bunyavira lacrosse, Bunyavira snow hare, Cornus herpesvirus, Chandipura virus, chikungunya virus, coxsacova (Cosavirus) A, vaccinia virus, coxsackievirus, Krima-Congo hemorrhagic fever virus, dengue virus, Doricavirus, Dubai virus, Duwenhage virus, eastern equine encephalitis virus, Ebola virus, echovirus, encephalomyocarditis virus, Epstein-Barr virus, European bat rabies virus, GB virus hepatitis C/G, Hantaan virus, Hendra virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Hepatitis E virus, hepatitis D virus, Marpox virus, human adenovirus, human astrovirus, human coronavirus, human cytomegalovirus, human enterovirus 68, human enterovirus 70, human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human membranous parvovirus, human T lymphovirus, human circovirus, influenza A virus, influenza B virus, influenza C virus, Israh's virus, JC polyoma virus, Japanese encephalitis virus, Hunnin coccus virus, KI polyoma virus, Kunjin virus, Lagues bat virus, Victoria lake Marburg virus, Victoria virus, Van, Langat virus, Lassa virus, Lorenter virus, skip disease virus, lymphocytic choriomeningitis virus, Markubo virus, Mariothis virus, MERS coronavirus, measles virus, mango encephalomyocarditis virus, Merck polyoma virus, Mocora virus, mollusk contact herpes virus, monkeypox virus, mumps virus, Murraya encephalitis virus, New York virus, Nipah virus, Norwalk virus, Olympic virus, aphtha virus, Eprosa virus, Pickerd virus, poliovirus, Pottatorus vein virus, Primala virus, rabies virus, rift valley fever virus, Rosa virus A, Ross river virus, rotavirus A, rotavirus B, rotavirus C, rubella virus, mountain virus, Saili virus (Salivirus) A, Sakuri virus, Murray fever Sicily virus, Muira virus, Semliki forest virus, hancheng virus, simian foamy virus, simian virus 5, sindbis virus, south ampelodon virus, st louis encephalitis virus, tick-borne powassan virus, torque ringworm virus, toscarnavirus, ewkunimi virus, vaccinia virus, varicella zoster virus, smallpox virus, venezuelan equine encephalitis virus, vesicular stomatitis virus, western equine encephalitis virus, WU polyoma virus, west nile virus, yama tumor virus, yabas disease virus, yellow fever virus, and saika virus. In certain embodiments, relative to a reference, a finger ring is sufficient to win and/or replace, e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, of a virus already present in a subject. In certain embodiments, the ring body is sufficient to compete with chronic or acute viral infections. In certain embodiments, the ring body can be administered prophylactically to protect against viral infection (e.g., to prevent a virus). In some embodiments, the amount of finger ring is sufficient to modulate (e.g., phenotype, viral level, gene expression, competition with other viruses, disease state, etc. is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more).

All references and publications cited herein are incorporated by reference.

The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it will be appreciated by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

Examples of the invention

Content listing

Example 1: preparing a dactylene body: design and Synthesis of synthetic Ring that inhibits Interferon (IFN) expression

Example 2: large scale production of finger ring bodies (finger ring bodies a and/or B): production and propagation of finger ring bodies

Example 3: in vitro action of the finger ring body (finger ring body a): in vitro assessment of expression of finger ring and effector functions such as expression of miRNA after cell infection

Example 4: immunological role of the finger ring body (finger ring body): in vivo effector function of the ring after administration, e.g. expression of miRNA

Example 5: preparation of synthetic finger ring: in vitro production of synthetic finger ring bodies

Example 6: assembly and infection of finger Ring bodies infectious finger Ring bodies were generated in vitro using the synthetic DNA sequences described in example 5

Example 7: selectivity of the finger ring body: the ability of in vitro generated synthetic finger rings to infect multiple tissue-derived cell lines

Example 8: identification and use of protein binding sequences: putative protein binding sites in the genome of the finger ring virus

Example 9: finger ring virus genome

Example 10: various lengths of nucleotides were inserted into the genome of the ring virus: adding DNA sequences of various lengths to the genome of the dactylovirus

Example 11: exemplary goods to be delivered: exemplary classes of nucleic acid and protein payloads in the Ring

Example 12: exemplary payload integration sites

Example 13: ring viruses of defined classes and conserved regions thereof

Example 14: replication-defective finger rings and helper viruses

Example 15: method for manufacturing ring body with copying capability

Example 16: the manufacturing method of the replication-defective finger ring body comprises the following steps: recovery and scale-up of replication-defective finger loops

Example 17: production of finger rings using suspension cells: production of finger rings in suspension cells.

Example 18: quantification of finger ring genome equivalents by qPCR: development of hydrolysis probe-based quantitative PCR assay to quantify finger rings

Example 19: expression of foreign proteins in mice using finger rings: expression of functional model proteins in vivo using finger ring bodies

Example 20: genome alignment to determine if the genomic DNA is integrated into the host genome

Example 21: evaluation of integration of finger Ring into host genome

Example 22: the ring body expressing the exogenous micro RNA sequence has the following functional functions: expression of functional nucleic acid effectors using finger ring surfaces

Example 23: preparation and production of finger rings expressing exogenous non-coding RNA: expression of exogenous small non-coding RNAs using finger loops

Example 24: conservation in the evolutionary branches of the ring virus: identification of seven evolved branches in the genus cyclopavirus A

Example 25: expression of endogenous miRNAs from the surface of the finger ring and deletion of endogenous miRNAs

Example 26: location of the ORF of the Ring Virus

Example 27: characterization of the area required for finger ring development

Example 28: finger ring in vivo delivery of exogenous proteins: this example demonstrates the in vivo effector function (e.g., protein expression) of the finger ring after administration

Example 29: identification of precursor miRNA (pre-nir) in dacryviruses: computational and experimental methods to identify novel precursor miRNAs encoded by various dactyloviruses

Example 30: determining endogenous targets of pre-miR of the ring virus: assays to determine endogenous targets and potential therapeutically relevant target pathways for Pre-miR encoded by various strains of Ring Virus

Example 31: making a ring encoding a native ring virus pre-miR: process for packaging replicative or non-replicative forms of finger rings expressing native pre-mirs of dactyloviruses

Example 32: in an in vitro cell culture model, using pre-miR of dactylovirus as a tumor suppressor: phenotypic effects of candidate pre-miRs identified as tumor suppressive from assays such as described in example 29

Example 33: using pre-miR of dactylovirus as an in vivo tumor suppressor: in vivo experiments to confirm tumor inhibition by tumor-inhibiting Ring Virus Pre-miR and cancer cell lines from in vitro assays as described in example 32

Example 34: tandem copies of the viral genome of the Ring

Example 35: in vitro circularized viral genome of the ring: construct comprising circular double stranded circular genomic DNA and minimal non-viral DNA

Example 36: ORF1 modeling and identification of conserved residues and domains: modeling and definition of putative Domain of the B-type Cycloviron ORF1 protein

Example 37: production of finger Ring containing chimeric ORF1 with a hypervariable Domain from a different Strain of the Cyclovirus

Example 38: production of chimeric ORF1 containing non-TTV proteins/peptides in place of the hypervariable domain

Example 39: ring delivery of secreted enzymes in vivo

Example 40: ring in vivo delivery of secreted antibodies

Example 41: design of finger Ring containing DNA payload

Example 42: transduction of finger rings encoding antibody transgenes

Example 43: tth8 and LY 2-based finger rings successfully transduce the EPO gene into lung cancer cells, respectively

Example 44: after intravenous (i.v.) administration, finger rings with therapeutic transgenes can be detected in vivo

Example 45: size distribution of coding sequences in finger ring viruses

Example 46: highly conserved motifs to characterize ORF2

Example 47: evidence for human full-length finger ring virus ORF1 mRNA

Example 48: in-vitro cyclized genome as input material for in-vitro production of finger ring body

Example 49: identification of conserved secondary structural motifs in ORF1 of finger-ring virus

Example 1: preparing a dactylene body:

this example describes the design and synthesis of synthetic finger rings that inhibit the expression of Interferon (IFN).

A finger ring (finger ring A) was designed which starts with 1) the DNA sequence encoding the capsid gene of the non-pathogenic packaging coat (Arch Virol (2007)152:1961-1975), accession number: A7XCE8.1(ORF11_ TTW 3); 2) DNA sequences encoding micrornas targeting host genes (e.g. IFN) (PLOS Pathogen [ public science library-Pathogen ] (2013),9(12), e1003818), accession numbers: AJ 620231.1; and 3) binding to a specific region in the capsid protein (e.g., having accession number: capsid specific region of Q99153.1) (Journal of Virology (2003),77(24), 13036-13041).

To this sequence was added a 1kb non-coding DNA sequence (referred to as loop B). The designed ring bodies (FIG. 2) were chemically synthesized to 3kb (total size) and sequence verified.

Ring sequences were transfected into human embryonic kidney 293T cells (every 10 on 12-well plates) using JetPEI reagent (Polyplus-transfection, Elgilich, France) according to the manufacturer's recommendations⁵1mg per cell). Control transfection contained only vector or cells transfected with JetPEI alone, and the transfection efficiency was optimized with a reporter plasmid encoding GFP. Fluorescence of control transfections was measured to ensure that cells were properly transfected. The transfected cultures were incubated overnight at 37 ℃ and 5% carbon dioxide.

After 18 hours, cells were washed 3 times with PBS before adding fresh medium. The supernatant was collected as follows for ultracentrifugation and harvesting of the finger rings. The medium was cleared by centrifugation at 4,000Xg for 30 minutes followed by centrifugation at 8,000Xg for 15 minutes to remove cells and cell debris. The supernatant was then filtered through a 0.45 μm pore size filter. The ring was pelleted by a 5% sucrose pad (5ml) at 27,000rpm for 1 hour, then resuspended in 1x Phosphate Buffered Saline (PBS) and 0.1% bacitracin (in 1/100 original volume). The concentrated finger ring bodies were centrifuged through a 20% to 35% sucrose gradient at 24,000rpm for 2 hours. Collecting the ring band at the gradient junction. The rings were then diluted with 1x PBS and precipitated at 27,000rpm for 1 hour. The ring pellet was resuspended in 1x PBS and further purified by a 20% to 35% continuous sucrose gradient.

Example 2: large Scale production of finger Ring bodies (finger Ring bodies A and/or B)

This example describes the production and propagation of finger ring bodies.

Purified finger rings as described in example 1 were prepared for large scale amplification in spinner flasks with suspension grown a549 cell production. A549 cells were maintained in F12K medium, 10% fetal bovine serum, 2mM glutamine, and antibiotics. After 24 hours incubation at 37 ℃ and 5% carbon dioxide, 10⁶Ring Loading of individual Ring bodies A549 cells were infected with Ring bodies, resulting in about 1X10⁷Each ring body particle. Cells were then washed 3 times with PBS and incubated with fresh medium for 6 hours.

For purification of the finger ring bodies, two ultracentrifugation steps based on cesium chloride gradients were performed as follows, followed by dialysis (Bio-Protocol [ biological Protocol ] (2012) Bio101: e 201). Cells were removed by centrifugation (6000x g, 10 min) and the supernatant was filtered through a 0.8 μm filter followed by a 0.2 μm filter. The filtrate was concentrated to a volume of 8ml through a filter (100,000 mw). The retentate was loaded into cesium sulfate solution and centrifuged at 247,000x g for 20 h. The ring band was removed, placed in a dialysis tube with a cut-off molecular weight of 14,000mw, and dialyzed. Further concentration may be carried out if desired.

Example 3: in vitro action of finger ring body (finger ring body A)

This example describes the in vitro assessment of expression of finger ring bodies and expression of effector functions such as mirnas after cell infection.

The effect of the purified finger ring body described in example 1 was evaluated in vitro by endogenous gene regulation (e.g., IFN signaling). HEK293T cells were transfected with dual luciferase plasmids (firefly luciferase with a promoter based on an Interferon Stimulated Response Element (ISRE) and a transfection control renilla luciferase with a constitutive promoter): the luciferase reporter mix (pcDNA3.1dsRluc and pISRE-Luc, 1:4 ratio (Clonetech)) was co-transfected (J Virol [ J. Virol ] (2008),82: 9823-.

HEK293T cells seeded in 6-well plates (2 sets of 3 control wells in triplicate and 3 experimental wells using finger ring a) were plated at 10⁷Multiple secondary infection administration refers to the ring body.

After 48 hours, the medium was replaced with new medium with or without 100u/ml universal type I interferon (PBL, Piscataway, NJ). Sixteen hours after IFN treatment, a dual luciferase assay (J Virol [ J. Virol ] (2008),82: 9823-. Firefly luciferase was normalized to renilla luciferase expression to control for transfection differences. Fold induction of the ISRE ffLuc reporter gene was calculated by dividing the equivalent experimental wells by the control wells and comparing the induction for each condition relative to the negative control.

In one embodiment, a decrease in luciferase signal in the ring treated group compared to the control will indicate that the ring reduces production of IFN in the cell.

Example 4: immunological action of finger ring body (finger ring body)

This example describes the in vivo effector functions of the finger ring, e.g., expression of mirnas, after administration.

Used in hundredfold dilutions from 10¹⁴Starting at one genome equivalent per kilogram and dropping to 0 genome equivalent per kilogram, purified finger rings prepared as described in examples 1 and 2 were administered intravenously to healthy pigs at various doses. To assess the effect on immune tolerance, pigs were injected daily with the above indicated dose of finger ring or vehicle control PBS for 3 days and sacrificed after 3 days.

Spleen, bone marrow and lymph nodes were harvested. Single cell suspensions were prepared from each tissue and stained with MHC-II, CD11c and extracellular markers of intracellular IFN. MHC +, CD11c +, IFN + antigen presenting cells from each tissue were analyzed by flow cytometry, for example, where cells positive for a given one of the above markers were cells that exhibited greater fluorescence than 99% of the cells in a negative control population lacking marker expression under the same conditions but otherwise similar to the cell analysis population.

In one embodiment, a decrease in the number of IFN + cells in the ring treatment group compared to the control will indicate that the ring reduces IFN production in the cells after administration.

Example 5: a synthetic finger ring body was prepared.

This example illustrates the in vitro production of synthetic finger ring bodies.

The DNA sequences of LY1 and LY2 lines from TTMiniV (Eur Respir J. [ European journal of respiration ] 8.2013; 42(2):470-9) between the EcoRV restriction sites were cloned into a kanamycin vector (Integrated DNA Technologies). In examples 6 and 7 and FIGS. 6A-10B, ring bodies including the DNA sequences of LY1 and LY2 strains from TTMiniV were referred to as Yuzhi ring body 1 (ring (Anello)1) and ring body 2 (ring 2), respectively. The cloned construct was transformed into 10-beta competent E.coli (New England Biolabs Inc.; New England Biolabs Inc.) according to the manufacturer's protocol, and the plasmid was purified (Qiagen, Inc.).

The DNA construct (fig. 3 and 4) was linearized with EcoRV restriction enzyme digestion (New England Biolabs Inc.)) at 37 ℃ for 6 hours according to the manufacturer's protocol, resulting in a double-stranded linear DNA fragment containing the TTMiniV genome, but excluding bacterial backbone elements (e.g., origin of replication and selectable markers). Agarose gel electrophoresis was then performed to excise a band of DNA of the correct size of the TTMiniV genomic fragment (2.9 kilobase pairs) and the DNA was gel purified from the excised agarose band using a gel extraction kit (Qiagen) according to the manufacturer's protocol.

Example 6: finger ring body assembly and infection

This example illustrates the successful in vitro production of infectious finger rings using the synthetic DNA sequences described in example 5.

Double-stranded linearized gel-purified genomic DNA of the ring virus (obtained in example 5) was transfected into HEK293T cells (human embryonic kidney cell line) or A549 cells (human lung cancer cell line) in intact plasmid or linearized form using lipofectin (Thermo Fisher Scientific). 6ug of plasmid or 1.5ug of linearized genomic DNA from the ring virus was used to transfect 70% confluent cells in T25 flasks. An empty vector backbone lacking viral sequences contained in the finger ring was used as a negative control. 6 hours after transfection, cells were washed twice with PBS and grown in fresh growth medium at 37 ℃ and 5% carbon dioxide. From the IDT, a DNA sequence encoding the human Ef1 α promoter followed by the YFP gene was synthesized. The DNA sequence was blunt-ended and ligated into a cloning vector (Thermo Fisher Scientific). The resulting vector was used as a control to assess transfection efficiency. YFP was detected 72 hours after transfection using a cell imaging system (Thermo Fisher Scientific). Transfection efficiencies of HEK293T and a549 cells were calculated as 85% and 40%, respectively (fig. 5).

Supernatants of 293T and A549 cells transfected with finger rings were harvested 96 hours post transfection. The harvested supernatant was centrifuged at 2000rpm for 10 minutes at 4 ℃ to remove any cell debris. Each harvested supernatant was used to infect new 293T and a549 cells, respectively, which were 70% confluent in wells of 24-well plates. After 24 hours incubation at 37 ℃ and 5% carbon dioxide, the supernatant was washed off, then twice with PBS and replaced with fresh growth medium. After incubating these cells for an additional 48 hours at 37 degrees and 5% carbon dioxide, the cells were harvested separately for genomic DNA extraction. Genomic DNA was collected for each sample using a genomic DNA extraction kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

To confirm that the in vitro generated finger rings successfully infected 293T and a549 cells, 100ng of genomic DNA harvested as described herein was used for quantitative polymerase chain reaction (qPCR) using primers specific for the sequence specific for the b-ringvirus or LY 2. qPCR was performed using SYBR green reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The normalization was performed using qPCR with primers specific for the genomic DNA sequence of GAPDH. The sequences of all primers used are listed in table 42.

Table 42:

as shown by the qPCR results shown in fig. 6A, 6B, 7A and 7B, finger rings produced in vitro and described in this example were infectious.

Example 7: selectivity of the ring body

This example illustrates the ability of synthetic finger rings generated in vitro to infect a variety of tissue-derived cell lines.

Supernatants with infectious TTMiniV finger rings (as described in example 5) were incubated with 70% confluent 293T, A549, Jurkat (acute T cell leukemia cell line), Raji (burkitt's lymphoma B cell line) and Chang cell line at 37 degrees and 5% carbon dioxide in wells of 24-well plates. 24 hours post infection, cells were washed twice with PBS and then replaced with fresh growth medium. The cells were then incubated for another 48 hours at 37 degrees and 5% carbon dioxide and then harvested for genomic DNA extraction. Genomic DNA was collected for each sample using a genomic DNA extraction kit (Thermo Fisher Scientific) according to the manufacturer's protocol.

To confirm that the finger rings produced in the previous examples successfully infected these cell lines, 100ng of genomic DNA harvested as described herein was used for quantitative polymerase chain reaction (qPCR) using primers specific for the sequence specific for the virus b or LY 2. qPCR was performed using SYBR green reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. The normalization was performed using qPCR with primers specific for the genomic DNA sequence of GAPDH. The sequences of all primers used are listed in table 42.

As shown by the qPCR results shown in fig. 6A-10B, ring bodies produced in vitro are not only infectious, but they are also capable of infecting a variety of cell lines, including epithelial cells, lung tissue cells, liver cells, cancer cells, lymphocytes, lymphoblasts, T cells, B cells, and kidney cells. It was also observed that synthetic finger rings were able to infect HepG2 cells (liver cell line), increasing more than 100-fold relative to controls.

Example 8: identification and use of protein binding sequences

This example describes putative protein binding sites in the genome of a finger ring virus that can be used, for example, to amplify and package effectors in the finger ring as described herein. In some cases, the protein binding site may be capable of binding an external protein, such as a capsid protein.

Two conserved domains in the genome of the ring virus are putative origins of replication: the 5' UTR conserved domain (5CD) and the GC-rich domain (GCR) (de Villiers et al, Journal of Virology 2011; Okamoto et al, Virology 1999). In one example, to confirm whether these sequences act as DNA replication sites or encapsidation signals, deletions were made in each region in a plasmid comprising TTMV-LY 2. A539 cells were transfected with pTTMV-LY2 Δ 5CD or pTTMV-LY2 Δ GCR. The transfected cells were incubated for four days, and then virus was isolated from the supernatant and cell pellet. A549 cells were infected with the virus, and four days later, the virus was isolated from the supernatant and the infected cell pellet. qPCR was performed to quantify viral genomes from the samples. Disruption of the origin of replication prevents the viral replicase from amplifying viral DNA, resulting in a reduction in the viral genome isolated from the transfected cell pellet as compared to wild-type virus. A small amount of virus is still packaged and can be found in the transfected supernatant and infected cell pellet. In some embodiments, disruption of the packaging signal will prevent viral DNA from being encapsulated by capsid proteins. Thus, in the examples, amplification of the viral genome is still present in the transfected cells, but is not found in the supernatant or infected cell pellet.

In another example, to characterize additional replication or packaging signals in DNA, a series of deletions across the entire TTMV-LY2 genome was used. The deletion was 100bp stepwise over the length of the sequence. Plasmids containing the deletion of TTMV-LY2 were transfected into A549 and tested as described above. In some embodiments, the deletion that disrupts viral amplification or packaging will comprise a potential cis regulatory domain.

Replication and packaging signals can be incorporated into the DNA sequence encoding the effector (e.g., in the genetic elements of the finger loop) to induce amplification and encapsulation. This is done in the context of larger regions of the genome of the finger genome (i.e. insertion of effectors into specific sites of the genome, or replacement of viral ORFs with effectors, etc.), or by incorporation of minimal cis-signals into the effector DNA. In the case of ring bodies lacking trans-replicating or packaging factors (e.g., replicase and capsid proteins, etc.), the trans-factors are supplied by the helper genes. The helper gene expresses all proteins and RNA sufficient to induce amplification and packaging, but lacks its own packaging signal. Meaning that the loop DNA is co-transfected with the helper gene, resulting in amplification and packaging of the effector rather than the helper gene.

Example 9: finger ring virus genome

This example describes deletions in the genome of a ring virus.

172 nucleotides (nt) (nt 3436 to 3607) were deleted in the non-coding region (NCR) of TTV-tth8 downstream of the ORF but upstream of the GC-rich region. A56-nt random sequence TTTGTGACACAAGATGGCCGACTTCCTTCCTCTTTAGTCTTCCCCAAAGAAGACAA (SEQ ID NO:696)) was inserted in the deletion. pTTV-tth8(3436-3707::56nt), a DNA plasmid containing an altered TTV-tth8 was generated. Mu.g of double-stranded circular plasmid or double-stranded SmaI linearized DNA (resulting in TTV-tth8 genomic fragment separated from bacterial backbone elements) was transfected in duplicate into HEK293 or A549 cells of 60% confluence in 6cm disks using lipofectamine 2000. At 96 hours post-transfection, virus was isolated from the cell pellet and supernatant by freeze-thawing, alternating three times between liquid nitrogen and a 37 ℃ water bath. The virus from the supernatant was used to re-infect cells (HEK293 cells were infected with the virus isolated from HEK293 and a549 cells were infected with the virus isolated from a 549). Virus was isolated from the cell pellet and supernatant by freeze-thawing 72 hours after infection. qPCR was performed on all samples. As shown in Table 43 below, TTV-tth8 was observed in both the cell pellet and the supernatant of the infected cells, indicating that pTTV-tth8 (3436-. Thus, TTV-tth8 is able to tolerate the deletion of nt 3436 to 3707.

Table 43: infection with TTV-tth8(3436-3707::56nt) in HEK293 and A549 resulted in viral amplification. The average genome equivalents from the experiments were repeated compared to negative control cells without plasmid or virus addition.

The engineered version of TTMV-LY2 was assembled, with deletions of nucleotides 574 to 1371 and 1432 to 2210(1577bp deletion), and with the insertion of the 513bp NanoLuc (nLuc) reporter ORF at the C-terminus of ORF1 (after nt 2609 of wild-type TTMV-LY 2). A plasmid (pVL46-015B) comprising the DNA sequence used to engineer TTMV-LY2 was transfected into a549 cells, and the virus was then isolated and used to infect new a549 cells, as described in example 17. nLuc luminescence was detected in the cell pellet and supernatant of infected cells, indicating viral replication (fig. 11A-11B). This indicates that TTMV-LY2 can tolerate deletions in the ORF region of at least 1577 bp.

To further characterize the viral genome, a series of deletions were made in TTMV-LY2 DNA. TTMV-LY2 with nt 574-1371 and 1432-2210 deletions but without nLuc insertions was prepared and tested for viral replication as described previously. Further deletions were made for TTMV-LY 2. DELTA.574- > 1371,. DELTA.1432- > 2210. Nt 1372 + 1431 was deleted to generate TTMV-LY2 Δ 574 + 2210. Furthermore, the sequence of ORF3 downstream of ORF1 was deleted (. DELTA.2610-2809). Finally, to test for deletions in the non-coding region, a series of 100bp deletions were performed sequentially throughout the NCR. All deletion mutants were tested for viral replication as described previously. Deletions leading to successful virus production (indicating that the deleted region is not essential for viral replication) were combined to make TTMV-LY2 variants with more deleted nucleotides. To identify viral genomes that can be amplified with helper genes, each deletion mutant that disrupts viral replication is tested along with helper genes that carry trans-replication and packaging elements. The deletion saved by the trans-expression of the replicating element indicates a region of the viral genome that can be deleted without preventing virus formation when the helper genes are provided from a separate source.

Example 10: insertion of nucleotides of various lengths into the genome of the dactylovirus

This example describes the addition of DNA sequences of various lengths to the genome of a finger ring virus, which in some cases can be used to produce finger rings as described herein.

The DNA sequence was cloned into a plasmid containing TTV-tth8(GenBank accession No. AJ620231.1) and TTMV-LY2(GenBank accession No. JX 134045.1). Insertions were made in the non-coding region (NCR)3 'of the open reading frame and 5' of the GC-rich region: after nucleotide 3588 of TTV-tth8 or nucleotide 2843 of TTMV-LY 2.

Random DNA sequences of the following lengths were inserted into the NCRs of TTV-tth8 and TTMV-LY 2: 100 base pairs (bp), 200bp, 500bp, 1000bp and 2000 bp. These sequences were designed to match the relative GC content of each viral genome: about 50% GC was used for insertion of TTV-tth8, and about 38% GC was used for TTMV-LY 2. In addition, several transgenes were inserted into the NCR. These include mirnas driven by the U6 promoter (351bp) (e.g., FF4 miRNA) and EGFP driven by the constitutive hEF1a promoter (2509 bp).

TTV-tth8 and TTMV-LY2 variants, including DNA inserts of various sizes, were transfected into mammalian cell lines, including HEK293 and a549, as previously described. The virus is isolated from the supernatant or cell pellet. The isolated virus was used to infect other cells. Production of virus from infected cells was monitored by quantitative PCR. In some embodiments, successful production of the virus will indicate tolerance of the insertion.

Example 11: exemplary goods to be delivered

This example describes exemplary classes of nucleic acid and protein payloads that can be delivered with, for example, finger rings described herein, e.g., finger rings based on finger ring viruses.

One example of a payload is mRNA for protein expression. The coding sequence of interest is transcribed from a viral promoter native to the source virus (e.g., a finger ring virus), or from a promoter introduced with the payload as part of a transgene. Alternatively, the mRNA is encoded in the open reading frame of the viral mRNA, resulting in a fusion between the viral protein and the protein of interest. When desired, cleavage domains such as 2A peptides or protease target sites can be used to separate the protein of interest from the viral protein.

Non-coding rna (ncrna) is another example of a payload. These RNAs are typically transcribed using RNA polymerase III promoters (e.g., U6 or VA). Alternatively, RNA polymerase II (e.g., a natural viral promoter or a regulatable synthetic promoter) is used to transcribe the ncRNA. When expressed from the RNA polymerase II promoter, the ncRNA is encoded as part of an mRNA exon, intron, or additional RNA transcribed downstream of the poly a signal. ncRNA is typically encoded as part of a larger RNA molecule, or cleaved using ribozymes or endoribonucleases. Ncrnas that can be encoded as cargo in the genome of the ring include microrna (miRNA), small interfering RNA (sirna), short hairpin RNA (shrna), antisense RNA, miRNA sponge, long noncoding RNA (lncrna), and guide RNA (grna).

DNA can be used as a functional element without RNA transcription. For example, DNA can be used as a template for homologous recombination. In another example, protein-binding DNA sequences can be used to drive packaging of a protein of interest into a capsid (e.g., to the outside of the protein referring to the loop). For homologous recombination, regions homologous to human genomic DNA are encoded into the vector DNA to serve as homology arms. Recombination can be driven by a targeting endonuclease (e.g., Cas9 with a gRNA, or a zinc finger nuclease), which can be expressed from a vector or from a separate source. Inside the cell, the single-stranded DNA genome is converted to double-stranded DNA, which then serves as a template for homologous recombination at the site of the genomic DNA break. To recruit the protein of interest, a protein binding sequence can be encoded into the finger ring DNA. The DNA binding protein of interest or a protein of interest fused to a DNA binding protein (e.g., Gal4) is bound to the ring body DNA. When the ring DNA is encapsulated by capsid proteins, the DNA binding proteins are also encapsulated and can be delivered to the cell with the ring.

Example 12: exemplary payload integration sites

This example describes exemplary sites in the genome of TTV-tth8(GenBank accession No. AJ620231.1) and TTMV-LY2(GenBank accession No. JX134045) into which a nucleic acid payload can be inserted.

Several strategies can be employed to insert the Open Reading Frame (ORF) regions of TTV-tth8 (nucleotides 336 to 3015) and TTMV-LY2 (nucleotides 424 to 2812). In one example, to tag a viral protein or generate a fusion protein, a payload is inserted in-frame into a particular ORF of interest. Alternatively, some or all of the ORF region is deleted, which may or may not disrupt viral protein function. The payload is then inserted into the deletion region. In addition, the hypervariable domain (HVD) in ORF1 of TTV-tth8 (between nucleotides 716 and 2362) or TTMV-LY2 (between nucleotides 724 and 2273) can be used as an insertion site. In some cases, insertions or nucleotide substitutions in the HVD may be better tolerated and/or disrupt viral function to a lesser extent.

Alternatively, the payload is inserted into a region of the vector corresponding to the non-coding region (NCR) of TTV-tth8 or TTMV-LY 2. In particular, insertions were made in the 5' NCR upstream of the TATA box, the 5' untranslated region (UTR), the 3' NCR downstream of the polya signal and upstream of the GC-rich region. In addition, an insertion was made in the miRNA region (nucleotides 3429 to 3506) of TTV-tth 8. For the 5' NCR region, an insertion was made upstream of the TATA box (between nucleotides 1 and 82 in TTV-tth8, between nucleotides 1 and 236 in TTMV-LY 2). In some embodiments, the transgene is inserted in the opposite direction to reduce promoter interference. For the 5' UTR, insertions were made downstream of the transcription start site (nucleotide 111 in TTV-tth8, nucleotide 267 in TTMV-LY 2) and upstream of the start codon of ORF2 (nucleotide 336 in TTV-tth8, nucleotide 421 in TTMV-LY 2). Insertion of the 5'UTR will add or replace nucleotides in the 5' UTR. As described in example 10, a 3' NCR insertion was made upstream of the GC-rich region, specifically after nucleotide 3588 in TTV-tth8 or nucleotide 2843 in TTMV-LY 2. The miRNA of TTV-tth8 was replaced with an alternative natural or synthetic miRNA hairpin.

Example 13: ring viruses of defined classes and conserved regions thereof

There are three genera of dacycloviruses in humans: torque teno virus (ringlet virus, TTV), torque teno virus (ringlet midsize virus, TTMDV) and torque teno virus (ringlet minivirus, TTMV). The type a torque viruse includes at least five (e.g., seven) well-supported phylogenetically evolved branches (fig. 11C). It is contemplated that any of these finger ring viruses may be used as the source virus (e.g., source of viral DNA sequences) for generating the finger rings described herein.

Of these sequences, the highest conservation was found in the 5' UTR domain (about 75% conservation) and the GC-rich domain (greater than 100 base pairs, GC content greater than 70%, about 70% conservation). In addition, the hypervariable domains (HVDs) in the sequence have very low conservation (about 30% conservation). All finger-ring viruses also contain regions in which all three reading frames are open. In some cases, the 5' UTR or GC-rich region may serve as an origin of replication.

Also provided herein are exemplary sequences of representative viruses from the TTV clade and each of TTMDV and TTMV, annotated with conserved regions (see, e.g., tables a1-a12, B1-B5, C1-C5, or 1-18).

Example 14: replication-defective finger rings and helper viruses

For the replication and packaging of the ring body, some elements may be provided in trans. These include proteins or non-coding RNAs that direct or support DNA replication or packaging. In some cases, the trans element may be obtained from a source that replaces the ring, such as a helper virus, plasmid, or from the cell genome.

The other elements are typically provided in cis. These elements may be, for example, sequences or structures in the finger loop DNA that serve as origins of replication (e.g., allowing amplification of the finger loop DNA) or packaging signals (e.g., binding proteins to load the genome into the capsid). Typically, a replication-defective virus or finger ring would lose one or more of these elements, such that the DNA cannot be packaged into infectious virions or finger rings even if the other elements are provided in trans.

Replication-defective viruses may be used as helper viruses, e.g., to control replication of finger loops (e.g., replication-defective or packaging-defective finger loops) in the same cell. In some cases, the helper virus will lack cis-replicating or packaging elements, but will express trans-elements, such as proteins and non-coding RNAs. Typically, a therapeutic ring body will lack some or all of these trans elements and thus will not replicate alone, but will retain the cis element. When co-transfected/infected into a cell, the replication-defective helper virus will drive amplification and packaging of the finger loop body. Thus, the packaged particles collected will consist only of the therapeutic ring body and will not be infected with helper virus.

To develop a replication-defective finger ring, conserved elements in non-coding regions of the finger ring virus are removed. In particular, the deletion of the conserved 5' UTR domain and the GC-rich domain will be tested separately or together. Both elements are believed to be important for viral replication or packaging. In addition, a series of deletions will be made throughout the non-coding region to identify a previously unknown region of interest.

Successful deletion of the replicating member will result in reduced amplification of finger ring DNA within the cell, e.g., as measured by qPCR, but will support certain infectious finger ring production, e.g., as monitored by assays on infected cells, which can include any or all of qPCR, western blot, fluorescent or luminescent assays. Successful deletion of the packaging element did not disrupt the finger loop DNA amplification, so an increase in finger loop DNA was observed in transfected cells by qPCR. However, the genome of the ring was not encapsulated, and thus infectious ring production was not observed.

Example 15: method for manufacturing ring body with copying capability

This example describes a method for recovering and expanding the production of replication-competent finger rings. When they encode in their genome all the essential genetic elements and ORFs required for replication in a cell, the ring is replication competent. Since these finger rings are replication-free, they do not require complementary activities provided in trans. However, they may require helper activity, such as transcription enhancers (e.g., sodium butyrate) or viral transcription factors (e.g., adenovirus E1, E2, E4, VA; HSV Vp16 and immediate early proteins).

In this example, double stranded DNA encoding the full sequence of the synthetic finger loop in linear or circular form was introduced into 5E +05 adherent mammalian cells in T75 flasks by chemical transfection or into 5E +05 suspension cells by electroporation. After an optimal period of time (e.g., 3-7 days post transfection), cells and supernatant are collected by scraping the cells into supernatant medium. Mild detergents (such as bile salts) were added to a final concentration of 0.5% and incubated at 37 ℃ for 30 minutes. Calcium chloride and magnesium chloride were added to final concentrations of 0.5mM and 2.5mM, respectively. Endonucleases (e.g., DNAse I, Benzonase) are added and incubated at 25 deg.C-37 deg.C for 0.5-4 hours. The ring suspension was centrifuged at 1000x g for 10 min at 4 ℃. The clarified supernatant was transferred to a new tube and diluted 1:1 with cryoprotectant buffer (also called stabilization buffer) and stored at-80 ℃ as needed. This will produce a ring body of generation 0 (P0). In order to keep the concentration of detergent below the safety limit for culturing cells, this inoculum was diluted at least 100-fold or more in Serum Free Medium (SFM) according to ring titer.

A fresh mammalian cell monolayer in a T225 flask was covered with a minimum volume sufficient to cover the culture surface and incubated at 37 ℃ and 5% carbon dioxide with gentle shaking for 90 minutes. The mammalian cells used in this step may be of the same or different cell type as used for P0 recovery. After incubation, the inoculum was replaced with 40ml serum-free, animal-origin-free medium. Cells were incubated at 37 ℃ and 5% carbon dioxide for 3-7 days. 4ml of a 10 Xsolution of the same mild detergent used previously was added to bring the final detergent concentration to 0.5%, and the mixture was then incubated at 37 ℃ for 30 minutes with gentle stirring. Add endonuclease and incubate at 25 ℃ -37 ℃ for 0.5-4 hours. The medium was then collected and centrifuged at 1000x g for 10 minutes at 4 ℃. The clear supernatant was mixed with 40ml of stabilization buffer and stored at-80 ℃. This will produce a seed stock or first generation finger ring (P1).

Depending on the titer of the stock, it was diluted no less than 100-fold in SFM and added to cells grown in multi-layer flasks of the desired size. Multiplicity of infection (MOI) and incubation time have been optimized on a smaller scale to ensure maximum finger ring production. After harvesting, the finger ring bodies can be purified and concentrated as desired. A schematic diagram showing the workflow, such as described in this example, is provided in fig. 12.

Example 16: method for producing replication-defective finger ring body

This example describes a method for recovering and expanding the generation of replication-defective finger loops.

The ring may be made replication deficient by deletion of one or more ORFs involved in replication (e.g., ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF2/3 and/or ORF2 t/3). Replication-deficient refers to loops that can be grown in a complementary cell line. Such cell lines constitutively express components that promote growth of the finger ring but are lost or non-functional in the genome of the finger ring.

In one example, one or more sequences of any one or more ORFs involved in ring propagation are cloned into a lentiviral expression system suitable for generating a stable cell line encoding a selectable marker, and a lentiviral vector is generated as described herein. Mammalian cell lines capable of supporting the propagation of finger ring bodies are infected with the lentiviral vector and subjected to selection pressure of a selection marker (e.g., puromycin or any other antibiotic) to select a population of cells that have stably integrated the cloned ORF. Once this cell line has been characterized and demonstrated to complement defects in engineered finger loops, supporting the growth and propagation of such finger loops, it can be expanded and stored in a low temperature storage. During expansion and maintenance of these cells, selection antibiotics are added to the medium to maintain the selection pressure. Once the finger ring is introduced into these cells, the selection antibiotic can be inhibited.

Once the cell line is established, growth and production of replication-defective finger loops is performed, for example, as described in example 15.

Example 17: production of finger ring bodies using suspension cells

This example describes the production of finger rings in suspension cells.

In this example, a549 or 293T producer cell line suitable for growth under suspension conditions was grown in animal component-free and antibiotic-free suspension medium (Thermo Fisher Scientific) in a WAVE bioreactor bag at 37 degrees and 5% carbon dioxide. Using lipofectamine 2000 (Thermo Fisher Scientific) under the current good manufacturing practice (cGMP), will be 1 × 10⁶Viable cells/mL of these cells inoculated are transfected with a plasmid containing the finger loop sequence and any complementary plasmid suitable or desirable for packaging of the finger loop (e.g., in the case of a replication-deficient finger loop, e.g., as described in example 16). In some cases, the complementing plasmid may encode a viral protein that has been deleted from the finger ring genome (e.g., a finger ring genome based on, for example, a viral genome such as described herein, e.g., a finger ring viral genome) but is used or desired for replication and packaging of the finger ring. Transfected cells in WAVE bioreactor The bags were grown and the supernatants were harvested at the following time points: 48 hours, 72 hours and 96 hours post transfection. The supernatant was separated from the cell pellet of each sample using centrifugation. The packaged finger ring particles were then purified from the harvested supernatant and lysed cell pellet using ion exchange chromatography.

Genomic equivalents in a purified preparation of finger loops can be determined, for example, by using a small aliquot of the purified preparation to harvest the finger loop genome using a viral genome extraction kit (Qiagen), followed by qPCR using primers and probes that target the finger loop DNA sequence, such as described in example 18.

The infectivity of the finger ring in the purified preparation can be quantified by serial dilution of the purified preparation to infect new a549 cells. These cells were harvested 72 hours after transfection and then qPCR assays were performed on genomic DNA using primers and probes specific for the finger ring DNA sequence.

Example 18: quantification of finger ring genome equivalents by qPCR

This example illustrates the development of a hydrolysis probe-based quantitative PCR assay to quantify finger rings. Primer and probe sets were designed based on the genomic sequences of the selected TTV (accession number AJ620231.1) and TTMV (accession number JX134045.1) using Geneious software and end-user optimization was performed. The primer sequences are shown in table 44 below.

Table 44: sequences of forward and reverse primers and hydrolysis probes were used to quantify TTMV and TTV genome equivalents by quantitative PCR.

TTMV		SEQ ID NO:
			Forward primer	5'-GAAGCCCACCAAAAGCAATT-3'	697
Reverse primer	5'-AGTTCCCGTGTCTATAGTCGA-3’	698
			Probe needle	5'-ACTTCGTTACAGAGTCCAGGGG-3'	699

TTV
			Forward primer	5'-AGCAACAGGTAATGGAGGAC-3’	700
Reverse primer	5'-TGGAAGCTGGGGTCTTTAAC-3’	701
			Probe needle	5'-TCTACCTTAGGTGCAAAGGGCC-3’	702

As a first step in the development process, qPCR was performed using TTV and TTMV primers and SYBR green chemistry to check primer specificity. FIG. 13 shows a different amplification peak for each primer pair.

The hydrolysis probe ordered is labeled at the 5 'end with the fluorophore 6FAM and at the 3' end with a minor groove binding non-fluorescent quencher (MGBNFQ). The PCR efficiency of the new primers and probes was then evaluated using two different commercial premixes, using purified plasmid DNA as the composition of the standard curve and increasing the concentration of primers. A standard curve was established by using purified plasmids containing the target sequences for the different primer-probe sets. Seventy-fold serial dilutions were performed to achieve a linear range within 7 logs and a quantitative lower limit of 15 copies per 20ul reaction. Premix No. 2 was able to produce a PCR efficiency of 90% -110%, which is an acceptable value for quantitative PCR (fig. 14). All primers for qPCR were purchased from IDT. Hydrolysis probes conjugated to fluorophore 6FAM and minor-groove binding non-fluorescent quencher (MGBNFQ) as well as all qPCR premixes were purchased from seimer feishel (Thermo Fisher). An exemplary amplification plot is shown in FIG. 15.

Using these primer-probe sets and reagents, genomic equivalents (GEq)/ml were quantified for the ring stock. The linear range was 1.5E +07-15GEq per 20ul reaction, which was then used to calculate GEq/ml, as shown in FIGS. 16A-16B. Samples at concentrations above the linear range can be diluted as required.

Example 19: expression of foreign proteins in mice using finger ring bodies

This example describes the use of finger rings in which the minicircovirus (TTMV) genome is engineered to express firefly luciferase protein in mice.

Plasmids encoding DNA sequences of engineered TTMV encoding the firefly luciferase gene were introduced into a549 cells (human lung cancer cell lines) by chemical transfection. 18ug of plasmid DNA was used to transfect 70% confluent cells in 10cm tissue culture plates. An empty vector backbone lacking TTMV sequence was used as a negative control. 5 hours after transfection, cells were washed twice with PBS and grown in fresh growth medium at 37 ℃ and 5% carbon dioxide.

Transfected a549 cells and their supernatants were harvested 96 hours after transfection. The harvested material was treated with 0.5% deoxycholate (vol. wt) at 37 ℃ for 1 hour, followed by endonuclease treatment. The ring particles were purified from the lysate using ion exchange chromatography. To determine the ring body concentration, samples of the ring body stock were run through a viral DNA purification kit and genome equivalents per ml were determined by qPCR using primers and probes targeting the ring body DNA sequence.

The dosage range of genome equivalents referring to the ring body in 1x phosphate buffer was performed in 8-10 weeks old mice by various injection routes (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular). Ventral and dorsal bioluminescence imaging was performed on each animal 3, 7, 10 and 15 days post-injection. Imaging was performed by intraperitoneal addition of luciferase substrate (Perkin-Elmer) to each animal, followed by in vivo imaging, at the indicated time points according to the manufacturer's protocol.

Example 20: genome alignment to determine if the genomic DNA is integrated into the host genome

This example describes a computational analysis to determine whether finger ring DNA can integrate into the host genome by examining whether a Torque Teno Virus (TTV) has integrated into the human genome.

The entire genome of representative TTV sequences from each of the five exemplary torque teno virus clades was aligned to human genomic sequences using the Basic Local Alignment Search Tool (BLAST) which found regions of local similarity between sequences. Representative TTV sequences shown in table 45 were analyzed:

table 45: representative TTV sequences

Branch of TTV evolution	NCBI accession number
		Evolution of branch A	AB064597.1
Evolution of branch B	AB028669.1
		Evolutionary branch C	AJ20231.1
Evolution of branch D	AF122914.3
		Evolution of branch E	AF298585.1

No significant similarity of the sequences from the aligned TTV to the human genome was found, indicating that the TTV has not integrated into the human genome.

Example 21: evaluation of integration of finger Ring into host genome

In this example, a549 cells (human lung cancer cell line) and HEK293T cells (human embryonic kidney cell line) were infected with the ring particles or AAV particles at an MOI of 5, 10, 30 or 50. 5 hours after infection, cells were washed with PBS and replaced with fresh growth medium. Cells were then grown at 37 degrees and 5% carbon dioxide. Five days after infection, cells were harvested and processed using a genomic DNA extraction kit (Qiagen) to harvest genomic DNA. Genomic DNA was also harvested from uninfected cells (negative control). A whole genome sequencing library was prepared for these harvested DNAs using Nextera DNA library preparation kit (Illumina) according to the manufacturer's protocol. The DNA library was sequenced using NextSeq 550 system (Illumina) according to the manufacturer's protocol. The sequencing data was assembled into reference genomes and analyzed for ligation between finger rings or AAV genomes and host genomes. In the case where ligation is detected, it will be verified in the original genomic DNA sample before sequencing library preparation by PCR. Primers are designed to amplify the region encompassing and surrounding the ligation. The frequency of ring integration into the host genome was determined by qPCR quantifying the number of ligations in the sample (representing the integration event) and the total number of ring copies. This ratio can be compared to the ratio of AAV.

Example 22: functional role of Ring expressing exogenous microRNA sequences

This example demonstrates the successful expression of exogenous mirnas from the genome of the finger ring using a native promoter (miR-625).

500ng of the following plasmid DNA was transfected into 60% confluent wells of HEK293T cells in 24-well plates:

i) empty plasmid skeleton

ii) a plasmid containing the TTV-tth8 genome in which the endogenous miRNA is Knocked Out (KO)

iii) TTV-tth8, wherein endogenous miRNAs are replaced by non-targeting, scrambled miRNAs

iv) TTV-tth8 in which the endogenous miRNA sequence is replaced by a miRNA encoding miR-625

72 hours after transfection, total miRNA were collected from transfected cells using Qiagen miRNeasy kit, followed by reverse transcription using miRNA Script RT II kit. Quantitative PCR was performed on the reverse transcribed DNA using primers that should specifically detect miRNA-625 or RNU6 small RNAs. RNU6 small RNA was used as a housekeeping gene and the data are plotted in fig. 17 as fold change relative to empty vector. As shown in FIG. 17, miR-625 refers to the loop causing an approximately 100-fold increase in miR-625 expression, whereas no signal is detected for empty vectors, miR-Knockouts (KO), and scrambled miRs.

Example 23: preparation and Generation of Ring expressing exogenous non-coding RNA

This example describes the synthesis and production of finger loops expressing exogenous small non-coding RNAs.

The DNA sequence from TTh8 strain from TTV was synthesized (Jelcic et al, Journal of Virology, 2004) and cloned into a vector containing a bacterial origin of replication and a bacterial antibiotic resistance gene. In this vector, the DNA sequence encoding the TTV miRNA hairpin is replaced with a DNA sequence encoding an exogenous small non-coding RNA (e.g., miRNA or shRNA). The engineered construct was then transformed into electrocompetent bacteria, followed by plasmid isolation using a plasmid purification kit according to the manufacturer's protocol.

Finger ring DNA encoding an exogenous small non-coding RNA was transfected into a eukaryotic production cell line to produce finger ring particles. Supernatants of transfected cells containing finger ring particles were harvested at various time points post transfection. The supernatant from filtration or purified finger ring particles are used for downstream applications, e.g., as described herein.

Example 24: conservation in the evolutionary branches of the Ring Virus

This example describes the identification of seven evolutionary branches in the genus cyclopavirus a. Representative sequences between these clades showed 54.7% pairwise identity throughout the sequence (figure 18). In the open reading frame, pairwise identity was lowest (about 48.8%), higher in the non-coding region (69.1% in 5'NCR and 74.6% in 3' NCR) (fig. 18). This suggests that the DNA sequence or structure of the non-coding region plays an important role in viral replication.

The amino acid sequences of the putative proteins in the type A torque teno virus were also compared. DNA sequences showed about 47% to 50% pairwise identity, while amino acid sequences showed about 32% to 38% pairwise identity (fig. 19). Interestingly, representative sequences from the clade of the type a torque teno virus were able to replicate successfully in vivo and were observed in the human population. This suggests that the amino acid sequence of the ring virus protein can vary widely while retaining functions such as replication and packaging.

The finger ring virus was found to have a locally highly conserved region in the non-coding region. The region downstream of the promoter is the conserved domain of the 71-bp 5' UTR, which has 95.2% pairwise identity in seven evolved branches of the A-type torque teno virus (FIG. 20). Downstream of the open reading frame of the 3' non-coding region of the type a torque teno virus, there are regions where there is substantial pairwise identity between representative sequences. Near the 3 'end of this 3' conserved non-coding region is a highly conserved sequence. The ring virus also included a GC-rich region with GC content over 70%, which showed 75.4% pairwise identity in their aligned region (fig. 21).

Example 25: expression of endogenous miRNAs from the surface of the finger ring and deletion of endogenous miRNAs

In one example, Raji B cells in culture were infected with finger rings containing a modified TTV-tth8 genome, wherein the TTV-tth8 genome was modified to delete the GC-rich region, as described in example 27. These finger loops contain sequences encoding the TTV-tth8 endogenous payload of the finger loop virus, a miRNA that targets the mRNA encoding the n-myc interacting protein (NMI), and enter the host cell by introduction of a plasmid containing the genome of the finger loop virus. NMI operates downstream of the JAK/STAT pathway to regulate transcription of various intracellular signals, including interferon-stimulated genes, proliferation and growth genes, and mediators of inflammatory responses. As shown in fig. 22, viral genomes were detected in target Raji B cells. Successful knock-down of NMI was also observed in target Raji B cells compared to control cells (fig. 23). The ring containing miRNA for NMI induced a more than 75% reduction in NMI protein levels compared to control cells. This example illustrates that finger ring with native finger ring virus mirnas can knock down target molecules in host cells.

In another example, an endogenous miRNA based on the ring of the ring virus is deleted. The resulting finger ring body (Δ miR) was then incubated with the host cells. The genomic equivalents of Δ miR ring genetic elements were then compared to the genomic equivalents of the corresponding ring that retained endogenous mirnas. As shown in figure 24, the ring genome deleted for the endogenous miRNA was detected in the cell at a level comparable to that observed for the ring genome in which the endogenous miRNA was still present. This example demonstrates that endogenous mirnas based on the dactylosome of the dactylovirus can be mutated or completely deleted and that the dactylosome genome can still be detected in the target cell.

Example 26: location of the ORF of the Ring Virus

This example describes the novel function of various putative ORFs of the dactylovirus. In this example, putative Open Reading Frame (ORF) sequences were designed downstream of the marker protein (i.e., the nanoluciferase) at the N-terminus of each ORF. Each ORF-nLuc plasmid was introduced into 5E +05 adherent cells (Vero or HEK293T) in 12-well plates by chemical transfection or into suspended 5E +05 cells by electroporation. After an optimal period of time (e.g., 3-7 days post-transfection), cells were fixed with 4% paraformaldehyde in PBS (cat #28908, zemer), permeabilized with 0.5% Triton X-100, and stained for nLuc with a rabbit polyclonal anti-nLuc antibody (gift from Promega), followed by goat anti-rabbit Alexa488 (cat # a-11008, zemer) conjugate secondary antibody. Nuclei were stained with DAPI (Cat # D3571, seimer feishell). Stained cells were visualized on a Zeiss axio vert a1 with a 20X objective and a monochromatic Axiocam 506 camera for cellular localization of the labeled proteins.

As shown in FIGS. 25A-25B, ORF2 was observed to localize in the cytoplasm and ORF1/1 was observed to localize in the nucleus in Vero and HEK293 cells. FIG. 25C shows the location of ORF1/2 and ORF 2/2.

Example 27: characterization of the area required for finger ring development

This example describes deletions in the genome of an dactylovirus to help characterize the portion of the genome sufficient for replication of the virus and production of the dactylovirus. A series of deletions were made in the non-coding region (NCR) of TTV-tth8 downstream of the ORF (nt 3016 to 3753). A36 nucleotide (nt) sequence (CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (SEQ ID NO:160)) was deleted from the GC region (labeled Δ 36nt (GC)). Furthermore, the 78-nt pre-microRNA sequence (CCGCCATCTTAAGTAGTTGAGGCGGACGGTGGCGTGAGTTCAAAGGTCACCATCAGCCACACCTACTCAAAATGGTGG (SEQ ID NO:161)) (labeled Δ 36nt (GC) Δ miR) was deleted from the 3' NCR. Finally, the 3'NCR at Δ 36nt (GC) was deleted for an additional 171 nt (CTTAAGTAGTTGAGGCGGACGGTGGCGTGAGTTCAAAGGTCACCATCAGCCACACCTACTCAAAATGGTGGACAATTTCTTCCGGGTCAAAGGTTACAGCCGCCATGTTAAAACACGTGACGTATGACGTCACGGCCGCCATTTTGTGACACAAGATGGCCGACTTCCTTCC (SEQ ID NO:162)) and labeled Δ 3' NCR (FIG. 26). Mu.g of the circular pTTV-tth8(WT), pTTV-tth8(Δ 36nt (GC)), pTTV-tth8(Δ 36nt (GC) Δ miR), pTTV-tth8(Δ 3'NCR) DNA plasmid containing the altered 3' NCR TTV-tth8, described above, respectively, were transfected into 60% confluent HEK293 in 12-well plates in triplicate using lipofectamine 2000. 48 hours after transfection, the cells were pelleted and lysed to isolate mRNA transcripts (RNeasy, Qiagen cat #74104) and converted to cDNA (high capacity cDNA reverse transcription kit, Saimer Feishel, cat # 4368814). qPCR was performed on all samples, and viral transcript expression was measured in each deletion case and normalized to internal control mRNA for GAPDH.

As shown in fig. 27A-27D, all three deletion mutants significantly inhibited viral transcript expression in vitro. Thus, the 3' NCR of TTV-tth8 is required for the production of finger rings for expression of transgenes.

TTV strain tth8, GeneBank accession No. AJ620231.1, deposited as a whole genome sequence. However, in the GC-rich region, a 36 nucleotide stretch was annotated as universal N. This region is highly conserved among TTV strains and may therefore be important for the biology of these viruses. The DNA sequences of hundreds of TTV strains were computationally aligned and used to generate a strong consensus sequence of these 36 nucleotides (CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (SEQ ID NO: 160)). The TTV-tth8 genomic sequence is referred to herein as the "wild-type" sequence, and thus the consensus sequence is inserted in place of the 36N segment listed in the publicly available TTV-tth8 sequence.

Example 28: ring delivery of exogenous proteins in vivo

This example demonstrates the effector function (e.g., expression of a protein) of the finger ring in vivo after administration.

Finger rings were prepared that included a transgene encoding a nanoluciferase (nLuc) (fig. 28A-28B). Briefly, a double stranded DNA plasmid containing the non-coding region of TTMV-LY2 and the nLuc expression cassette was transfected into HEK293T cells along with a double stranded DNA plasmid encoding the entire TTMV-LY2 genome as a trans-replication and packaging factor. After transfection, cells were cultured to allow production of finger rings and finger ring material was harvested and enriched by nuclease treatment, ultrafiltration/diafiltration and sterile filtration. Additional HEK293T cells were transfected with: a non-replicating DNA plasmid comprising an nLuc expression cassette and a TTMV-LY2 ORF transfection cassette, but lacking the non-coding domains necessary for replication and packaging to serve as a "non-viral" negative control. Non-viral samples were prepared according to the same protocol as the ring material.

Three healthy mice were injected intramuscularly and monitored by IVIS luminea imaging (Bruker) over nine days (fig. 29A). As a non-viral control, a non-replicating formulation was administered to three additional mice (fig. 29B). On day 0, 25 μ L of finger ring or non-viral formulation was injected into the left hind leg and the right hind leg was re-injected on day 4 (see arrows in fig. 29A and B). After 9 days of IVIS imaging, more nLuc luminescent signal was observed in mice injected with the ring formulation (fig. 29A) compared to the non-viral formulation (fig. 29B), consistent with in vivo trans gene expression following ring transduction.

Example 29: identification of precursor miRNA (Pre-mIR) in finger ring virus

This example describes various computational and experimental methods to identify various novel precursor mirnas encoded by dactyloviruses.

Calculation method

Finger ring virus strains are very different from each other at the nucleotide sequence level. However, finger-ring virus strains, especially those within the same evolutionary branch, may exhibit significant similarities in genomic organization of various components, such as promoters, GC-rich regions, non-coding and coding regions (see, e.g., fig. 29D). Described herein is a method in which pre-miR sequences of various finger-ring virus strains (for which the pre-miR sequences are unknown) are predicted by alignment with finger-ring virus strains that have been experimentally validated for pre-miR sequences.

Briefly, various publicly available small RNA sequencing datasets of small RNAs from cell lines and various human samples were mined to discover novel pre-miR sequences encoded by various finger-ring virus strains. Publicly available computational tools and algorithms based on structural prediction or machine learning classification, such as mFold programs, miRANDA algorithm, miRScan, miRanalyzer, miRDeep (https:// www.ncbi.nlm.nih.gov/PMC/articles/PMC1559940/, https:// www.frontiersin.org/articles/10.3389/fbioe.2015.00007/full) are used to predict novel mirnas encoded by various ring viruses. The expression of the novel mirnas was then confirmed, validated and quantified using northern blotting with probes designed for specific miRNA sequences and/or RT-qPCR with specific primers for mirnas.

Experimental methods

In one example, high throughput small RNA sequencing is performed on human tissue or blood samples infected with dactylovirus to discover novel pre-mirnas encoded by dactylovirus. For this purpose, RNA is collected from homogenized human tissue samples or human blood samples. Small RNA libraries were prepared and sequenced using Illumina kit and sequencing platform. Sequencing reads were stored, aligned and analyzed on a BaseSpace Sequence Hub (Illumina).

In a second example, high throughput small RNA sequencing was performed on various cell lines treated with the following conditions to find novel pre-mirnas encoded by dactylviruses: (a) a cell line infected with a naturally occurring dactylovirus, a cell line transfected with an in vitro synthesized genome of the dactylovirus, and (c) a cell line infected with a dactylovirus packaged in vitro using the synthesized genome. The expression of novel mirnas was confirmed, validated and quantified using northern blotting with probes designed for specific miRNA sequences and/or RT-qPCR with specific primers for mirnas.

Example 30: determining endogenous targets for pre-miR of dactylovirus

This example describes an assay to determine endogenous targets and potential treatment-related target pathways for pre-mirs encoded by various finger-ring virus strains.

Computationally predicted and/or experimentally validated single pre-miRNA sequences encoded by various dactyloviruses were cloned into lentiviral vectors, driven by the U6 promoter. Non-targeted scrambled miRNA sequences driven by the U6 promoter were also cloned in a similar manner and used as controls. The lentiviral plasmid was cloned such that when packaged, its genome would comprise (i) the pre-miRNA sequence driven by the U6 promoter, (ii) the puromycin resistance gene driven by the SV40 promoter, and (iii) the Green Fluorescent Protein (GFP) gene driven by the CMV promoter. Each of these lentiviral plasmids was separately co-transfected into HEK-293T cells along with lentiviral helper plasmids to package the virus. Six hours after transfection, the medium of the transfected cells was aspirated, washed once with PBS and replaced with fresh medium. Lentiviral-containing medium was collected 72 hours after transfection. The medium is filtered through a 0.4um filter to remove any cells and then used to infect the cell type of interest, such as HeLa, Raji and THP1, in triplicate. Cells containing the integrated lentiviral genome were selected by starting treatment with puromycin 3 days after infection. RNA was harvested from stably selected cell lines using an RNA extraction kit (qiagen) and then reverse transcribed to cDNA using a reverse transcriptase kit (seimer feishell technologies). The cDNA samples were processed to generate an indexed short read library. Using the Illumina sequencing platform, the uniquely indexed short read library was multiplexed sequenced, yielding approximately 2000 million reads per sample. Sequencing reads were stored, aligned and analyzed using BaseSpace Sequence Hub (Illumina). The target of each individual candidate pre-miR is determined by comparing gene expression in cell lines expressing the candidate pre-miR to gene expression in cell lines expressing a scrambled pre-miR. The Ingenuity Pathway analysis was performed to test whether pre-mirnas target specific pathways, in particular therapy-related pathways. The workflow diagram described in this example is shown in fig. 30.

Example 31: manufacture of Ring encoding Pre-miR of Natural Ring Virus

This example describes the process of packaging a replicative or non-replicative form of a finger ring body expressing a native pre-miR of a dactylovirus.

The genome of the synthetic non-replicating form of the ring body comprises the following components: (i) an origin of replication, (ii) a sequence encoding a pre-miRNA for an finger virus, (III) an RNA polymerase III, e.g. U6 or H1, driving expression of the pre-miRNA, and (iv) a packaging signal. The genome is packaged by transfection into a helper cell line that stably expresses all the proteins required for viral packaging. Transfected cells were harvested 7 days post-transfection and processed to make finger ring preparations as described herein. Genomic equivalent titers of the finger ring preparations were determined by performing qPCR as described herein. The appropriate dose of the ring preparation was then used for downstream application.

The genome may be synthesized as a replicative form of the finger loop, for example, by generating a natural ring virus, except that expression of the pre-miRNA sequence is manipulated using an exogenous promoter such as U6 or a tissue-specific promoter. The genome was packaged by transfection into HEK-293T cells. Transfected cells were harvested 7 days post-transfection and processed to make finger ring preparations as described herein. Genomic equivalent titers of the finger ring preparations were determined by performing qPCR as described herein. The appropriate dose of the ring preparation was used for downstream application.

Example 32: use of pre-miR of dactylovirus as tumor suppressor in vitro cell culture model

This example describes a study to confirm the phenotypic effect of candidate pre-mirs identified as tumor suppressive from an assay, e.g., as described in example 29.

Candidate pre-mirnas with tumor-inhibiting effect were identified based on the analysis as described in example 29. A replicate form of the finger ring that encodes these candidate pre-mirnas as well as the scrambled pre-mirnas was prepared as described in example 31. Cancer cell lines from the NCI-60 cancer cell line panel were seeded in 96-well plates. When 30% confluent, these cell lines were treated with finger rings containing candidate pre-mirs or scrambled pre-mirs at a dose of five genome equivalents per cell. Medium containing the finger ring bodies was aspirated five hours after infection, then washed twice with PBS and replaced with fresh medium. Three days after treatment, the treated cells were subjected to an Alamar blue assay to determine which pre-mirs inhibited proliferation of cancer cell lines.

Example 33: use of pre-miR of dactylovirus as in vivo tumor suppressor

This example describes an in vivo experiment to confirm tumor inhibition of candidate tumor-inhibiting ring virus pre-mirs and cancer cell lines from screening in vitro assays as described in example 32.

Xenografts were prepared by subcutaneous injection of screened cancer cell lines from the analysis described in example 32 together with matrigelInto the flank of athymic mice. Once the xenograft tumor became accessible, 3x10 was performed⁶Local tumor injection of genome-equivalent finger rings encoding tumor-inhibiting pre-mirnas or scrambled pre-mirnas. The effect of loop injection on tumor growth was determined by routine tumor growth measurements over three weeks, tumor weight measurements of xenograft tumors at the end of the experiment, and by BrdU incorporation assay.

Example 34: tandem copies of the viral genome of the Ring

This example describes a plasmid-based expression vector that contains two copies of a single circovirus genome, arranged in tandem such that the GC-rich region of the upstream genome is close to the 5' region of the downstream genome (fig. 31A).

Ring viruses replicate by rolling circle, in which replicase (Rep) proteins bind to the genome at the origin of replication and initiate DNA synthesis around the circle. For the finger ring viral genome to be contained in the plasmid backbone, this requires either replication of the entire plasmid length, which is longer than the native viral genome, or recombination of the plasmid results in a smaller loop containing the genome with the least backbone. Therefore, replication of the virus from the plasmid may be inefficient. To increase the efficiency of viral genome replication, the plasmid was engineered to have tandem copies of TTV-tth8 and TTMV-LY 2. These plasmids present every possible circular arrangement of the genome of the ring virus: wherever the Rep protein binds, it drives replication of the viral genome from upstream to downstream of the origin of replication. A similar strategy has been used for the production of porcine finger ring viruses (Huang et al, 2012, Journal of Virology 86(11) 6042-.

The tandem TTV-tth8 was assembled by cloning copies of the genome sequentially into a plasmid backbone, leaving 12bp of non-viral DNA between the two sequences. Several TTV-tth8 variants were assembled into a tandem plasmid, including the wild-type and TTV-tth8(Δ 36GC) (i.e., the TTV-tth8 genome was engineered to include a 36 nucleotide GC-rich sequence as described herein), which deleted 36 base pairs from the GC-rich region. Tandem TTMV-LY2 was assembled by gold gate (Golden-gate) assembly, while incorporating two copies of the genome into the backbone, and leaving no additional nucleotides between the genomes.

A plasmid carrying tandem copies of TTV-tth8 (. DELTA.36 GC) was transfected into HEK239T cells. Cells were cultured for five days, then lysed using 0.1% Triton X-100 and nuclease treated to digest viral capsid unprotected DNA. qPCR was then performed on TTV-tth8 genomic sequence and plasmid backbone using Taqman probe. The TTV-tth8 genomic copy was normalized to the backbone copy. As shown in fig. 31B, the tandem TTV-tth8 produced more than four times the number of viral genomes than plasmids carrying a single copy. When the number of TTV-tth8 genomic sequences is considered to double, each transfected copy of the tandem plasmid produces more than twice as many copies of the new synthetic genome. These data indicate that engineering tandem dactylovirus genomes can increase viral genome replication and can be used as a strategy to increase dactylovirus yield.

Example 35: in vitro circularized viral genomes

This example describes a construct comprising circular double stranded circular genomic DNA and minimal non-viral DNA. These circovirus genomes are more closely matched to double-stranded DNA intermediates found during replication of wild-type finger-ring viruses. When introduced into a cell, such circular double-stranded with minimal non-viral DNA refers to the fact that the genomic DNA of a ring virus can undergo rolling circle replication to produce, for example, the genetic elements described herein.

In one example, the plasmid carrying the TTV-tth8 variant and TTMV-LY2 was digested with a restriction endonuclease that recognizes sites flanking the genomic DNA. The resulting linearized genomes are then ligated to form circular DNA. These ligation reactions were performed with different DNA concentrations to optimize intramolecular ligation. The ligated loops are either transfected directly into mammalian cells or further processed to remove non-circular genomic DNA by digestion with restriction endonucleases to cut the plasmid backbone and exonucleases to degrade linear DNA. For TTV-tth8, XmaI endonuclease was used to linearize the DNA; the ligated loop contained 53bp of non-viral DNA between the GC-rich region and the 5' non-coding region. For TTMV-LY2, a viral genomic DNA circle free of non-viral DNA was generated using type IIS restriction enzyme Esp 3I. This protocol was adapted from the previously published TTV-tth8 cyclization (Kincaid et al, 2013, PLoS Pathologens [ public science library-Pathogens ]9(12): e 1003818). To demonstrate the improvement in finger ring virus yield, circularised TTV-tth8 and TTMV-LY2 were transfected into HEK293T cells. After 7 days of incubation, cells were lysed and qPCR was performed to compare the level of the dactylovirus genome between circularized and plasmid-based dactylovirus genomes. The increase in the level of the genome of the dactylovirus indicates that circularization of the viral DNA is a useful strategy for increasing the yield of the dactylovirus.

In another example, the plasmid TTMV-LY2 (pVL46-240) and TTMV-LY2-nLuc were linearized with Esp3I or EcoRV-HF, respectively. The digested plasmid was purified on a 1% agarose gel, then electroeluted or Qiagen column purified and ligated with T4 DNA ligase. The circularised DNA was concentrated on a 100kDa UF/DF membrane prior to transfection. Cyclization was confirmed by gel electrophoresis as shown in fig. 31C. One day prior to lipofection with Lipofectamine 2000, HEK293T was applied at 3x10⁴Individual cell/cm²Inoculated into a T-225 flask. Nine micrograms of circularized TTMV-LY2 DNA and 50. mu.g of circularized TTMV-LY2-nLuc were co-transfected one day after flask inoculation. For comparison, additional T-225 flasks were co-transfected with 50. mu.g of linearized TTMV-LY2 and 50. mu.g of linearized TTMV-LY 2-nLuc.

Ring production was performed for eight days prior to harvesting cells in Triton X-100 harvest buffer. Generally, the finger ring bodies can be enriched by, for example, lysis of host cells, clarification of lysates, filtration, and chromatography. In this example, harvested cells were nuclease treated prior to sodium chloride conditioning and 1.2 μm/0.45 μm normal flow filtration. The clarified harvest was concentrated on a 750kDa MWCO mPES hollow fiber membrane and the buffer was exchanged into PBS. The TFF retentate was filtered through a 0.45 μm filter and loaded onto a Sephacryl S-500HR SEC column pre-equilibrated in PBS. The finger ring body was treated through a SEC column at 30 cm/hr. As shown in fig. 31D, each fraction was collected by qPCR and analyzed for viral genome copy number and transgene copy number. From the void volume of the SEC chromatogram, fraction 7, viral genome and transgene copies were observed starting. A residual plasmid peak was observed at fraction 15. The copy numbers of the TTMV-LY2 genome and the TTMV-LY2-nLuc transgene were very consistent with the finger ring generated at fractions 7-10 using circularized input DNA, indicating that the packaged finger ring contains the nLuc transgene. The SEC fractions were combined and concentrated using a 100kDa MWCO PVDF membrane, then filtered 0.2 μm prior to in vivo administration.

Circularization of input finger loop DNA resulted in a three-fold increase in the percent recovery of nuclease protected genomes throughout purification compared to linearized finger loop DNA, indicating that use of circularized input finger loop DNA improves manufacturing efficiency, as shown in table 46.

TABLE 46 purification Process yields

Example 36: ORF1 modeling and identification of conserved residues and domains

This example describes in silico modeling of ORF1 protein of torque virus b and defines putative domains based on structural motifs and amino acid conservation/similarity.

The ORF1 protein was predicted to be the major capsid protein of finger ring viruses based on the presence of an arginine-rich region and the high presence of beta-sheet in secondary structure prediction using PSIpred (II) ((II))http://bioinf.cs.ucl.ac.uk/ psipred/)。RaptorX(http://raptorx.uchicago.edu/) Is used for carrying out structure prediction and contact prediction on the sequences of eight kinds of B-type torque teno viruses. The torque b virus ORF1 sequences were used because they were shorter (about 650 amino acids) than the torque a virus (about 750 amino acids), which was predicted to have fewer unstructured regions. Five of the predicted structures contained elements with similarities for the identification of the putative domain of ORF1 (fig. 33). ORF1 is divided into five regions-an arginine-rich region, a putative core (jelly roll domain), a hypervariable region, an N22 region, and a C-terminal domain.

Structural models of the leptovirus type b strain CBS203 are used to show residues/structural regions with some conservation in the leptovirus type b family. To analyze conserved residues, 110 torque B virus ORF1 sequences were aligned in Geneius using the ClustalW alignment algorithm. Residues were then assessed for conservation by percent identity and similarity using the BLOSUM62 matrix with a threshold of 1. Residues with greater than 60% similarity for all strains in the alignment are highlighted on the structural model (figure 34). A total of 26 residues (about 4%) share amino acid similarity with 100% of the aligned sequences. The 80% and 60% cutoffs contained 23.7% and 36.7% of the total residues, respectively.

A similar alignment algorithm and similarity determination was performed on 258 strains of the type A torque viruse. Similarity and identity are shown in the consensus sequences from the alignment, and the putative domains were assigned based on alignment with the primary sequence of the b-type torque teno virus (figure 35). The torque ringvirus a has 29 residues (3.9%), which are 100% similar and in good agreement with the observations of torque ringvirus b. Interestingly, the a-type torque ringvirus has a higher percentage of residues compared to b-type torque ringviruses with at least 80% (30.9% residues) or 60% (42.9% residues) similarity.

Example 37: production of finger Ring containing chimeric ORF1 with a hypervariable Domain from a different Strain of the Cyclovirus

This example describes domain swapping of the hypervariable region of ORF1 to generate a chimeric finger loop comprising the arginine-rich region of ORF1 of one TTV strain, the jelly roll domain, the N22 and C-terminal domains, and the hypervariable domain of ORF1 protein from a different TTV strain.

The full-length genomic LY2 strain of torque virus b has been cloned into an expression vector for expression in mammalian cells. The genome was mutated to remove the hypervariable domain of LY2 and replace it with the hypervariable domain of the distantly philic b torque teno virus (fig. 36). The plasmid containing the LY2 genome with the exchanged hypervariable price domain (pTTMV-LY2-HVRa-z) was then linearized and circularized using the previously published method (Kincaid et al, PLoS Pathologens [ public science library-Pathogens ] 2013). HEK293T cells were transfected with the circularized genome and incubated for 5-7 days to allow for ring production. After the incubation period, finger loops were purified from the supernatant and cell pellet of transfected cells by gradient ultracentrifugation.

To determine whether the chimeric finger is still infectious, isolated viral particles were added to uninfected cells. Cells were incubated for 5-7 days to allow virus replication. After incubation, the ability of the chimeric finger ring to establish infection will be monitored by immunofluorescence, western blot and qPCR. The structural integrity of the chimeric virus was assessed by negative staining and cryoelectron microscopy. The chimeric finger ring body can be further tested for its ability to infect cells in vivo. The establishment of the ability to generate functional chimeric finger loops through hypervariable domain exchange may allow virus engineering to alter tropism and possibly evade immunodetection.

Example 38: production of chimeric ORF1 containing non-TTV proteins/peptides in place of the hypervariable domain

This example describes the replacement of the hypervariable region of ORF1 with other proteins or peptides of interest to produce a chimeric ORF1 protein comprising an arginine-rich region, a jelly roll domain, N22, and a C-terminal domain of one TTV strain, and a non-TTV protein/peptide in place of the hypervariable domain.

As shown in example B, the hypervariable domain of LY2 was deleted from the genome and the protein or peptide of interest could be inserted into this region (fig. 37). Examples of types of sequences that may be incorporated into this region include, but are not limited to, affinity tags, single chain variable regions (scfvs) of antibodies, and antigenic peptides. The mutant genome in the plasmid (pTTMV-LY2- Δ HVR-POI) was linearized and circularized as described in example B. The circularized genome was transfected into HEK293T cells and incubated for 5-7 days. After incubation, the chimeric finger ring body containing the POI is purified from the supernatant and the cell pellet by ultracentrifugation and/or affinity chromatography, where appropriate.

The ability to generate functional chimeric finger rings containing POIs was evaluated using various techniques. First, purified virus is added to uninfected cells to determine whether the chimeric ring can replicate and/or deliver a payload to naive cells. In addition, the structural integrity of the chimeric finger ring body was evaluated using electron microscopy. For the functional chimeric finger rings in vitro, the ability to replicate/deliver the payload in vivo was also assessed.

Example 39: ring delivery of secreted enzymes in vivo

This example illustrates the in vivo effector function of secreted enzymes delivered by the finger ring after administration.

Finger rings were prepared that contained a transgene encoding ADAMTS13 (a depolymerizing element with thrombospondin type 1 motif and a metalloprotease, member 13). Briefly, five constructs were generated: construct a-TTMV-LY2 vector ± ADAMTS 13; construct B-ADAMTS13 protein and TTMV-LY2 ORF; construct C-plasmid for production of TTMV-LY2 vector; construct D-the plasmid used to produce ADAMTS13 protein and TTMV-LY2 ORF; and construct E-sterile PBS. Construct a and construct B were produced in HEK-293T cells and purified by nuclease treatment, ultrafiltration/diafiltration and sterile filtration. Construct C and construct D were produced in e.coli, purified by MaxiPrep, then diluted to the target copy number in PBS, and then sterile filtered. Construct E was generated by sterile filtration of PBS. HEK-293T cells were expanded from thawing to passage 4 in DMEM + 10% FBS on a three day and four day passage schedule. At passage 5, cells were at approximately 5x10⁴Individual cell/cm²Inoculation for transfection the next day. One day after inoculation, cells were co-transfected with the construct using Lipofectamine 2000. After transfection, cells were incubated to allow production of finger loops and finger loops were harvested.

25uL of finger ring preparation or appropriate control was administered intravenously to genetically engineered VWD type 2B mice. Blood was drawn from each animal daily to determine hemolysis and thrombocytopenia, and the secretion of ADAMTS13 in the blood was measured: (520 ADAMTS13 Activity assay, fluorescence assay Anaspec Inc. (Anaspec, Inc.)

The presence of ADAMTS13 signals was measured each of days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21. In addition, at the same time point, an in vitro hemolysis assay protocol was performed. Briefly, blood is centrifuged and the absorbance of the supernatant, which includes plasma and lysed red blood cells, is measured. Percent lysis was calculated from a standard curve of lysed erythrocytes (Triton X-100). The presence of increased ADAMTS13 and decreased hemolysis will demonstrate the in vivo expression and activity of ADAMTS13 delivered via finger ring.

Example 40: ring in vivo delivery of secreted antibodies

This example illustrates the in vivo effector function of secreted antibodies delivered by the finger ring after administration.

Finger rings containing transgenes encoding anti-VEGF monoclonal antibodies (bevacizumab) were prepared. Briefly, five constructs were generated: construct a-TTMV-LY2 vector ± bevacizumab; construct B-bevacizumab protein and TTMV-LY2 ORF; construct C-plasmid for production of TTMV-LY2 vector; construct D-plasmid for production of bevacizumab protein and TTMV-LY2 ORF; and construct E-sterile PBS. Construct a and construct B were produced in HEK-293T cells and purified by nuclease treatment, ultrafiltration/diafiltration and sterile filtration. Construct C and construct D were produced in e.coli, purified by MaxiPrep, then diluted to the target copy number in PBS, and then sterile filtered. Construct E was generated by sterile filtration of PBS. HEK-293T cells were expanded from thawing to passage 4 in DMEM + 10% FBS on a three day and four day passage schedule. At passage 5, cells were at approximately 5x10 ⁴Individual cell/cm²Inoculation for transfection the next day. One day after inoculation, cells were co-transfected with the construct using Lipofectamine 2000. After transfection, cells were incubated to allow production of finger loops and finger loops were harvested.

25uL of finger ring preparation or appropriate control was administered intravenously to CD1 immunocompromised mice bearing metastatic colorectal cancer xenografts. Tumor volume measurements and blood draws were performed daily for each animal to use bevacizumabThe (mAb-based) ELISA test kit (Eagle Biosciences) measures the expression of bevacizumab protein. In the 0 th, 1 th, 2 th, 3 th, 4 th, 5 th, 6 th, 7 th, 8 th, 9 th, 10 th, 11 th,12. The presence of mAb signal was measured each of days 13, 14, 15, 16, 17, 18, 19, 20, and 21. Mice were sacrificed on day 22. In some embodiments, the detected reduction in tumor volume and large amounts of bevacizumab will demonstrate the in vivo expression and activity of bevacizumab delivered by finger ring.

Example 41: designing finger ring body containing payload

This example describes the design of an exemplary finger ring genetic element comprising a trans gene. The genetic element consists of the essential cis-replicating and packaging domains from members of the dacyriviridae family, as well as a non-dactylovirus payload, which may include, for example, a protein or a non-coding RNA-expressing gene. The ring lacks the trans-protein elements necessary for replication and packaging, and requires proteins provided by other sources (e.g., helper viruses, such as replication viruses, expression plasmids, or genomic integration) for rolling circle replication and encapsidation.

In one set of examples, the entire protein-encoding DNA sequence was deleted, from the first start codon to the last stop codon (fig. 38). For TTV-tth8, nucleotides 336 to 3015 from the start codon of ORF2 to the stop codon of ORF3 were deleted. For TTMV-LY2, 424 to 2813 from the start codon of ORF2 to the stop codon of ORF3 were deleted. The resulting DNA retains viral non-coding regions (NCRs), including the viral promoter, 5'UTR conserved domain, 3' UTR (encoding miRNA in some strains of finger ring virus, e.g., TTV-tth8), and GC-rich regions. The finger ring NCR contains the necessary cis-domains, including the viral origin of replication and the capsid binding domain. However, absent an open reading frame encoding a ring virus protein, the ring is unable to express the necessary protein factors required for DNA replication and encapsidation, and therefore cannot be amplified or packaged unless these elements are provided in trans.

Payload DNA, including but not limited to protein coding sequences, the entire trans gene (including non-dactylovirus promoter sequences) and the non-coding RNA gene, is incorporated into the dactylovirus genetic element by insertion into a site of the deleted dactylovirus open reading frame (fig. 38). For example, expression of a protein coding sequence can be driven by a native viral promoter or a synthetic promoter incorporated as a trans gene.

Replication-defective or replication-incompetent refers to a genetic element of a genome (e.g., as described herein) that may lack protein-coding sequences for viral replication and/or capsid factors. Thus, the packaged finger ring body is produced by co-transfecting cells with finger ring DNA and viral protein-encoding DNA as described in this example. The viral proteins are expressed from a wild-type viral genome with replication capacity, a non-replicating plasmid comprising the viral proteins under the control of a viral promoter, or a plasmid comprising the viral proteins under the control of a strong constitutive promoter.

Example 42: transduction of finger rings encoding transgenes

In this example, finger ring LY 2-Immunoadhesin (IA) was made using finger virus LY2, isolated from a lung sample, and then engineered to deliver human immunoadhesin. Double-stranded circular LY2-IA was designed to be a finger genomic DNA (which includes the LY2 noncoding region (5' UTR, GC-rich region) and the IA coding cassette, but does not include a finger genomic ORF (e.g., as described in example 41)), and then generated by in vitro circularization, as described herein. The ring virus ORF is provided in trans and is located in a separate in vitro circularised DNA. Both DNAs were co-transfected into HEK293T cells in two biological replicates (shown as "a" and "B" in figure 39). Two biological replicates were also tested, each negative control (mock transfection) and positive control (IA expression cassette in plasmid). Transduction of the ring preparations into the lung derived human cell lines EKVX and a549 resulted in detection of secreted immunoadhesins by ELISA (fig. 39; see bar graph on right). In addition, immunofluorescence analysis of LY2-IA transduced EKVX cells revealed cells positive for immunoadhesion expression.

Example 43: tth8 and LY 2-based finger rings successfully transduce the EPO gene into lung cancer cells, respectively

In this example, a non-small cell lung cancer cell line (EKVX) was transduced using two different rings carrying an erythropoietin gene (EPO). As described herein, finger rings are produced by in vitro cyclization and include two types of finger rings based on the LY2 or tth8 backbones (e.g., as described in tables 15 and 16 or tables 5 and 6, respectively). Each of the LY2-EPO and tth8-EPO finger loops includes genetic elements that include the EPO-encoding cassette and the non-coding regions (5' UTR, GC-rich regions) of the LY2 or tth8 genomes, respectively, but do not include a ring virus ORF, e.g., as described in example 41. Cells were inoculated with purified finger rings or positive controls (high dose or same dose as finger rings with AAV2-EPO) and incubated for 7 days. The ring virus ORF is provided in trans and is located in a separate in vitro circularised DNA. Culture supernatants were sampled at 3, 5.5 and 7 days post inoculation and assayed for EPO using a commercial ELISA kit. LY2-EPO and tth8-EPO indicate that both rings successfully transduced cells, EPO titers were significantly higher (P <0.013 at all time points) compared to untreated (negative) control cells (fig. 40).

Example 44: after intravenous (i.v.) administration, finger rings with therapeutic transgenes can be detected in vivo

In this example, after intravenous (i.v.) administration, the ring body encoding human growth hormone (hGH) was detected in vivo. Replication-deficient based on the LY2 backbone and encoding exogenous hGH (LY2-hGH) means that the loop body is produced by in vitro cyclization as described herein. LY2-hGH refers to the genetic elements of the loop body including the LY2 noncoding region (5' UTR, GC-rich region) and the hGH coding cassette, but not to the ring virion virus ORF, e.g., as described in example 41. LY2-hGH was administered intravenously to mice. The ring virus ORF is provided in trans and is located in a separate in vitro circularised DNA. Briefly, ring bodies (LY2-hGH) or PBS (n-4 mice/group) were injected intravenously on day 0. The finger rings were administered to independent groups of animals with 4.66E +07 finger ring genomes per mouse.

In a first example, a genomic DNA copy of a ring virus is detected. On day 7, blood and plasma were collected and the hGH DNA amplicons were analyzed by qPCR. LY2-hGH ring bodies were present in the cell fraction of whole blood 7 days after infection in vivo (fig. 41A). Furthermore, the absence of finger rings in plasma demonstrates that these finger rings cannot replicate in vivo (fig. 41B).

In a second example, hGH mRNA transcripts were detected after in vivo transduction. On day 7, blood was collected and analyzed for hGH mRNA transcript amplicons by qRT-PCR. GAPDH was used as a control house-keeping gene. hGH mRNA transcripts were measured in the cell fraction of whole blood. mRNA from the transgene encoded by the finger ring was detected in vivo (figure 42).

Example 45: size distribution of coding sequences in finger ring viruses

The coding sequence (CDS) length of all ring viruses was evaluated using an extensive catalogue of internally identified wild-type strains. CDS lengths of dactyloviruses were plotted, three strains of human dactyloviruses (a, a; b, β; and c, γ) were compared, and the publicly available genomic sequence lengths were compared to those assembled internally (internally) by the inventors. The average CDS length for all ring viruses is about 2100 nucleotides. TTV in the genus circovirus a is greater than that from the genera circovirus b and circovirus c (TTV miniand TTV mesogenes, respectively). Specifically, the average CDS observed in the type A torque teno virus TTV was 2237 nucleotides, ranging from 1800 + 2541 nucleotides. The mean CDS length for type B torque teno virus was observed to be 2011 nucleotides, ranging from 1803-2229 nucleotides. The mean CDS length of the type c torque teno virus was observed to be 2012 nucleotides in the range 1812-.

Example 46: highly conserved motifs to characterize ORF2

As shown by the exemplary genome in fig. 43A, ring virus ORF2 likely encodes a non-structural protein with possible phosphatase activity and a role in viral replication and host immune regulation. The presence of the conserved ORF2 amino acid motif was examined in a broad library of viral sequences (fig. 43B). This motif was then used to identify over 1,000 finger ring virus ORF2 sequences in the internal and public sequences. This ORF2 motif was found to remain conserved among a large number of human finger ring virus strains as well as all non-human finger ring viruses examined (rodent, porcine and primate finger ring viruses, and chicken anemia viruses), making it the most highly conserved finger ring virus motif identified to date. Structural modeling of ORF2 was also performed, indicating that conserved residues in the ORF2 motif remain in the helix-turn-helix structure, the orientation of which indicates the possible presence of a metal binding domain (fig. 43C). Interestingly, phylogenetic trees of ORF1 compared to ORF2 (fig. 43D) showed a breakdown of similar genus levels of torque ringworm a, torque ringworm b and torque c, indicating that ORF2 is genus specific.

Example 47: evidence for human full-length finger ring virus ORF1 mRNA

The finger ring virus expresses at least three alternatively spliced mRNAs in vitro, the longest of which (about 2.2kb) is expected to encode the full-length ORF 1. In this example, ORF1 mRNA transcription was assessed in vivo.

For this purpose, publicly available RNA Seq tissue data from GTEx (genotype-tissue expression) project were examined. The goal was to identify human tissue samples that contained sufficient ring virus RNA reads to classify the viral transcripts. 104 tissue samples with a ring virus RNA read (2.4% of all tissues and 19% of blood samples) were identified; of these 7 samples had more than 20 ring virus RNA reads, allowing for viral transcriptome analysis. 3 of these 7 dactylovirus positive samples also had matching WGS data from which the corresponding dactylovirus DNA genomes could be assembled for accurate read mapping (fig. 44A). The ring virus diversity prohibits informative RNA read mapping due to the lack of the corresponding viral reference genome. RNA reads mapped to the region of ORF1 were detected in three donors (two blood samples and one lung tissue sample). In one donor blood sample, finger ring viral RNA reads encompassing the full-length ORF1 region were identified (fig. 44B, grey bars represent read pairs). This is the first in vivo confirmation of full-length dactylovirus transcripts using RNA Seq data.

Example 48: in-vitro cyclized genome as input material for in-vitro production of finger ring body

This example demonstrates that In Vitro Circularized (IVC) double-stranded dactylovirus DNA, as the source material for the dactylovirus genetic elements described herein, is more robust than dactylovirus genomic DNA in a plasmid to produce a desired density of packaged dactylovirus genomes.

1.2E +07 HEK293T cells (human embryonic kidney cell line) in T75 flasks were transfected with 11.25ug of either: (i) an in vitro circularized double stranded TTV-tth8 genome (IVC TTV-tth8), (ii) a TTV-tth8 genome in the plasmid backbone, or (iii) a plasmid comprising only the ORF1 sequence of TTV-tth8 (non-replicating TTV-tth 8). Cells were harvested 7 days post transfection, lysed with 0.1% Triton, and treated with 100 units/ml Benzonase. Lysate was used for cesium chloride density analysis; the density was measured and the TTV-tth8 copy quantification was performed on each fraction of the cesium chloride linear gradient. As shown in figure 45, IVC TTV-tth8 produced significantly more copies of the viral genome at the expected density of 1.33 compared to the TTV-tth8 plasmid.

1E +07Jurkat cells (human T lymphocyte cell line) were nucleofected with the LY2 genome circularized in vitro (LY2IVC) or the LY2 genome in a plasmid. Cells were harvested 4 days post transfection and lysed using a buffer containing 0.5% tritium and 300mM sodium chloride, followed by two rounds of instant freeze-thawing. The lysate was treated with 100 units/ml benzonase and then subjected to cesium chloride density analysis. Density measurements and LY2 genomic quantification were performed on each fraction of the cesium chloride linear gradient. As shown in figure 46, transfection of the LY2 genome circularized in vitro in Jurkat cells resulted in a spike at the expected density compared to plasmid transfection containing the LY2 genome (which did not show a detectable peak in figure 46).

Example 49: identification of conserved secondary structural motifs in ORF1 of finger-ring virus

In this example, computational models were used to identify conserved motifs in the secondary structure of the ORF1 protein of the finger ring virus. Secondary structure prediction was performed on individual strains using the JPred program.

Typically, the jelly roll domain of human TTV is about 200 Amino Acids (AA) ± 3 AA in length. The secondary structure of the exemplary jelly roll domain begins with a 5-7 AA beta chain, followed by a 3-5 AA random coil, a 15-16 AA beta chain, a 26-28 AA random coil, a 15-17 AA alpha helix, a 2 AA random coil, a 3-4 AA beta chain, an 8 AA random coil, a 10-11 AA beta chain, a 5-6 AA random coil, a 6-7 AA beta chain, an 8-14 AA random coil, an 8-14 AA alpha helix (which may be split into 2 smaller helices in some cases), a 3-4 AA random coil, a 4-5 AA beta chain, a 10 AA random coil, a 5-6 AA beta chain, a 20-21 AA random coil, a 7-9 AA beta chain, a 14-16 AA random coil, a, 5-7 AA beta chain. FIG. 47 shows an alignment of exemplary finger ring virus ORF1 secondary structures from evolved branches of A, B and C viruses.

YNPX in the N22 domain of ORF1²DXGX²The secondary structure of the N (SEQ ID NO:829) motif also has a conserved secondary structure around it. Starting from the 5-6 AA beta chain that is cleaved after the tyrosine (Y) at position 1 of the motif, most of the motif lines up in an 8-9 AA random coil until the terminal asparagine (N), where another 7-8 AA beta chain begins. An alignment of exemplary finger ring virus ORF 1N 22 motif sequences is shown in FIG. 48. Tyrosine in the motif breaks the beta chain, with the second beta chain starting from the terminal asparagine of the motif.

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