阅读说明：本技术 用于治疗前蛋白转化酶枯草杆菌蛋白酶/Kexin 9型(PCSK9)相关的病症的组合物和方法 (Compositions and methods for treating proprotein convertase subtilisin/Kexin type 9 (PCSK9) -associated disorders ) 是由 A.S.伦德伯格 S.库尔卡尼 L.克莱因 Y.S.阿拉泰恩 R.L.博戈拉德 H.K.帕 于 2018-02-15 设计创作，主要内容包括：本申请提供了用于离体或体内治疗具有与PCSK9有关的一种或多种病况的患者的材料和方法。此外,本申请提供了通过基因组编辑来编辑和/或调节细胞中PCSK9基因的表达的材料和方法。(The present application provides materials and methods for ex vivo or in vivo treatment of patients having one or more conditions associated with PCSK 9. In addition, the present application provides materials and methods for editing and/or modulating the expression of the PCSK9 gene in a cell by genome editing.)

1. a method for editing a proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene in a cell by genome editing, comprising the steps of: introducing one or more deoxyribonucleic acid (DNA) endonucleases into a cell to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

2. An ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of:

(a) Isolating hepatocytes from the patient;

(b) Editing within or near the proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene or other DNA sequence encoding a regulatory element of the PCSK9 gene of hepatocytes; and

(c) Implanting the genome-edited hepatocytes into a patient.

3. The method of claim 2, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases to the hepatocyte to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

4. An ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of:

(a) Generating patient-specific induced pluripotent stem cells (ipscs);

(b) Edits are made within or near proprotein convertase subtilisin/Kexin type 9 (PCSK9) genes of ipscs or other DNA sequences encoding regulatory elements of the PCSK9 gene;

(c) Differentiating the genome-edited ipscs into hepatocytes; and

(d) Implanting the hepatocytes into a patient.

5. the method of claim 4, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases to the iPSC to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or adjacent to the PCSK9 gene or the PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

6. An ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of:

(a) Isolating mesenchymal stem cells from the patient;

(b) Editing within or near the proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene or other DNA sequence encoding a regulatory element of the PCSK9 gene of mesenchymal stem cells;

(c) differentiating the genome-edited mesenchymal stem cells into hepatocytes; and

(d) the hepatocytes are implanted into the patient.

7. The method of claim 6, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the mesenchymal stem cell to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or adjacent to the PCSK9 gene or the PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

8. An in vivo method for treating a patient having a PCSK 9-associated disorder, comprising the steps of: editing proprotein convertase subtilisin/Kexin type 9 (PCSK9) genes in cells of patients.

9. The method of claim 8, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the cell to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

10. The method of any one of claims 8-9, wherein the cell is a hepatocyte.

11. The method of claim 10, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are delivered to the hepatocytes by local injection, systemic infusion, or a combination thereof.

12. A method of altering a contiguous genomic sequence of a PCSK9 gene in a cell, comprising contacting the cell with one or more deoxyribonucleic acid (DNA) endonucleases to achieve one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs).

13. The method of claim 12, wherein the change in contiguous genomic sequence occurs in one or more exons of the PCSK9 gene.

14. The method of any one of claims 1-13, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are selected from any of those listed in SEQ ID NOs 1-620, and variants having at least 70% homology to any of those listed in SEQ ID NOs 1-620.

15. The method of claim 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more proteins or polypeptides.

16. The method of claim 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more polynucleotides encoding one or more DNA endonucleases.

17. The method of claim 16, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

18. The method of claim 17, wherein the one or more ribonucleic acids (RNAs) are one or more chemically modified RNAs.

19. The method of claim 18, wherein the one or more ribonucleic acids (RNAs) are chemically modified in the coding region.

20. The method of any one of claims 16-19, wherein the one or more polynucleotides or one or more ribonucleic acids (RNAs) are codon optimized.

21. The method of any one of claims 1-20, wherein the method further comprises introducing one or more grnas or one or more sgrnas into the cell.

22. The method of claim 21, wherein the one or more grnas or one or more sgrnas comprise a spacer sequence that is complementary to a DNA sequence within or near the PCSK9 gene.

23. The method of claim 21, wherein the one or more grnas or one or more sgrnas comprise a spacer sequence complementary to a sequence flanking the PCSK9 gene or other sequence encoding a regulatory element of the PCSK9 gene.

24. The method of any one of claims 21-23, wherein the one or more grnas or one or more sgrnas are chemically modified.

25. The method of any one of claims 21-24, wherein the one or more grnas or one or more sgrnas are pre-complexed with one or more deoxyribonucleic acid (DNA) endonucleases.

26. The method of claim 25, wherein the pre-complexing involves covalent attachment of the one or more grnas or one or more sgrnas to one or more deoxyribonucleic acid (DNA) endonucleases.

27. The method of any one of claims 14-26, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle.

28. The method of any one of claims 21-26, wherein one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle that further comprises one or more grnas or one or more sgrnas.

29. the method of any one of claims 12 or 21-23, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are encoded in an AAV vector particle, wherein the AAV vector serotype is selected from the group consisting of SEQ ID NO 4,734-5,302 and those listed in table 2.

30. The method of any one of claims 21-23, wherein the one or more grnas or one or more sgrnas are encoded in an AAV vector particle, wherein the AAV vector serotype is selected from the group consisting of SEQ ID NOs 4,734-5,302 and those listed in table 2.

31. The method of any one of claims 21-23, wherein one or more deoxyribonucleic acid (DNA) endonucleases are encoded in an AAV vector particle that further encodes one or more grnas or one or more sgrnas, wherein the AAV vector serotype is selected from those listed in SEQ ID NO 4,734-5,302 and table 2.

32. Single molecule guide RNA comprising at least one spacer sequence, which is an RNA sequence corresponding to any one of SEQ ID NO 5,305-28, 696.

33. The single molecule guide polynucleotide of claim 32, wherein the single molecule guide polynucleotide further comprises a spacer extension region.

34. The single molecule guide polynucleotide of claim 32, wherein the single molecule guide polynucleotide further comprises a tracrRNA extension region.

35. The single molecule guide polynucleotide of claims 32-34, wherein the single molecule guide polynucleotide is chemically modified.

36. The single molecule guide RNA of any one of claims 32-35, which is pre-complexed with an RNA endonuclease.

37. The single molecule guide RNA of claim 36, wherein the DNA endonuclease is a Cas9 or Cpf1 endonuclease.

38. The single molecule guide RNA of claim 37, wherein the Cas9 or Cpf1 endonuclease is selected from the group consisting of streptococcus pyogenes Cas9, staphylococcus aureus Cas9, neisseria meningitidis Cas9, streptococcus thermophilus CRISPR1 Cas9, streptococcus thermophilus CRISPR 3 Cas9, treponema denticola Cas9, cphioplasma denticola Cas 1, and combinations thereof,L. bacteriumND2006 Cpf1 and aminoacidococcus species BV3L6 Cpf1 and variants having at least 70% homology to said enzymes.

39. the single molecule guide RNA of claim 38, wherein the Cas9 or Cpf1 endonuclease comprises one or more Nuclear Localization Signals (NLS).

40. The single molecule guide RNA of claim 39, wherein at least one NLS is at or within 50 amino acids of the amino terminus of the Cas9 or Cpf1 endonuclease and/or at least one NLS is at or within 50 amino acids of the carboxy terminus of the Cas9 or Cpf1 endonuclease.

41. DNA encoding the single-molecule guide RNA of any one of claims 31-34.

Brief Description of Drawings

The various aspects of the materials and methods disclosed and described in this specification can be better understood by reference to the drawings, in which:

Fig. 1A-B depict a type II CRISPR/Cas system:

Fig. 1A is a depiction of a type II CRISPR/Cas system that includes a gRNA; and is

Fig. 1B is another depiction of a type II CRISPR/Cas system that includes sgrnas.

Figures 2, 3 and 4 depict the efficiency of gRNA cleavage by streptococcus pyogenes Cas9 targeting the PCSK9 gene in HEK293T cells.

Figure 5 depicts the cleavage efficiency of grnas targeting PCSK9 in HuH7 cells.

Figures 6A and 6B depict the cleavage efficiency of grnas of PCSK9 targeting human primary hepatocytes isolated from pigs, monkeys, and humans.

Figure 7A depicts the cleavage efficiency of grnas targeting PCSK9 in primary hepatocytes.

Fig. 7B depicts the cleavage efficiency of grnas targeting C3 in primary hepatocytes.

Figure 7C depicts the effect of gene editing by grnas on PCSK9 protein secretion in primary hepatocytes.

Brief description of the sequence listing

1-620 is a Cas endonuclease ortholog sequence.

621-631 is an intentional blank.

SEQ ID NO 632-4,715 is a microRNA sequence.

SEQ ID NO 4,716-4,733 is intentionally blank.

SEQ ID NO 4,734-5,302 is an AAV serotype sequence.

5,303 is a PCSK9 nucleotide sequence.

5,304 is a gene sequence comprising 1-5 kilobase pairs upstream and/or downstream of the PCSK9 gene.

5,305-5,365 is a 20 bp spacer sequence for targeting with a treponema denticola Cas9 endonuclease within or near the PCSK9 gene or other DNA sequence encoding a regulatory element of the PCSK9 gene.

5,366-5,545 is a 20 bp spacer sequence within or near the other DNA sequence used to target the PCSK9 gene or a regulatory element encoding the PCSK9 gene with the Streptococcus thermophilus Cas9 endonuclease.

5,546-6,579 is a 20 bp spacer sequence for targeting to the PCSK9 gene or other DNA sequence encoding a regulatory element of the PCSK9 gene with a Staphylococcus aureus Cas9 endonuclease.

6,580-7,269 is a 20 bp spacer sequence for targeting to the PCSK9 gene or other DNA sequence encoding a regulatory element of the PCSK9 gene with a Neisseria meningitidis Cas9 endonuclease.

7,270-18,791 is a 20 bp spacer sequence within or near the other DNA sequence used to target the PCSK9 gene or regulatory element encoding the PCSK9 gene with the Streptococcus pyogenes Cas9 endonuclease.

18,792-28,696 is a 22 bp spacer sequence for targeting the PCSK9 gene or other DNA sequences encoding the regulatory elements of the PCSK9 gene with the amino acid coccus, Lachnospiraceae and Francisella neoinland Cpf1 endonucleases.

28,697-28,726 is an intentional blank.

28,727 is a sample guide rna (grna) for streptococcus pyogenes Cas9 endonuclease.

28,728-28730 show the sgRNA sequences of the samples.

Detailed Description

I. Introduction to the design reside in

Genome editing

The present invention provides strategies and techniques for targeted, specific alteration of genetic information (genomes) of living organisms. As used herein, the term "alteration" or "alteration of genetic information" refers to any alteration in the genome of a cell. In the context of treating a genetic disorder, alterations may include, but are not limited to, insertions, deletions, and corrections. The term "insertion" as used herein refers to the addition of one or more nucleotides in a DNA sequence. Insertions may range from small insertions of a few nucleotides to insertions of large fragments (such as cDNA or genes). The term "deletion" refers to the loss or removal of one or more nucleotides in a DNA sequence or the loss or removal of the function of a gene. In some cases, a deletion may include, for example, a loss of several nucleotides, exons, introns, gene segments, or the entire sequence of a gene. In some cases, a deletion of a gene refers to an elimination or reduction of function or expression of the gene or its gene product. This can be caused not only by deletion of sequences within or near the gene, but also by other events (e.g., insertions, nonsense mutations) that disrupt the expression of the gene. The term "correcting" as used herein refers to a change in one or more nucleotides of a genome in a cell, whether by insertion, deletion or substitution. Such corrections may result in a genotypic or phenotypic outcome that is more structurally or functionally favorable to the corrected genomic locus. One non-limiting example of "correcting" includes correcting a mutant or defective sequence to a wild-type sequence that restores the structure or function of the gene or its gene product. Depending on the nature of the mutation, correction may be achieved via various strategies disclosed herein. In one non-limiting example, missense mutations can be corrected by replacing the region containing the mutation with the wild-type counterpart. As another example, repetitive mutations in a gene can be corrected by removing additional sequences (e.g., repetitive amplification).

In some aspects, the alteration can also include a gene knock-in, knock-out, or knock-down. As used herein, the term "knock-in" refers to the addition of a DNA sequence or fragment thereof to a genome. Such DNA sequences to be knocked in may include one or more entire genes, may include regulatory sequences associated with the gene or any portion or fragment of the foregoing. For example, a cDNA encoding a wild-type protein can be inserted into the genome of a cell carrying a mutant gene. The knock-in strategy does not necessarily replace the defective gene in whole or in part. In some cases, the knock-in strategy can further involve replacing an existing sequence with a provided sequence, e.g., replacing a mutant allele with a wild-type copy. On the other hand, the term "knock-out" refers to the elimination of a gene or the expression of a gene. For example, a gene may be knocked out by deleting or adding nucleotide sequences that result in disruption of the reading frame. As another example, a gene may be knocked out by replacing a portion of the gene with an unrelated sequence. Finally, the term "knock-down" as used herein refers to a reduction in the expression of a gene or its gene product. As a result of gene knockdown, protein activity or function may be reduced, or protein levels may be reduced or eliminated.

Genome editing generally refers to the process of altering the nucleotide sequence of a genome, preferably in an accurate or predetermined manner. Examples of methods of genome editing described herein include methods of cleaving deoxyribonucleic acid (DNA) at a precise target location of a genome using a positional nuclease, thereby creating a single-stranded or double-stranded DNA break at a specific location within the genome. Such breaks can, and often are, repaired by natural endogenous cellular processes such as Homology Directed Repair (HDR) and end joining of different sources (NHEJ), as recently in Cox et al,Those reviewed in Nature Medicine 21(2), 121-31 (2015). These two major DNA repair processes consist of a family of alternative pathways. NHEJ is directly ligated to DNA ends resulting from double strand breaks, sometimes with the loss or addition of nucleotide sequences that can disrupt or enhance gene expression. HDR uses homologous or donor sequences as templates for insertion of defined DNA sequences at break points. The homologous sequence may be in an endogenous genome (such as a sister chromatid). Alternatively, the donor may be an exogenous nucleic acid, such as a plasmid, single-stranded oligonucleotide, double-stranded oligonucleotide, duplex oligonucleotide, or virus, which has a region of high homology to a nuclease-cleaved locus, but which may also contain additional sequences or sequence changes, including deletions, which may be incorporated into the cleaved target locus. A third repair mechanism may be microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ", where the gene outcome is similar to NHEJ, asSmall deletions and insertions may occur at the cleavage site. MMEJ can use several base pairs of homologous sequences flanking the DNA break site to drive more favorable DNA end-joining repair results, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); kent et al,Nature Structural and Molecular Biology, adv. Online doi:10.1038/nsmb.2961(2015); Mateos-Gomez et al,Nature 518, 254-57 (2015); Ceccaldi et al,Nature 528, 258-62 (2015). In some cases, possible repair outcomes may be predicted based on potential micro-homology analysis at the site of DNA break.

Each of these genome editing mechanisms can be used to establish the desired genome alteration. One step in the genome editing process may be to create one or two DNA breaks in the target locus near the intended mutation site, either as double-stranded breaks or as two single-stranded breaks. This can be achieved by using a targeting polypeptide, as described and explained herein.

CRISPR endonuclease system

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genomic loci can be present in the genomes of many prokaryotes (e.g., bacteria and archaea). In prokaryotes, CRISPR loci encode products that: it functions as a class of immune system to help protect the prokaryote from foreign invaders such as viruses and bacteriophages. There are three phases of CRISPR locus function: integration of the new sequence into the CRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreign invading nucleic acids. Five classes of CRISPR systems (e.g., type I, type II, type III, type U, and type V) have been identified.

CRISPR loci comprise many short repeats, called "repeats". When expressed, the repeated sequences may form secondary structures (e.g., hairpins) and/or comprise unstructured single-stranded sequences. The repeated sequences often occur in clusters and frequently differ between species. The repeated sequences are regularly spaced by unique insertion sequences (called "spacers") to create a repeated sequence-spacer-repeated sequence locus architecture. The spacer is identical or has high homology to known foreign invasion sequences. The spacer-repeat unit encodes criprpr rna (crrna), which is processed into the mature form of the spacer-repeat unit. crrnas contain "seed" or spacer sequences (in a naturally occurring form in prokaryotes, which target foreign invading nucleic acids) that are involved in targeting a target nucleic acid. Spacer sequences are located at the 5 'or 3' end of the crRNA.

The CRISPR locus also comprises a polynucleotide sequence encoding a CRISPR-associated (Cas) gene. The Cas gene encodes an endonuclease involved in the biogenic and interfering stages of crRNA function in prokaryotes. Some Cas genes contain homologous secondary and/or tertiary structures.

Type II CRISPR system

the origin of crRNA in type II CRISPR systems in nature requires trans-activated CRISPR RNA (tracrRNA). Non-limiting examples of type II CRISPR systems are shown in fig. 1A and 1B. The tracrRNA may be modified by endogenous rnase III and then hybridized to the crRNA repeats in the pre-crRNA array. Endogenous rnase III may be recruited to cleave pre-crRNA. Exonuclease trimming of the cleaved crRNA can be performed to produce a mature crRNA form (e.g., 5' trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and crRNA bind to a localization polypeptide (e.g., Cas 9). The crRNA of the crRNA-tracrRNA-Cas9 complex can direct the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid in a type II CRISPR system is called a Protospacer Adjacent Motif (PAM). In nature, the PAM is necessary to facilitate binding of the localization polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also known as Nmeni or CASS4) are further subdivided into types II-A (CASS4) and II-B (CASS4 a). Jinek et al,Science, 337(6096) 816-821 (2012) show that the CRISPR/Cas9 system can be used for RNA-programmable genome editing and is internationalPatent application publication No. WO2013/176772 provides numerous examples and applications of CRISPR/Cas endonuclease systems for site-specific gene editing.

V-type CRISPR system

The type V CRISPR system has several important differences relative to the type II system. For example, Cpf1 is a single RNA-guided endonuclease that lacks tracrRNA compared to type II systems. Indeed, Cpf 1-related CRISPR arrays can be processed into mature crRNAs without the need for additional trans-activated tracrrnas. V-type CRISPR arrays can be processed into short mature crrnas of 42-44 nucleotides in length, where each mature crRNA starts at 19 nucleotides of the direct repeat sequence followed by 23-25 nucleotides of the spacer sequence. In contrast, mature crRNA in a type II system may start at 20-24 nucleotides of the spacer sequence, followed by about 22 nucleotides of the direct repeat sequence. Also, Cpf1 may utilize a T-rich protospacer-adjacent motif, such that the Cpf1-crRNA complex efficiently cleaves the target DNA behind a short T-rich PAM, unlike G-rich PAM behind target DNA for type II systems. Thus, the V-type system cuts at points distant from the PAM, whereas the II-type system cuts at points adjacent to the PAM. In addition, unlike type II systems, Cpf1 cleaves DNA by staggered DNA double strand breaks with 4 or 5 nucleotide 5' overhangs. Type II systems cleave by blunt double strand breaks. Similar to the type II system, Cpf1 contains a predicted RuvC-like endonuclease domain, but lacks the second HNH endonuclease domain, unlike the type II system.

Cas gene/polypeptide and protospacer adjacent motif

Exemplary CRISPR/Cas polypeptides include, e.g., Fonfara et al,Nucleic Acids Research, 422577-2590 (2014). As the Cas gene is found, the CRISPR/Cas gene naming system has undergone multiple rewrites. Fonfara et al also provide PAM sequences for Cas9 polypeptides from different species (see also SEQ ID NO: 1-620).

Compositions and methods of the invention

Provided herein are cellular, ex vivo and in vivo methods for using genome engineering tools to generate permanent changes to the genome by deleting or mutating the PCSK9 gene or other DNA sequences encoding regulatory elements of the PCSK9 gene. Such methods use endonucleases, such as CRISPR-associated (Cas9, Cpf1, etc.) nucleases, to permanently edit within or near the genomic locus of the PCSK9 gene or other DNA sequences encoding the regulatory elements of the PCSK9 gene. In this manner, the examples set forth in this disclosure may help reduce or eliminate the expression of the PCSK9 gene with a single treatment (rather than providing potential therapy for the life of the patient).

Localizing polypeptides (endonucleases, enzymes)

A targeting polypeptide is a nuclease used to cleave DNA during genome editing. The targeting polypeptide can be administered to the cell or patient as one or more polypeptides or one or more mrnas encoding the polypeptides. Any of the enzymes or orthologs listed in SEQ ID NO 1-620 or disclosed herein may be used in the methods herein.

In the context of CRISPR/Cas9 or CRISPR/Cpf1 systems, a localization polypeptide can bind to a guide RNA that in turn specifies a site in the target DNA to which the polypeptide is directed. In the CRISPR/Cas9 or CRISPR/Cpf1 systems disclosed herein, the localization polypeptide can be an endonuclease, such as a DNA endonuclease.

The localization polypeptide can comprise multiple nucleic acid cleavage (i.e., nuclease) domains. Two or more nucleic acid cleavage domains may be linked together via a linker. For example, the joint may comprise a flexible joint. A linker may comprise a length of 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, 30, 35, 40 or more amino acids.

the naturally occurring wild-type Cas9 enzyme comprises two nuclease domains: an HNH nuclease domain and a RuvC domain. Herein, the term "Cas 9" denotes a naturally occurring and recombinant Cas 9. The Cas9 enzymes contemplated herein may comprise an HNH or HNH-like nuclease domain and/or a RuvC or RuvC-like nuclease domain.

The HNH or HNH-like domain comprises an McrA-like fold. The HNH or HNH-like domain comprises two antiparallel β -strands and one α -helix. The HNH or HNH-like domain comprises a metal binding site (e.g., a divalent cation binding site). The HNH or HNH-like domain can cleave one strand of the target nucleic acid (e.g., the complementary strand of the crRNA targeting strand).

The RuvC or RuvC-like domain comprises an RNase H or RNase H-like fold. The RuvC/rnase H domain is involved in a number of series of nucleic acid-based functions, including acting on RNA and DNA. The rnase H domain comprises 5 β -strands surrounded by multiple α -helices. The RuvC/rnase H or RuvC/rnase H-like domain comprises a metal binding site (e.g., a divalent cation binding site). A RuvC/RNase H or RuvC/RNase H-like domain can cleave one strand of a target nucleic acid (e.g., the non-complementary strand of a double-stranded target DNA).

The localization polypeptide can introduce a double-stranded break or a single-stranded break in a nucleic acid (e.g., genomic DNA). The double-strand break may stimulate the endogenous DNA-repair pathway of the cell (e.g., homology-dependent repair (HDR) or NHEJ or alternative nonhomologous end joining (a-NHEJ) or microhomology-mediated end joining (MMEJ)). NHEJ can repair cleaved target nucleic acids without the need for a cognate template. This can sometimes result in a small deletion or insertion (insertion/deletion) at the cleavage site in the target nucleic acid, and can result in disruption or alteration of gene expression. HDR can occur when a homologous repair template or donor is available. The homologous donor template can comprise a sequence that is homologous to a sequence flanking the target nucleic acid cleavage site. Sister chromatids can be used by cells as repair templates. However, for genome editing purposes, the repair template may be supplied as an exogenous nucleic acid (such as a plasmid, duplex oligonucleotide, single stranded oligonucleotide, or viral nucleic acid). For exogenous donor templates, additional nucleic acid sequences (such as transgenes) or modifications (such as single or multiple base changes or deletions) may be introduced between the flanking regions of homology such that the additional or altered nucleic acid sequences also become incorporated into the target locus. MMEJ can lead to gene outcomes similar to NHEJ, as small deletions and insertions can occur at the cleavage site. MMEJ can use several base pairs of homologous sequences flanking the cleavage site to drive favorable end-ligated DNA repair results. In some cases, possible repair outcomes may be predicted based on potential micro-homology analysis in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert an exogenous polynucleotide sequence into a target nucleic acid cleavage site. One exogenous polynucleotide sequence is referred to herein as a donor polynucleotide (or donor sequence). The donor polynucleotide, a portion of the donor polynucleotide, one copy of the donor polynucleotide, or a portion of one copy of the donor polynucleotide can be inserted into a target nucleic acid cleavage site. The donor polynucleotide can be an exogenous polynucleotide sequence, i.e., a sequence that does not occur naturally at the target nucleic acid cleavage site.

Modification of the target DNA by NHEJ and/or HDR can result, for example, in mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, translocation, and/or gene mutation. Methods of deleting genomic DNA and incorporating non-natural nucleic acids into genomic DNA are examples of genome editing.

The localization polypeptide can comprise a sequence ID number 8 or Sapranauskaskaskaskask et al, as compared to a wild-type exemplary localization polypeptide [ e.g., Cas9, US2014/0068797 sequence from Streptococcus pyogenes, Nucleic Acids Res, 39(21): 9275-9282 (2011)]And a plurality of other localization polypeptides having at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% amino acid sequence identity. The localization polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 consecutive amino acids to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra). The localization polypeptide can comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity over 10 consecutive amino acids to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra)And (4) sex. The localization polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra) over 10 consecutive amino acids in the HNH nuclease domain of the localization polypeptide. The localization polypeptide can comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra) over 10 consecutive amino acids in the HNH nuclease domain of the localization polypeptide. The localization polypeptide can comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra) over 10 contiguous amino acids of a RuvC nuclease domain of the localization polypeptide. The localization polypeptide can comprise up to 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra) over 10 contiguous amino acids of a RuvC nuclease domain of the localization polypeptide.

The localization polypeptide can comprise a modified form of a wild-type exemplary localization polypeptide. The modified form of the wild-type exemplary localization polypeptide can comprise a mutation that reduces the nucleic acid cleavage activity of the localization polypeptide. The modified form of the wild-type exemplary localization polypeptide can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type exemplary localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra). The modified form of the localization polypeptide may not have substantial nucleic acid cleavage activity. When the localizing polypeptide is in a modified form having no substantial nucleic acid cleaving activity, it is referred to herein as "enzymatically inactive".

The modified form of the localization polypeptide can comprise a mutation such that it can induce a single-stranded break (SSB) on the target nucleic acid (e.g., by cleaving only one of the sugar-phosphate backbones of the double-stranded target nucleic acid). In certain aspects, the mutation can result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% nucleic acid cleavage activity of one or more of the plurality of nucleic acid cleavage domains of the wild-type localization polypeptide (e.g., Cas9 from streptococcus pyogenes, supra). In certain aspects, the mutation can result in one or more of the plurality of nucleic acid cleavage domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid cleavage domains retaining the ability to cleave the non-complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of the target nucleic acid. For example, residues in a wild-type exemplary streptococcus pyogenes Cas9 polypeptide (such as Asp10, His840, Asn854, and Asn856) are mutated to inactivate one or more of a plurality of nucleic acid cleavage domains (e.g., nuclease domains). The residue to be mutated can correspond to residues Asp10, His840, Asn854, and Asn856 (e.g., as determined by sequence and/or structural alignment) in a wild-type exemplary streptococcus pyogenes Cas9 polypeptide. Non-limiting examples of mutations include D10A, H840A, N854A, or N856A. One skilled in the art will recognize that mutations other than alanine substitutions may be suitable.

In certain aspects, the D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce a localization polypeptide that substantially lacks DNA cleavage activity. The H840A mutation may be combined with one or more of the D10A, N854A, or N856A mutations to produce a localization polypeptide that substantially lacks DNA cleavage activity. The N854A mutation may be combined with one or more of the H840A, D10A, or N856A mutations to produce a localization polypeptide that substantially lacks DNA cleavage activity. The N856A mutation may be combined with one or more of the H840A, N854A, or D10A mutations to produce a localization polypeptide that substantially lacks DNA cleavage activity. A localization polypeptide comprising a substantially inactive nuclease domain is referred to as a "nickase".

Nickase variants of RNA-guided endonucleases (e.g., Cas9) can be used to increase the specificity of CRISPR-mediated genome editing. Wild-type Cas9 is typically guided by a single guide RNA, which guideThe guide RNA is designed to hybridize to a specified about 20 nucleotide sequence (such as an endogenous genomic locus) in the target sequence. However, several mismatches can be tolerated between the guide RNA and the target locus, effectively reducing the length of homology required in the target site to, for example, as little as 13 nucleotides of homology, and thereby leading to an increased likelihood of binding and double-stranded nucleic acid cleavage (also referred to as off-target cleavage) of the CRISPR/Cas9 complex elsewhere in the target genome. Because the nickase variants of Cas9 cleave only one strand each in order to create a double-strand break, a pair of nickases must be bound in close proximity to and on the surface of the opposite strands of the target nucleic acid, thereby creating a pair of nicks, which are the equivalent of a double-strand break. This requires that two separate guide RNAs (each directed against a nickase) must be bound in close proximity to and on the surface of opposite strands of the target nucleic acid. This requirement essentially doubles the minimum length of homology required for double strand breaks to occur, thereby reducing the likelihood of: double-stranded cleavage events will occur elsewhere in the genome where the two guide RNA sites (if they are present) cannot be sufficiently close to each other for a double-stranded break to form. As described in the art, a nicking enzyme may also be used to facilitate HDR relative to NHEJ. HDR can be used to introduce selected changes to target sites in a genome by using specific donor sequences that effectively mediate the desired changes. Descriptions of various CRISPR/Cas systems for use in gene editing can be found, for example, in international patent application publication nos. WO2013/176772, andNature Biotechnology 32347-355 (2014), and references cited therein.

Contemplated mutations may include substitutions, additions and deletions, or any combination thereof. The mutation converts the mutated amino acid to alanine. The mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine, lysine, or arginine). The mutation converts the mutated amino acid to an unnatural amino acid (e.g., selenomethionine). The mutation converts the mutated amino acid into an amino acid mimic (e.g., a phosphate mimic). The mutation may be a conservative mutation. For example, the mutation converts the mutated amino acid into an amino acid that is similar in size, shape, charge, polarity, conformation, and/or rotamer to the mutated amino acid (e.g., cysteine/serine mutation, lysine/asparagine mutation, histidine/phenylalanine mutation). The mutation may result in a transfer of the reading frame and/or the establishment of an immature stop codon. Mutations can cause changes in the regulatory regions of a gene or locus that affect the expression of one or more genes.

The localization polypeptide (e.g., a variant, mutant, enzymatically inactive, and/or conditionally enzymatically inactive localization polypeptide) can be targeted to a nucleic acid. The localization polypeptide (e.g., a variant, mutant, enzymatically inactive and/or conditionally enzymatically inactive endoribonuclease) can target DNA. The targeting polypeptide (e.g., a variant, mutant, enzymatically inactive and/or conditionally enzymatically inactive endoribonuclease) can target an RNA.

The localization polypeptide can comprise one or more non-native sequences (e.g., the localization polypeptide is a fusion protein).

The localization polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes), one nucleic acid binding domain, and two nucleic acid cleavage domains (i.e., one HNH domain and one RuvC domain).

The localization polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes) and two nucleic acid cleavage domains (i.e., one HNH domain and one RuvC domain).

the localization polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes) and two nucleic acid cleavage domains, wherein one or both of the nucleic acid cleavage domains have at least 50% amino acid identity to a nuclease domain of Cas9 from a bacterium (e.g., streptococcus pyogenes).

The localization polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes), two nucleic acid cleavage domains (i.e., one HNH domain and one RuvC domain), and a non-native sequence (e.g., a nuclear localization signal) or a linker that links the localization polypeptide to a non-native sequence.

The localization polypeptide can comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes), two nucleic acid cleavage domains (i.e., one HNH domain and one RuvC domain), wherein the localization polypeptide comprises a mutation in one or both of the nucleic acid cleavage domains that reduces the cleavage activity of the nuclease domain by at least 50%.

The localization polypeptide may comprise an amino acid sequence having at least 15% amino acid identity to Cas9 from a bacterium (e.g., streptococcus pyogenes) and two nucleic acid cleavage domains (i.e., one HNH domain and one RuvC domain), wherein one of the nuclease domains comprises a mutation of aspartate 10, and/or wherein one of the nuclease domains may comprise a mutation of histidine 840, and wherein the mutation reduces cleavage activity of the nuclease domains by at least 50%.

The one or more localization polypeptides (e.g., DNA endonucleases) can include two nickases that together effect one double-strand break at a particular locus of the genome, or four nickases that together effect or cause two double-strand breaks at a particular locus of the genome. Alternatively, a localization polypeptide (e.g., a DNA endonuclease) can effect or cause a double-strand break at a particular locus in the genome.

Non-limiting examples of Cas9 orthologs from other bacterial strains include, but are not limited to, Cas proteins identified in:Acaryochloris marina MBIC 11017; acetobacter arabinosus DSM 5501; acidithiobacillus caldus; acidithiobacillus ferrooxidans ATCC 23270; alicyclobacillus acidocaldarius LAA 1; acid thermal alicyclic acid thermalThe seed DSM 446;Allochromatium vinosum DSM 180；Ammonifex degensii KC 4; anabaena ATCC 29413; arthrospira maxima CS-328; spirulina platensisstr. Paraca(ii) a Arthrospira species PCC 8005; bacillus pseudomycoides DSM 12442; bacillus selenide MLS 10; the bacterium Burkholderia 1_1_ 47;Caldicelulosiruptor becscii DSM 6725；Candidatus Desulforudis audaxviator MP104C；Caldicellulosiruptor hydrothermalis108; clostridial phage c-st; clostridium botulinumA3 str. Loch Maree(ii) a Clostridium botulinum Ba4 str.657; clostridium difficile QCD-63q 42;Crocosphaera watsonii WH 8501; cyanobacteria species ATCC 51142; cyanobacteria species CCY 0110; cyanobacteria species PCC 7424; cyanobacteria species PCC 7822;Exiguobacterium sibiricum 255-15; large fengold bacteria ATCC 29328;Ktedonobacter racemifer DSM 44963; lactobacillus delbrueckii subspecies bulgaricus PB 2003/044-T3-4; lactobacillus salivarius ATCC 11741; listeria innocua; c, Lymphenium species PCC 8106; marinobacter species ELB 17;Methanohalobium evestigatum Z-7303; microcystis phage Ma-LMM 01; microcystis aeruginosa NIES-843;Microscilla marina ATCC 23134; prototype micrococcus PCC 7420; neisseria meningitidis; nitrosococcus halophilus Nc 4; nocardia darwinia subspecies DSM 43111; nodularia cystokiniana CCY 9414; candida species PCC 7120; oscillatoria species PCC 6506;Pelotomaculum_thermopropionicumA _ SI; shipao motoga SJ 95;Polaromonas naphthalenivorans CJ 2; polar region monad species JS 666; tetrodotoxin pseudoalteromonas TAC 125; streptomyces pristinaespiralis ATCC 25486; streptomyces pristinaespiralis ATCC 25486; streptococcus thermophilus; streptomyces viridochromogenes DSM 40736; streptomyces roseus DSM 43021; synechococcus species PCC 7335; and pyrenophora africana TCF52B (chynski et al,RNA Biol., 2013; 10(5): 726-737)。

In addition to Cas9 orthologs, other Cas9 variants, such as inactivated dCas9 and fusion proteins with effector domains of different functions, can serve as platforms for genetic regulation. Any of the foregoing enzymes may be used in the present invention.

Additional examples of endonucleases that can be used in the present invention are given in SEQ ID NO 1-620. These proteins may be modified prior to use, or may be encoded in a nucleic acid sequence such as DNA, RNA, or mRNA, or in a vector construct such as a plasmid or AAV vector as taught herein. In addition, they may be codon optimized.

1-620 discloses a non-exhaustive list of endonuclease sequences.

Genome-targeted nucleic acids

The present invention provides a genome-targeted nucleic acid that can direct the activity of a polypeptide of interest (e.g., a localization polypeptide) against a particular target sequence within a target nucleic acid. The genome-targeted nucleic acid may be RNA. The genome-targeted RNA is referred to herein as a "guide RNA" or "gRNA". The guide RNA can comprise at least a spacer sequence and a CRISPR repeat that hybridizes to a target nucleic acid sequence of interest. In type II systems, the gRNA also contains a second RNA called a tracrRNA sequence. In type II guide rna (grna), CRISPR repeats and tracrRNA sequences hybridize to each other to form duplexes. In type V guide rna (grna), crRNA forms a duplex. In both systems, the duplex can bind to the localization polypeptide such that the guide RNA and localization polypeptide form a complex. The genome-targeted nucleic acid can provide target specificity to the complex through its binding to a localization polypeptide. The genome-targeted nucleic acid can thus direct the activity of the targeting polypeptide.

Exemplary guide RNAs include spacer sequences of 15-200 bases, wherein the genomic position is based on the GRCh38 human genome component. Exemplary guide RNAs include spacer sequences based on the RNA version of the DNA sequence presented in SEQ ID NO 5,305-28, 696. One of ordinary skill in the art will appreciate that each guide RNA can be designed to include a spacer sequence that is complementary to its genomic target sequence. For example, each spacer sequence, e.g., the RNA version of the DNA sequence presented in SEQ ID NO:5,305-28,696, can be placed into a single RNA chimera or crRNA (along with the corresponding tracrRNA). See Jinek et al, Science, 337, 816-821 (2012) And Deltcheva et al, Nature, 471, 602-607 (2011)。

The genome-targeted nucleic acid can be a bimolecular guide RNA. The genome-targeted nucleic acid may be a single-molecule guide RNA.

The bimolecular guide RNA can comprise two RNA strands. The first strand comprises in the 5 'to 3' direction an optional spacer extension sequence, a spacer sequence and a minimal CRISPR repeat. The second strand may comprise a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat), a 3' tracrRNA sequence, and optionally a tracrRNA extension sequence.

The single guide rna (sgrna) in a type II system may comprise in the 5' to 3' direction an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat, a single guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single molecule guide linker may link a minimal CRISPR repeat and a minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension may comprise one or more hairpins.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. The sgRNA can comprise a spacer sequence of less than 20 nucleotides at the 5' end of the sgRNA sequence. The sgRNA can comprise more than 20 nucleotide spacer sequences at the 5' end of the sgRNA sequence. The sgRNA can comprise a spacer sequence of variable length of 17-30 nucleotides at the 5' end of the sgRNA sequence (see table 1).

The sgRNA can contain NO uracil at the 3' end of the sgRNA sequence, such as in SEQ ID NO:28,729 of table 1. The sgRNA can include one or more uracils at the 3' end of the sgRNA sequence, such as in SEQ ID NO:28,730 of table 1. For example, the sgRNA can include 1 uracil (U) at the 3' end of the sgRNA sequence. The sgRNA can comprise 2 uracils (UUs) at the 3' end of the sgRNA sequence. The sgRNA can comprise 3 uracils (UUUs) at the 3' end of the sgRNA sequence. The sgRNA can comprise 4 uracils (uuuuuu) at the 3' end of the sgRNA sequence. The sgRNA can comprise 5 uracils (UUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 6 uracils (UUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 7 uracils (UUUUUUU) at the 3' end of the sgRNA sequence. The sgRNA can comprise 8 uracils (UUUUUUUU) at the 3' end of the sgRNA sequence.

The sgrnas can be unmodified or modified. For example, a modified sgRNA can comprise one or more 2' -O-methyl phosphorothioate nucleotides.

TABLE 1

The single molecule guide rna (sgrna) in a type V system can comprise a minimal CRISPR repeat and a spacer sequence in the 5 'to 3' direction.

By way of illustration, guide RNAs or other smaller RNAs used in CRISPR/Cas9 or CRISPR/Cpf1 systems can be readily synthesized by chemical means, as exemplified below and described in the art. Although chemical synthesis procedures continue to increase, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond around 100 nucleotides. One approach for preparing RNA of greater length is to produce two or more molecules linked together. Much longer RNAs, such as those encoding Cas9 or Cpf1 endonuclease, are more easily prepared by enzymatic methods. Different types of RNA modifications may be introduced during or after chemical synthesis and/or enzymatic preparation of RNA, for example, modifications that increase stability, reduce the likelihood or extent of an innate immune response, and/or enhance other properties as described in the art.

Spacer extension sequences

in certain examples of genome-targeted nucleic acids, spacer extension sequences can alter activity, provide stability, and/or provide a location for modifying the genome-targeted nucleic acid. The spacer extension sequence may alter activity or specificity at (on-target) or off-target. In certain examples, a spacer extension sequence may be provided. The spacer extension sequence may have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000 or 7000 or more nucleotides. The spacer extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or more nucleotides. The spacer extension sequence may have a length of less than 10 nucleotides. The spacer extension sequence may have a length of between 10-30 nucleotides. The spacer extension sequence may have a length of between 30-70 nucleotides.

The spacer extension sequence may comprise another moiety (e.g., a stability control sequence, an endoribonuclease binding sequence, a ribozyme). The moiety may decrease or increase the stability of the nucleic acid targeting the nucleic acid. The portion may be a transcription terminator segment (i.e., a transcription termination sequence). The moiety may function in a eukaryotic cell. The moiety may function in prokaryotic cells. The moiety may function in eukaryotic and prokaryotic cells. Non-limiting examples of suitable moieties include: a 5' cap (e.g., 7-methylguanylate cap (m 7G)), a riboswitch sequence (e.g., to allow for modulated stability and/or modulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., directly conjugated to a fluorescent molecule, conjugated to a moiety that facilitates fluorescence detection, a sequence that allows for fluorescence detection, etc.), and/or a modification or sequence that provides a binding site for a protein (e.g., a protein that functions on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.).

Spacer sequence

The spacer sequence hybridizes to a sequence in the target nucleic acid of interest. Spacers of nucleic acids that target a genome can interact with a target nucleic acid in a sequence-specific manner by hybridization (i.e., base pairing). The nucleotide sequence of the spacer may vary with the sequence of the target nucleic acid of interest.

In the CRISPR/Cas system herein, the spacer sequence can be designed to hybridize to the target nucleic acid located 5' to the PAM of the Cas9 enzyme used in the system. The spacer may be perfectly matched to the target sequence or may have a mismatch. Each Cas9 enzyme has a specific PAM sequence that it recognizes in the target DNA. For example, streptococcus pyogenes recognizes a PAM in a target nucleic acid comprising the sequence 5' -NRG-3', wherein R comprises a or G, wherein N is any nucleotide, and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

the target nucleic acid sequence may comprise 20 nucleotides. The target nucleic acid can comprise less than 20 nucleotides. The target nucleic acid may comprise more than 20 nucleotides. The target nucleic acid can comprise at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30 or more nucleotides. The target nucleic acid may comprise up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30 or more nucleotides. The target nucleic acid sequence may comprise 20 bases immediately 5' to the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3' (SEQ ID NO:28727), the target nucleic acid can comprise a sequence corresponding to N, where N is any nucleotide and the underlined NRG sequence is Streptococcus pyogenes PAM. The target nucleic acid sequence is generally referred to as a PAM strand, while the complementary nucleic acid sequence is generally referred to as a non-PAM strand. One skilled in the art will recognize that the spacer sequence hybridizes to a non-PAM strand of the target nucleic acid (fig. 1A and 1B).

The spacer sequence that hybridizes to the target nucleic acid can have a length of at least about 6 nucleotides (nt). The spacer sequence may be at least about 6 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides or at least about 40 nucleotides, from about 6 nucleotides to about 80 nucleotides, from about 6 nucleotides to about 50 nucleotides, from about 6 nucleotides to about 45 nucleotides, from about 6 nucleotides to about 40 nucleotides, from about 6 nucleotides to about 35 nucleotides, from about 6 nucleotides to about 30 nucleotides, from about 6 nucleotides to about 25 nucleotides, from about 6 nucleotides to about 20 nucleotides, from about 6 nucleotides to about 19 nucleotides, from about 10 nucleotides to about 50 nucleotides, from about 10 nucleotides to about 45 nucleotides, from about 10 nucleotides to about 40 nucleotides, or a mixture thereof, From about 10 nucleotides to about 35 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 19 nucleotides, from about 19 nucleotides to about 25 nucleotides, from about 19 nucleotides to about 30 nucleotides, from about 19 nucleotides to about 35 nucleotides, from about 19 nucleotides to about 40 nucleotides, from about 19 nucleotides to about 45 nucleotides, from about 19 nucleotides to about 50 nucleotides, from about 19 nucleotides to about 60 nucleotides, from about 20 nucleotides to about 25 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 20 nucleotides to about 35 nucleotides, from about 20 nucleotides to about 40 nucleotides, from about 20 nucleotides to about 45 nucleotides, from about 20 nucleotides to about 50 nucleotides, or from about 20 nucleotides to about 60 nucleotides. The spacer sequence may comprise 20 nucleotides. The spacer may comprise 19 nucleotides. In certain examples, the spacer can comprise 18 nucleotides. In certain examples, the spacer can comprise 22 nucleotides.

In certain examples, the percent complementarity between the spacer sequence and the target nucleic acid is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In certain examples, the percent complementarity between the spacer sequence and the target nucleic acid is at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 97%, at most about 98%, at most about 99%, or 100%. In certain examples, the percent complementarity between the spacer sequence and the target nucleic acid is 100% over the 6 consecutive nucleotides on the 5' -most side of the target sequence of the complementary strand of the target nucleic acid. The percent complementarity between the spacer sequence and the target nucleic acid can be at least 60% over about 20 consecutive nucleotides. The spacer sequence and the target nucleic acid may differ in length by 1-6 nucleotides, which may be considered as one or more lobes.

Spacer sequences can be designed or selected using computer programming. The computer program may use variables such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic background, chromatin accessibility,% GC, frequency of genome occurrence (e.g., belonging to the same or similar sequence but varying in one or more spots due to mismatches, insertions or deletions), methylation status, presence of SNPs, and the like.

Minimal CRISPR repeat

In certain aspects, the minimal CRISPR repeat is a sequence that has at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference CRISPR repeat (e.g., a crRNA from streptococcus pyogenes).

In certain aspects, the minimal CRISPR repeat comprises a nucleotide that can hybridize to a minimal tracrRNA sequence in a cell. The minimal CRISPR repeat and the minimal tracrRNA sequence may form a duplex, i.e. a base-paired double-stranded structure. The minimum CRISPR repeat and the minimum tracrRNA sequence may together bind a localization polypeptide. At least a portion of the minimal CRISPR repeat hybridizes to a minimal tracrRNA sequence. In certain aspects, at least a portion of the minimum CRISPR repeat comprises at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the minimum tracrRNA sequence. At least a portion of the smallest CRISPR repeat may comprise up to about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the smallest tracrRNA sequence.

The minimal CRISPR repeat can have a length of about 7 nucleotides to about 100 nucleotides. For example, the length of the minimum CRISPR repeat is from about 7 nucleotides (nt) to about 50 nucleotides, from about 7 nucleotides to about 40 nucleotides, from about 7 nucleotides to about 30 nucleotides, from about 7 nucleotides to about 25 nucleotides, from about 7 nucleotides to about 20 nucleotides, from about 7 nucleotides to about 15 nucleotides, from about 8 nucleotides to about 40 nucleotides, from about 8 nucleotides to about 30 nucleotides, from about 8 nucleotides to about 25 nucleotides, from about 8 nucleotides to about 20 nucleotides, from about 8 nucleotides to about 15 nucleotides, from about 15 nucleotides to about 100 nucleotides, from about 15 nucleotides to about 80 nucleotides, from about 15 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 40 nucleotides, from about 15 nucleotides to about 30 nucleotides, or from about 15 nucleotides to about 25 nucleotides. In certain aspects, the minimum CRISPR repeat is about 9 nucleotides in length. In certain aspects, the minimum CRISPR repeat is about 12 nucleotides in length.

The minimum CRISPR repeat can be at least about 60% identical to a reference minimum CRISPR repeat (e.g., a wild-type crRNA from streptococcus pyogenes) over a stretch of at least 6,7, or 8 contiguous nucleotides. For example, the minimal CRISPR repeat can have at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 98% identity, at least about 99% identity, or 100% identity over a stretch of at least 6,7, or 8 contiguous nucleotides to a reference minimal CRISPR repeat.

Minimum tracrRNA sequence

The minimum tracrRNA sequence may be a sequence having at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a wild-type tracrRNA from streptococcus pyogenes).

The minimal tracrRNA sequence may comprise nucleotides that hybridize to the minimal CRISPR repeat in the cell. The minimal tracrRNA sequence and the minimal CRISPR repeat form a duplex, i.e. a base-paired double-stranded structure. The minimal tracrRNA sequence and the minimal CRISPR repeat can together bind a localization polypeptide. At least part of the minimal tracrRNA sequence may hybridize to the minimal CRISPR repeat. The minimum tracrRNA sequence may have at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% complementarity to the minimum CRISPR repeat.

The minimum tracrRNA sequence may have a length of about 7 nucleotides to about 100 nucleotides. For example, the minimum tracrRNA sequence may be about 7 nucleotides (nt) to about 50 nucleotides, about 7 nucleotides to about 40 nucleotides, about 7 nucleotides to about 30 nucleotides, about 7 nucleotides to about 25 nucleotides, about 7 nucleotides to about 20 nucleotides, about 7 nucleotides to about 15 nucleotides, about 8 nucleotides to about 40 nucleotides, about 8 nucleotides to about 30 nucleotides, a length of about 8 nucleotides to about 25 nucleotides, about 8 nucleotides to about 20 nucleotides, about 8 nucleotides to about 15 nucleotides, about 15 nucleotides to about 100 nucleotides, about 15 nucleotides to about 80 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 30 nucleotides, or about 15 nucleotides to about 25 nucleotides. The minimum tracrRNA sequence may be about 9 nucleotides in length. The minimum tracrRNA sequence may be about 12 nucleotides. The minimum tracrRNA may consist of tracrRNA nucleotides 23-48 as described in Jinek et al, supra.

The minimum tracrRNA sequence may be at least about 60% identical to a reference minimum tracrRNA (e.g., a wild-type tracrRNA from streptococcus pyogenes) sequence over a stretch of at least 6, 7, or 8 consecutive nucleotides. For example, the minimum tracrRNA sequence may have at least about 65% identity, about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90% identity, about 95% identity, about 98% identity, about 99% identity, or 100% identity over a stretch of at least 6, 7, or 8 contiguous nucleotides with a reference minimum tracrRNA sequence.

The duplex between the smallest CRISPR RNA and the smallest tracrRNA may comprise a double helix. The duplex between the smallest CRISPR RNA and the smallest tracrRNA can comprise at least about 1,2, 3,4, 5,6, 7,8, 9, or 10 or more nucleotides. The duplex between the smallest CRISPR RNA and the smallest tracrRNA can contain up to about 1,2, 3,4, 5,6, 7,8, 9, or 10 or more nucleotides.

The duplex may contain mismatches (i.e., the two strands of the duplex are not 100% complementary). The duplex may contain at least about 1,2, 3,4, or 5 or more mismatches. The duplex may contain up to about 1,2, 3,4, or 5 or more mismatches. The duplex may contain no more than 2 mismatches.

Projection

In certain instances, a "bulge" may be present in the duplex between the smallest CRISPR RNA and the smallest tracrRNA. The bulge is an unpaired region of nucleotides within the duplex. The projections may facilitate binding of the duplex to a localization polypeptide. The bulge may comprise an unpaired 5'-XXXY-3' (SEQ ID NO:28,731) (wherein X is any purine and Y comprises nucleotides which can form wobble pairs with nucleotides on the opposite strand) on one side of the duplex and an unpaired nucleotide region on the other side of the duplex. The number of unpaired nucleotides on both sides of the duplex may be different.

In one example, the bulge can comprise an unpaired purine (e.g., adenine) on the smallest CRISPR repeat strand of the bulge. In certain examples, a protuberance can comprise the unpaired 5'-AAGY-3' of the smallest tracrRNA sequence strand of the protuberance, wherein Y comprises a nucleotide that can form a wobble pair with a nucleotide on the smallest CRISPR repeat sequence strand.

The bulge on the minimal CRISPR repeat side of the duplex may comprise at least 1,2, 3,4, or 5 or more unpaired nucleotides. The bulge on the minimal CRISPR repeat side of the duplex may comprise at most 1,2, 3,4, or 5 or more unpaired nucleotides. The bulge on the minimal CRISPR repeat side of the duplex may comprise 1 unpaired nucleotide.

The bulge on the smallest tracrRNA sequence side of the duplex may comprise at least 1,2, 3,4, 5,6, 7,8, 9, or 10 or more unpaired nucleotides. The bulge on the smallest tracrRNA sequence side of the duplex may comprise up to 1,2, 3,4, 5,6, 7,8, 9 or 10 or more unpaired nucleotides. The bulge on the second side of the duplex (e.g., the smallest tracrRNA sequence side of the duplex) may comprise 4 unpaired nucleotides.

The protrusion may comprise at least one wobble pair. In some examples, the protrusion may comprise at most one wobble pair. The projections may comprise at least one purine nucleotide. The projections may comprise at least 3 purine nucleotides. The bulge sequence may comprise at least 5 purine nucleotides. The bulge sequence may comprise at least one guanine nucleotide. The bulge sequence may comprise at least one adenine nucleotide.

Hair clip

In various examples, one or more hairpins can be located 3 'to the smallest tracrRNA in the 3' tracrRNA sequence.

The hairpin may begin at least about 1,2, 3,4, 5,6, 7,8, 9, 10, 15, or 20 or more nucleotides 3' to the last pairing nucleotide in the minimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpin may begin at most about 1,2, 3,4, 5,6, 7,8, 9, or 10 or more nucleotides 3' to the last paired nucleotide in the duplex of the minimum CRISPR repeat and the minimum tracrRNA sequence.

Hairpins can comprise at least about 1,2, 3,4, 5,6, 7,8, 9, 10, 15, or 20 or more contiguous nucleotides. Hairpins can comprise up to about 1,2, 3,4, 5,6, 7,8, 9, 10, 15, or more contiguous nucleotides.

The hairpin may comprise a CC dinucleotide (i.e., two consecutive cytosine nucleotides).

A hairpin may comprise nucleotides that form a duplex (e.g., nucleotides in the hairpin that hybridize together). For example, the hairpin may comprise CC dinucleotides that hybridize to GG dinucleotides in a hairpin duplex of a 3' tracrRNA sequence.

One or more hairpins can interact with a guide RNA-interacting region of a localization polypeptide.

In some examples, there are two or more hairpins, and in other examples, there are three or more hairpins.

3' tracrRNA sequence

The 3' tracrRNA sequence may comprise a sequence having at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequence identity to a reference tracrRNA sequence (e.g., a tracrRNA from streptococcus pyogenes).

The 3' tracrRNA sequence may have a length of about 6 nucleotides to about 100 nucleotides. For example, the 3' tracrRNA sequence may have from about 6 nucleotides (nt) to about 50 nucleotides, from about 6 nucleotides to about 40 nucleotides, from about 6 nucleotides to about 30 nucleotides, from about 6 nucleotides to about 25 nucleotides, from about 6 nucleotides to about 20 nucleotides, from about 6 nucleotides to about 15 nucleotides, from about 8 nucleotides to about 40 nucleotides, from about 8 nucleotides to about 30 nucleotides, from about 8 nucleotides to about 25 nucleotides, a length of about 8 nucleotides to about 20 nucleotides, about 8 nucleotides to about 15 nucleotides, about 15 nucleotides to about 100 nucleotides, about 15 nucleotides to about 80 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 30 nucleotides, or about 15 nucleotides to about 25 nucleotides. The 3' tracrRNA sequence may have a length of about 14 nucleotides.

The 3' tracrRNA sequence may be at least about 60% identical to a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from streptococcus pyogenes) over a stretch of at least 6, 7, or 8 consecutive nucleotides. For example, the 3' tracrRNA sequence may have at least about 60% identity, about 65% identity, about 70% identity, about 75% identity, about 80% identity, about 85% identity, about 90% identity, about 95% identity, about 98% identity, about 99% identity, or 100% identity over a stretch of at least 6, 7, or 8 contiguous nucleotides with a reference 3' tracrRNA sequence (e.g., a wild-type 3' tracrRNA sequence from streptococcus pyogenes).

The 3' tracrRNA sequence may comprise more than one duplex-forming region (e.g., hairpin, hybrid region). The 3' tracrRNA sequence may comprise two duplex forming regions.

The 3' tracrRNA sequence may comprise a stem-loop structure. The stem loop structure in the 3' tracrRNA may comprise at least 1,2, 3,4, 5,6, 7,8, 9, 10, 15 or 20 or more nucleotides. The stem-loop structure in the 3' tracrRNA may comprise up to 1,2, 3,4, 5,6, 7,8, 9 or 10 or more nucleotides. The stem-loop structure may comprise a functional moiety. For example, the stem-loop structure may comprise an aptamer, ribozyme, protein-interacting hairpin, CRISPR array, intron, or exon. The stem-loop structure may comprise at least about 1,2, 3,4 or 5 or more functional moieties. The stem-loop structure may comprise up to about 1,2, 3,4 or 5 or more functional moieties.

The hairpin in the 3' tracrRNA sequence may comprise a P-domain. In certain examples, the P-domain may comprise a double-stranded region in a hairpin.

tracrRNA extension sequences

An extension sequence of tracrRNA can be provided, whether in the context of single molecule targeting or double molecule targeting. the tracrRNA extension sequence may have a length of about 1 nucleotide to about 400 nucleotides. the tracrRNA extension sequence may have a length of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 nucleotides. the tracrRNA extension sequence may have a length of about 20 to about 5000 nucleotides or more. the tracrRNA extension sequence may have a length of more than 1000 nucleotides. the tracrRNA extension sequence may have a length of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or more nucleotides. the tracrRNA extension sequence may have a length of less than 1000 nucleotides. the tracrRNA extension sequence may comprise a length of less than 10 nucleotides. the tracrRNA extension sequence may be 10-30 nucleotides in length. the tracrRNA extension sequence may be 30-70 nucleotides in length.

The tracrRNA extension sequence may comprise a functional portion (e.g., a stability control sequence, a ribozyme, an endoribonuclease binding sequence). The functional portion may comprise a transcription terminator segment (i.e., a transcription termination sequence). The functional moiety can have a total length of about 10 nucleotides (nt) to about 100 nucleotides, about 10 nucleotides to about 20 nucleotides, about 20 nucleotides to about 30 nucleotides, about 30 nucleotides to about 40 nucleotides, about 40 nucleotides to about 50 nucleotides, about 50 nucleotides to about 60 nucleotides, about 60 nucleotides to about 70 nucleotides, about 70 nucleotides to about 80 nucleotides, about 80 nucleotides to about 90 nucleotides or about 90 nucleotides to about 100 nucleotides, about 15 nucleotides to about 80 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 30 nucleotides, or about 15 nucleotides to about 25 nucleotides. The functional moiety may function in a eukaryotic cell. The functional moiety may function in prokaryotic cells. The functional moiety may function in eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moieties include a 3' poly-adenylated tail, a riboswitch sequence (e.g., to allow for modulated stability and/or modulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplast, etc.), a modification or sequence that provides tracking (e.g., directly conjugated to a fluorescent molecule, conjugated to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.), and/or a modification or sequence that provides a binding site for a protein (e.g., a protein that functions on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.). the tracrRNA extension sequence may comprise a primer binding site or a molecular indicator (e.g., a barcode sequence). The tracrRNA extension sequence may comprise one or more affinity tags.

Single molecule leader linker sequence

The linker sequence of the single molecule guide nucleic acid may have a length of about 3 nucleotides to about 100 nucleotides. In Jinek et al, supra, for example, simple 4 nucleotide "tetracycle" (-GAAA-), Science, 337(6096): 816-. Exemplary linkers have a length of about 3 nucleotides (nt) to about 90 nucleotides, about 3 nucleotides to about 80 nucleotides, about 3 nucleotides to about 70 nucleotides, about 3 nucleotides to about 60 nucleotides, about 3 nucleotides to about 50 nucleotides, about 3 nucleotides to about 40 nucleotides, about 3 nucleotides to about 30 nucleotides, about 3 nucleotides to about 20 nucleotides, about 3 nucleotides to about 10 nucleotides. For example, the linker may have a length of about 3 nucleotides to about 5 nucleotides, about 5 nucleotides to about 10 nucleotides, about 10 nucleotides to about 15 nucleotides, about 15 nucleotides to about 20 nucleotides, about 20 nucleotides to about 25 nucleotides, about 25 nucleotides to about 30 nucleotides, about 30 nucleotides to about 35 nucleotides, about 35 nucleotides to about 40 nucleotides, about 40 nucleotides to about 50 nucleotides, about 50 nucleotides to about 60 nucleotides, about 60 nucleotides to about 70 nucleotides, about 70 nucleotides to about 80 nucleotides, about 80 nucleotides to about 90 nucleotides, or about 90 nucleotides to about 100 nucleotides. The linker for the single molecule guide nucleic acid may be between 4-40 nucleotides. The linker may be at least about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides. The linker may be up to about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

The linker may comprise any of a wide variety of sequences, although in certain examples the linker will not comprise a sequence with a broad region of homology to other parts of the guide RNA that may cause intramolecular binding that interferes with other functional regions of the guide. In Jinek et al (supra), a simple 4 nucleotide sequence-GAAA-, Science, 337(6096): 816-.

The linker sequence may comprise a functional moiety. For example, the linker sequence may comprise one or more features including an aptamer, ribozyme, protein-interacting hairpin, protein binding site, CRISPR array, intron, or exon. The linker sequence may comprise at least about 1,2, 3,4 or 5 or more functional moieties. In certain examples, the linker sequence may comprise up to about 1,2, 3,4, or 5 or more functional moieties.

Nucleic acid modification (chemical and structural modification)

In certain aspects, a polynucleotide introduced into a cell may comprise one or more modifications that may be used alone or in combination, e.g., to enhance activity, stability, or specificity, alter delivery, reduce an innate immune response in a host cell, or for other enhancements, as further described herein and known in the art.

In certain examples, the modified nucleotides may be used in CRISPR/Cas9 or CRISPR/Cpf1 systems, in which case the guide RNA (single or double molecule guide) and/or DNA or RNA encoding Cas9 or Cpf1 endonuclease introduced into the cell may be modified, as described and exemplified below. Such modified nucleotides may be used in CRISPR/Cas9 or CRISPR/Cpf1 systems to edit any one or more genomic loci.

Using CRISPR/Cas9 or CRISPR/Cpf1 systems for purposes of non-limiting illustration of such applications, modification of the guide RNA can be used to enhance the formation or stability of CRISPR/Cas9 or CRISPR/Cpf1 genome editing complexes comprising the guide RNA (which can be a single molecule guide or a bilayer) and Cas9 or Cpf1 endonuclease. Modification of the guide RNA may also or alternatively be used to enhance the initiation, stability or kinetics of the interaction between the genome editing complex and the target sequence in the genome, which may be used, for example, to enhance activity at the target. Modification of the guide RNA can also or alternatively be used to enhance specificity, e.g., the relative ratio of genome editing at a target site relative to effects at other (off-target) sites.

The modification may also or alternatively be used to increase the stability of the guide RNA, for example by increasing its resistance to degradation by a ribonuclease (rnase) present in the cell, thereby causing its half-life in said cell to be increased. In aspects in which Cas9 or Cpf1 endonuclease is introduced into the cell to be edited by RNA (which requires translation to generate the endonuclease), modifications that enhance the half-life of the guide RNA can be particularly useful because increasing the half-life of the guide RNA introduced simultaneously with the RNA encoding the endonuclease can be used to increase the time for the guide RNA and the encoded Cas9 or Cpf1 endonuclease to co-exist in the cell.

Modifications may also or alternatively be used to reduce the likelihood or extent that an RNA introduced into a cell elicits an innate immune response. Such responses, which have been characterized definitively in the context of RNA interference (RNAi), including small interfering RNA (sirna), as described below and in the art, tend to be associated with reduced RNA half-life and/or the induction of cytokines or other factors associated with the immune response.

It is also possible to subject the coding endonuclease to an introduction into a cellOne or more types of modifications made to the RNA of the enzyme, including, but not limited to, modifications that enhance the stability of the RNA (such as by increasing its degradation by RNases present in the cell), enhancing the resulting product(s) ((ii))Namely, it isEndonuclease), and/or modifications that reduce the likelihood or extent to which RNA introduced into the cell elicits an innate immune response.

Combinations of modifications, such as the foregoing and others, may also be used. In the case of CRISPR/Cas9 or CRISPR/Cpf1, for example, one or more types of modifications can be made to the guide RNA (including those exemplified above), and/or one or more types of modifications can be made to the RNA encoding the Cas endonuclease (including those exemplified above).

By way of illustration, guide RNAs or other smaller RNAs used in CRISPR/Cas9 or CRISPR/Cpf1 systems can be readily synthesized by chemical means, thereby enabling many modifications to be readily incorporated, as exemplified below and described in the art. Although chemical synthesis procedures continue to increase, purification of such RNAs by procedures such as high performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond around 100 nucleotides. One approach that can be used to prepare chemically modified RNAs of greater length is to produce two or more molecules linked together. Much longer RNAs, such as those encoding Cas9 endonuclease, are more easily prepared by enzymatic methods. Although fewer types of modifications are available for use in enzymatically produced RNA, there are modifications that can be used, for example, to enhance stability, reduce the likelihood or extent of an innate immune response, and/or enhance other attributes, as described further below and in the art; and new types of modifications are being formally developed.

As an illustration of different types of modifications, particularly those often used with smaller chemically synthesized RNAs, the modifications may comprise one or more nucleotides modified at the 2' position of the sugar, in certain aspects, 2' -O-alkyl, or 2' -fluoro-modified nucleotides. In certain aspects, the RNA modification comprises a2 '-fluoro, 2' -amino, or 2 'O-methyl modification on a pyrimidine ribose, abasic residue, or an inverted base located at the 3' terminus of the RNA. Such modifications are routinely incorporated into oligonucleotides, and these oligonucleotides have been demonstrated to have a higher Tm for a given target (i.e., higher target binding affinity) than 2' -deoxyoligonucleotides.

Many nucleotide and nucleoside modifications have been demonstrated to render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than the natural oligonucleotides; these modified oligonucleotides survive intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising a modified backbone (e.g., phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatom or heterocyclic intersugar linkages). Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, in particular CH₂-NH-O-CH₂、CH、~N(CH₃)~O~CH₂(referred to as methylene (methylimino) or MMI backbone), CH₂ --O--N (CH₃)-CH₂、CH₂ -N (CH₃)-N (CH₃)-CH₂And O-N (CH)₃)- CH₂ -CH₂A backbone, wherein the natural phosphodiester backbone is represented as O-P-O-CH); amide backbones [ see De Mesmaeker et al, Ace. Chem. Res., 28:366-374 (1995)](ii) a Morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide Nucleic Acid (PNA) backbones (in which the phosphodiester backbone of an oligonucleotide is replaced by a polyamide backbone, said nucleotide being directly or indirectly bound to the aza nitrogen atom of the polyamide backbone, see Nielsen et al,Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates (including 3 'alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -amino phosphoramidate and aminoalkyl phosphoramidate), thionocarbamates, thionochlorophosphonates, thionochloroalkylphosphotriesters, and substituted phosphorothionates having one or more pendant groupsBoranophosphate esters of normal 3'-5' linkages, 2'-5' linked analogs of these and those having inverted polarity wherein adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5 '-2'; see U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361; and 5,625,050.

Oligomeric compounds based on morpholino are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Vol.30, No. 3, (2001); Heasman, Dev. biol.,243: 209-214 (2002); Nasevicius et al,Nat, Genet, 26:216-220 (2000); Lacerra et al,Proc. Natl. Acad. Sci., 97: 9591-; and U.S. Pat. No. 5,034,506 issued on 23/7/1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al,J. Am. chem. Soc., 122: 8595-.

The modified oligonucleotide backbone, excluding the phosphorus atom therein, has a backbone formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); a siloxane backbone; sulfide, sulfoxide and sulfone backbones; a methylacetyl and thiomethyloyl backbone; methylene and thio-methyl acetyl backbones; a backbone comprising an olefin; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and has N, O, S and CH mixed₂Other backbones that make up the moiety; see U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437; and 5,677,439.

One or more substituted sugar moieties may also be included, for example, one of the following at the 2' position: OH, SH, SCH₃、F、OCN、OCH₃ OCH₃、OCH₃ O(CH₂)_n CH₃、O(CH₂)_n NH₂Or O (CH)₂)_n CH₃Wherein n is from 1 to about 10; c₁To C₁₀Lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; cl; br; CN; CF (compact flash)₃；OCF₃(ii) a O-, S-or N-alkyl; o-, S-or N-alkenyl; SOCH₃；SO₂ CH₃；ONO₂；NO₂；N₃；NH₂(ii) a A heterocycloalkyl group; a heterocycloalkylaryl group; an aminoalkylamino group; a polyalkylamino group; a substituted silyl group; an RNA cleavage group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or groups for improving the pharmacodynamic properties of the oligonucleotide, and other substituents with similar properties. In certain aspects, the modification comprises 2 '-methoxyethoxy (2' -O-CH)₂CH₂OCH₃Also known as 2' -O- (2-methoxyethyl)) (Martin et al,Heiv. chim. Acta, 1995, 78, 486). Other modifications include 2 '-methoxy (2' -O-CH)₃) 2 '-propoxy (2' -OCH)₂ CH₂CH₃) And 2 '-fluoro (2' -F). Similar modifications can also be made at other positions of the oligonucleotide, particularly at the 3 'position of the sugar on the 3' terminal nucleotide and at the 5 'position of the 5' terminal nucleotide. The oligonucleotide may also have a sugar mimetic, such as cyclobutyl in place of the furanosyl pentasaccharide.

In certain examples, the sugar and internucleoside linkages (i.e., the backbone) of the nucleotide units can be replaced with novel groups. The base units may be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound (an oligonucleotide mimetic that has been demonstrated to have excellent hybridization properties) is known as Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide may be replaced with an amide-containing backbone (e.g., an aminoethylglycine backbone).The nucleobases may be retained and bound directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082, 5,714,331 and 5,719,262. Additional teachings of PNA compounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991)。

The guide RNA may additionally or alternatively include nucleobase (often referred to simply as "base" in the art) modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases that are only rarely or transiently found in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also known as 5-methyl-2' deoxycytidine and often referred to in the art as 5-Me-C), 5-Hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other heterosubstituted alkyl adenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine and 2, 6-diaminopurine. Kornberg, A., DNA Replication, W.H. Freeman&Co., San Francisco, pp.75-77 (1980); Gebeyehu et al,Nucl. Acids Res. 15:4513 (1997). "universal" bases known in the art, e.g., inosine, may also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 deg.C (Sanghvi, Y. S., see crook, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, p. 276 and 278), and are an aspect of base substitution.

the modified nucleobases may comprise other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-thioalkyl, and optionally substituted thiouracil, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine (quinine) and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

The nucleobases may further comprise those disclosed in U.S. Pat. No. 3,687,808, in The convention Encyclopedia of Polymer Science And Engineering, pp.858-859, Kroschwitz, J.I., ed. John Wiley&Sons, 1990 by Englisch et al,Angewandle Chemie, International Edition ', 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications', page 289, page 302, crook, S.T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ℃ (Sanghvi, Y.S., crook, S.T. and Lebleu, B., eds. ' Antisense Research and Applications ', CRC Press, Boca Raton,1993, p. 276) and are aspects of base substitution, even more particularly when combined with 2' -O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Pat. Nos. 3,687,808, and 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 5,763,588, 5,830,653, 6,005,096; and U.S. patent application publication Opening 2003/0158403.

Thus, the term "modified" refers to a non-natural sugar, phosphate, or base that is incorporated into a guide RNA, endonuclease, or both guide RNA and endonuclease. Not all positions in a given oligonucleotide are necessarily uniformly modified, and in fact, more than one of the foregoing modifications may be incorporated into a single oligonucleotide, or even a single nucleoside within an oligonucleotide.

The guide RNA and/or mRNA (or DNA) encoding the endonuclease can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to: lipid moieties such as cholesterol moieties [ Letsinger et al, Proc. Natl. Acad. Sci. USA, 86: 6553-6556 (1989)](ii) a Cholic acid [ Manoharan et al, Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)](ii) a Thioethers, e.g. hexyl-S-trityl mercaptan [ Manohara et al,Ann. N.Y. Acad. Sci., 660: 306-309 (1992) and Manoharan et al, Bioorg. Med. Chem. Let., 3: 2765-2770 (1993)](ii) a Thiocholesterol Oberhauser et al, Nucl. Acids Res., 20: 533-538 (1992)](ii) a Aliphatic chains, e.g. dodecanediol or undecyl residues [ Kabanov et al,FEBS Lett., 259: 327-, Biochimie, 75: 49- 54 (1993)](ii) a Phospholipids, e.g. dicetyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate [ Manohara et al, Tetrahedron Lett., 36:3651-3654 (1995) and Shea et al, Nucl. Acids Res., 18: 3777-3783 (1990)](ii) a Polyamine or polyethylene glycol chains [ Mancharan et al, Nucleosides & Nucleotides, 14: 969-973 (1995)](ii) a Adamantane acetic acid [ Manoharan et al, Tetrahedron Lett., 36: 3651-3654 (1995)](ii) a Palm-based moiety [ (Mishra et al), Biochim. Biophys. Acta, 1264: 229-237 (1995)](ii) a Or octadecyl amine or hexylamino-carbonyl-t-hydroxycholesterol moieties [ crook et al, J. Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, 5,112,963, 5,214,136, 5,245,022, 5,254,469, 5,258,506, 5,262,536, 5,272,250, 5,292,873, 5,317,098, 5,371,241, 5,391,723, 5,416,203, 5,451,463, 5,510,475, 5,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587, 723, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,941.

Sugars and other moieties can be used to target nucleotide-containing proteins and complexes (such as cationic polysomes and liposomes) to specific sites. For example, hepatocyte-directed transfer may be mediated by the asialoglycoprotein receptor (ASGPR); see, e.g., Hu, et al,Protein Pept Lett. 21(10) 1025-30 (2014). Other systems known in the art and formally developed can be used to target the biomolecules and/or complexes thereof used in the present context to specific target cells of interest.

These targeting moieties or conjugates can include a conjugate group covalently bound to a functional group, such as a primary or secondary hydroxyl group. The conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In the context of the present invention, groups that enhance pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or enhance sequence-specific hybridization with a target nucleic acid. In the context of the present invention, groups that enhance pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the invention. Representative conjugate groups are disclosed in International patent application No. PCT/US92/09196 (filed 10/23.1992, published as WO 1993007883) and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to: lipid moieties such as cholesterol moieties, cholic acids, thioethers (e.g. hexyl-5-tritylthiol), thiocholesterol, aliphatic chains (e.g. dodecanediol or undecyl residues), phospholipids (e.g. dihexadecyl-rac-glycerol or triethylammonium l, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate), polyamine or polyethylene glycol chains or adamantane acetic acid, palmityl moieties or octadecyl amine or hexylamino-carbonyl-oxycholesterol moieties. See, for example, U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603, 5,512,439, 5,578,718, 5,371,241, 5,391,723, 5,416,203, 5,451,463, 5,578,718, 36552, 5,565,552, 5,567,810, 5,574, 5,142, 5,481,585, 5,587,6972, 5,578,718, 36697,697,6972, 5,578,718, 36697,6972, and 5,578,718.

longer polynucleotides, which are less amenable to chemical synthesis and are typically produced by enzymatic synthesis, can also be modified in a variety of ways. Such modifications may include, for example, the introduction of certain nucleotide analogs, the incorporation of specific sequences or other moieties at the 5 'or 3' end of the molecule, and other modifications. By way of illustration, mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modification of mRNA can be used, for example, to increase its translation or stability (such as by increasing its resistance to cellular degradation), or to reduce the tendency of RNA to elicit an innate immune response that is often observed in cells following introduction of exogenous RNA, particularly longer RNAs such as the one encoding Cas 9.

Numerous such modifications have been described in the art, such as polya tails, 5' Cap analogs (e.g., Anti Reverse Cap Analogs (ARCA) or m7G (5 ') ppp (5 ') g (mcap)), modified 5' or 3' untranslated regions (UTR), use of modified bases (such as pseudo-UTP, 2-thio-UTP, 5-methylcytidine-5 ' -triphosphate (5-methyl-CTP), or N6-methyl-ATP), or phosphatase treatment to remove the 5' terminal phosphate. These and other modifications are known in the art, and new modifications of RNA are under formal development.

There are numerous commercial suppliers of modified RNA including, for example, TriLink Biotech, AxoLabs, Bio-Synthesis inc, Dharmacon, and many others. As described by TriLink, for example, 5-methyl-CTP can be used to confer desirable characteristics, such as increased nuclease stability, increased translation, or reduced interaction of innate immune receptors with in vitro transcribed RNA. It has also been demonstrated that 5-methylcytidine-5' -triphosphate (5-methyl-CTP), N6-methyl-ATP, and pseudo-UTP and 2-thio-UTP reduce innate immune stimulation in culture and in vivo, while enhancing translation, as exemplified by the below-mentioned publications of Kormann et al and Warren et al.

It has been demonstrated that chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic effects; referring to the description of the preferred embodiment,Example (b) such as, for example,Kormann et al,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used, for example, to increase the stability of an RNA molecule and/or to reduce its immunogenicity. Using chemical modifications such as pseudo-U, N6-methyl-a, 2-thio-U, and 5-methyl-C, it was found that substitution of only 1/4 uridine and cytidine residues with 2-thio-U and 5-methyl-C, respectively, resulted in a significant reduction in toll-like receptor (TLR) -mediated mRNA recognition in mice. By reducing activation of the innate immune system, these modifications can be used to effectively increase mRNA stability and longevity in vivo; see, e.g., Kormann et al (supra).

It has also been demonstrated that repeated administration of synthetic messenger RNAs (which incorporate modifications designed to bypass the innate anti-viral response) can reprogram differentiated human cells to pluripotency. See, e.g., Warren, et al,Cell Stem Cell, 7(5) 618-30 (2010). Such modified mRNA functioning as a major reprogramming protein may be a reprogramming of a variety of human cellsEfficient tools for cell types. Such cells are called induced pluripotent stem cells (ipscs), and it was found that enzymatically synthesized RNA comprising 5-methyl-CTP, pseudo-UTP and an anti-inversion cap analogue (ARCA) can be used to effectively avoid the antiviral response of the cells; see, e.g., Warren et al (supra).

Other modifications of the polynucleotides described in the art include, for example, the use of poly-A tails, the addition of 5 'cap analogs such as m7G (5') ppp (5 ') G (mCAP)), modifications of the 5' or 3 'untranslated region (UTR), or phosphatase treatment to remove the 5' terminal phosphate, and new protocols are being formally developed.

Modification in conjunction with RNA interference (RNAi), including small interfering RNA (sirna), a number of compositions and techniques have been developed that are suitable for preparing modified RNA for use herein. sirnas present specific in vivo challenges because their effect on gene silencing through mRNA interference is often transient, which may require repeated administrations. In addition, siRNA is double stranded rna (dsRNA), and mammalian cells have an immune response that has evolved to detect and neutralize dsRNA, which is often a byproduct of viral infection. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase) and potentially retinoic acid-inducible gene I (RIG-I) which can mediate cellular responses to dsRNA, as well as Toll-like receptors such as TLR3, TLR7 and TLR8 which can trigger the induction of cytokines in response to such molecules; see, for example, a review of the following documents: angart et al,Pharmaceuticals (Basel) 6(4) 440-468 (2013); kanasty et al,Molecular Therapy 20(3), 513 and 524 (2012); burnett et al,biotechnol J.6 (9) 1130-46 (2011); judge and MacLachlan, Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A variety of modifications have been developed and used to enhance RNA stability, reduce innate immune responses, and/or achieve other benefits that may be useful with respect to the introduction of polynucleotides into human cells, as described herein; see, for example, a review of the following documents: whitehead KA et al, Annual Review of Chemical and Biomolecular Engineering,2: 77-96 (2011), Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010), Chernolovskaya et al,Curr Opin Mol The, 12(2):158-67 (2010); Deleavey et al,Curr Protic Nucleic Acid Chem Chapter 16: Unit 16.3 (2009), Behlke, Oligonucleotides 18(4):305-19 (2008), Fucini et al,Nucleic Acid Ther 22(3): 205-, Front Genet 3:154 (2012)。

As noted above, there are many commercial suppliers of modified RNA, many of which have been adept at modifications designed to improve the effectiveness of siRNA. Based on various findings reported in the literature, various schemes are provided. For example, Dharmacon notes that substitution of non-bridging oxygens with sulfur (phosphorothioate, PS) has been widely used to improve nuclease resistance of siRNA as reported by Kole, Nature Reviews Drug Discovery 11: 125-. Modification of the 2' -position of ribose has been reported to improve nuclease resistance of internucleotide phosphate linkages, while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. The combination of modest PS backbone modifications with small well tolerated 2 '-substitutions (2' -O-methyl, 2 '-fluoro, 2' -hydrogen) has been associated with highly stable sirnas for in vivo applications, such as Soutschek et al.Nature 432:173-178 (2004); and 2' -O-methyl modifications have been reported to be effective in improving stability as reported by Volkov, Oligonucleotides 19:191-202 (2009). With respect to reducing the induction of innate immune responses, it has been reported that modification of specific sequences with 2' -O-methyl, 2' -fluoro, 2' -hydrogen reduces TLR7/TLR8 interactions while generally retaining silencing activity; see, e.g., Judge et al,Mol. ther. 13:494-505 (2006); and Cekaite et al,J. Mol. biol. 365:90-108 (2007). Additional modifications (such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine) have also been shown to minimize immune effects mediated by TLR3, TLR7, and TLR 8; see, e.g., Kariko, K. et al, Immunity 23:165-175 (2005)。

As is also known in the art and commercially available, many conjugates can be applied to the polynucleotides (such as RNA) used herein, which can enhance their cellular delivery and/or uptake, including, for example, cholesterol, tocopherol, and folate, lipids, peptides, polymers, linkers, and aptamers; see, for example, reviews by Winkler, ther. deliv. 4:791-809 (2013), and references cited therein.

Codon optimization

Polynucleotides encoding the targeting polypeptides can be codon optimized according to standard methods known in the art for expression in cells containing the target DNA of interest. For example, if the intended target nucleic acid is in a human cell, a human codon-optimized Cas 9-encoding polynucleotide is envisioned for use in producing Cas9 polypeptides.

Complexes of genome-targeted nucleic acids and targeting polypeptides

The genome-targeted nucleic acid interacts with a localization polypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), thereby forming a complex. The genome-targeted nucleic acid directs the localization polypeptide to the target nucleic acid.

Ribonucleoprotein complex (RNP)

The targeting polypeptide and the genome-targeting nucleic acid can each be administered to a cell or patient, respectively. In another aspect, the targeting polypeptide can be pre-complexed with one or more guide RNAs, or one or more crrnas and tracrRNA together. The pre-complexed material may then be administered to the cells or patient. Such a pre-complexed substance is called ribonucleoprotein particles (RNPs). The localization polypeptide in RNP may be, for example, Cas9 endonuclease or Cpf1 endonuclease. The localization polypeptide may be flanked at the N-terminus, C-terminus, or both the N-terminus and C-terminus by one or more Nuclear Localization Signals (NLS). For example, the Cas9 endonuclease can be flanked by two NLS, one at the N-terminus and a second at the C-terminus. The NLS may be any NLS known in the art, such as SV40 NLS. The weight ratio of the genome-targeting nucleic acid to the localization polypeptide in the RNP can be 1: 1. For example, the weight ratio of sgRNA to Cas9 endonuclease in RNP can be 1: 1.

Nucleic acids encoding system components

The invention provides nucleic acids comprising a nucleotide sequence encoding a genome-targeted nucleic acid of the invention, a localization polypeptide of the invention, and/or any nucleic acid or proteinaceous molecule necessary to perform aspects of the methods of the invention.

The nucleic acid encoding the genome-targeted nucleic acid of the invention, the localization polypeptide of the invention, and/or any nucleic acid or proteinaceous molecule necessary to perform aspects of the methods of the invention can comprise a vector (e.g., a recombinant expression vector).

The term "vector" denotes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which represents a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

In certain examples, the vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors", or more simply "expression vectors", which serve equivalent functions.

The term "operably linked" refers to the linkage of a nucleotide sequence of interest to a regulatory sequence in a manner that allows for expression of the nucleotide sequence. The term "regulatory sequence" is intended to include, for example, promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel;. Gene Expression Technology: Methods in Enzymology 185, Academic Press, san Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). One skilled in the art will appreciate that the design of an expression vector can depend on factors such as the selection of the target cell, the desired level of expression, and the like.

Contemplated expression vectors include, but are not limited to, viral vectors and other recombinant vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, retroviruses (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentiviruses, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). Other vectors contemplated for eukaryotic target cells include, but are not limited to, vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). Other vectors may be used so long as they are compatible with the host cell.

In certain examples, the vector may comprise one or more transcriptional and/or translational control elements. Depending on the host/vector system utilized, any of a number of suitable transcriptional and translational control elements may be used in the expression vector, including constitutive and inducible promoters, transcriptional enhancer elements, transcriptional terminators, and the like. The vector may be a self-inactivating vector which inactivates viral sequences or components or other elements of the CRISPR mechanism.

Non-limiting examples of suitable eukaryotic promoters (i.e., promoters functional in eukaryotic cells) include those from Cytomegalovirus (CMV) immediate early, Herpes Simplex Virus (HSV) thymidine kinase, early and late SV40, Long Terminal Repeats (LTR) from retroviruses, human elongation factor-1 promoter (EF1), hybrid constructs comprising a Cytomegalovirus (CMV) enhancer fused to a chicken β -actin promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1 locus Promoter (PGK), and mouse metallothionein-I.

For expression of small RNAs, including guide RNAs used in association with Cas endonucleases, various promoters such as RNA polymerase III promoters (including, for example, U6 and H1) may be advantageous. In connection with enhancementDescriptions and parameters of the use of such promoters are known in the art, and additional information and protocols are being formally described; see, e.g., Ma, h, et al, Molecular Therapy - Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12。

The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also comprise appropriate sequences for amplifying expression. The expression vector can also include a nucleotide sequence encoding a non-native tag (e.g., a histidine tag, a hemagglutinin tag, a green fluorescent protein, etc.) fused to a localization polypeptide, thereby producing a fusion protein.

The promoter may be an inducible promoter (e.g., a heat shock promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, etc.). The promoter may be a constitutive promoter (e.g., CMV promoter, UBC promoter). In some cases, the promoter can be a spatially and/or temporally limited promoter (e.g., a tissue-specific promoter, a cell-type specific promoter, etc.).

Nucleic acids encoding the genome-targeting nucleic acids and/or localization polypeptides of the invention can be packaged inside or on the surface of a delivery vehicle for delivery to a cell. Delivery vehicles contemplated include, but are not limited to, nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycol particles, hydrogels, and micelles. As described in the art, a variety of targeting moieties can be used to enhance the preferential interaction of such agents with a desired cell type or location.

Introduction of the complexes, polypeptides and nucleic acids of the invention into cells may occur by viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nuclear transfection, calcium phosphate precipitation, Polyethyleneimine (PEI) mediated transfection, DEAE-dextran mediated transfection, liposome mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle mediated nucleic acid delivery, and the like.

Method of treatment

Provided herein are methods for treating a patient having dyslipidemia. One aspect of such methods is ex vivo cell-based therapy. For example, a biopsy of the patient's liver is performed. Liver-specific progenitor cells or primary hepatocytes are then isolated from the biopsied material, e.g., by biopsy. These progenitor or primary hepatocytes are then corrected for chromosomal DNA using the materials and methods described herein. Finally, the progenitor cells or primary hepatocytes are transplanted into the patient. Any cell source or type can be used as progenitor cells.

Another aspect of such methods is ex vivo cell-based therapy. For example, patient-specific induced pluripotent stem cells (ipscs) can be generated. The chromosomal DNA of these iPS cells can then be edited using the materials and methods described herein. Next, the genome-edited ipscs can differentiate into other cells. Finally, the differentiated cells are implanted into the patient.

Yet another aspect of such methods is ex vivo cell-based therapy. For example, mesenchymal stem cells may be isolated from a patient, which may be isolated from the bone marrow or peripheral blood of the patient. Next, chromosomal DNA of these mesenchymal stem cells can be edited using the materials and methods described herein. The genome-edited mesenchymal stem cells can then be differentiated into any type of cell, e.g., a hepatocyte. Finally, these differentiated cells, e.g., hepatocytes, are transplanted into the patient.

One advantage of ex vivo cell therapy protocols is the ability to conduct a comprehensive analysis of treatment prior to administration. Nuclease-based therapeutics may have some level of off-target effect. Performing gene editing ex vivo allows one to characterize the edited cell population prior to implantation. The invention includes sequencing the entire genome of the edited cell to ensure that off-target effects, if any, can be at genomic locations associated with minimal risk to the patient. In addition, populations of specific cells, including clonal populations, can be isolated prior to implantation.

Another advantage of ex vivo cell therapy relates to genetic modification in ipscs compared to other primary cell sources. ipscs are prolific, making it easy to obtain a large number of cells that would be required for cell-based therapy. Furthermore, ipscs are an ideal cell type for performing clonal isolation. This allows for the screening of correct genome modifications without risking a reduction in viability. In contrast, other primary cells (such as hepatocytes) survive only a few passages and are difficult to clonally propagate. Thus, ipscs for the treatment of dyslipidemia will be much cheaper to handle and the amount of time required to produce the desired genetic modification can be shortened.

The methods may also include in vivo based therapies. The chromosomal DNA of the cells in the patient is edited using the materials and methods described herein.

Although certain cells present attractive targets for ex vivo therapy and therapy, increased delivery efficacy may allow for direct in vivo delivery to such cells. Ideally, targeting and editing is directed to the relevant cells. Cleavage in other cells can also be prevented by using promoters that are active only in certain cells and/or developmental stages. Additional promoters are inducible and thus can be temporally controlled if the nuclease is delivered as a plasmid. The amount of time that delivered RNA and protein remain in the cell can also be modulated using treatments or domains added to alter half-life. In vivo processing would eliminate many processing steps, but a lower delivery rate may require a higher editing rate. In vivo treatment can eliminate problems and losses from ex vivo treatment and transplantation.

One advantage of in vivo gene therapy may be the ease of manufacture and administration of therapeutic agents. The same treatment regimen and therapy will have the potential to be used to treat more than one patient (e.g., many patients with the same or similar genotype or allele). In contrast, ex vivo cell therapy typically requires the use of the patient's own cells, which are isolated, manipulated and returned to the same patient.

Also provided herein are cellular methods of editing the PCSK9 gene in a cell by genome editing. For example, cells can be isolated from a patient or animal. The chromosomal DNA of the cell can then be edited using the materials and methods described herein.

The methods provided herein, whether cellular methods or ex vivo methods or in vivo methods, may involve reducing (knocking down) or eliminating (knocking out) the expression of the PCSK9 gene by introducing one or more insertions, deletions, or mutations within or near the PCSK9 gene or other DNA sequences encoding regulatory elements of the PCSK9 gene.

For example, a knock-down or knock-out strategy may involve disrupting the reading frame of the PCSK9 gene by introducing random insertions or deletions (insertions/deletions) due to the imprecise NHEJ repair pathway. This can be achieved by: one single-strand break or double-strand break is induced in the target gene with one or more CRISPR endonucleases and a gRNA (e.g., crRNA + tracrRNA, or sgRNA), or two or more single-strand breaks or double-strand breaks are induced in the target gene with two or more CRISPR endonucleases and two or more sgrnas. This approach may require the development and optimization of sgrnas for the PCSK9 gene.

Alternatively, the knock-down or knock-out strategy may also involve deletion of one or more segments within or near the PCSK9 gene or other DNA sequences encoding regulatory elements of the PCSK9 gene. This deletion strategy requires at least one pair of a gRNA (e.g., crRNA + tracrRNA, or sgRNA) and one or more CRISPR endonucleases capable of binding to two different sites within or near the PCSK9 gene. A CRISPR endonuclease configured with two grnas can induce two double-strand breaks at desired positions. After cleavage, both ends, whether blunt or overhanging, can be ligated by NHEJ, resulting in deletion of the inserted segment. The NHEJ repair pathway may result in insertions, deletions or mutations at the junction.

In addition to the above genome editing strategies, another strategy involves modulating the expression, function or activity of PCSK9 by editing in the regulatory sequence.

In addition to the editing options listed above, Cas9 or similar proteins can be used to target the effector domain to the same target site that can be identified for editing, or to additional target sites within the scope of the effector domain. A series of chromatin modifying enzymes, methylases or demethylases may be used to alter the expression of a target gene. One possibility is to reduce the expression of the PCSK9 protein if the mutation results in an undesired activity. These types of epigenetic regulation have some advantages, in particular because they are limited in possible off-target effects.

In addition to mutations in the coding and splicing sequences, there may be many types of genomic target sites.

Regulation of transcription and translation involves many different kinds of sites of interaction with cellular proteins or nucleotides. It is often possible to target the DNA binding sites of transcription factors or other proteins for mutations or deletions to study the effect of the sites, although they may also be targeted to alter gene expression. Sites can be added by non-homologous end joining (NHEJ) or direct genome editing (by Homology Directed Repair (HDR)). The use of whole genome studies with increased genome sequencing, RNA expression and transcription factor binding has increased our ability to identify how the site leads to developmental or temporal gene regulation. These control systems may be direct, or may involve extensive coordinated regulation, which may require integration of activity from multiple enhancers. Transcription factors typically bind to degenerate DNA sequences of 6-12 bases in length. The low level of specificity provided by each site suggests that complex interactions and rules are involved in the binding and functional outcome. Binding sites with lower degeneracy may provide a simpler means of modulation. Artificial transcription factors can be designed to specify longer sequences with sequences of lower similarity in the genome and with lower potential for off-target cleavage. Any of these types of binding sites can be mutated, deleted or even created to effect changes in gene regulation or expression (cancer, m.c. et al),Nature(2015))。

Another type of gene regulatory region with these characteristics is the microrna (mirna) binding site. mirnas are non-coding RNAs that play a key role in post-transcriptional gene regulation. mirnas can regulate the expression of 30% of all genes encoding mammalian proteins. The specificity achieved by double-stranded RNA (RNAi) is found to beEfficient gene silencing, and additionally small non-coding RNAs (cancer, M.C. et al),Nature(2015)). The largest type of non-coding RNA important for gene silencing is miRNA. In mammals, mirnas are first transcribed into long RNA transcripts, which may be individual transcription units, portions of introns, or other transcripts. Long transcripts are called primary mirnas (primary-mirnas), which include imperfectly base-paired hairpin structures. These primary-mirnas can be cleaved by the microprocessor (protein complex in the nucleus, related to Drosha) into one or more shorter precursor mirnas (pre-mirnas).

Pre-mirnas are short stem loops of about 70 nucleotides in length with 2-nucleotide 3' -overhangs that are exported as mature 19-25 nucleotide miRNA-miRNA duplexes. miRNA strands (guide strands) with lower base-pairing stability can be loaded onto the RNA-induced silencing complex (RISC). Passenger chains (labeled with x) may be functional, but are often degraded. Mature mirnas link RISC to partially complementary sequence motifs in target mrnas that are predominantly present within the 3' untranslated region (UTR) and induce post-transcriptional gene silencing (Bartel, d.p.).Cell 136, 215-233 (2009); Saj, A. & Lai, E.C. Curr Opin Genet Dev 21, 504-510 (2011))。

mirnas can be important in the development, differentiation, cell cycle and growth control of mammals and other multicellular organisms, as well as in substantially all biological pathways. mirnas may also be involved in cell cycle control, apoptosis and stem cell differentiation, hematopoiesis, hypoxia, muscle development, neurogenesis, insulin secretion, cholesterol metabolism, aging, viral replication, and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, although many different mirnas can target a single miRNA transcript. More than 28645 microRNAs have been annotated in the latest version of miRBase (v.21). Some mirnas may be encoded by multiple loci, some of which may be expressed from clusters that are co-transcribed in tandem. This feature allows for a complex regulation network with multiple approaches and feedback control. mirnas may be part of these feedback and regulatory loops, and mayTo help regulate gene expression by keeping protein production within limits (Herranz, H.& Cohen, S.M. Genes Dev 24, 1339-1344 (2010); Posadas, D.M. & Carthew, R.W. Curr Opin Genet Dev 27, 1-6 (2014))。

mirnas may also be important in a number of human diseases associated with aberrant miRNA expression. This association underscores the importance of miRNA regulatory pathways. Recent studies of miRNA deletions have linked miRNA regulation of immune response (Stern-gingossar, n,Science 317, 376-381 (2007))。

mirnas also have a strong link to cancer and can play a role in different types of cancer. Mirnas have been found to be down-regulated in many tumors. mirnas can be important in the regulation of key cancer-related pathways, such as cell cycle control and DNA damage response, and thus can be used in diagnostics and can be clinically targeted. Micrornas can subtly regulate the balance of angiogenesis such that experiments removing all micrornas inhibit tumor angiogenesis (Chen, s. et al),Genes Dev 28, 1054-1067 (2014))。

As has been demonstrated for protein-encoding genes, miRNA genes can also exhibit epigenetic changes that occur with cancer. Many miRNA loci can be associated with CpG islands, increasing the chances that they are regulated by DNA methylation (Weber, B., streesemann, c., Brueckner, B).& Lyko, F. Cell Cycle6, 1001-1005 (2007)). Most studies have used chromatin-remodeling drugs for treatment to reveal epigenetically silenced mirnas.

In addition to their role in RNA silencing, mirnas can also activate translation (Posadas, D.M.& Carthew, R.W.Curr Opin Genet Dev27, 1-6 (2014)). Knocking out miRNA sites can lead to reduced expression of the targeted gene, while introducing these sites can increase expression.

By mutating seed sequences that may be important for binding specificity (bases 2-8 of microrna), single mirnas can be knocked out most efficiently. Cleavage at this region, followed by repair of the error by NHEJ, can be effective by blocking binding to the target siteEliminating miRNA function. Mirnas can also be inhibited by specific targeting of specific loop regions adjacent to palindromic sequences. Catalytically inactive Cas9 may also be used to inhibit shRNA expression (Zhao, y, et al),Sci Rep4, 3943 (2014)). In addition to targeting mirnas, binding sites can be targeted and mutated to prevent silencing by mirnas.

In accordance with the present disclosure, any microrna (mirna) or binding site thereof may be incorporated into the compositions of the present invention.

The composition may have regions such as, but not limited to, regions comprising the sequences of any of the microRNAs listed in SEQ ID NO:632-4,715, the reverse complement of the microRNA listed in SEQ ID NO:632-4,715, or the microRNA anti-seed region of any of the microRNAs listed in SEQ ID NO:632-4,715.

The compositions of the invention may comprise one or more microrna target sequences, microrna sequences or microrna seeds. Such sequences may correspond to any known microRNA, such as those taught in U.S. publication US2005/0261218 and U.S. publication US 2005/0059005. As a non-limiting embodiment, the known microRNAs in the human genome, their sequences and their binding site sequences are set forth below in SEQ ID NO. 632-4,715.

The microrna sequence comprises a "seed" sequence, i.e. a sequence in the region of positions 2-8 of the mature microrna, which has perfect watson-crick complementarity with the miRNA target sequence. The microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some aspects, a microrna seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of a mature microrna), with the seed complementary site in the corresponding miRNA target flanked by adenine (a) opposite position 1 of the microrna. In some aspects, a microrna seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of a mature microrna), with the seed complementary site in the corresponding miRNA target flanked by adenine (a) opposite position 1 of the microrna. See, e.g., Grimson A, Farh KK, Johnston WK, Garrett-Engel P, Lim LP, Bartel DP; Mol cell. 2007 Jul 6;27(1): 91-105. The bases of the microRNA seeds have complete complementarity with the target sequence.

The identification of microRNAs, microRNA target regions, their expression patterns and their role in biology have been reported (Bonauer et al, Curr Drug Targets 201011: 943-.

For example, if a composition is not intended to be delivered to the liver, but terminates at those, miR-122 (a microrna abundant in the liver) can inhibit expression of the delivered sequence if one or more target sites of miR-122 are engineered as a polynucleotide encoding the target sequence. The introduction of one or more binding sites of different micrornas can be engineered to further reduce longevity, stability, and protein translation, thus providing an additional layer of toughness.

As used herein, the term "microrna site" refers to a microrna target site or microrna recognition site, or any nucleotide sequence to which a microrna binds or associates. It is understood that "binding" may follow traditional Watson-Crick hybridization rules, or may reflect any stable association of microRNAs with target sequences at or near microRNA sites.

In contrast, for the purposes of the compositions of the present invention, microrna binding sites can be engineered to have sequences (i.e., removed from) that naturally occur to increase protein expression in a particular tissue. For example, the miR-122 binding site can be removed to improve protein expression in the liver.

Specifically, micrornas are known to be differentially expressed in immune cells (also referred to as hematopoietic cells), such as Antigen Presenting Cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, and the like. Immune cell specific micrornas are implicated in immunogenicity, autoimmunity, immune response to infection, inflammation, and unwanted immune responses following gene therapy and tissue/organ transplantation. Immune cell-specific micrornas also regulate many aspects of the development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are expressed only in immune cells, and are particularly abundant in myeloid dendritic cells. Introduction of a miR-142 binding site into the 3' -UTR of a polypeptide of the invention can selectively inhibit gene expression in antigen presenting cells through miR-142 mediated mRNA degradation, limit antigen presentation in professional APCs (e.g., dendritic cells), and thereby prevent antigen-mediated immune responses following gene delivery (see, Annoni a et al, blood, 2009, 114, 5152-.

In one embodiment, microrna binding sites known to be expressed in immune cells, particularly antigen presenting cells, can be engineered into polynucleotides to inhibit expression of the polynucleotides in APCs, inhibiting antigen-mediated immune responses, through microrna-mediated RNA degradation, while expression of the polynucleotides is maintained in non-immune cells in which immune cell-specific micrornas are not expressed.

Many microrna expression studies have been conducted and described in the art to analyze differential expression of micrornas in various cancer cells/tissues and other diseases. Some micrornas are abnormally high expressed in some cancer cells, while others are under-expressed. For example, micrornas are differentially expressed in: cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO 2011/157294); cancer stem cells (US 2012/0053224); pancreatic cancer and diseases (US2009/0131348, US2011/0171646, US2010/0286232, US 8389210); asthma and inflammation (US 8415096); prostate cancer (US 2013/0053264); hepatocellular carcinoma (WO2012/151212, US2012/0329672, WO2008/054828, US 8252538); lung cancer cells (WO2011/076143, WO2013/033640, WO2009/070653, US 2010/0323357); cutaneous T cell lymphoma (WO 2013/011378); colorectal cancer cells (WO2011/0281756, WO 2011/076142); cancer-positive lymph nodes (WO2009/100430, US 2009/0263803); nasopharyngeal carcinoma (EP 2112235); chronic obstructive pulmonary disease (US2012/0264626, US 2013/0053263); thyroid cancer (WO 2013/066678); ovarian cancer cells (US2012/0309645, WO 2011/095623); breast cancer cells (WO2008/154098, WO2007/081740, US2012/0214699), leukemias and lymphomas (WO2008/073915, US2009/0092974, US2012/0316081, US2012/0283310, WO 2010/018563).

Non-limiting examples of microRNA sequences and tissues and/or cells to be targeted are described in SEQ ID NOs:632-4,715.

Genome engineering strategies

In certain aspects, the methods of the invention may comprise editing one or both of the alleles. Modification of allele in gene editing has the advantage of permanently altering the target gene or gene product.

The steps of the ex vivo method of the invention may comprise using genome engineering editing of hepatocytes isolated from the patient. Alternatively, the steps of the ex vivo method of the invention may comprise editing patient-specific ipscs or mesenchymal stem cells. Likewise, the steps of the in vivo method of the invention comprise editing cells in a dyslipidemia patient using genomic engineering. Similarly, a step in the cellular methods of the invention may comprise editing the PCSK9 gene in a human cell by genome engineering.

Any CRISPR endonuclease can be used in the methods of the invention, each having its own associated PAM, which may or may not be disease specific.

For example, expression of the PCSK9 gene may be disrupted or eliminated by introducing random insertions or deletions (indels) due to an imprecise NHEJ repair pathway. The target region may be the coding sequence (i.e., exon) of the PCSK9 gene. Insertion or deletion of nucleotides in the coding sequence of a gene can cause a "frame shift" in which the normal 3-letter codon pattern is disrupted. In this way, gene expression and thus protein production may be reduced or eliminated. The method may also be used to target any intron, intron exon junction or regulatory DNA element of the PCSK9 gene, where sequence changes may interfere with expression of the PCSK9 gene.

As another example, NHEJ can also be used to delete segments within or near genes, either directly or by altering splice donor or acceptor sites (by cleavage of one or several grnas targeting several positions). This may be useful if small random insertions/deletions do not effectively knock down the target gene. Guide chain pairs have been used for this type of deletion.

In the absence of a donor, ends from DNA breaks or ends from different breaks can be ligated using several non-homologous repair pathways, where DNA ends are ligated with little or no base pairing at the junction. In addition to canonical NHEJ, similar repair mechanisms exist, such as alt-NHEJ. If there are two breaks, the insertion section can be deleted or inverted. The NHEJ repair pathway may result in insertions, deletions or mutations at the linker.

NHEJ can also lead to homology-independent target integration. For example, inclusion of a nuclease target site on the donor plasmid can facilitate integration of the transgene into the chromosome double strand break following in vivo nuclease cleavage of both the donor and chromosome (Cristea., Biotechnol Bioeng.2013 Mar;110(3): 871-80).

Following nuclease cleavage, a 15-kb inducible gene expression cassette was inserted into a defined locus in a human cell line using NHEJ. (see, e.g., Maresca, m., Lin, v.g., Guo, N.& Yang, Y., Genome Res23,539-Nature, 540, 144-149 (2016)). The integrated sequence may disrupt the reading frame of the PCSK9 gene or alter the structure of the gene.

as a further alternative, Homologous Direct Repair (HDR) may also be used to knock out a gene or alter gene function. HDR is basically an error-free mechanism that uses supplied homologous DNA sequences as templates in DSB repair processes. The ratio of HDR is a function of the distance between the mutation and the cleavage site, so it is important to select overlapping or nearest target sites. The template may include additional sequences flanked by homologous regions, or may contain sequences other than genomic sequences, thereby allowing sequence editing.

For example, an HDR knockout strategy can include disrupting the structure or function of the PCSK9 gene by inserting the gene or replacing portions of the gene with a non-functional or unrelated sequence. This can be achieved by: one single-strand break or double-strand break is induced in a target gene with one or more CRISPR endonucleases and grnas (e.g., crRNA + tracrRNA, or sgrnas), or two or more single-strand breaks or double-strand breaks are induced in a target gene with one or more CRISPR endonucleases and two or more grnas, in the presence of a donor DNA template (which can be a short single-strand oligonucleotide, a short double-strand oligonucleotide, a long single-strand or double-strand DNA molecule) exogenously introduced to direct a cellular DSB response to HDR. This approach may require the development and optimization of gRNA and donor DNA molecules for the PCSK9 gene.

Homology directed repair is a cellular mechanism for repairing DSBs. The most common form is homologous recombination. Additional HDR pathways exist, including single strand annealing and alternative HDR. Genome engineering tools allow researchers to manipulate homologous recombination pathways of cells to establish site-specific modifications to the genome. It has been found that cells can repair double strand breaks using synthetic donor molecules provided in trans. And 1/10⁶Specific cleavage increased the HDR ratio by more than 1,000 fold compared to the ratio of cells receiving the cognate donor alone. The ratio of homology-directed repair (HDR) at a particular nucleotide is a function of the distance to the cleavage site, so it is important to select an overlapping or closest target site.

The donor supplied for HDR editing varies significantly, but may contain the expected sequence with small or large flanking homology arms to allow annealing to genomic DNA. The homologous region flanking the introduced gene change may be 30 base pairs or less, or as large as several thousand base cassettes which may contain promoters, cDNAs, etc. Both single-stranded and double-stranded oligonucleotide donors have been used. These oligonucleotides range in size from less than 100 nucleotides to over many kb, although longer ssDNA can also be prepared and used. Double stranded donors including PCR amplicons, plasmids and mini-loops may be used. In general, it has been found that AAV vectors can be a very efficient way to deliver donor templates, although the packaging limit for various donors is <5 kb. Active transcription of the donor increased HDR by 3-fold, indicating that the inclusion of a promoter can increase transformation. In contrast, CpG methylation of the donor decreases gene expression and HDR.

In addition to wild-type endonucleases (such as Cas9), there are nickase variants that have one or other nuclease domains inactivated, resulting in cleavage of only one DNA strand. HDR can be directed from various Cas nickases or using pairs of nickases flanking the target region. The donor may be single stranded, nicked or dsDNA.

The donor DNA may be supplied with a nuclease, or independently by a number of different methods, for example by transfection, nanoparticles, microinjection or viral transduction. Many connectivity options have been proposed to increase the availability of donors for HDR. Examples include attaching the donor to a nuclease, to a DNA binding protein that binds nearby, or to a protein involved in DNA end binding or repair.

Repair pathway selection can be guided by a number of culture conditions (such as those affecting the cell cycle) or by DNA repair and targeting of related proteins. For example, to increase HDR, key NHEJ molecules, such as KU70, KU80, or DNA ligase IV, may be inhibited.

In addition to genome editing by NHEJ or HDR, site-specific gene insertion using NHEJ pathway and HR has been performed.

The PCSK9 gene contains a number of exons as shown in table 3. Any one or more of these exons or nearby introns may be targeted to create one or more insertions or deletions that disrupt the reading frame and ultimately abolish PCSK9 protein activity.

In some embodiments, the methods can provide gRNA pairs that are deleted by cleaving the gene twice at positions flanking the unwanted sequences. The sequence may include one or more exons, introns, intron-exon junctions, other DNA sequences encoding regulatory elements of the PCSK9 gene, or combinations thereof. The cleavage can be accomplished by a pair of DNA endonucleases (each of which produces a DSB in the genome) or a plurality of nickases (which together produce a DSB in the genome).

Alternatively, the method can provide a gRNA to perform a double-stranded cleavage within an encoding or splicing sequence. The double-stranded nicks may be generated by a single DNA endonuclease or multiple nicking enzymes that together generate a DSB in the genome.

The splice donor and acceptor are typically within 100 base pairs of adjacent introns. In certain examples, the methods can provide grnas that cleave at approximately ± 100-3100 base pairs for each exon/intron junction of interest.

For any genome editing strategy, gene editing can be confirmed by sequencing or PCR analysis.

Target sequence selection

The transfer in the position of the 5 'boundary and/or the 3' boundary relative to a particular reference locus may be used to facilitate or enhance a particular application of gene editing, depending in part on the endonuclease system selected for the editing, as further described and exemplified herein.

In the first non-limiting example of such target sequence selection, many endonuclease systems have rules or criteria that can direct the initial selection of potential target sites for cleavage, such as the requirement for a PAM sequence motif at a specific position adjacent to the DNA cleavage site in the case of CRISPR type II or type V endonucleases.

In another non-limiting example of target sequence selection or optimization, the frequency of "off-target" activity (i.e., the frequency of DSBs occurring at sites other than the selected target sequence) for a particular combination of target sequence and gene editing endonuclease can be assessed relative to the frequency of target activity. In some cases, cells that have been correctly edited at a desired locus may have selective advantages over other cells. Illustrative, but non-limiting examples of selective advantages include the attainment of attributes such as enhanced replication rate, persistence, resistance to certain conditions, enhanced rate of successful graft implantation or persistence in vivo after introduction into a patient, and other attributes related to maintenance, or increased number or viability of such cells. In other cases, cells that have been correctly edited at a desired locus may be positively selected by one or more screening methods for identifying, classifying, or otherwise selecting cells that have been correctly edited. Selective advantage and targeted selection methods can exploit phenotypes associated with alterations. In some cases, the cells may be edited two or more times in order to establish a second modification that results in a new phenotype for selection or purification of the desired cell population. Such a second modification can be established by adding a second gRNA for a selectable or screenable marker. In some cases, using a DNA fragment containing cDNA along with a selectable marker, cells can be properly edited at a desired locus.

Regardless of whether any selectivity advantages apply or any targeted selection applies to a particular situation, target sequence selection may also be guided by off-target frequency considerations in order to enhance the effectiveness of the application and/or reduce the potential for unwanted changes at sites other than the desired target. As further described and exemplified herein and in the art, the occurrence of off-target activity can be influenced by a number of factors, including the similarity and dissimilarity between the target site and the various off-target sites, as well as the particular endonuclease used. Bioinformatic tools are available which aid in the prediction of off-target activity, and such tools can often also be used to identify the most likely site of off-target activity which can then be assessed in an experimental setting to assess the relative frequency of off-target activity to on-target activity, thereby allowing the selection of sequences with higher relative on-target activity. Illustrative examples of such techniques are provided herein, and others are known in the art.

Another aspect of target sequence selection involves homologous recombination events. Sequences sharing regions of homology may serve as the focus of homologous recombination events leading to deletion of the inserted sequence. Such recombination events occur during normal replication of chromosomes and other DNA sequences, and also at other times when DNA sequences are synthesized, such as in the case of repair of Double Strand Breaks (DSBs) that occur periodically in the normal cell replication cycle, but can also be enhanced by the occurrence of different events such as uv light and other inducers of DNA breaks, or the presence of certain agents such as different chemical inducers. Many of these inducers cause DSBs to occur indiscriminately in the genome, and DSBs can often be induced and repaired in normal cells. During repair, the original sequence can be reconstructed with full fidelity, but in some cases small insertions or deletions (called "insertions/deletions") are introduced at the DSB sites.

DSBs can also be specifically induced at specific locations, as in the case of the endonuclease systems described herein, which can be used to cause targeted or preferential genetic modification events at selected chromosomal locations. The tendency of homologous sequences to recombine in the context of DNA repair (and replication) can be exploited in many cases and is the basis for one application of gene editing systems such as CRISPR, where homology-directed repair is used to insert a sequence of interest (which is provided by the use of a "donor" polynucleotide) into a desired chromosomal location.

Desired deletions can also be achieved using regions of homology between particular sequences (which may be small regions of "micro homology", which may comprise as few as 10 base pairs or less). For example, a single DSB may be introduced at a site that exhibits little homology to nearby sequences. During normal repair of such DSBs, the consequence of this high frequency is deletion of the inserted sequence, due to the fact that the DSBs and the accompanying cellular repair process promote recombination.

However, in certain cases, selection of target sequences within the region of homology may also result in much larger deletions, including gene fusions (when the deletion is in the coding region), which may or may not be desirable in particular cases.

The examples provided herein further illustrate the selection of different target regions for the establishment of DSBs designed to induce insertions, deletions or mutations that result in a reduction or elimination of PCSK9 protein activity, and the selection of specific target sequences within such regions designed to minimize off-target events relative to on-target events.

Human cells

To ameliorate dyslipidemia or any disorder associated with PCSK9, the primary target of gene editing is human cells as described and explained herein. For example, in an ex vivo method, the human cells may be somatic cells, which, after modification using the described techniques, may give rise to differentiated cells, such as hepatocytes or progenitor cells. For example, in an in vivo method, the human cells may be hepatocytes, kidney cells, or cells from other affected organs.

By performing gene editing in autologous cells that originate from a patient in need thereof and that have thus been perfectly matched to said patient, it is possible to generate cells that: which can be safely reintroduced into the patient and which is effective to produce a population of cells that will effectively ameliorate one or more clinical conditions associated with the patient's disease.

Stem cells are capable of multiplying and producing more progenitor cells, which in turn have the ability to produce large numbers of blasts that in turn can produce differentiated or differentiable daughter cells. The daughter cells themselves can be induced to propagate and produce progeny that subsequently differentiate into one or more mature cell types while also retaining one or more cells with parental developmental potential. The term "stem cell" then denotes a cell: which in certain cases have the ability or potential to differentiate into a more specialized or differentiated phenotype, and which retain the ability to reproduce substantially undifferentiated in certain cases. In one aspect, the term progenitor or stem cell refers to a generalized mother cell, the progeny (progeny) of which are specialized (often in different orientations) by differentiation, e.g., by fully acquiring individual characteristics, as occurs in the progressive diversification of embryonic cells and tissues. Differentiation of cells is a complex process that typically occurs through many cell divisions. Differentiated cells may be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells can be considered a stem cell, the range of cell types that can be produced by each can vary considerably. Some differentiated cells also have the ability to produce cells with greater developmental potential. Such ability may be natural or may be induced manually after treatment with various factors. In many biological cases, stem cells can also be "pluripotent" in that they can produce progeny of more than one distinct cell type, but this is not required for "stem-ness".

Self-renewal can be another important aspect of stem cells. In theory, self-renewal can occur by either of two major mechanisms. Stem cells can divide asymmetrically, with one progeny retaining the stem state and the other expressing some other specific function and phenotype that is unique. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thereby maintaining some stem cells in the population as a whole, while other cells in the population produce only differentiated progeny. Typically, a "progenitor cell" has a more primitive (i.e., at an earlier step along the developmental pathway or progression than a fully differentiated cell) cell phenotype. Progenitor cells also often have significant or very high proliferative potential. Progenitor cells can give rise to a variety of different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

In the context of cellular ontogeny, the adjective "differentiated" or "differentiating" is a relative term. A "differentiated cell" is a cell that: it has progressed further down the developmental pathway relative to the cells to which it is compared. Thus, stem cells can differentiate into lineage restricted precursor cells (such as myocyte progenitor cells), which in turn can further differentiate down the pathway into other types of precursor cells (such as myocyte precursors), and then into terminally differentiated cells, such as myocytes, that play a unique role in certain tissue types and may or may not retain the ability to further proliferate.

Induced pluripotent stem cells

The genetically engineered human cells described herein are induced pluripotent stem cells (ipscs). One advantage of using ipscs is that the cells can be derived from the same individual to whom the progenitor cells are administered. That is, somatic cells can be obtained from an individual, reprogrammed to induced pluripotent stem cells, and then re-differentiated into progenitor cells (e.g., autologous cells) to be administered to the individual. Because the progenitor cells are substantially derived from an autologous source, the risk of graft rejection or allergic response may be reduced compared to using cells from another individual or group of individuals. In addition, the use of ipscs would eliminate the need for cells derived from embryonic sources. Thus, in one aspect, the stem cells used in the disclosed methods are not embryonic stem cells.

Although differentiation is generally irreversible in a physiological context, several methods have recently been developed to reprogram somatic cells to ipscs. Exemplary methods are known to those skilled in the art and are briefly described below.

The term "reprogramming" refers to a process of altering or reversing the differentiation state of a differentiated cell (e.g., a somatic cell). In other words, reprogramming refers to the process of driving cells back into differentiation into more undifferentiated or primitive cell types. It should be noted that placing many primary cells in culture can result in some loss of fully differentiated characteristics. Thus, simply culturing such cells, as included in the term differentiated cells, does not render these cells undifferentiated (e.g., undifferentiated) or pluripotent. The shift of differentiated cells to pluripotency requires reprogramming of the stimulation beyond that which results in a partial loss of differentiation characteristics in culture. Reprogrammed cells are also characterized by the ability to be passaged for long periods without loss of growth potential, relative to the primary cell parent (which typically has the ability to divide only a limited number of times in culture).

The cells to be reprogrammed may be allowed to partially or eventually differentiate prior to reprogramming. Reprogramming may include a complete reversal of the differentiated state of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. Reprogramming may include a complete or partial reversal of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming may result in the expression of a particular gene by the cell, the expression of which further facilitates reprogramming. In certain examples described herein, reprogramming of a differentiated cell (e.g., a somatic cell) can cause the differentiated cell to assume an undifferentiated state (e.g., to be an undifferentiated cell). The resulting cells are referred to as "reprogrammed cells" or "induced pluripotent stem cells (ipscs or iPS cells)".

Reprogramming may involve alteration (e.g., reversal) of at least some heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic blots, etc., that occur during cell differentiation. Reprogramming is different from simply maintaining an existing undifferentiated state of cells that are already pluripotent or maintaining an existing less than fully differentiated state of cells that are already pluripotent (e.g., myogenic stem cells). Reprogramming is also different from promoting self-renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods described herein may also be used for such purposes in certain instances.

Many methods are known in the art that can be used to generate pluripotent stem cells from somatic cells. Any such method of reprogramming somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

Reprogramming methods to generate pluripotent cells using defined combinations of transcription factors have been described. Through direct transduction of Oct4, Sox2, Klf4, and c-Myc, mouse somatic cells can be transformed into ES cell-like cells with expanded developmental potential; see, for example, Takahashi and Yamanaka, Cell 126(4): 663-76 (2006). ipscs are similar to ES cells in that they restore pluripotency-related transcriptional lines and many epigenetic views. In addition, the mouse iPSC meets all standard determinations of pluripotency: specifically, Cell types that differentiate into three germ layers in vitro, teratoma formation, contribute to chimerism, germline transmission [ see, e.g., Maherali and Hochedlinger, Cell Stem Cell. 3(6):595-605 (2008) ], and tetraploid complementation.

Human ipscs are available using similar transduction methods, and the transcription factor triplet group OCT4, SOX2 and NANOG have been established as a core set of transcription factors that control pluripotency; see, e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57 (2014); Barrett et al, Stem Cells Trans Med3:1-6 sctm.2014-0121 (2014); Focosi et al,Blood Cancer Journal 4: e211 (2014); and references cited therein. Historically, iPSC production has been achieved by introducing nucleic acid sequences encoding stem cell-associated genes into adult somatic cells using viral vectors.

Ipscs can be produced or derived from terminally differentiated somatic cells, as well as from adult stem cells or adult stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming. In such cases, it may not be necessary to include as many reprogramming factors as are needed to reprogram terminally differentiated cells. In addition, reprogramming can be induced by non-viral introduction of a reprogramming factor, e.g., by introducing the protein itself, or by introducing a nucleic acid encoding a reprogramming factor, or by introducing messenger RNA that produces a reprogramming factor post-translationally (see, e.g., Warren et al,Cell Stem Cell, 7(5) 618-30 (2010). Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-associated genes including, for example, Oct-4 (also known as Oct-3/4 or Pouf51), Sox2, Sox3, Sox15, Sox18, NANOG, Klfl, Klf2, Klf4, Klf5, NR5a2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN 28. Reprogramming using the methods and compositions described herein may further comprise introducing into the somatic cell one or more of the following: oct-3/4, a member of the Sox family, a member of the Klf family, and a member of the Myc family. The methods and compositions described herein may also comprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, where cells differentiated from reprogrammed cells are to be used in, for example, human therapy, in one aspect, the reprogramming is not achieved by a method of altering the genome. Thus, in such instances, reprogramming can be achieved, for example, without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a starting cell population may be enhanced by the addition of various agents (e.g., small molecules), as demonstrated in: shi et al,Cell-Stem Cell 2:525-,Nature Biotechnology 26(7):795-,Cell-Stem Cell 3: 132-135 (2008). Thus, an agent or combination of agents that enhances the efficiency or rate of induced pluripotent stem cell production may be used to produce patient-specific or disease-specific ipscs. Some non-limiting examples of agents that enhance reprogramming efficiency include soluble Wnt, Wnt conditioned media, BIX-01294(G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, Histone Deacetylase (HDAC) inhibitors, valproic acid, 5' -azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and Trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancers include: suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, Vorinostat) and other hydroxamic acids), BML-210, Depudecin (e.g., (-) -Depudecin), HC toxin, Nullscript (4- (l, 3-dioxo-lH, 3H-benzo [ de ] isoquinolin-2-yl) -N-hydroxybutyramide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VP A) and other short chain fatty acids), Scriptaid, suramin sodium, trichostatin A (TSA), APHA compounds 8, Apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamide (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (meta-carboxy cinnamic acid dioxymic acid), JNJ16241199, Tubacin, A-161906, progxamide, oxamflatin, 3-Cl-UCHA (e.g., 6- (3-chlorophenylureido) capronic hydroxamic acid), AOE (2-amino-8-oxo-9, 10-epoxydecanoic acid), CHAP31, and CHAP 50. Other reprogramming enhancers include, for example, dominant negative forms of HDACs (e.g., catalytically inactive forms), siRNA inhibitors of HDACs, and antibodies that specifically bind HDACs. Such inhibitors are available, for example, from BIOMOL International, Fukasawa, Merck biosciences, Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for expression of stem cell markers. Such expression in cells derived from somatic cells identifies the cells as induced pluripotent stem cells. The stem cell marker may be selected from the non-limiting set comprising SSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto, Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, cells expressing Oct4 or Nanog were identified as pluripotent. Methods for detecting the expression of such markers may include, for example, RT-PCR and immunological methods of detecting the presence of the encoded polypeptide, such as western blot or flow cytometric analysis. Detection may involve not only RT-PCR, but also detection of protein markers. Intracellular markers can be optimally identified by RT-PCR or protein detection methods such as immunocytochemistry, whereas cell surface markers are easily identified by e.g. immunocytochemistry.

The pluripotent stem cell characteristics of the isolated cells can be confirmed by a test that evaluates the ability of ipscs to differentiate into cells of each of the three germ layers. As an example, teratoma formation in nude mice can be used to evaluate the pluripotent properties of isolated clones. The cells may be introduced into nude mice, and histology and/or immunohistochemistry may be performed on tumors arising from the cells. For example, the growth of a tumor comprising cells from all three germ layers further indicates that the cells are pluripotent stem cells.

Liver cell

In certain aspects, the genetically engineered human cell described herein is a hepatocyte. Hepatocytes are cells of the main parenchymal tissue of the liver. The liver cells account for 70-85% of the mass of the liver. These cells are involved: protein synthesis; protein storage; the conversion of carbohydrates; synthesis of cholesterol, bile salts and phospholipids; detoxification, modification and excretion of exogenous and endogenous substances; as well as initiation and secretion of bile formation.

Preparation of patient-specific iPSC

One step of the ex vivo method of the invention may comprise preparing patient-specific iPS cells, patient-specific iPS cells or a patient-specific iPS cell line. There are many established methods in the art for making patient-specific iPS cells, as described in the following references: takahashi and Yamanaka 2006, Takahashi, Tanabe et al.2007. For example, the preparing step may include: a) isolating somatic cells, such as skin cells or fibroblasts, from the patient; and b) introducing a set of pluripotency-associated genes into said somatic cells to induce said cells to become pluripotent stem cells. The group of pluripotency-associated genes is one or more genes selected from the group consisting of: OCT4, SOX1, SOX2, SOX3, SOX15, SOX18, NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT, and LIN 28.

Performing a biopsy or aspiration of the liver or bone marrow of a patient

A biopsy or aspirate is a sample of tissue or fluid that is removed from the body. There are many different kinds of biopsies or aspirations. They almost exclusively involve the use of sharp tools to remove small amounts of tissue. If the biopsy is to be on the skin or other sensitive area, the anesthesia machine may be applied first. The biopsy or aspiration may be performed according to any of the methods known in the art. For example, in a biopsy, a needle is injected through the abdominal skin into the liver, thereby capturing the liver tissue. For example, in bone marrow aspiration, a large needle is used to enter the pelvic bone to collect bone marrow.

Isolation of liver-specific progenitor or primary hepatocytes

Liver-specific progenitor cells and primary hepatocytes can be isolated according to any method known in the art. For example, human hepatocytes are isolated from fresh surgical specimens. Healthy liver tissue is used to isolate hepatocytes by collagenase digestion. The resulting cell suspension was filtered through a 100-mm nylon mesh and centrifuged at 50g for 5 minThe pellet is precipitated, resuspended, and washed 2-3 times in cold wash medium. Human hepatic stem cells are obtained by culturing hepatic cells obtained from fresh liver preparations under stringent conditions. Hepatocytes plated on collagen-coated plates were cultured for 2 weeks. After 2 weeks, surviving cells were removed and characterized for expression of stem cell markers (Herrera et al), STEM CELLS 2006;24: 2840 -2850)。

Isolation of mesenchymal Stem cells

Mesenchymal stem cells may be isolated according to any method known in the art, such as from the bone marrow or peripheral blood of a patient. For example, bone marrow aspirate may be collected into a syringe with heparin. Cells can be washed and centrifuged on Percoll. Cells can be cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Low glucose) containing 10% Fetal Bovine Serum (FBS) (Pitttinger MF, Mackay AM, Beck SC et al, Science 1999; 284:143-147)。

Genetically modified cells

The term "genetically modified cell" means a cell comprising at least one genetic modification introduced by genome editing (e.g., using CRISPR/Cas9 or CRISPR/Cpf1 systems). In certain ex vivo examples herein, the genetically modified cell can be a genetically modified progenitor cell. In certain in vivo examples herein, the genetically modified cell can be a genetically modified hepatocyte. Genetically modified cells comprising exogenous genome-targeting nucleic acids and/or exogenous nucleic acids encoding genome-targeting nucleic acids are contemplated herein.

The term "control treatment population" describes a population of cells that have been treated with the same medium, viral induction, nucleic acid sequence, temperature, confluence, flask size, pH, etc., except for the addition of genome editing components. Any method known in the art may be used to measure PCSK9 gene transcription or protein expression or activity, e.g., western blot analysis of PCSK9 protein or real-time PCR for quantification of PCSK9 mRNA.

The term "isolated cell" means a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cells can be cultured in vitro, e.g., under defined conditions, or in the presence of other cells. Optionally, the cell may be introduced later into a second organism or reintroduced into the organism from which it was isolated (or its source cell).

The term "isolated population" with respect to an isolated cell population means a cell population that has been removed and isolated from a mixed or heterogeneous cell population. In certain instances, the isolated population may be a substantially pure population of cells as compared to a heterogeneous population from which the cells are isolated or enriched. In certain instances, the isolated population can be an isolated population of human progenitor cells, e.g., a substantially pure population of human progenitor cells as compared to a heterogeneous population of cells comprising human progenitor cells and source cells of human progenitor cells.

The term "substantially enhanced" with respect to a specific cell population means a cell population wherein the occurrence of a specific type of cell is increased at least 2 fold, at least 3-, at least 4-, at least 5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, at least 20-, at least 50-, at least 100-, at least 400-, at least 1000-, at least 5000-, at least 20000-, at least 100000-fold or more relative to a pre-existing level or a reference level, depending on e.g. the desired level of such cells for ameliorating dyslipidemia.

The term "substantially enriched" with respect to a particular cell population means a cell population that is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or more relative to the cells that make up the total cell population.

The term "substantially enriched" or "substantially pure" with respect to a particular cell population means a cell population that is at least about 75%, at least about 85%, at least about 90%, or at least about 95% pure relative to the cells comprising the total cell population. That is, the term "substantially pure" or "substantially purified" with respect to a population of progenitor cells means a population of cells that contains less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, or less than 1% of cells that are not progenitor cells as defined by the term herein.

Genomic edited ipscs differentiate into other cell types

Another step of the ex vivo method of the invention may comprise differentiating the genome-edited ipscs into hepatocytes. The differentiation step may be performed according to any method known in the art. For example, hipscs are differentiated into definitive endoderm using a variety of treatments, including activin and B27 complement (Life Technology). Causing further differentiation of definitive endoderm into hepatocytes, the treatment comprising: FGF4, HGF, BMP2, BMP4, oncostatin M, dexamethasone (Dexametason), etc. (Duan et al, STEM CELLS; 2010;28: 674-one 686, Ma et al, STEM CELLS TRANSLATIONAL MEDICINE)2013; 2:409-419)。

Genome-edited differentiation of mesenchymal stem cells into hepatocytes

Another step of the ex vivo method of the present invention may comprise differentiating the genome-edited mesenchymal stem cells into hepatocytes. The differentiation step may be performed according to any method known in the art. For example, hMSC (Sawitza I et al) is treated with various factors and hormones including insulin, transferrin, FGF4, HGF, bile acids, Sci Rep. 2015; 5: 13320)。

Transplanting cells into a patient

another step of the ex vivo method of the invention may comprise transplanting said hepatocytes into a patient. This implantation step can be accomplished using any implantation method known in the art. For example, the genetically modified cells can be injected directly into the blood of a patient or otherwise administered to a patient.

Another step of the ex vivo method of the invention comprises transplanting said progenitor cells or primary hepatocytes into a patient. This implantation step can be accomplished using any implantation method known in the art. For example, the genetically modified cells can be injected directly into the liver of a patient or otherwise administered to a patient.

Formulation and delivery

Pharmaceutically acceptable carriers

Ex vivo methods of administering progenitor cells to an individual contemplated herein include the use of therapeutic compositions comprising progenitor cells.

The therapeutic composition may contain a physiologically tolerable carrier as well as the cellular composition and optionally at least one additional biologically active agent as described herein dissolved or dispersed therein as an active ingredient. In certain instances, the therapeutic composition is not substantially immunogenic when administered to a mammalian or human patient for therapeutic purposes, unless so desired.

in general, progenitor cells described herein can be administered as a suspension containing a pharmaceutically acceptable carrier. One skilled in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservatives, preservatives or other agents in amounts that substantially interfere with the viability of the cells to be delivered to an individual. The cell-containing formulation may include, for example, osmotic buffers that allow for maintenance of cell membrane integrity, and optionally, nutrients that maintain cell viability or enhance graft implantation after administration. Such formulations and suspensions are known to those skilled in the art and/or may be suitable for use with progenitor cells as described herein using routine experimentation.

The cell composition may also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients in amounts suitable for use in the methods of treatment described herein.

The additional agent included in the cell composition may include a pharmaceutically acceptable salt of the component therein. Pharmaceutically acceptable salts include the acid addition salts formed with the following acids (formed with the free amino groups of the polypeptide): inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no substances other than the active ingredient and water, or containing buffering agents such as sodium phosphate at physiological pH, physiological saline, or both, such as phosphate buffered saline. Still further, the aqueous carrier may contain more than one buffering salt, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also contain a liquid phase in addition to and excluding water. Examples of such further liquid phases are glycerol, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cellular composition effective to treat a particular disorder or condition may depend on the nature of the disorder or condition and may be determined by standard clinical techniques.

Guide RNA preparation

The guide RNAs of the present invention may be formulated with pharmaceutically acceptable excipients such as carriers, solvents, stabilizers, adjuvants, diluents, and the like, depending on the particular mode of administration and dosage form. The guide RNA composition can be formulated to achieve a physiologically compatible pH, and a range from a pH of about 3 to a pH of about 11, about pH 3 to about pH 7, depending on the formulation and route of administration. In some cases, the pH may be adjusted to a range of about pH 5.0 to about pH 8. In certain instances, the composition may comprise a therapeutically effective amount of at least one compound as described herein, and one or more pharmaceutically acceptable excipients. Optionally, the composition may comprise a combination of compounds described herein, or may include a second active ingredient useful for treating or preventing bacterial growth (such as, but not limited to, an antibacterial or antimicrobial agent), or may include a combination of agents of the present invention.

Suitable excipients include, for example, carrier molecules comprising large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles. Other exemplary excipients may include antioxidants (such as, but not limited to, ascorbic acid), chelating agents (such as, but not limited to, EDTA), carbohydrates (such as, but not limited to, dextrins, hydroxyalkyl celluloses, and hydroxyalkyl methylcelluloses), stearic acid, liquids (such as, but not limited to, oils, water, saline, glycerol, and ethanol), wetting or emulsifying agents, pH buffering substances, and the like.

Delivery of

The guide RNA polynucleotide (RNA or DNA) and/or endonuclease polynucleotide (RNA or DNA) can be delivered by viral or non-viral delivery vehicles known in the art. Alternatively, the endonuclease polypeptide may be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles. In a further alternative aspect, the DNA endonuclease may be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crrnas and tracrRNA together.

The polynucleotides may be delivered by non-viral delivery vehicles including, but not limited to, nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, Gene therapy, 18: 1127-.

For the polynucleotides of the invention, the agent may be selected from any of those taught, for example, in international application PCT/US 2012/069610.

Polynucleotides such as guide RNAs, sgrnas, and mRNA encoding endonucleases can be delivered to cells or patients by Lipid Nanoparticles (LNPs).

LNP denotes any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm or 25 nm. Alternatively, the nanoparticles may be in the size range of 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic or neutral lipids. Neutral lipids such as fusogen (fusogenic) phospholipid DOPE or membrane component cholesterol may be included in LNPs as 'helper lipids' to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy due to poor stability and rapid clearance, and the development of inflammatory or anti-inflammatory responses.

LNPs can also comprise hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids known in the art can be used to produce LNPs. Examples of lipids used for the production of LNP are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE and GL 67A-DOPE-DMPE-polyethylene glycol (PEG). Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1 and 7C 1. Examples of neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are: PEG-DMG, PEG-CerC14 and PEG-CerC 20.

The lipids can be combined in any number of molar ratios to produce LNP. In addition, the polynucleotides can be combined with lipids in a wide range of molar ratios to produce LNPs.

As previously described, the targeting polypeptide and the genome-targeted nucleic acid can each be administered to a cell or patient, respectively. In another aspect, the targeting polypeptide may be pre-complexed with one or more guide RNAs, or one or more crrnas and tracrRNA together. The pre-complexed material may then be administered to a cell or patient. Such a pre-complexed substance is called ribonucleoprotein particles (RNPs).

RNA is capable of forming specific interactions with RNA or DNA. Although this property is used in many biological processes, it is accompanied by the risk of promiscuous interactions in a nucleic acid-rich cellular environment. One solution to this problem is the formation of ribonucleoprotein particles (RNPs) in which the RNA is pre-complexed with an endonuclease. Another benefit of the RNP is protection of the RNA from degradation.

The endonuclease in the RNP may be modified or unmodified. Likewise, the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerous modifications are known in the art and may be used.

The endonuclease and sgRNA are typically combined in a 1:1 molar ratio. Alternatively, the endonuclease, crRNA and tracrRNA are typically combined in a 1:1:1 molar ratio. However, a wide range of molar ratios can be used to produce RNP.

AAV (adeno-associated virus)

Recombinant adeno-associated virus (AAV) vectors can be used for delivery. Techniques for producing rAAV particles are standard in the art, wherein the AAV genome to be packaged (which includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions) is provided to the cell. Production of rAAV generally requires that the following components be present within a single cell (referred to herein as a packaging cell): rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not within), and helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype other than the rAAV genomic ITRs, including, but not limited to, the AAV serotypes described herein. Production of pseudotyped rAAV is disclosed, for example, in international patent application publication No. WO 01/83692.

AAV serotypes

An AAV particle packaged with a polynucleotide encoding a composition of the invention (e.g., an endonuclease, donor sequence, or RNA guide molecule of the invention) can comprise or be derived from any natural or recombinant AAV serotype. According to the invention, the AAV particle may utilize or be based on a serotype selected from any of the following serotypes and variants thereof, including, but not limited to, AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60, AAV161.6/hu.61, AAV 7-7/rh.53948, AAV1-8/rh.49, AAV2, AAV AAV2.5T, AAV 4662-15/hu.62, AAV 1.223, AAV 1.6/hu.223, AAV 2-223, AAV 2/hu.223, AAV 2-1.223, AAV 2/hu.5, AAV 639, AAV 2.223, AAV 2/hu.5, AAV 2.5/hu.5, AAV2, AAV 639, AAV 2/hu.5/5/61, AAV 2.223.5/hu.5, AAV2, AAV 2.5/hu.5/5/61, AAV 2.223.5, AAV2, AAV 2.5/hu.5/5, AAV2, AAV 2.5/hu.5/61, AAV2, AAV 2.223.5, AAV 2.5/61, AAV-11/rh.53, AAV-3, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV-9/rh.52, AAV3, AAV-19/rh.55, AAV42.12, AAV-10, AAV-11, AAV-12, AAV-13, AAV-15, AAV-1 b, AAV-2, AAV-3 a, AAV-3 b, AAV-4, AAV-5 a, AAV-5 b, AAV-6 b, AAV-8, AAV-aa, AAV-1, AAV-12, AAV-20, AAV-21, AAV-23, AAV-25, AAV-5, AAV-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV-8/r 11.64, AAV 8/rh.64, rh-9.64, AAV 20/rh.52, AAV-8/hu.54, AAV-5, AAV-10, AAV-11, AAV-12, AAV-1, AAV-13, AAV-3, AAV-, AAV52/hu.19, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV 9.7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV DJVA 3.3.4, AAV 3.5, AAV 3.7.45, AAV9.47, AAV5, AAV2 VVHV 1, AAV2, hEVhEaVhEaVhEaVhEaV 1.7, hEaVhEaVhEaVhEaVhEaVhEaV 1.7, AAV 2-7, AAV2 VhEvhEvhEvhEvhEv7.15, AAV 2-7.15, AAV2-5, AAV2 vhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEv7.7.7, AAV2 vhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEvhEv7.7.7.7.7.7.7, AAV2 vhEvhEvhEvhEvhEvhEvhEvhEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEvEv, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, Vhu.37, AAVhu.39, Vhu.4, AAVhu.40, Vhu.41, AAVhu.42, AAVhu.43, Vhu.44, Vhu.1, Vhu.Vhu.2, AAVhu-4858, AAVhu-48, AAVhu-4435, AAVhu-35, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, Vhu.41, AAVhu.42, AAVhu-AAVhu.43, AAVhu-8, AAVhu-V-7, AAVhu-V-2, AAVhu-3, AAVhu-2, AAVhu-V-2, AAVhu-V-8, AAVhu-2, AAVhu-V-2, AAVhu-8, AAVhu-V-2, AAVhu-V-2, AAVhu-3-V, AAAAV-LK 18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, Vrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAh.24, AAh.25, AAh.2R, AAh.31, AAh.32, AAVrh.33, AAVrh.34, Vrh, Vrh.38, AAVrh 52, AAVrh 52, AAVrh 52, AAVrh 52, AAVrh 52, AAVrh 52, AAVrh 52, AAVrh, AA, AAV-LK16, AAAV, AAV shuttle 100-1, AAV shuttle 100-2, AAV shuttle 100-3, AAV shuttle 100-7, AAV shuttle 10-2, AAV shuttle 10-6, AAV shuttle 10-8, AAV SM 100-10, AAV SM 100-3, AAVSM 10-1, AAV SM 10-2, and/or AAV SM 10-8.

In some aspects, the AV serotype may be or have a mutation in the AAV9 sequence as described by N pulcherla et al (Molecular Therapy 19(6):1070 and 1078 (2011)), such as but not limited to AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV 9.84.

In some aspects, the AAV serotype may be or have a sequence as described in U.S. patent No. US 6156303, such as but not limited to AAV3B (SEQ ID NOs: 1 and 10 of US 6156303), AAV6 (SEQ ID NOs: 2,7 and 11 of US 6156303), AAV2 (SEQ ID NOs: 3 and 8 of US 6156303), AAV3A (SEQ ID NOs: 4 and 9 of US 6156303) or derivatives thereof.

In some aspects, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al (Journal of Virology 82(12): 5887-, (2) R587Q, wherein arginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln), and (3) R590T, wherein arginine (R; Arg) at amino acid 590 is changed to threonine (T; Thr).

In some embodiments, the AAV serotype may be or have a sequence as described in international publication number WO2015121501, such as, but not limited to, a true AAV (ttava) (SEQ ID NO:2 of WO 2015121501), "UPenn AAV 10" (SEQ ID NO:8 of WO 2015121501), "japanese AAV 10" (SEQ ID NO:9 of WO 2015121501), or variants thereof.

In accordance with the present invention, AAV capsid serotype selection or use can be from a variety of species. In one embodiment, the AAV may be an avian AAV (aaav). The AAAV serotype can be or have a sequence as described in U.S. patent No. US 9238800, such as but not limited to AAAV (SEQ ID NOs: 1,2, 4,6, 8, 10, 12 and 14 of US9,238,800) or variants thereof.

In one embodiment, the AAV may be bovine AAV (baav). The BAAV serotype may be or have a sequence as described in US patent No. US9,193,769, such as but not limited to BAAV (SEQ ID NOs: 1 and 6 of US 9193769) or variants thereof. The BAAV serotype may be or have a sequence as described in US patent No. US7427396, such as but not limited to BAAV (SEQ ID NOs: 5 and 6 of US 7427396) or variants thereof.

In one embodiment, the AAV may be a goat AAV. The goat AAV serotype can be or have a sequence as described in U.S. Pat. No. US7427396, such as but not limited to goat AAV (SEQ ID NO:3 of US 7427396) or a variant thereof.

In other embodiments, the AAV may be engineered as a hybrid AAV from two or more parental serotypes. In one embodiment, the AAV may be AAV2G9, comprising sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be or have a sequence as described in U.S. patent publication No. US 20160017005.

In one embodiment, the AAV may be a serotype produced from an AAV9 capsid library having mutations in amino acids 390-627 (numbering VP 1), as described by Pulichela et al (Molecular Therapy 19(6):1070 1078 (2011.) the serotype and corresponding nucleotide and amino acid substitutions may be, but are not limited to, AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F36417), AAV 9.1765 (A1233672, A1314T, A1642T, C T; Q36412, Q T, T T, A587T, AAV9.6 (T12336447; T T, AAV T; AAV 361772; AAV T, AAV 361772; AAV T, AAV T; AAV T, AAV T, 361772, AAV T, V T, AAV T, V3617, V T, V3617, V T, V36, AAV9.24 (T1507, T1521; W503), AAV9.26 (A1337, A1769; Y446, Q590), AAV9.33 (A1667; D556), AAV9.34(A1534, C1794; N512), AAV9.35 (A1289, T1450, C1494, A1515, C1794, G1816; Q430, Y484, N98, V606), AAV9.40 (A1694, E565), AAV9.41 (A1348, T1362; T450), AAV9.44 (A1684, A1701, A1737; N562, K567), AAV9.45(A1492, C; N498, L602), AAV9.46 (G1441, T1525, T9; G481, W509, L517), 9.47 (G1, G1248, A1669, C1745, S1749, S1441, G1441, T1525, T1529, T1639, W482, L517), AAV1, G1638, G1669, C3553, C3559, AAV 3253, AAV 3259, AAV 3253, AAV 3259, AAV3, AAV 3253, AAV3, C3553, AAV 3270, AAV3, C3553, AAV3, l537), AAV9.55 (T1605; F535), AAV9.58 (C1475, C1579; T492, H527), AAV.59 (T1336; Y446), AAV9.61 (A1493; N498), AAV9.64 (C1531, A1617; L511), AAV9.65 (C1335, T1530, C1568; A523), AAV9.68 (C1510; P504), AAV9.80 (G1441; G481), AAV9.83 (C1402, A1500; P468, E500), AAV9.87 (T1464, T1468; S490), AAV9.90 (A1196; Y399), AAV9.91 (T1316, A1583, C1782, T1806; L439, K528), AAV9.93 (A1273, A1421, A1718, C1712, G2, A1734, A1832, A611, S578, C611, T578, T611, T578, K528), AAV9.93 (A1273, A1631, A1635, P578, P582, and AAV 9.95).

In one embodiment, the AAV may be a serotype comprising at least one AAV capsid CD8+ T-cell epitope. As a non-limiting example, the serotype may be AAV1, AAV2, or AAV 8.

In one example, the AAV may be a variant, such as PHP.A or PHP.B as described in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

In one example, the AAV can be a serotype selected from the group consisting of SEQ ID NO 4,734-5,302 and any of those found in Table 2.

In one example, the AAV can be encoded by a sequence, fragment or variant as disclosed in SEQ ID NO, 734-5,302 and Table 2.

General principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, curr. Topics in Microbiological. andImmunol, 158: 97-129). Various schemes are described in Ratschin et al,Mol, cell, biol, 4:2072 (1984); Hermonat et al,Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al,Mo1 cell biol 5:3251 (1985); McLaughlin et al,J. Virol, 62:1963 (1988); and Lebkowski et al,1988 mol, cell, biol., 7:349 (1988), Samulski et al(1989, j. virol, 63: 3822-; U.S. Pat. No. 5,173,414, WO 95/13365 and corresponding U.S. Pat. No. 5,658.776, WO 95/13392, WO 96/17947, PCT/US98/18600, WO 97/09441 (PCT/US 96/14423); WO 97/08298 (PCT/US 96/13872); WO 97/21825 (PCT/US 96/20777); WO 97/06243 (PCT/FR 96/01064); WO 99/11764, Perrin et al(1995) Vaccine 13:1244-(1993) Human Gene Therapy 4:609-615, Clark et al(1996) Gene Therapy 3: 1124-; U.S. patent nos. 5,786,211; U.S. patent nos. 5,871,982; and U.S. Pat. No. 6,258,595.

AAV vector serotypes can be matched to target cell types. For example, the following exemplary cell types can be transduced with, inter alia, a designated AAV serotype.

TABLE 2 tissue/cell types and serotypes

Tissue/cell type	Serotype
		Liver disease	AAV3、AAV5、AAV8、AAV9
Skeletal muscle	AAV1、AAV7、AAV6、AAV8、AAV9
		Central nervous system	AAV5、AAV1、AAV4、AAV9
RPE	AAV5、AAV4
		Photoreceptor cell	AAV5
Lung (lung)	AAV9
		Heart and heart	AAV8
Pancreas gland	AAV8
		Kidney (Kidney)	AAV2、AAV8
Hematopoietic stem cells	AAV6

In addition to adeno-associated viral vectors, other viral vectors can be used. Such viral vectors include, but are not limited to, lentiviruses, alphaviruses, enteroviruses, pestiviruses, baculoviruses, herpesviruses, epstein-barr viruses, papovaviruses, poxviruses, vaccinia viruses, and herpes simplex viruses.

In certain aspects, Cas9 mRNA, sgRNA targeting one or both loci in the PCSK9 gene, and donor DNA can each be formulated separately into lipid nanoparticles, or both can be co-formulated into one lipid nanoparticle.

In certain aspects, Cas9 mRNA can be formulated into lipid nanoparticles, while sgrnas and donor DNA can be delivered in AAV vectors.

Options are available to deliver Cas9 nuclease as a DNA plasmid, as mRNA, or as a protein. The guide RNA may be expressed from the same DNA, or may also be delivered as RNA. The RNA may be chemically modified to alter or increase its half-life, or to reduce the likelihood or extent of an immune response. The endonuclease protein can form a complex with the gRNA prior to delivery. Viral vectors allow for efficient delivery; the cleaved form of Cas9 and the smaller ortholog of Cas9 can be packaged into AAV as can the donor of HDR. There are also a number of non-viral delivery methods that can deliver each of these components, or non-viral and viral methods can be employed in tandem. For example, nanoparticles can be used to deliver proteins and guide RNAs, while AAV can be used to deliver donor DNA.

Administration and administration

The terms "administering," "introducing," and "transplanting" are used interchangeably in the context of placing cells (e.g., progenitor cells) into an individual by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, to produce a desired effect. Cells (e.g., progenitor cells) or their differentiated progeny may be administered by any suitable route that results in delivery to a desired location in an individual where at least a portion of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to an individual may be as short as a few hours, e.g., 24 hours, to several days, to as long as several years, or even the survival of the patient, i.e., long-term graft implantation. For example, in certain aspects described herein, an effective amount of hepatic progenitors is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

The terms "individual", "subject", "host" and "patient" are used interchangeably herein and refer to any individual for whom diagnosis, treatment or therapy is desired. In certain aspects, the subject is a mammal. In certain aspects, the subject is a human.

When provided prophylactically, the progenitor cells described herein can be administered to an individual prior to any symptoms of dyslipidemia. Thus, prophylactic administration of hepatic progenitor cell populations is useful for preventing dyslipidemia.

The progenitor cell population administered according to the methods described herein can comprise allogeneic progenitor cells obtained from one or more donors. Such progenitor cells may be of any cell or tissue origin, such as liver, muscle, heart, etc. By "allogeneic" is meant progenitor cells or a biological sample comprising progenitor cells obtained from one or more different donors of the same species (where the genes at one or more loci are not the same). For example, the hepatic progenitor population administered to an individual may be derived from a more unrelated donor individual, or from one or more non-identical siblings. In certain cases, syngeneic progenitor cell populations may be used, such as those obtained from genetically identical animals or from monozygotic twins. The progenitor cells can be autologous cells; that is, the progenitor cells are obtained or isolated from one individual and administered to the same individual, i.e., the donor and recipient are the same person.

The term "effective amount" means the amount of the population of progenitor cells or their progeny required to prevent or reduce at least one or more signs or symptoms of dyslipidemia, and means an amount of the composition sufficient to provide the desired effect (e.g., treating an individual with dyslipidemia). The term "therapeutically effective amount" thus denotes an amount of progenitor cells or a composition comprising progenitor cells that is sufficient to promote a particular effect when administered to a typical individual, such as an individual having or at risk of dyslipidemia. An effective amount also includes an amount sufficient to prevent or delay the development of, alter the progression of (e.g., without limitation, slow the progression of, or reverse the symptoms of) a disease. It will be appreciated that the appropriate "effective amount" for any given case may be determined by one of ordinary skill in the art using routine experimentation.

For use in the various aspects described herein, an effective amount of progenitor cells comprises at least 10²progenitor cells, at least 5X10²Progenitor cell, at least 10³Progenitor cells, at least 5X10³Progenitor cell, at least 10⁴Progenitor cells, at least 5X10⁴Progenitor cell, at least 10⁵Progenitor cells, at least 2X 10⁵Progenitor cells, at least 3X 10⁵Progenitor cells, at least 4X 10⁵Progenitor cells, at least 5X10⁵Progenitor cells, at least 6X 10⁵Progenitor cells, at least 7X 10⁵Progenitor cells, at least 8X 10⁵Progenitor cells, at least 9X 10⁵Progenitor cell, at least 1X 10⁶Progenitor cells, at least 2X 10⁶Progenitor cells, at least 3X 10⁶Progenitor cells, at least 4X 10⁶Progenitor cells, at least 5X10⁶Progenitor cells, at least 6X 10⁶Progenitor cells, at least 7X 10⁶Progenitor cells, at least 8X 10⁶Progenitor cells, at least 9X 10⁶Individual progenitor cells or multiples thereof. The progenitor cells may be derived from one or more donors, or may be obtained from an autologous source. In certain examples described herein, the progenitor cells can be expanded in culture prior to administration to an individual in need thereof.

A modest and incremental reduction in the level of PCSK9 expressed in cells of patients with PCSK 9-related disorders may be beneficial for ameliorating one or more symptoms of the disease, for increasing long-term survival, and/or for reducing side effects associated with other therapies. The presence of progenitor cells that produce reduced levels of PCSK9 is beneficial after administration of such cells to a human patient. In certain instances, effective treatment of an individual results in a reduction in PCSK9 levels of at least about 3%, 5%, or 7% relative to total PCSK9 in the treated individual. In certain examples, the reduction in PCSK9 will be at least about 10% of total PCSK 9. In certain examples, the reduction in PCSK9 will be at least about 20% to 30% of total PCSK 9. Similarly, the introduction of even a relatively limited subpopulation of cells with significantly reduced levels of PCSK9 may be beneficial in different patients, as in some cases normalized cells will have a selective advantage over diseased cells. However, even modest levels of progenitor cells with reduced PCSK9 levels may be beneficial for improving one or more aspects of dyslipidemia in a patient. In certain examples, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the hepatic progenitors in a patient to which such cells are administered are producing reduced levels of PCSK 9.

By "administering" is meant delivering the progenitor cell composition into an individual by a method or route that results in at least partial localization of the cell composition at the desired site. The cellular composition may be administered by any suitable route that results in effective treatment in the individual, i.e., administration results inDelivery to a desired location in an individual, wherein at least a portion (i.e., at least 1 x 10) of the composition delivered⁴Individual cells) are delivered to the desired site for a certain period of time.

In one aspect of this method, the pharmaceutical composition may be administered via a route such as, but not limited to: enteral (into the intestine), parenteral, epidural (into the dura), oral (by mouth), transdermal, epidural, intracerebral (into the brain), intracerebroventricular (into the ventricle), epidermal (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), intranasal (through the nose), intravenous (into the vein), intravenous bolus, intravenous drip, intraarterial (into the artery), intramuscular (into the muscle), intracardial (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eyes), intracavitary injection (into the pathological cavity), intracavitary (into the base of the penis), intravaginal administration, intrauterine, extraamniotic administration, transdermal (through diffusion of intact skin for systemic distribution), transmucosal (diffusion through the mucosa), transvaginal, insufflation (snuff), sublingual, sublabial, enema, eye drop (onto conjunctiva), ear drop, ear (in or through the ear), cheek (pointing to the cheek), conjunctiva, skin, tooth (to one or more teeth), electroosmosis, intracervical, intranasal sinus, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarticular, intrabiliary, intrabronchial, bursa, intrachondral (intracartilaginous), caudate (intracisternal), intracisternal (intracorneal), intracoronary (intracoronary), intracapsular (intracoronary), intracavernosaloplasmic (intracapsular space of the penis), intradiscal (intraspinal disc), intraductal (intraglandular), intraduodenal (intraduodenal), intraduodenal (in or below dural), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), intragingival (in the gum), intraileal (in the distal part of the small intestine), intralesional (in the local focus or direct introduction of the local focus), intracavitary (in the lumen), intralymphatic (in the lymph), intramedullary (in the medullary cavity of the bone), intracerebroventricular (in the brain membrane), intramyocardial (in the heart muscle), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardium), intrathoracic (in the chest), intraprostatic (in the prostate), intrapulmonary (in the lung or its bronchi), intranasal (in the nose or periorbital sinus), intraspinal (in the spine), intrasynovial (in the synovial cavity of the joints), intratendinous (in the tendons), intratesticular (in the testis), intrathecal (in the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (in the chest), intrarenal (in the kidney), intratumoral (intratumoral), intratympanic (intratympanic), intravascular (one or more blood vessels), intraventricular (intraventricular), iontophoretic (by means of an electric current, in which ions of soluble salts migrate to the tissues of the body), lavage (washing or rinsing of open wounds or body cavities), laryngeal (directly on the larynx), nasogastric (through the nose and into the stomach), occlusive dressing techniques (topical route application, which then covers the dressing covering of the area), ocular (to the outer eye), oropharyngeal (directly to the oral cavity and pharynx), parenteral, transdermal, periarticular, epidural, perinervous, periodontal, rectal, respiratory (by oral or nasal inhalation in the respiratory tract for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (into the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, locally, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic membrane (across or through the tympanic cavity), ureter (to the ureter), urethral (to the urethra), vaginal, sacral block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopenetration, and spinal cord.

Modes of administration include injection, infusion, instillation, and/or ingestion. "injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerobrospinal and intrasternal injection and infusion. In certain examples, the route is intravenous. For delivery of cells, administration by injection or infusion can be made.

The cells may be administered systemically. The phrases "systemic administration," "administered systemically," "administered peripherally," and "administered peripherally" refer to administration of a population of progenitor cells other than directly into a target location, tissue, or organ, such that it instead enters the circulatory system of the individual and is thus subject to metabolism and other similar processes.

The efficacy of a treatment comprising a composition for treating dyslipidemia can be determined by a skilled clinician. However, a treatment is considered to be an "effective treatment" if any or all of the signs or symptoms (but as an example, the levels thereof) of PCSK9 are altered in a beneficial manner (e.g., by at least 10%) or other clinically acceptable symptoms or markers that improve or ameliorate the disease. Efficacy may also be measured by the failure of an individual to worsen, as assessed by hospitalization or the need for medical intervention (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indices are known to those skilled in the art and/or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals), and includes: (1) inhibiting the disease, e.g., arresting or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptoms occurring.

Treatment according to the invention may ameliorate one or more symptoms associated with dyslipidemia by reducing or altering the amount of PCSK9 in an individual.

Features and characteristics of proprotein convertase subtilisin/Kexin type 9 (PCSK9) genes

PCSK9 is associated with diseases and conditions such as, but not limited to, abetalipoproteinemia, adenoma, arteriosclerosis, atherosclerosis, cardiovascular disease, cholelithiasis, coronary arteriosclerosis, coronary heart disease, non-insulin dependent diabetes mellitus, hypercholesterolemia, familial hypercholesterolemia, hyperinsulinemia, hyperlipidemia, familial combined hyperlipidemia, hypobetalipoproteinemia, chronic kidney failure, liver disease, liver tumor, melanoma, myocardial infarction, lethargy, tumor metastasis, wilms tumor, obesity, peritonitis, pseudoxanthomatosis elastosis, cerebrovascular accident, vascular disease, xanthomatosis, peripheral vascular disease, myocardial ischemia, dyslipidemia, impaired glucose tolerance, xanthoma, polygenic hypercholesterolemia, secondary malignant liver tumors, dementia, overweight, hepatitis c, chronic, carotid atherosclerosis, type IIa hyperlipoproteinemia, intracranial atherosclerosis, ischemic stroke, acute coronary syndrome, aortic calcification, cardiovascular disease, type IIb hyperlipoproteinemia, peripheral artery disease, type II familial hyperaldosteronism, familial hypolipoproteinemia, autosomal recessive hypercholesterolemia, autosomal dominant hypercholesterolemia 3, coronary artery disease, liver cancer, ischemic cerebrovascular accident, and arteriosclerotic cardiovascular disease NOS. Editing the PCSK9 gene using any of the methods described herein can be used to treat, prevent, and/or alleviate the symptoms of the diseases and disorders described herein.

The activity of PCSK9 is primarily limited to the liver, and PCSK9 is associated with dyslipidemia, PCSK 9-associated familial hypercholesterolemia, hypercholesterolemia (familial), papillary adenocarcinoma of the stomach, homozygous familial hypercholesterolemia, and nasopharyngitis. PCSK 9-associated familial hypercholesterolemia is an inherited disorder (autosomal dominant) in which the body develops dangerous blood cholesterol levels due to a deficiency in low density lipoprotein cholesterol receptors. PCSK 9-associated familial hypercholesterolemia affects 1 of 500 heterozygous to 1,000,000 homozygous throughout the world and is more common in south african white, french canadian, libasian governor and finnish populations. Common symptoms of PCSK 9-associated familial hypercholesterolemia include elevated circulating cholesterol contained in low density lipoproteins alone or also in very low density lipoproteins. Current treatments for PCSK 9-related familial hypercholesterolemia include the administration of statins to inhibit hydroxymethylglutaryl-coenzyme a reductase (HMG-CoA-reductase) in the liver. Another option for treating PCSK 9-associated familial hypercholesterolemia is ezetimibe to inhibit cholesterol absorption in the intestinal tract.

Dyslipidemia is a genetic disease characterized by elevated blood lipid levels that contribute to the development of arterial obstruction (atherosclerosis). These lipids include plasma cholesterol, triglycerides or high density lipoproteins. Dyslipidemia increases the risk of heart attack, stroke, or other circulatory system concerns. Current regulations include lifestyle changes, such as exercise and dietary changes, and the use of lipid lowering drugs (such as statins). Non-statin lipid lowering drugs include bile acid sequestrants, cholesterol absorption inhibitors, drugs for homozygous familial hypercholesterolemia, fibrates, niacin, omega-3 fatty acids, and/or combination products. Treatment options often depend on the particular lipid abnormality, although different lipid abnormalities often co-exist. Treatment of children is more challenging because dietary changes may be difficult to implement and lipid lowering therapies have not proven effective.

In one embodiment, the target tissue of the compositions and methods described herein is liver tissue.

In one embodiment, the gene is proprotein convertase subtilisin/Kexin type 9 (PCSK9), which may also be referred to as subtilisin/Kexin-like protease PC 9. PCSK9 has a cytogenetic location of 1p32.3, and the genomic coordinates are at position 55,039,548-55,064,852 on the forward strand on chromosome 1. The nucleotide sequence of PCSK9 is shown as SEQ ID NO:5,303. BSND is an upstream gene of PCSK9 on the forward chain, and RP11-101C11.1 is a downstream gene of PCSK9 on the forward chain. PCSK9 has NCBI gene ID of 255738, Uniprot ID of Q8NBP7, and Ensembl gene ID of ENSG 00000169174. PCSK9 has 2045 SNPs, 18 introns and 20 exons. The exon identifiers from Ensembl and the start/stop sites of introns and exons are shown in Table 3.

TABLE 3 Intron and exons of PCSK9

Table 4 provides information on all transcripts of the PCSK9 gene based on the Ensembl database. The transcript ID from Ensembl and the corresponding NCBI RefSeq ID for that transcript, the translation ID from Ensembl and the corresponding NCBI RefSeq ID for that protein are provided in table 4, as are the biotypes of the transcript sequences classified by Ensembl and the exons and introns in the transcript based on the information in table 3.

TABLE 4 transcript information for PCSK9

Rs 38K 9 has 2045 SNPs, and the PCSK9 gene has the NCBI rs number and/or the Uniprot VAR number of rs10465831, rs10465832, rs10585118, rs10674598, rs10888896, rs10888897, rs 108342911, rs111400659, rs111427099, rs 111565670724, rs111705971, rs111830949, rs111976283, rs11206513, rs 06514, rs11206515, rs11206516, rs 06517, rs112071856, rs 096465, rs 1129191496 48, rs 269712513877631251387763, rs 140138776313871455648, rs 140138776368, rs 14013877704742, rs 14013877704705, rs 120355648, rs 140563756375637563756375648, rs 3235729, rs 140704708, rs 32563756375637563756375637563756375648, rs 37563756375637563756375637563756375637563756375637567, rs 3756375637563756375637563756375637563756375637567, rs 3756375637563756375637563756375637567, rs 37563756375637563756375637563756375637563756375637567, rs 1745, rs 17563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs 1745, rs 729, rs72, rs140641462, rs140837910, rs140838551, rs140865730, rs 140911961969, rs141135099, rs141407779, rs141438059, rs141502002, rs141593516, rs141855360, rs141867978, rs141901482, rs 141707042, rs 14196319671, rs141995194, rs142118418, rs 1421491451491451491451491457920, rs 2956351455639, rs 29561489, rs 2914556351455620, rs 2956351455620, rs 2914556351451455620, rs 29563514556351455620, rs 2956351455620, rs 1711435620, rs 171143351455620, rs 1711455620, rs 171143351455620, rs 14556351455620, rs 351453514556351455620, rs 3514535145351455620, rs 182145563514556351455620, rs 35145351453514535145351455620, rs 351453514535145351455620, rs 182145355620, rs 355620, rs 1821453556357235729, rs 3514535145355620, rs 182145357235729, rs 182145355620, rs 17240, rs 172, rs 17240, rs 178, rs 17355620, rs 1772355620, rs 177235723572357235723572357235723572357235729, rs 178, rs 18214535723572357235723572357235723572357235729, rs 178, rs 1821451, rs 182145357235723572357235723572357235729, rs 1821451, rs 182145357235723572357235729, rs 18214535723572357235729, rs 1828, rs 1821451, rs 1821453572357235729, rs No 2, rs No. 2, rs No. 2, rs No. 2, RS No. 2, rs No. 2, RS No. 2, RS No. RS, rs184303022, rs184518483, rs184548073, rs 18456367, rs184568630, rs184816632, rs185003821, rs185361730, rs185365167, rs185392267, rs 185581611617, rs185710397, rs185840193, rs185905805, rs185927385, rs186112040, rs 250869, rs186329941, rs 1865404540433, rs 186657537530, rs186669805, rs 186359, rs 186186799, rs 1699, rs 3007967799, rs 300799, rs 30056325637799, rs 300563756375637799, rs 300799, rs 300794354799, rs 3005637563756375637563756375637799, rs 300799, rs 3005637563756375637563756375637563756375637563756375637569, rs 3005637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 3003756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs 2005445, rs 1699, rs 1563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 1699, rs 156375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637567, rs 200rs 172, rs 15637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637569, rs 1699, rs 1692, rs 1563756375637569, rs 1699, rs 1692, rs 1699, rs 200rs 179, rs 1699, rs 179, rs 15637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637563756375637, rs28362205, rs28362206, rs28362207, rs28362213, rs28362214, rs28362218, rs28362219, rs28362220, rs28362221, rs28362222, rs 28229, rs 2822569, rs28362259, rs 2836579, rs 28229, rs 282228229, rs 2822289, rs289, rs289,362259, rs 28579, rs289,362259, rs 28229, rs289,289,289,289,289,28229,28229,289,289,289,9,9,289,289,28362252,28362239,289,289,289,289,289,2822289,289,rs 282228369,289,289,289,28369,2822289,289,2822289,289,289,28362252,rs 28369,rs 28369,289,28369,289,289,289,28369,2828369,2828289,rs 28282828369,28289,rs 2828282828362252,rs 289,rs 282828289,rs 289,28282828289,28282828289,289,28282828289,289,282828289,28282828282828282828282828289,28289,289,289,289,289,rs 28282828282828289,rs 289,2828282828282828282828289,289,2828282828289,289,28282828282828282828289,282828289,282828289,rs 2828282828289,289,28282828282828289,289,rs 282828282828282828282828289,2828282828282828282828289,rs 2828282828282828282828289,282828289,rs 289, rs 282828289,rs 282828282828289,rs 28282828289, rs 28289,289,289,282828282828289, rs 28282828289, rs 28282828282828282828282828289, rs 282828282828282828282828289,289,282828289,289,289,28282828289, rs 28282828282828282828282828282828289,289, rs289,2828289, rs 2828282828362252,289,289,28282828282828289,2828282828289,289,289,2828289,289,289,289,2828282828282828282828289, rs289,289,289, rs289,289,289,289,282828289,289,, rs367785668, rs367912777, rs368103912, rs368156218, rs368257906, rs368282130, rs 367335, rs368406783, rs368511429, rs 3685169937, rs 368618619039, rs368899514, rs369013756, rs 3693736961446, rs 36937066144, rs 06369787856, rs 369118118118118278, rs 370373698204, rs 7283728383, rs 369799, rs369793427, rs 36937373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373757, rs 37373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373757, the 1, 373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373757, the 1, 3737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373757, 3737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373737373757, the 1, rs 373737373737373737373737373737373737373737, rs41294819, rs41294821, rs41294823, rs41294825, rs41297881, rs41297883, rs41297885, rs4275490, rs45439391, rs45448095, rs45454392, rs 4547898, rs45479392, rs45487891, rs45490396, rs 454553898, rs45508296, rs 4553530931, rs 455345534553455345539, rs 53525352534553455345534553455345539, rs 5352534553455345534553455345534553455345534553455346, rs 535253455345569, rs 53525345525345534553455345534553455345534553455345534553455345534553455346, rs 455253525352535253455253455253455345569, rs 53525352535253455345534553455345534553455345569, rs 535253525345569, rs 535253525352535253525345569, rs 5352535253525345569, rs 5352535253525352534556300, rs 535253525352535253525345569, rs 5352535253455637569, rs 53525352535353455637569, rs 535253525352535253535353535345569, rs 5352535253525345569, rs 535253525352535253535345569, rs 535352535253535353535353535345569, rs 5345569, rs 535253525353535353535352535253535345563756375637569, rs 5352535253525345569, rs 535253525353525352535253525352535253455637569, rs 535253525345569, rs 5353534556375637569, rs 5352535253455637569, rs 5352535253525353535352535253525352535253535353525345563756375637569, rs 53525345569, rs 5353535353535345569, rs 5352535345569, rs 53535253525352535353535353535345569, rs 535253535352535253525345569, rs 53455637569, rs 53535353535253535353535345569, rs 535253525345569, rs 5353535353525352535352535253525345569, rs 5345563756375637563756375637569, rs 5345569, rs 53525352535253525352535253535345569, rs 53535352535353535353535353535353535353535353535345569, rs 5345569, rs 535253535253525352534556375637563756375637569, rs 5352535253455637569, rs 53525352535253525345, rs534885728, rs535214548, rs 53535359199, rs535471, rs535518776, rs 5355586, rs535856008, rs535914324, rs536037529, rs536224897, rs536292091, rs536492847, rs536502255, rs536512341, rs536565868, rs537026366, rs 5314569, rs537281156, rs 7305205256, rs 5328570, rs537483343, rs 537609, rs 5382328, rs538316722, rs 535252399 399, rs 535284399, rs54779, rs538886648, rs 538954779, rs 547754779, rs 54775477547754779, rs 5477547754775477547754779, rs 54775477547754775477547754779, rs 5477547754775477547754779, rs 547754775477547754775477547754779, rs 5477547754775477547754775646, rs 547754775646, rs 5477547756355477569, rs 547754775477543554355435543554355435543554355435543554355435543554355646, rs 54355646, rs54779, rs 543554779, rs 5435543554355477569, rs 5435543554355435569, rs 5435543554355435543554355435567, rs 5435543554355435543554355435543554355435543554355435543554355435543554355435543554355435543554355435543554355435567, rs 1745, rs 5435543554355648, rs 1745, rs 54355435543554355435543554355435543554355435543554355435567, rs 1745, rs 54355435567, rs 53543554355435543554355648, rs 1745, rs 535435543554355435543554355435569, rs 1745, rs 5354355648, rs 1745, rs 5354355435543554355435543554355435567, rs 535435567, rs 5354355648, rs 1745, rs 535435569, rs 5354355648, rs 1745, rs 53543554355435543554355435543554355648, rs 1745, rs 53543554355435569, rs 1745, rs 5354355435543554355435569, rs 1745, rs 5354355648, rs 535435569, rs 5354355435543554355435543554355435569, rs 1745, rs 535435569, rs 53543556, rs 5507306, rs550263135, rs550300315, rs550366748, rs550413887, rs550549291, rs550562137, rs550577460, rs550679929, rs550791152, rs550944404, rs 5509470701315846, rs551379054, rs551467121, rs551502518, rs 5585858579, rs 162550200200201, rs 551631632342342347770, rs 55181551815555755563755557, rs 5556555575556375545563755798, rs 5555755455637554556375545563755557, rs 55555575556375545563755557, rs 55557554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637559, rs 5555555555555555554556375545563755455637554556375545554555455637554556375545563755455637554555455637554556375545563755455637554556375545554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455545554555455545563755455637554556375545563755455545554555455637554556375545563755455545554556375545563755455637554555455545563755455545554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455637554556375545563755455545554555455637554556375545563755455545563755455637575, rs 554555455545554555455545554555455545554555575, rs55575, rs 55555555555555575, rs 555555555555555555575, rs55575, rs 55555555555555555557rs 555555555555555545554555455545554555455555555555554555455545554555455555555545554555555555455545554555575, rs 555745, rs 5545555745, rs 555555555545554555455545554555455545554555455545554555575, rs 55455545555545555555555545554555455545554555555555554555455545554555455555555545554555455545554555455545554555455545554555455545555745, rs, rs562480265, rs562491023, rs562829799, rs56295417, rs562957212, rs 5656024336, rs 56099655, rs 5656114423, rs 56166632, rs 56231860, rs 56562926262676, rs 5656641, rs 5666566656665666569, rs 56565656575703, rs 56565757579, rs 565756575757575756579, rs 565657565757575657575657575756579, rs 565656575757565757565756575757575657575657565756579, rs 565656565657575756575757575756575657575757579, rs 5656565657565757575657575757565757575757575757575756579, rs 5656565657565756575657565756575657565756575756575657565756579, rs 565656565656565656575757575657565756575657565757565756575757575657569, rs 5656565656565656565656575657565756575657565756575657565756575657569, rs 56565656565656565656565656565656565656565656565656565656565656565756575657575657565657569, rs 565656565656565656565656565656565656565656575757575656575757575756575757575757575757575756575657565756575757575657565756575657565756575657569, rs 5657565756575657565756575657565657565756575657569, rs 56565656565656565656565656565756575656575657565756565656575657565657565756575657565756575657565756575657565756575657565756575657565756575657565756575657569, rs 56575657565656575657565756575657565756575657569, rs 565656565656575657565656565656565657565756575657565756575756575657569, rs 565656565656565656565656565657565756565657565756575657565756575657565756575657565756575657565756573 575657569, rs 565656575657565756575657565756575657565756575657565756575657565756575657565756575657565756575657565756573, rs569, rs 56575656565656575657569, rs 56575657565756575657565756575657565756575657565756575657565756575657565656565656575657565756575657569, rs 5656573, rs 565656573, rs 56565657565656565656565657, rs575583588, rs 5755958, rs575827041, rs575974291, rs576035975, rs576103835, rs576302293, rs 57647298018, rs576535214, rs576552402, rs576575389, rs 576639, rs 57667383821, rs576806431, rs 578517, rs577339951, rs 5745763, rs 745774577472610, rs 5777333, rs 57762327675, rs 57768376612, rs577832979, rs 57785778799, rs 57799, rs 57816281154610, rs 575746, rs 585540, rs584626, rs 58516531, rs58667756, rs587776545, rs 5767590, rs 6472659, rs 657265726572659, rs 647265726572657265729, rs 64726572657265729, rs 6472657265729, rs 64727272727272727265729, rs 647265726572657265729, rs 64727265727272729, rs 64726572657265729, rs 6472657265729, rs 6472727272727265729, rs 647265726572657265729, rs 647265729, rs 64726572657272727272727272657265729, rs 64726572657265726572657265729, rs 64726572657265727272726572657265729, rs 6472657265729, rs 64729, rs 6472657272647265729, rs 6472657265729, rs 64729, rs 647265726572657265729, rs 6472657265729, rs 647265729, rs 647265726572657265729, rs 64726472647264729, rs 6472647264726472647264726572657265729, rs 64729, rs 647264726472647264726472647265729, rs 647265726572657265726572657265729, rs 64729, rs 64726572657265726572657265726572657265729, rs 64729, rs 647265729, rs 64726572647264729, rs 64729, rs 6472659, rs 64726572657265729, rs 64729, rs 6472657265729, rs 6472657265726572657265726572657265726572657265726572659, rs 6472657265729, rs 64729, rs 6472647264729, rs 64729, rs 64726472647264726472647264726572657265726572657265726572657265726572657265729, rs 647265729, rs 64729, rs746423690, rs746438392, rs746442570, rs746457760, rs746504242, rs 746529029062, rs746639289, rs746695481, rs746705490, rs 7467676723269, rs 746767729, rs 7468687074683567706792857, rs 746868357479387, rs 747039779, rs 74796792679267794354799, rs74779, rs 74757475794375794354779, rs 747556779, rs 7475567779435477794354779, rs 74755677794354779, rs 7475567779435477799, rs 7475563779849, rs 747556377943547779849, rs 7556377943547779849, rs 7556377943547779435477799, rs 755637794354779, rs 7574755637563779435477175637799, rs 755637798, rs 7475747574757475747574757475747574755637798, rs 757475747574755637798, rs 757475747574757475747574755637798, rs 75563779175637798, rs 7574757475747574757475747574755637798, rs 74757475747574757475747574757475747574757475747574757475747574799, rs 75747574755637798, rs 75747574757475747574755637798, rs 75747574757475747574798, rs 7574798, rs 747574757475747574755637798, rs 74757475747574755637798, rs 7475747574757475849, rs 74757475747574757475747574757475747574757475849, rs 7475747574757475849, rs 7475849, rs 75747574757475747574757475747574757475849, rs 747574757475849, rs 7475849, rs 74757475747574757475849, rs 7475849, rs 7475747574757475747574757475849, rs 74798, rs 747556300, rs 74757475747574757475849, rs 74757475747574757475747556300, rs 7475849, rs 7475747556300, rs 747556300, rs 7556300, rs 747556300, rs 74755637792, rs 7475747556300, rs 7475747574757475747574755637792, rs 74755637793, rs 74755637792, rs 7475849, rs 74755637792, rs 74757475747574757475747574757475747574757475747574757475747556300, rs 7475747517799, rs 747574757475747556300, rs 7475747556300, rs 747556300, rs 7475747574757475747574757475, rs7530425, rs753062243, rs753086395, rs753141052, rs753308448, rs7533186, rs753332916, rs753353734, rs 7533535359110, rs 753505065056, rs 75352523652363643728, rs 75753603237, rs 753657553757996, rs753695505, rs 7535856645, rs753857795, rs 96617, rs754143671 671, rs 7541799, rs 757979799, rs 75755679799, rs 75567556799, rs 75757556755637799, rs 7556755637799, rs 755637799, rs 75755637755637792, rs 755637755637793, rs 757575755637793, 757575757575757575755637799, 75757575755637793, rs 757575757575757575757575755637793, 75757575757575757575757575755637793, 75757575757575757575757575757575729, 75757575757575757575729, rs 757575757572793, 75757575757575757575757575757575729, 75757575757575757575729, rs 757575757575757575757572757575757572793, 75757575757575757575729, rs 75757275729, rs 75757575757575757575757575757575757575757275729, rs 75729, rs 7575757575757575757575729, 757275757575757275729, rs 75729, rs 757575757575757575727572757575729, rs 75757575729, rs 7575757575757575757575757575729, 757575757575757575757575757575757275729, 7575757575757572757575757275727575757575757575757575757575757575729, 75729, rs 75727572757275727572757275729, rs 757275729, rs 75729, rs 75757575757575757575757575757275757575757575757575757575757575729, 75757575757275727575729, 75729, rs 757572757575757575729, 75757575729, 757572757275727572757575757575757575757275729, 757572757275757575757575757575729, 7575757572757275757575729, 7575757575757575757575757575757575727575757575727572757275727572757575757275727575757575757575757575757575729, rs 75757575729, 7575757575757275729, rs 75729, 7575757575729, 757275, rs759503095, rs759519174, rs759545385, rs759554053, rs759557001, rs759590927, rs759618377, rs759688652, rs 7597943, rs 759818018065, rs 75989676198761913, rs760011800 1800, rs 761800 1800, rs 767676771800, rs 7676767676767776777677767748, rs 76767676767676777677767776777626, rs 7676767677767776777677767776779, rs 767776777677767776777677767776777677767776779, rs 7677767776777677767776777677767776779, rs 76767676767676767676767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776777677767776779, rs 767776777677767776777677767776779, rs 7677767776777677767776777677767776777677767776777677767776777677767776779, rs 76777677767776777677767776777677767776779, rs 7677767776777677767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776777677767776779, rs 76777677767776777677767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776777677767776779, rs 76777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776776476779, rs 7677767776777677767776777677767776779, rs 76777677767776779, rs 76777677767776777677767776777677767776777677767776777677767776777677767776777677767776776476776476777677767776779, rs 767776777677767776777677767776779, rs 767776777677767776779, rs 76777677767776777677767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776777677767776779, rs 767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776777677767776779, rs 7677767776777677, rs765330219, rs765583923, rs765626863, rs765668920, rs765733763, rs765737080, rs 76573959572, rs765744739, rs765760837, rs765777205, rs765789797, rs 765835055055055055055055055055051, rs765918391, rs765939807, rs766010409, rs 766349 349, rs 028813, rs 76676767677779, rs 250777777779, rs 2507777925495, rs 7664770, rs 7663131313131313131313131313131315, rs766 7676767676767676767676779, rs 766767676767676779, rs766 77779, rs 7777777777779, rs 7777777777777777779, rs 64767777777777777777777777777777779, rs 767676767676767676767676767676767676767676767676767676767676767676767676767676767676779, rs766 777777777777779, rs 64777777777777777777777777777777779, rs 6477777777777777777777777777777777779, rs 647777777777777777777777777777777777777777567777779, rs 6477777777775677567756775677567756775677567756775677569, rs766 77567756775677567756775677567756775677567756775677569, rs766 775677567756775677567756775677569, rs766 77567756775677567756775677567756775677567756775677567756775677567756775677569, rs 6476569, rs766 775676567656765676767656765676567656765676567656765676569, rs766 77569, rs 7676567656765676567656765676567656765676567656765676567776765676567677567677567677569, rs 7656765676569, rs 76567656765676567656767756775676777677767777777777569, rs 765676569, rs 767676767676767756775677777777777777767776569, rs 765676567676767656767776777777567756765676569, rs 76569, rs 7676767776569, rs766 767777777777777777777776777656767777777776569, rs 7777765676567656767777777777775676567656765676569, rs 76569, rs 7656775676567656765676569, rs766 76569, rs 765676569, rs 765676567777777777569, RS 6476567677777756765677569, RS 647677777777775676569, RS 76569, RS 64765676567656765676567656, rs771069624, rs771070411, rs771108863, rs771143407, rs 771192192192382, rs771238985, rs771257845, rs771274473, rs771359523, rs771421073, rs771479424, rs771532186, rs771594920, rs771601056, rs 771631241241241241241241241241241241241241241241241241241241241241244, rs771641933, rs 7716868682047716820420420477204204204204204204204204773, rs 771777777777777777777777777779, rs 777777777777777777777777779, rs 7712477124777777777777779, rs 7777777777777777777777777777779, rs 77777777777777777777777777777777777777777777777777777777779, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 77777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 77777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777, rs777645981, rs777706463, rs 77777734307, rs 7778429, rs777843526, rs777860859, rs777931489, rs7779865 8655, rs 7777777777777777777777777777777777779, rs 7743694, rs 77916491649, rs778104784, rs778117521, rs 77815788885, rs 77777777777777779, rs 7777777777777777779, rs 7777777777777777777777781, rs 77777777777777777777781, rs 777777777791779, rs 77777777777777777756777791779, rs 7777777777777777777777777777779, rs 777777777777777777777777777777777777777777777777777777779, rs 777777777777777777777777777777777777777777779179567, rs 777777777777777777777777777777777777777777777777777777569, rs 777777777777777777777777777777777777777777777777777777777777777777569, rs 7777777777777777777777777777777777777777777777777777777777777777569, rs 777777777777777777777777777777777777777777777777777777777777779179569, rs 77777777777777777777777777777777777777777777777777777777569, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777777777569, rs 777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777569, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777777777569, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777779, rs 7777779, rs 777777777777777777777777777777777777777777777777777777777777777777777791799, rs 7777777777777777777777777777777777777777777777777777777777777777777777777777, VAR _058531, VAR _058532, VAR _058533, VAR _058534, VAR _058535, VAR _058536, VAR _058537, VAR _067282, VAR _067351, and VAR _ 073657.

In one example, a guide RNA for use in the present invention can comprise at least one 20 nucleotide (nt) target nucleic acid sequence listed in table 5. The gene symbols and sequence identifiers of the genes (gene SEQ ID NOs) are provided in table 5, including the gene sequences of 1-5 base pairs upstream and/or downstream of the target gene (extended gene SEQ ID NOs) and the 20 nt target nucleic acid sequences (20 nt target sequence SEQ ID NOs). In the sequence Listing, the respective target gene, the strand used to target the gene (represented by either the (+) strand or the (-) strand in the sequence Listing), the associated PAM type and PAM sequence are described for each of the 20 nt target nucleic acid sequences (SEQ ID NO:5,305-28, 696). It is understood in the art that the spacer sequence (where "T" is "U") may be an RNA sequence corresponding to the 20 nt sequence listed in table 5.

TABLE 5 nucleic acid sequences

gene symbol	Gene SEQ ID NO	Extended gene SEQ ID NO	20 nt target sequence SEQ ID NO
				PCSK9	5,303	5,304	5,305-28,696

In one embodiment, the guide RNA used in the present invention may comprise at least one spacer sequence, which (where "T" is "U") may be an RNA sequence corresponding to a 20 nucleotide (nt) target sequence, such as, but not limited to, any of SEQ ID NOS: 5,305-28, 696.

In one embodiment, the guide RNA used in the present invention may comprise at least one spacer sequence, which (where "T" is "U") is an RNA sequence corresponding to a 20 nt sequence, such as, but not limited to, any of SEQ ID NOS: 5,305-28, 696.

In one embodiment, the guide RNA can comprise a 20 nucleotide (nt) target nucleic acid sequence associated with a PAM type, such as, but not limited to, NAAAAC, NNAGAAW, NNGRRT, nnnnnnght tt, NRG, or YTN. As one non-limiting example, a 20 nt target nucleic acid sequence of a particular target gene and a particular PAM type, where "T" is "U", can be an RNA sequence corresponding to any of the 20 nt nucleic acid sequences in table 6.

TABLE 6 nucleic acid sequences by PAM type

In one embodiment, the guide RNA may comprise a 22 nucleotide (nt) target nucleic acid sequence associated with the YTN PAM type. As a non-limiting example, a 22 nt target nucleic acid sequence of a particular target gene can comprise a 20 nt nuclear sequence, wherein the 20 nt nuclear sequence, where "T" is "U", can be an RNA sequence corresponding to SEQ ID NO 18,792-28,696. As another non-limiting example, the 22 nt target nucleic acid sequence of a particular target gene may comprise a core sequence, wherein the core sequence, in the case where "T" is "U", may be a fragment, segment or region of an RNA sequence corresponding to any one of SEQ ID NOS 18,792-28,696.

VI. Other possible treatment regimens

Gene editing can be performed using nucleases engineered to target specific sequences. To date, there are 4 major classes of nucleases: meganucleases and their derivatives, Zinc Finger Nucleases (ZFNs), transcription activators such as effector nucleases (TALENs), and CRISPR-Cas9 nuclease systems. Nuclease platforms differ in design difficulty, targeting density, and mode of action, particularly because the specificity of ZFNs and TALENs is through protein-DNA interactions, while RNA-DNA interactions primarily direct Cas 9.

CRISPR endonucleases (such as Cas9) can be used in the methods of the invention. However, the teachings described herein, such as therapeutic target sites, can be applied to other forms of endonucleases, such as ZFNs, TALENs, HE or MegaTAL, or using a combination of nucleases. However, in order to apply the teachings of the present invention to such endonucleases, one needs to engineer proteins specific to a particular target site, among other things.

Additional binding domains can be fused to the Cas9 protein to increase specificity. The target site of these constructs may map to the site designated by the identified gRNA, but may require additional binding motifs, such as for the zinc finger domain. In the case of Mega-TAL, meganucleases can be fused to the TALE DNA-binding domain. Meganuclease domains can increase specificity and provide cleavage. Similarly, an inactivated or dead Cas9 (dCas9) can be fused to the cleavage domain and requires a sgRNA/Cas9 target site and an adjacent binding site for the fused DNA-binding domain. This may require some protein engineering of dCas9 in addition to catalytic inactivation to reduce binding in the absence of additional binding sites.

Zinc finger nucleases

Zinc Finger Nucleases (ZFNs) are modular proteins comprising an engineered zinc finger DNA binding domain linked to the catalytic domain of the type II endonuclease fokl. Because FokI functions only as a dimer, a pair of ZFNs must be engineered to bind to homologous target "half-site" sequences on opposite DNA strands with precise spacing between them to enable the formation of catalytically active FokI dimers. Following dimerization of the FokI domain (which is not sequence specific by itself), a DNA double strand break is created between the ZFN half-sites as a starting step in genome editing.

The DNA-binding domain of each ZFN typically comprises 3-6 zinc fingers of the abundant Cys2-His2 architecture, each finger recognizing primarily a triplet of nucleotides on one strand of the target DNA sequence, although cross-strand interaction with the fourth nucleotide may also be important. Changes in the amino acid of a finger in a position that makes critical contact with DNA will alter the sequence specificity of the given finger. Thus, a four-finger zinc finger protein will selectively recognize a12 base pair target sequence, where the target sequence is a complex of triplet preferences contributed by each finger, although triplet preferences may be affected to varying degrees by adjacent fingers. An important aspect of ZFNs is that they can be easily retargeted to almost any genomic address simply by modifying individual fingers, although extensive expert experience is also required to achieve this. In most applications of ZFNs, proteins of 4-6 fingers are used, recognizing 12-18 base pairs respectively. Thus, a pair of ZFNs typically recognizes a 24-36 base pair combined target sequence, excluding the typical 5-7 base pair spacer between half-sites. The binding sites may be further separated by larger spacers (including 15-17 base pairs). Target sequences of this length may be unique in the human genome, provided that repeat sequences or gene homologs are not included in the design process. Although, as such, ZFN protein-DNA interactions are not absolute in their specificity, off-target binding and cleavage events actually occur, whether as heterodimers between two ZFNs or as homodimers of one or the other ZFNs. The latter possibility has been effectively eliminated as follows: the dimerization interface of FokI domains is engineered to establish "positive" and "negative" variants, also referred to as obligate heterodimer variants, that can only dimerize with each other and not with themselves. Forcing obligate heterodimers prevents homodimer formation. This has greatly enhanced the specificity of ZFNs, as well as any other nucleases employing these FokI variants.

A variety of ZFN-based systems have been described in the art, modifications of which have been formally reported, and numerous references describe rules and parameters for guiding the design of ZFNs; see, e.g., Segal et al,Proc Natl Acad Sci USA 96(6) 2758-63 (1999); dreier B et al,J Mol Biol. 303(4) 489-502 (2000); liu Q et al,J Biol Chem. 277(6) 3850-6 (2002); dreier et al,J Biol Chem 280(42) 35588-97 (2005); and Dreier et al,J Biol Chem. 276(31):29466-78 (2001)。

Transcription activator-like effector nucleases(TALEN)

TALENs represent another form of modular nuclease whereby, like ZFNs, an engineered DNA binding domain is linked to a FokI nuclease domain and a pair of TALENs operate in tandem to achieve targeted DNA cleavage. The main difference with ZFNs is the nature of the DNA binding domain and the associated target DNA sequence recognition performance. The TALEN DNA binding domain is derived from a TALE protein, originally described in the plant bacterial pathogen xanthomonas (Xanthomonas sp.) in (b). TALEs comprise a tandem array of 33-35 amino acid repeats, each of which recognizes a single base pair in a target DNA sequence, typically up to 20 base pairs in length, resulting in a total target sequence length of up to 40 base pairs. The nucleotide specificity of each repeat sequence was determined by the Repeat Variable Diresidue (RVD) comprising exactly 2 amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are mainly recognized by the four RVDs respectively: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly. This constitutes a much simpler recognition code than zinc fingers and thus represents an advantage over the latter in terms of nuclease design. Nevertheless, like ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefited from the use of obligate heterodimer variants of fokl domains to reduce off-target activity.

Additional variants of FokI domains have been prepared which are inactivated with respect to their catalytic function. If half of the TALEN or ZFN pair contains an inactive FokI domain, only single-stranded DNA cleavage (nicking) will occur at the target site, not the DSB. The results are comparable to the use of CRISPR/Cas9 or CRISPR/Cpf1 "nickase" mutants, where one of the Cas9 cleavage domains has been inactivated. DNA nicking can be used to drive genome editing by HDR, but with less efficiency than using DSBs. The main benefit is that unlike DSBs (which are prone to NHEJ-mediated error repair), off-target incisions are repaired quickly and accurately.

Various TALEN-based systems have been described in the art and modifications thereof have been formally reported; see, for example, Boch,Science 326(5959) 1509-12 (2009); mak et al,Science 335(6069) 716-9 (2012); and Moscou et al,Science 326(5959):1501 (2009). Several groups have described the use of TALENs or cloning schemes based on the "Golden Gate" platform; see, e.g., Cermak et al,Nucleic Acids Res. 39(12) E82 (2011); li et al,Nucleic Acids Res. 39(14) 6315-25 (2011); weber et al,PLoS One. 6(2) E16765 (2011); wang et al,J Genet Genomics 41(6) 339-47, 2014, 5 months and 17 days electronic publication (2014); and Cerak T et al,Methods Mol Biol. 1239:133-59 (2015)。

Homing endonucleases

Homing Endonucleases (HEs) are sequence-specific endonucleases that have long recognition sequences (14-44 base pairs) and cleave DNA with high specificity, often at sites unique to the genome. There are at least 6 known HE families classified by their structure, including GIY-YIG, His-Cis box, H-N-H, PD- (D/E) xK, and Vsr-like, which are derived from a wide range of hosts, including eukaryotes, protists, bacteria, archaea, cyanobacteria, and bacteriophages. As with ZFNs and TALENs, HE can be used to establish DSBs at the target locus as an initial step in genome editing. In addition, some natural and engineered HEs cleave only a single strand of DNA, thereby acting as site-specific nickases. The large target sequences of HE and the specificity they provide have made them attractive candidates for the establishment of site-specific DSBs.

Various HE-based systems have been described in the art, and modifications thereof have been formally reported; see, e.g., Steentoft et al,Glycobiology 24(8) 663-80 (2014), Belfort and Bonocora,Methods Mol Biol. 11231-26 (2014), Hafez and Hausner,Genome 55(8) 553-69 (2012); and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev

As other examples of hybrid nucleases, the Megatal platform and the Tev-mTALEN platform utilize a fusion of a TALE DNA binding domain and a catalytically active HE, with TALDNA binding and specificity modulated by E, and cleavage sequence specificity by HE; see, e.g., Boissel et al, NAR 422591-2601 (2014); kleinstein et al,G3 41155-65 (2014); and Boissel and Scharenberg,Methods Mol. Biol. 1239: 171-96 (2015)。

In another variation, the MegaTev architecture is a fusion of a meganuclease (Mega) with a nuclease domain (Tev) derived from the GIY-YIG homing endonuclease I-TevI, the two active sites are ~ 30 base pairs apart at positions on the DNA substrate and produce two DSBs with incompatible sticky ends, see, e.g., Wolfs et al,NAR 42, 8816-29 (2014). It is anticipated that other combinations of existing nuclease-based protocols will evolve and can be used to achieve the targeted genomic modifications described herein.

dCas9-FokI or dCpf1-Fok1 and other nucleases

Combining the structural and functional properties of the nuclease platforms described above would provide another genome editing scheme that could potentially overcome some of the inherent drawbacks. As one example, CRISPR genome editing systems typically use a single Cas9 endonuclease to create DSBs. The specificity of targeting is driven by a 20 or 24 nucleotide sequence in the guide RNA that undergoes watson-crick base pairing with the target DNA (in the case of Cas9 from streptococcus pyogenes, plus an additional 2 bases in the adjacent NAG or NGG PAM sequence). Such sequences are of sufficient length to be unique in the human genome, however, the specificity of the RNA/DNA interaction is not absolute and can sometimes tolerate significant confusion, particularly in the 5' half of the target sequence, effectively reducing the number of bases driving specificity. One solution to this has been to completely inactivate Cas9 or Cpf1 catalytic functions-retaining only RNA-guided DNA binding functions-and alternatively to fuse the fokl domain to an inactivated Cas 9; see, e.g., Tsai et al, Nature Biotech 32:569-76 (2014); and Guilinger et al,Nature Biotech. 32:577-82 (2014). Because FokI must dimerize to become catalytically active, two guide RNAs are required to join two FokI fusions in close proximity to each otherDimer formation and DNA cleavage. This essentially doubles the number of bases in the combined target site, thereby increasing the targeting stringency of the CRISPR-based system.

As other examples, fusion of a TALE DNA binding domain to a catalytically active HE (such as I-TevI) takes advantage of the regulatable DNA binding and specificity of TALEs, as well as the cleavage sequence specificity of I-TevI, with the exception that off-target cleavage can be further reduced.

VII. kit

The present invention provides kits for carrying out the methods described herein. The kit may include one or more of: a genome-targeted nucleic acid, a polynucleotide encoding a genome-targeted nucleic acid, a localization polypeptide, a polynucleotide encoding a localization polypeptide, and/or any nucleic acid or proteinaceous molecule required to implement aspects of the methods described herein, or any combination thereof.

The kit may comprise: (1) a vector comprising a nucleotide sequence encoding a genome-targeted nucleic acid, (2) a targeting polypeptide or vector comprising a nucleotide sequence encoding a targeting polypeptide, and (3) an agent for reconstituting and/or diluting said vector and/or polypeptide.

The kit may comprise: (1) a vector comprising (i) a nucleotide sequence encoding a nucleic acid that targets a genome, and (ii) a nucleotide sequence encoding a localization polypeptide, and (2) an agent for reconstituting and/or diluting the vector.

In the above kit, the kit may comprise a single molecule guide nucleic acid targeting a genome. In the above kit, the kit may comprise a genome-targeted bimolecular nucleic acid. In the above kit, the kit may comprise two or more kinds of bimolecular guidance or monomolecular guidance. The kit may comprise a vector encoding a nucleic acid targeting the nucleic acid.

In the above kit, the kit may further comprise a polynucleotide to be inserted to achieve a desired genetic modification.

The components of the kit may be in separate containers or combined in a single container.

The kit described above may further comprise one or more additional reagents, wherein such additional reagents are selected from buffers, buffers for introducing the polypeptide or polynucleotide into a cell, wash buffers, control reagents, control vectors, control RNA polynucleotides, reagents for producing the polypeptide in vitro from DNA, adapters for sequencing, and the like. The buffer may be a stabilization buffer, a reconstitution buffer, a dilution buffer, or the like. The kit may also comprise one or more components that may be used to facilitate or enhance the binding or cleavage of DNA at the target by endonucleases, or to improve the specificity of targeting.

In addition to the components described above, the kit may contain instructions for using the components of the kit to practice the method. Instructions for practicing the methods may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate (such as paper or plastic) or the like. The instructions may be presented in the kit as a package instruction, in a label for the container of the kit or components thereof (i.e., accompanying the package or sub-package), etc. The instructions may be presented as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, flash memory, etc.). In some cases, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) may be provided. An example of this is a kit containing a web site where instructions can be viewed and/or from which instructions can be downloaded. As with the instructions, the manner in which the instructions are obtained can be recorded on a suitable substrate.

Specific methods and compositions of the invention

The invention therefore relates in particular to the following non-limiting methods according to the invention: in a first method (method 1), the present invention provides a method for editing a proprotein convertase subtilisin/Kexin 9 type (PCSK9) gene in a cell by genome editing, comprising the steps of: introducing one or more deoxyribonucleic acid (DNA) endonucleases into a cell to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions, or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

In another method (method 2), the invention provides an ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of: isolating hepatocytes from the patient; editing within or near the proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene or other DNA sequence encoding a regulatory element of the PCSK9 gene of hepatocytes; and implanting the genome-edited hepatocytes into the patient.

In another method (method 3), the invention provides the method of method 2, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the hepatocyte to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

In another method (method 4), the invention provides an ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of: generating patient-specific induced pluripotent stem cells (ipscs); edits are made within or near proprotein convertase subtilisin/Kexin type 9 (PCSK9) genes of ipscs or other DNA sequences encoding regulatory elements of the PCSK9 gene; differentiating the genome-edited ipscs into hepatocytes; and implanting the hepatocytes into the patient.

In another method (method 5), the invention provides the method of method 4, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases to the ipscs to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

In another method (method 6), the invention provides an ex vivo method for treating a patient having a PCSK 9-associated condition or disorder comprising the steps of: isolating mesenchymal stem cells from the patient; editing within or near the proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene or other DNA sequence encoding a regulatory element of the PCSK9 gene of mesenchymal stem cells; differentiating the genome-edited mesenchymal stem cells into hepatocytes; and implanting the hepatocytes into the patient.

In another method (method 7), the invention provides the method of method 6, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the mesenchymal stem cells to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

In another method (method 8), the invention provides an in vivo method for treating a patient having a PCSK 9-associated disorder, comprising the steps of: editing proprotein convertase subtilisin/Kexin type 9 (PCSK9) genes in cells of patients.

In another method (method 9), the invention provides the method of method 8, wherein the editing step comprises introducing one or more deoxyribonucleic acid (DNA) endonucleases into the cell to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs) within or adjacent to the PCSK9 gene or PCSK9 regulatory element that result in one or more permanent insertions, deletions or mutations of at least one nucleotide within or adjacent to the PCSK9 gene, thereby reducing or eliminating expression or function of the PCSK9 gene product.

In another method (method 10), the invention provides the method of any one of methods 8-9, wherein the cell is a hepatocyte.

In another approach (method 11), the invention provides the method of method 10, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are delivered to the hepatocytes by local injection, systemic infusion, or a combination thereof.

In another method (method 12), the invention provides a method of altering the continuous genomic sequence of the PCSK9 gene in a cell, comprising contacting the cell with one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more Single Strand Breaks (SSBs) or Double Strand Breaks (DSBs).

In another approach (method 13), the invention provides the method of method 12, wherein the alteration of the contiguous genomic sequence occurs in one or more exons of the PCSK9 gene.

In another method (method 14), the invention provides the method of any one of methods 1-13, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are selected from any of those listed in SEQ ID NOs 1-620, and variants having at least 70% homology to any of those listed in SEQ ID NOs 1-620.

In another method (method 15), the invention provides the method of method 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more proteins or polypeptides.

In another method (method 16), the invention provides the method of method 14, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more polynucleotides encoding the one or more DNA endonucleases.

In another method (method 17), the invention provides the method of method 16, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases.

In another method (method 18), the invention provides the method of method 17, wherein the one or more ribonucleic acids (RNAs) are one or more chemically modified RNAs.

In another approach (method 19), the invention provides the method of method 18, wherein the one or more ribonucleic acids (RNAs) are chemically modified in the coding region.

In another method (method 20), the invention provides the method of any one of methods 16-19, wherein the one or more polynucleotides or one or more ribonucleic acids (RNAs) are codon optimized.

In another method (method 21), the invention provides the method of any one of methods 1-20, wherein the method further comprises introducing one or more grnas or one or more sgrnas into the cell.

In another approach (method 22), the invention provides the method of method 21 in which one or more grnas or one or more sgrnas comprise a spacer sequence complementary to a segment of the coding sequence of the PCSK9 gene.

In another method (method 23), the invention provides a method of any one of methods 21-22, wherein the one or more grnas or one or more sgrnas are chemically modified.

In another method (method 24), the invention provides a method of any one of methods 21-23, wherein one or more grnas or one or more sgrnas are pre-complexed with one or more deoxyribonucleic acid (DNA) endonucleases.

In another approach (method 25), the invention provides the method of method 24, wherein the pre-complexing involves covalent attachment of one or more grnas or one or more sgrnas to one or more deoxyribonucleic acid (DNA) endonucleases.

In another approach (method 26), the invention provides the method of any one of methods 14-25, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle.

In another approach (method 27), the invention provides a method of any one of methods 21-25, wherein one or more deoxyribonucleic acid (DNA) endonucleases are formulated in a liposome or lipid nanoparticle that further comprises one or more grnas or one or more sgrnas.

In another method (method 28), the invention provides the method of any one of methods 12 or 21-22, wherein the one or more deoxyribonucleic acid (DNA) endonucleases are encoded in an AAV vector particle, wherein the AAV vector serotype is selected from those listed in tables 4 and 5.

In another method (method 29), the invention provides a method of any one of methods 21-22, wherein the one or more grnas or one or more sgrnas are encoded in an AAV vector particle, wherein the AAV vector serotype is selected from those listed in tables 4 and 5.

In another method (method 30), the invention provides the method of any one of methods 21-22, wherein one or more deoxyribonucleic acid (DNA) endonucleases are encoded in an AAV vector particle that also encodes one or more grnas or one or more sgrnas, wherein the AAV vector serotype is selected from those listed in tables 4 and 5.

The present invention also provides a composition (composition 1) comprising a single guide RNA comprising at least one spacer sequence which is an RNA sequence corresponding to any one of SEQ ID NO 5,305-28, 696.

In another composition (composition 2), the invention provides the single guide RNA of composition 1, wherein the single guide RNA further comprises a spacer extension region.

in another composition (composition 3), the invention provides the single guide RNA of composition 1, wherein the single guide RNA further comprises a tracrRNA extension.

In another composition (composition 4), the invention provides the single guide RNA of compositions 1-3, wherein the single guide RNA is chemically modified.

In another composition (composition 5), the present invention provides a non-naturally occurring CRISPR/Cas system comprising a polynucleotide encoding a Cas9 or Cpf1 enzyme and at least one single-molecule guide RNA of compositions 1-4.

In another composition (composition 6), the invention provides the CRISPR/Cas system of composition 5, wherein the polynucleotide encoding Cas9 or Cpf1 enzyme is selected from streptococcus pyogenes Cas9. Staphylococcus aureus Cas9, Neisseria meningitidis Cas9, Streptococcus thermophilus CRISPR1 Cas9, Streptococcus thermophilus CRISPR 3 Cas9, treponema dentiger Cas9,L. bacteriumND2006 Cpf1 and aminoacidococcus species BV3L6 Cpf1 and variants having at least 70% homology to these enzymes.

In another composition (composition 7), the present invention provides the CRISPR/Cas system of composition 6, wherein the polynucleotide encoding Cas9 or Cpf1 enzyme comprises one or more Nuclear Localization Signals (NLS).

In another composition (composition 8), the present invention provides the CRISPR/Cas system of composition 7, wherein at least one NLS is at or within 50 amino acids of the amino terminus of the polynucleotide encoding the Cas9 or Cpf1 enzyme, and/or at least one NLS is at or within 50 amino acids of the carboxy terminus of the polynucleotide encoding the Cas9 or Cpf1 enzyme.

In another composition (composition 9), the present invention provides the CRISPR/Cas system of composition 8, wherein the polynucleotide encoding Cas9 or Cpf1 enzyme is codon optimized for expression in eukaryotic cells.

In another composition (composition 10), the invention provides DNA encoding a single guide RNA of compositions 1-3.

In another composition (composition 11), the invention provides DNA encoding the CRISPR/Cas system of compositions 7-9.

In another composition (composition 12), the invention provides a vector comprising the DNA of the composition of 10 or 11.

In another composition (composition 13), the invention provides the vector of composition 12, wherein the vector is a plasmid.

in another composition (composition 14), the invention provides the vector of composition 12, wherein the vector is an AAV vector particle, and the AAV vector serotype is selected from the group consisting of SEQ ID NOs 1-620 and those listed in Table 2.

IX. Definition of

The terms "comprising" or "including" are used with reference to the compositions, methods, and respective components necessary for the present invention, and remain open to unspecified elements whether or not necessary.

The term "consisting essentially of … …" means that those elements are required for a given aspect. The terms allow for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the aspects of the invention.

The term "consisting of … …" means the compositions, methods, and respective components thereof described herein, excluding any elements not listed in the description of this aspect.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Certain numerical values presented herein are preceded by the term "about". The term "about" refers to a number within ± 10% of the recited number.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. 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. In case of conflict, the present specification will control.

Any numerical range recited in this specification describes all sub-ranges subsumed within that range with the same numerical precision (i.e., having the same number of assigned numbers). For example, the range "1.0 to 10.0" describes all sub-ranges between (and including) the minimum value of 1.0 and the maximum value of 10.0, such as, for example, "2.4 to 7.6", even though a range of "2.4 to 7.6" is not explicitly described in the text of the specification. The applicants reserve the right to modify the specification (including the claims) to specifically recite any sub-ranges of equal numerical precision to the extent that such sub-ranges are included within the ranges explicitly recited in the specification. All such ranges are inherently described in this specification such that modifications to explicitly describe any such subranges would comply with the written description, sufficiency of description, and over-range requirements, including the requirements under the 35 u.s.c. § 112(a) and 123(2) EPC regulations. In addition, unless explicitly stated or the context requires otherwise, all numerical parameters described in this specification (such as those expressing values, ranges, amounts, percentages, and so forth) may be read as if the antecedent "about," even if the antecedent "about" does not explicitly appear before the number. In addition, numerical parameters described in this specification should be construed in light of the number of reported significant digits, the accuracy of the value, and by applying ordinary rounding techniques. It will also be appreciated that the numerical parameters described in this specification will necessarily have inherent variability characteristics of the underlying measurement techniques used to determine the value of the parameter.

The details of one or more aspects of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural forms unless the context clearly dictates otherwise. 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 disclosure belongs. In case of conflict, the present specification will control.

The invention is further illustrated by the following non-limiting examples.

X. Examples

The present invention will be more fully understood by reference to the following examples, which provide exemplary, non-limiting aspects of the invention.

The examples describe the use of the CRISPR system as an exemplary genome editing technique for establishing a defined therapeutic genomic deletion, insertion or substitution (referred to herein as a "genome modification") in the PSCK9 gene that results in a permanent deletion or mutation of the PSCK9 gene that reduces or eliminates PSCK9 protein activity. The introduction of identified therapeutic modifications represents a novel therapeutic strategy for potential improvement of dyslipidemia, as described and explained herein.

Example 1 CRISPR/SpCas9 target site of PCSK9 Gene

Scanning a region of the PCSK9 gene for a target site. Each region was scanned for a Protospacer Adjacent Motif (PAM) with sequence NRG. The gRNA 20 bp spacer sequence corresponding to PAM was then identified as shown in SEQ ID NO 7,270-18,791 of the sequence Listing.