阅读说明：本技术 诱导外显子跳读的核酸-多肽组合物和方法 (Nucleic acid-polypeptide compositions and methods for inducing exon skipping ) 是由 亚瑟·A·莱文 安德鲁·约翰·吉尔 迈克尔·卡拉米安·科克伦 黄汉华 文卡塔·拉马纳·多帕拉 于 2018-09-21 设计创作，主要内容包括：本文公开了在错误剪接的mRNA转录物中诱导插入、缺失、复制或改变以诱导外显子跳读或外显子包含的分子和药物组合物。本文描述的还包括治疗疾病或病症的方法,其包括在错误剪接的mRNA转录物中诱导插入、缺失、复制或改变以诱导外显子跳读或外显子包含的分子或药物组合物。(Disclosed herein are molecules and pharmaceutical compositions that induce insertions, deletions, duplications, or alterations in mis-spliced mRNA transcripts to induce exon skipping or exon inclusion. Also described herein are methods of treating a disease or disorder comprising a molecule or pharmaceutical composition that induces insertions, deletions, duplications or alterations in a mis-spliced mRNA transcript to induce exon skipping or exon inclusion.)

1. A polynucleic acid conjugate comprising a target cell-binding moiety bound to at least one polynucleotide molecule that hybridizes to a target region of a pre-mRNA transcript of the DMD gene, wherein the at least one polynucleic acid molecule induces splicing out of an exon from the pre-mRNA transcript to generate an mRNA transcript encoding a functional dystrophin protein.

2. The polynucleic acid conjugate of claim 1, wherein the functional dystrophin protein is a truncated form of a dystrophin protein.

3. The polynucleic acid conjugate according to claim 1, wherein said target region is at an exon-intron junction, wherein said exon is an exon that is to be spliced out.

4. The polynucleic acid conjugate according to claim 3, wherein said exon is exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53 or 55.

5. The polynucleic acid conjugate of claim 3, wherein said exon-intron junction is located 5' to the exon to be spliced out.

6. The polynucleic acid conjugate of claim 5, wherein the target region is an intron region upstream of the exon-intron junction.

7. The polynucleic acid conjugate of claim 5 or 6, wherein the target region is located about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides upstream of the exon-intron junction.

8. The polynucleic acid conjugate of claim 3, wherein said exon-intron junction is located 3' to the exon to be spliced out.

9. The polynucleic acid conjugate according to claim 8, wherein the target region is an intron region downstream of the exon-intron junction.

10. The polynucleic acid conjugate according to claim 8 or 9, wherein the target region is located about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides downstream of the exon-intron junction.

11. The polynucleic acid conjugate according to any one of claims 1-10, wherein the target cell binding moiety binds to two or more, three or more, four or more, five or more, six or more or eight or more polynucleic acid molecules.

12. The polynucleic acid conjugate according to any of claims 1-10, wherein the polynucleic acid molecule is from about 10 to about 50 nucleotides in length.

13. The polynucleic acid conjugate according to any of claims 1 to 12, wherein the polynucleic acid molecule has about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID No 964-1285.

14. The polynucleic acid conjugate according to any of claims 1 to 13, wherein the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 964 and 1285.

15. The polynucleic acid conjugate according to any of claims 1-14, wherein the polynucleic acid molecule further comprises 1, 2, 3 or 4 mismatches.

16. The polynucleic acid conjugate according to any of claims 1 to 15, wherein the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-.

17. The polynucleic acid conjugate according to claim 16, wherein said polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 1056 and 1076.

18. The polynucleic acid conjugate according to claim 16, wherein said polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 1077-1094.

19. The polynucleic acid conjugate according to claim 16, wherein said polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1147-1162.

20. The polynucleic acid conjugate according to claim 16, wherein said polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 1173-1211.

21. The polynucleic acid conjugate according to any of claims 1-19, wherein the binding moiety comprises an antibody.

22. The polynucleic acid conjugate of claim 21, wherein said antibody comprises an anti-transferrin antibody.

23. The polynucleic acid conjugate according to any of claims 1-19, wherein the binding moiety comprises a plasma protein.

24. The polynucleic acid conjugate according to any of claims 1-23, wherein the polynucleic acid conjugate comprises:

A-(X¹-B)_n

formula (V)

Wherein the content of the first and second substances,

a comprises the binding moiety;

b consists of the polynucleic acid molecule;

X¹consisting of a bond or a first non-polymeric linker; and is

n is an average value selected from 1 to 12.

25. The polynucleic acid conjugate according to any of claims 1-24, wherein the polynucleic acid molecule comprises a passenger strand and a guide strand.

26. The polynucleic acid conjugate according to claim 25, wherein the guide strand comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5' -vinylphosphonate modified non-natural nucleotide, or a combination thereof.

27. The polynucleic acid conjugate according to claim 25, wherein the guide strand comprises about 2, 3, 4, 5, 6, 7, 8 or 9 phosphorothioate-modified non-natural nucleotides.

28. The polynucleic acid conjugate according to claim 25, wherein the guide strand comprises 1 phosphorothioate-modified non-natural nucleotide.

29. The polynucleic acid conjugate of any of claims 26-28, wherein the phosphorothioate modified non-natural nucleotide is located at an internucleotide linkage of the polynucleotide.

30. The polynucleic acid conjugate according to claim 26, wherein the at least one 5 '-vinylphosphonate modified non-natural nucleotide is located about 1, 2, 3, 4 or 5 bases from the 5' end of the guide strand.

31. The polynucleic acid conjugate according to claim 26 or 30, wherein the at least one 5 '-vinylphosphonate-modified non-natural nucleotide is further modified at the 2' -position.

32. The polynucleic acid conjugate according to claim 31, wherein the 2 ' -modification is selected from the group consisting of 2 ' -O-methyl, 2 ' -O-methoxyethyl (2 ' -O-MOE), 2 ' -deoxy, T-deoxy-2 ' -fluoro, 2 ' -O-aminopropyl (2 ' -O-AP), 2 ' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl (2 ' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE) or 2 ' -O-N-methylacetamido (2 ' -O-NMA) modified nucleotides.

33. The polynucleic acid conjugate according to claim 25, wherein the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorodiamidate morpholino oligomer modified non-natural nucleotides.

34. The polynucleic acid conjugate of claim 25, wherein the passenger strand comprises 100% phosphorodiamidate morpholino oligomer modified non-natural nucleotides.

35. The polynucleic acid conjugate of claim 25, wherein the passenger strand is shorter in length than the guide strand, thereby creating a 5 'overhang, a 3' overhang, or a combination thereof.

36. The polynucleic acid conjugate of claim 25, wherein the length of the passenger strand is equal to the length of the guide strand, thereby creating blunt ends at each end of the polynucleic acid molecule.

37. The polynucleic acid conjugate according to claim 35 or 36, wherein the polynucleic acid molecule is a phosphorodiamidate morpholino oligo/RNA heteroduplex.

38. The polynucleic acid conjugate according to claim 25, wherein the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptide nucleic acid modified non-natural nucleotides.

39. The polynucleic acid conjugate of claim 25, wherein the passenger strand comprises 100% peptide nucleic acid modified non-natural nucleotides.

40. The polynucleic acid conjugate of claim 25, wherein the passenger strand is shorter in length than the guide strand, thereby creating a 5 'overhang, a 3' overhang, or a combination thereof.

41. The polynucleic acid conjugate of claim 25, wherein the length of the passenger strand is equal to the length of the guide strand, thereby creating blunt ends at each end of the polynucleic acid molecule.

42. The polynucleic acid conjugate according to claim 40 or 41, wherein the polynucleic acid molecule is a peptide nucleic acid/RNA heteroduplex.

43. The polynucleic acid conjugate of claim 25, wherein the passenger strand is conjugated to a-X¹。

44. The polynucleic acid conjugate according to claim 43, wherein A-X¹Conjugated to the 5' end of the passenger strand.

45. The polynucleic acid conjugate according to claim 43, wherein A-X¹Conjugated to the 3' end of the passenger strand.

46. The polynucleic acid conjugate according to any of claims 24 or 43-45, wherein X¹Is a bond.

47. The polynucleic acid conjugate according to any of claims 24 or 43-45, wherein X ¹Is C₁-C₆An alkyl group.

48. The polynucleic acid conjugate according to any of claims 24 or 43-45, wherein X¹Is a homobifunctional or heterobifunctional linker, optionally conjugated to C₁-C₆An alkyl group.

49. The polynucleic acid conjugate according to claim 1, further comprising C.

50. The polynucleic acid conjugate of claim 49, wherein C is polyethylene glycol.

51. The polynucleic acid conjugate according to any of claims 24-50, wherein C is via X²Conjugated directly to B.

52. The polynucleic acid conjugate of claim 51, wherein X²Consisting of a bond or a second non-polymeric linker.

53. The polynucleic acid conjugate of claim 52, wherein X²Is a bond.

54. The polynucleic acid conjugate of claim 52, wherein X²Is C₁-C₆An alkyl group.

55. The polynucleic acid conjugate of claim 52, wherein X²Is a homobifunctional or heterobifunctional linker, optionally conjugated to C₁-C₆An alkyl group.

56. The polynucleic acid conjugate of any one of claims 1-55, wherein the passenger strand is conjugated to A-X¹And X²-C。

57. The polynucleic acid conjugate according to any of claims 1 to 56, wherein A-X¹Is conjugated to the 5' end of the passenger strand, and X ²-C is conjugated to the 3' end of the passenger strand.

58. The polynucleic acid conjugate according to any of claims 1 to 56, wherein X²-C is conjugated to the 5' end of the passenger strand, and A-X¹Conjugated to the 3' end of the passenger strand.

59. The polynucleic acid conjugate of any of claims 1-58, wherein the polynucleic acid conjugate comprises:

A-X¹-(B-X²-C)_n

formula (VI)

Wherein the content of the first and second substances,

a comprises the binding moiety;

b consists of the polynucleic acid molecule;

c consists of a polymer;

X¹consisting of a bond or a first non-polymeric linker;

X²by a bond or a second group of non-polymeric linkersForming; and is

n is an average value selected from 1 to 12.

60. The polynucleic acid conjugate according to claim 1, further comprising D.

61. The polynucleic acid conjugate according to claim 60, wherein D is an endosomolytic moiety.

62. A polynucleic acid molecule comprising at least 23 consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 1056-1058 or 1087-1089, wherein said polynucleic acid molecule comprises not more than 50 nucleotides in length.

63. A polynucleic acid molecule comprising SEQ ID NO 1056-1058, wherein said polynucleic acid molecule comprises NO more than 50 nucleotides in length.

64. A polynucleic acid molecule comprising SEQ ID NO 1087-1089, wherein said polynucleic acid molecule comprises NO more than 50 nucleotides in length.

65. A pharmaceutical composition comprising:

the polynucleic acid conjugate of claims 1-61 or the polynucleic acid molecule of claims 62-64; and

a pharmaceutically acceptable excipient.

66. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition is formulated for systemic delivery.

67. The pharmaceutical composition of claim 65 or 66, wherein the pharmaceutical composition is formulated for parenteral administration.

68. A method of treating a disease or condition characterized by a defective mRNA in a subject in need thereof, comprising:

administering to the subject the polynucleic acid conjugate of claims 1-61 or the polynucleic acid molecule of claims 62-64 to induce exon skipping that results in the production of processed mRNA encoding a functional protein from the defective mRNA, thereby treating the disease or condition in the subject.

69. The method of claim 68, wherein the disease or condition is a neuromuscular disease, a genetic disease, a cancer, a genetic disease, or a cardiovascular disease.

70. The method of claim 69, wherein the neuromuscular disease is muscular dystrophy.

71. The method of claim 70, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy.

72. A method of treating muscular dystrophy in a subject in need thereof comprising:

administering to the subject the polynucleic acid conjugate of claims 1-61 or the polynucleic acid molecule of claims 62-64, thereby treating the muscular dystrophy in the subject.

73. The method of claim 72, wherein the muscular dystrophy is Duchenne muscular dystrophy.

74. The method of any one of the preceding claims, wherein the subject is a human.

75. A kit comprising the polynucleic acid conjugate of claims 1 to 61 or the polynucleic acid molecule of claims 62 to 64.

Background

Modulation of RNA function is an area of ongoing therapeutic interest. Drugs that affect mRNA stability, such as antisense oligonucleotides and short interfering RNAs, are one way to modulate RNA function. Another set of oligonucleotides can modulate RNA function by altering pre-mRNA processing to include or exclude specific regions of pre-mRNA from the final gene product, the encoded protein. Oligonucleotide therapeutics therefore represent a means of modulating protein expression of disease states and therefore have utility as therapeutics.

Disclosure of Invention

In certain embodiments, disclosed herein are molecules and pharmaceutical compositions for modulating RNA processing. Also disclosed herein, in some embodiments, are molecules and pharmaceutical compositions for treating muscular dystrophy.

In certain embodiments, disclosed herein are methods of treating a disease or disorder caused by mis-spliced mRNA transcripts in a subject in need thereof, the method comprising: administering to the subject a polynucleic acid molecule conjugate; wherein the polynucleic acid molecule conjugate is conjugated to a cellular targeting binding moiety; wherein the polynucleotide optionally comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; wherein the polynucleic acid molecule conjugate induces insertions, deletions, duplications or alterations in the mis-spliced mRNA transcript to induce exon skipping (exon skipping) or exon inclusion (exon inclusion) in the mis-spliced mRNA transcript, thereby generating a fully processed mRNA transcript; and wherein the fully processed mRNA transcript encodes a functional protein, thereby treating the disease or disorder in the subject. In some embodiments, the disease or disorder is further characterized by one or more mutations in the mRNA. In some embodiments, the disease or disorder comprises a neuromuscular disease, a genetic disease, a cancer, a genetic disease, or a cardiovascular disease. In some embodiments, the disease or disorder is muscular dystrophy. In some embodiments, the disease or disorder is duchenne muscular dystrophy. In some embodiments, the exon skipping is exon skipping of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some embodiments, the exon skipping is an exon skipping of exon 23 of the DMD gene. In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (I):

A-X-B

Formula I

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide; and is

X is a bond or a first linker.

In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (II):

A-X-B-Y-C

formula II

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (III):

A-X-C-Y-B

formula III

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In some embodiments, the at least one 2 'modified nucleotide comprises morpholino, 2' -O-methyl, 2 '-O-methoxyethyl (2' -O-MOE), 2 '-O-aminopropyl, 2' -deoxy, t-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modified nucleotides. In some embodiments, the at least one 2' modified nucleotide comprises Locked Nucleic Acid (LNA), Ethylene Nucleic Acid (ENA), or Peptide Nucleic Acid (PNA). In some embodiments, the at least one 2' modified nucleotide comprises a morpholino. In some embodiments, the at least one inverted abasic moiety is At least one terminal end. In some embodiments, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleic acid molecule is at least about 10 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule has a length of at least one of: about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides. In some embodiments, the polynucleic acid molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the polynucleic acid molecule comprises at least one of: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification . In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 30% modification and about 20% to about 30% modification. In some embodiments, the polynucleic acid molecule comprises from about 10% to about 20% modifications. In some embodiments, the polynucleic acid molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% of the modifications. In some embodiments, the polynucleic acid molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications. In some embodiments, the polynucleic acid molecule comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications. In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides. In some embodiments, the polynucleic acid molecule comprises a single strand. In some embodiments, the polynucleic acid molecule comprises two or more strands. In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide that hybridizes to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the second polynucleotide comprises at least one modification. In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X and Y are independently a bond, a degradable linker, non-degradable A linker, a cleavable linker, or a non-polymeric linker group. In some embodiments, X is a bond. In some embodiments, X is C₁-C₆An alkyl group. In some embodiments, Y is C₁-C₆An alkyl group. In some embodiments, X is a homobifunctional or heterobifunctional linker, optionally conjugated to C₁-C₆An alkyl group. In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the binding moiety is an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5000 Da. In some embodiments, a-X is conjugated to the 5 'end of B and Y-C is conjugated to the 3' end of B. In some embodiments, Y-C is conjugated to the 5 'end of B and a-X is conjugated to the 3' end of B. In some embodiments, A-X, Y-C or a combination thereof is conjugated to an internucleotide linkage. In some embodiments, the method further comprises D. In some embodiments, D is conjugated to C or a. In some embodiments, D is conjugated to the molecular conjugate of formula (II) according to formula (IV):

(A-X-B-Y-C_c)-L-D

Formula IV

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker;

y is a bond or a second linker;

l is a bond or a third linker;

d is an endosomolytic (endosomolytic) moiety; and is

c is an integer of 0 to 1; and is

Wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or an inverted abasic moiety; and D is conjugated anywhere on A, B or C.

In some embodiments, D is INF7 or melittin. In some embodiments, L is C₁-C₆An alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the method further comprises at least a second binding moiety a. In some embodiments, the at least second binding moiety a is conjugated to A, B or C.

In some embodiments, disclosed herein is a method of inducing an insertion, deletion, duplication, or alteration in a mis-spliced mRNA transcript to induce exon skipping or exon inclusion in a mis-spliced mRNA transcript, the method comprising: contacting a target cell with a polynucleic acid molecule conjugate, wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety; hybridizing the polynucleic acid molecule conjugate to a mis-spliced mRNA transcript in a target cell to induce insertions, deletions, duplications or alterations in the mis-spliced mRNA transcript, thereby inducing exon skipping or exon inclusion, wherein the mis-spliced mRNA transcript is capable of encoding a functional form of a protein; and translating the functional form of the protein from the fully processed mRNA transcript of the previous step. In some embodiments, the target cell is a target cell of a subject. In some embodiments, the mis-spliced mRNA transcript further induces a disease or disorder. In some embodiments, the disease or disorder is further characterized by one or more mutations in the mRNA. In some embodiments, the disease or disorder comprises a neuromuscular disease, a genetic disease, a cancer, a genetic disease, or a cardiovascular disease. In some embodiments, the disease or disorder is muscular dystrophy. In some embodiments, the disease or disorder is duchenne muscular dystrophy. In some embodiments, the exon skipping is exon skipping of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some embodiments, the exon skipping is an exon skipping of exon 23 of the DMD gene. In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (I):

A-X-B

Formula I

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide; and is

X is a bond or a first linker.

In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (II):

A-X-B-Y-C

formula II

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In some embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (III):

A-X-C-Y-B

formula III

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In some embodiments, the at least one 2 'modified nucleotide comprises a morpholino, 2' -O-methyl, 2 '-O-methoxyethyl (2' -O-MOE), 2 '-O-aminopropyl, 2' -deoxy, T-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modified nucleotides. In some embodiments, the at least one 2' modified nucleotide comprises Locked Nucleic Acid (LNA), Ethylene Nucleic Acid (ENA), Peptide Nucleic Acid (PNA). In some embodiments, the at least one 2' modified nucleotide comprises a morpholino. In some embodiments, the at least one inverted abasic moiety is at least one terminus. In some embodiments, the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some embodiments, the polynucleic acid molecule is at least about 10 to about 30 nucleotides in length. In some embodiments, the polynucleic acid molecule has a length of at least one of: about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides. In some embodiments, the polynucleic acid molecule is at least about 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the polynucleic acid molecule comprises at least one of: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% modification % to about 70%, about 50% to about 70% and about 60% to about 70%. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification. In some embodiments, the polynucleic acid molecule comprises at least one of: about 10% to about 30% modification and about 20% to about 30% modification. In some embodiments, the polynucleic acid molecule comprises from about 10% to about 20% modifications. In some embodiments, the polynucleic acid molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% of the modifications. In some embodiments, the polynucleic acid molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications. In some embodiments, the polynucleic acid molecule comprises at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications. In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides. In some embodiments, the polynucleic acid molecule comprises a single strand. In some embodiments, the polynucleic acid molecule comprises two or more strands. In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide that hybridizes to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some embodiments, the The second polynucleotide comprises at least one modification. In some embodiments, the first polynucleotide and the second polynucleotide are RNA molecules. In some embodiments, the first polynucleotide and the second polynucleotide are siRNA molecules. In some embodiments, X and Y are independently a bond, a degradable linker, a non-degradable linker, a cleavable linker, or a non-polymeric linker group. In some embodiments, X is a bond. In some embodiments, X is C₁-C₆An alkyl group. In some embodiments, Y is C₁-C₆An alkyl group. In some embodiments, X is a homobifunctional or heterobifunctional linker, optionally conjugated to C₁-C₆An alkyl group. In some embodiments, Y is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the binding moiety is an antibody or binding fragment thereof. In some embodiments, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof. In some embodiments, C is polyethylene glycol. In some embodiments, C has a molecular weight of about 5000 Da. In some embodiments, a-X is conjugated to the 5 'end of B and Y-C is conjugated to the 3' end of B. In some embodiments, Y-C is conjugated to the 5 'end of B and a-X is conjugated to the 3' end of B. In some embodiments, A-X, Y-C or a combination thereof is conjugated to the internucleotide linkage. In some embodiments, the method further comprises D. In some embodiments, D is conjugated to C or a. In some embodiments, D is conjugated to the molecular conjugate of formula (II) according to formula (IV):

(A-X-B-Y-C_c)-L-D

Formula IV

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker;

y is a bond or a second linker;

l is a bond or a third linker;

d is an endosomolytic moiety; and is

c is an integer of 0 to 1; and is

Wherein the polynucleotide comprises at least one 2' modified nucleotide, at least one modified internucleotide linkage, or an inverted abasic moiety; and D is conjugated anywhere on A, B or C.

In some embodiments, D is INF7 or melittin. In some embodiments, L is C₁-C₆An alkyl group. In some embodiments, L is a homobifunctional linker or a heterobifunctional linker. In some embodiments, the method further comprises at least a second binding moiety a. In some embodiments, the at least second binding moiety a is conjugated to A, B or C. In some embodiments, the method is an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the subject is a human.

In certain embodiments, disclosed herein are pharmaceutical compositions comprising a molecule obtained by any one of the methods disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle formulation. In some embodiments, the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration.

In certain embodiments, disclosed herein are compositions comprising a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate comprises a polynucleotide comprising a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 45-963. In certain embodiments, disclosed herein are compositions comprising a polynucleic acid molecule conjugate, wherein the polynucleic acid molecule conjugate comprises a polynucleotide comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 45-963. In certain embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (I):

A-X-B

formula I

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide; and is

X is a bond or a first linker.

In certain embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (II):

A-X-B-Y-C

formula II

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In certain embodiments, the polynucleic acid molecule conjugate is a conjugate of formula (III):

A-X-C-Y-B

Formula III

Wherein the content of the first and second substances,

a is a binding moiety;

b is a polynucleotide;

c is a polymer;

x is a bond or a first linker; and is

Y is a bond or a second linker.

In certain embodiments, the at least one 2 'modified nucleotide comprises morpholino, 2' -O-methyl, 2 '-O-methoxyethyl (2' -O-MOE), 2 '-O-aminopropyl, 2' -deoxy, t-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) or 2 '-O-N-methylacetamido (2' -O-NMA) modified nucleotides. In some embodiments, the at least one 2' modified nucleotide comprises a morpholino.

In certain embodiments, disclosed herein is a polynucleic acid conjugate comprising a target cell-binding moiety bound to at least one polynucleotide molecule that hybridizes to a target region of a pre-mRNA transcript of the DMD gene, wherein the at least one polynucleic acid molecule induces splicing out of an exon from the pre-mRNA transcript to generate an mRNA transcript encoding a functional dystrophin protein. In some embodiments, the functional dystrophin protein is a truncated form of a dystrophin protein. In some embodiments, the target region is at an exon-intron junction, wherein the exon is an exon that is to be spliced out. In some embodiments, the exon is exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53 or 55. In some embodiments, the exon-intron junction is located 5' to the exon to be spliced out. In some embodiments, the target region is an intron region upstream of the exon-intron junction. In some embodiments, the target region is located about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides upstream of the exon-intron junction. In some embodiments, the exon-intron junction is located 3' to the exon to be spliced out. In some embodiments, the target region is an intron region downstream of the exon-intron junction. In some embodiments, the target region is located about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides downstream of the exon-intron junction. In some embodiments, the target cell-binding moiety binds to two or more, three or more, four or more, five or more, six or more, or eight or more polynucleic acid molecules. In some embodiments, the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some embodiments, the polynucleic acid molecule has about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO 964-1285. In some embodiments of the present invention, the substrate is, The polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule further comprises 1, 2, 3 or 4 mismatches. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-1094, 1147-1162 or 1173-1211. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1173-1211. In some embodiments, the binding moiety comprises an antibody. In some embodiments, the antibody comprises an anti-transferrin antibody. In some embodiments, the binding moiety comprises a plasma protein. In some embodiments, the polynucleic acid conjugate comprises a- (X) ¹-B)_n: formula (V), wherein a comprises the binding moiety; b consists of the polynucleic acid molecule; x¹Consisting of a bond or a first non-polymeric linker; n is an average value selected from 1 to 12. In some embodiments, the polynucleic acid molecule comprises a passenger strand and a guide strand. In some embodiments, the guide strand comprises at least one modified internucleotide linkage, at least one inverted abasic moiety, at least one 5' -vinylphosphonate modified non-natural nucleotide, or a combination thereof. In some embodiments, the guide strand comprises about 2, 3, 45, 6, 7, 8, or 9 phosphorothioate modified non-natural nucleotides. In some embodiments, the guide strand comprises 1 phosphorothioate modified non-natural nucleotide. In some embodiments, the phosphorothioate modified non-natural nucleotide is located at an internucleotide linkage of the polynucleotide. In some embodiments, the at least one 5 '-vinylphosphonate modified non-natural nucleotide is located about 1, 2, 3, 4, or 5 bases from the 5' terminus of the guide strand. In some embodiments, the at least one 5 '-vinylphosphonate modified non-natural nucleotide is further modified at the 2' -position. In some embodiments, the 2 ' -modification is selected from a 2 ' -O-methyl, 2 ' -O-methoxyethyl (2 ' -O-MOE), 2 ' -deoxy, T-deoxy-2 ' -fluoro, 2 ' -O-aminopropyl (2 ' -O-AP), 2 ' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl (2 ' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), or 2 ' -O-N-methylacetamido (2 ' -O-NMA) modified nucleotide. In some embodiments, the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorodiamidate morpholino oligomer modified non-natural nucleotides. In some embodiments, the passenger strand comprises 100% phosphorodiamidate morpholino oligomer modified non-natural nucleotides. In some embodiments, the passenger strand is shorter in length than the guide strand, thereby creating a 5 'overhang, a 3' overhang, or a combination thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby creating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a phosphorodiamidate morpholino oligomer/RNA heteroduplex. In some embodiments, the passenger strand comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptide nucleic acid modified non-natural nucleotides. In some embodiments, the passenger strand comprises 100% peptide nucleic acid modified non-natural nucleotides. In some embodiments, the passenger strand is shorter in length than the guide strand, thereby creating a 5' overhang, 3' overhangs or combinations thereof. In some embodiments, the passenger strand is equal in length to the guide strand, thereby creating blunt ends at each end of the polynucleic acid molecule. In some embodiments, the polynucleic acid molecule is a peptide nucleic acid/RNA heteroduplex. In some embodiments, the passenger strand is conjugated to a-X¹. In some embodiments, A-X¹Conjugated to the 5' end of the passenger strand. In some embodiments, A-X¹Conjugated to the 3' end of the passenger strand. In some embodiments, X¹Is a bond. In some embodiments, X¹Is C₁-C₆An alkyl group. In some embodiments, X¹Is a homobifunctional or heterobifunctional linker, optionally conjugated to C₁-C₆An alkyl group. In some embodiments, the polynucleic acid conjugate further comprises C. In some embodiments, C is polyethylene glycol. In some embodiments, C is via X²Conjugated directly to B. In some embodiments, X²Consisting of a bond or a second non-polymeric linker. In some embodiments, X²Is a bond. In some embodiments, X²Is C₁-C₆An alkyl group. In some embodiments, X²Is a homobifunctional or heterobifunctional linker, optionally conjugated to C ₁-C₆An alkyl group. In some embodiments, the passenger strand is conjugated to a-X¹And X²-C. In some embodiments, A-X¹Is conjugated to the 5' end of the passenger strand, and X²-C is conjugated to the 3' end of the passenger strand. In some embodiments, X²-C is conjugated to the 5' end of the passenger strand, and A-X¹Conjugated to the 3' end of the passenger strand. In some embodiments, the polynucleic acid conjugate comprises: A-X¹-(B-X²-C)_n(ii) a Formula (VI), wherein a comprises the binding moiety; b consists of the polynucleic acid molecule; c consists of a polymer; x¹Consisting of a bond or a first non-polymeric linker; x²Consisting of a bond or a second non-polymeric linker; and n is an average value selected from 1 to 12. In some embodiments, theThe polynucleic acid conjugate further comprises D. In some embodiments, D is an endosomolytic moiety.

In certain embodiments, disclosed herein is a polynucleic acid molecule comprising at least 23 consecutive bases of a base sequence selected from the group consisting of SEQ ID NOs 1056-1058 or 1087-1089, wherein said polynucleic acid molecule comprises NO more than 50 nucleotides in length.

In certain embodiments, disclosed herein is a polynucleic acid molecule comprising SEQ ID NO 1056-1058, wherein said polynucleic acid molecule comprises NO more than 50 nucleotides in length.

In certain embodiments, disclosed herein is a polynucleic acid molecule comprising SEQ ID NO 1087-1089, wherein said polynucleic acid molecule comprises NO more than 50 nucleotides in length.

In certain embodiments, disclosed herein is a pharmaceutical composition comprising: a polynucleic acid conjugate as described herein or a polynucleic acid molecule as described herein; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

In certain embodiments, disclosed herein is a method of treating a disease or condition characterized by a defective mRNA in a subject in need thereof, comprising: administering to the subject a polynucleic acid conjugate as described herein or a polynucleic acid molecule as described herein to induce exon skipping that results in the production of processed mRNA encoding a functional protein from the defective mRNA, thereby treating the disease or condition in the subject. In some embodiments, the disease or condition is a neuromuscular disease, a genetic disease, a cancer, a genetic disease, or a cardiovascular disease. In some embodiments, the neuromuscular disease is a muscular dystrophy. In some embodiments, the muscular dystrophy is duchenne muscular dystrophy, becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some embodiments, the subject is a human.

In certain embodiments, disclosed herein is a method of treating muscular dystrophy in a subject in need thereof, comprising: administering to the subject a polynucleic acid conjugate as described herein or a polynucleic acid molecule as described herein, thereby treating the muscular dystrophy of the subject. In some embodiments, the muscular dystrophy is duchenne muscular dystrophy. In some embodiments, the subject is a human.

In certain embodiments, disclosed herein are kits comprising a polynucleic acid conjugate described herein or a polynucleic acid molecule described herein.

In certain embodiments, disclosed herein are kits comprising a molecule obtained by any one of the methods disclosed herein.

Drawings

Figure 1 depicts the sequence of a terminal nucleotide extended Phosphorodiamidate Morpholino Oligo (PMO).

Figure 2A depicts the terminal nucleotide extended phosphorothioate antisense oligonucleotide (PS ASO) sequence.

FIG. 2B depicts a fully extended phosphorothioate antisense oligonucleotide (PS ASO) sequence.

Figure 3 depicts the method of quantifying the DMD mRNA read-out in total RNA using Taqman qPCR.

FIG. 4 depicts a chromatogram of an anti-CD 71 mAb-PMO reaction mixture generated using Hydrophobic Interaction Chromatography (HIC) method 2.

Fig. 5A depicts a chromatogram of anti-CD 71 mAb generated using Size Exclusion Chromatography (SEC) method 1.

Figure 5B depicts a chromatogram of anti-CD 71 mAb-PMO DAR1, 2 generated using Size Exclusion Chromatography (SEC) method 1.

FIG. 5C depicts a chromatogram of anti-CD 71 mAb-PMO DAR >2 generated using Size Exclusion Chromatography (SEC) method 1.

Fig. 6A depicts a chromatogram of anti-CD 71 mAb generated using Hydrophobic Interaction Chromatography (HIC) method 2.

Fig. 6B depicts a chromatogram of a purified anti-CD 71 mAb- PMODAR 1,2 conjugate produced using Hydrophobic Interaction Chromatography (HIC) method 2.

Figure 6C depicts a chromatogram of a purified anti-CD 71 mAb-PMODAR >2 conjugate produced using Hydrophobic Interaction Chromatography (HIC) method 2.

FIG. 7A depicts a chromatogram of a rapid protein liquid chromatography (FPLC) purification of anti-CD 71 Fab-PMO using Hydrophobic Interaction Chromatography (HIC) method 3.

Fig. 7B depicts a chromatogram of an anti-CD 71 Fab produced using SEC method 1.

Figure 7C depicts a chromatogram of an anti-CD 71 Fab-PMO DAR1 conjugate produced using SEC method 1.

Figure 7D depicts a chromatogram of an anti-CD 71 Fab-PMO DAR 2 conjugate produced using SEC method 1.

Figure 7E depicts a chromatogram of an anti-CD 71 Fab-PMO DAR 3 conjugate produced using SEC method 1.

Fig. 7F depicts a chromatogram of anti-CD 71 Fab produced using HIC method 4.

FIG. 7G depicts a chromatogram of an anti-CD 71 Fab-PMO DAR 1 conjugate produced using HIC method 4.

FIG. 7H depicts a chromatogram of an anti-CD 71 Fab-PMO DAR 2 conjugate produced using HIC method 4.

FIG. 7I depicts a chromatogram of an anti-CD 71 Fab-PMO DAR 3 conjugate produced using HIC method 4.

Fig. 8A depicts a chromatogram of an anti-CD 71 mAb-PS ASO reaction mixture generated using SAX method 2.

Fig. 8B depicts a chromatogram of anti-CD 71 mAb generated using SEC method 1.

Fig. 8C depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 1 conjugate generated using SEC method 1.

Fig. 8D depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 2 conjugate generated using SEC method 1.

Fig. 8E depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 3 conjugate produced using SEC method 1.

Figure 8F depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 1 conjugate produced using SAX method 2.

Figure 8G depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 2 conjugate produced using SAX method 2.

Figure 8H depicts a chromatogram of an anti-CD 71 mAb-PS ASO DAR 3 conjugate produced using SAX method 2.

FIG. 9 depicts an agarose gel of nested PCR using PMO and anti-CD 71 mAb-PMO conjugate to detect exon 23 skipping in differentiated C2C12 cells.

FIG. 10 depicts an agarose gel of nested PCR using PMO, anti-CD 71 mAb-PMO and anti-CD 71 Fab-PMO conjugates to detect exon 23 skipping in differentiated C2C12 cells.

Figure 11 depicts nested PCR agarose gels using PMO, ASO, conjugated anti-CD 71 mAb-ASO of DAR1 ("ASC-DAR 1"), conjugated anti-CD 71 mAb-ASO of DAR2 ("ASC-DAR 2"), and conjugated anti-CD 71 mAb-ASO of DAR3 ("ASC-DAR 3") to detect exon 23 skipping in differentiated C2C12 cells.

Fig. 12A depicts an agarose gel of nested PCR to detect exon 23 reads in gastrocnemius of wild-type mice administered a single intravenous injection of anti-CD 71 mAb-PMO conjugate.

FIG. 12B is a quantitative plot of PCR products from gastrocnemius.

Figure 12C is a graph of in vivo exon skipping from the gastrocnemius muscle of wild type mice quantified using Taqman qPCR.

Figure 13A depicts an agarose gel of nested PCR that detects exon 23 skipping in wild-type mouse myocardium after a single intravenous injection.

FIG. 13B is a quantitative plot of PCR products from myocardium.

Figure 14 depicts sequencing data of DNA fragments from jumped and wild type PCR products.

Figure 15 shows exon skipping activity of exon skipping PMOs targeting different lengths of exon 45 in human DMD pre-mRNA in transfected primary human skeletal muscle cells.

Figure 16 shows the binding of htfr1.mab-PMO conjugate to human transferrin receptor in vitro.

Figure 17 shows exon skipping activity of htfr1.mab-PMO conjugates in primary human skeletal muscle cells.

Figure 18 shows exon skipping activity of htfr1.mab-PMO conjugates in myotubes of primary and immortalized human skeletal muscle cells.

Detailed Description

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high selectivity and specificity. However, in some cases, nucleic acid therapy is also hindered by poor intracellular uptake, insufficient intracellular concentrations in the target cell, and low potency. To address these issues, various modifications of nucleic acid compositions have been explored, for example, novel linkers for better stabilization and/or reduced toxicity, optimization of binding moieties for improved target specificity and/or target delivery, and nucleic acid polymer modifications for improved stability and/or reduced off-target effects.

In some cases, one such area of use of oligonucleotides is for the treatment of muscular dystrophy. Muscular dystrophy includes several diseases affecting muscles. Duchenne muscular dystrophy is a severe form of muscular dystrophy and is caused by mutations in the DMD gene. In some cases, mutations in the DMD gene disrupt the translational reading frame and result in a nonfunctional dystrophin (dystrophin).

In certain embodiments, described herein are methods and compositions relating to nucleic acid therapy to induce insertions, deletions, duplications or alterations in mis-spliced mRNA transcripts to induce exon skipping or exon inclusion, which are used to restore the translational reading frame. In some embodiments, also described herein are methods and compositions for treating a disease or disorder characterized by a mis-processed mRNA transcript, wherein upon removal of an exon, the mRNA is capable of encoding a functional protein, thereby treating the disease or disorder. In additional embodiments, described herein include pharmaceutical compositions and kits for treating the disease or disorder.

RNA processing

RNA plays an important role in the regulation of gene expression and cell physiology. Proper processing of RNA is critical for translation of functional proteins. Alterations in RNA processing, such as the result of mis-splicing of RNA, can lead to disease. For example, mutation of a splice site results in the exposure of a premature stop codon, the loss of an exon, or the inclusion of an intron. In some cases, the alteration in RNA processing results in insertion, deletion, or replication. In some cases, the alteration in RNA processing results in insertion, deletion, or replication of an exon. In some cases, the alteration in RNA processing results in insertion, deletion, or replication of an intron.

Exon skipping

Exon skipping is a form of RNA splicing. In some cases, exon skipping occurs when an exon is skipped or spliced out of the processed mRNA. As a result of exon skipping, the processed mRNA does not contain skipped exons. In some cases, exon skipping results in the expression of altered products.

In some cases, Antisense Oligonucleotides (AONs) are used to induce exon skipping. In some cases, an AON is a short nucleic acid sequence that binds to a particular mRNA or pre-mRNA sequence. For example, AONs bind to splice sites or exon enhancers. In some cases, binding of an AON to a particular mRNA or pre-mRNA sequence generates a double-stranded region. In some cases, the formation of the double-stranded region occurs at a site where the spliceosome or a protein associated with the spliceosome will normally bind and cause the exon to be skipped. In some cases, skipping of exons results in restoration of the reading frame of the transcript and allows for the production of partially functional proteins.

Exon inclusion

In some cases, mutations in the RNA result in exon skipping. In some cases, the mutation is at least one of at a splice site, near a splice site, and at a distance from a splice site. In some cases, the mutation results in inactivation or attenuation of a splice site, disruption of at least one of an exon splicing enhancer or an intron splicing enhancer, and production of an exon splicing silencer or an intron splicing enhancer. In some cases, the mutation alters the secondary structure of the RNA. In some cases, mutations alter the secondary structure of RNA resulting in disruption of the accessibility of signals critical for exon recognition.

In some cases, the use of AONs results in inclusion of skipped exons. In some cases, the AON binds to at least one of a splice site, a site near the splice site, and a site remote from the splice site. In some cases, AONs bind at sites in RNA to prevent disruption of exonic or intronic splicing enhancers. In some cases, the AON binds at a site in the RNA to prevent the production of exon splicing silencers or intron splicing silencers.

Indications of

In some embodiments, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat diseases or disorders characterized by defective mRNA. In some embodiments, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat a disease or disorder by inducing insertion, deletion, duplication or alteration in a mis-spliced mRNA transcript to induce exon skipping or exon inclusion.

Most human protein-encoding genes are alternatively spliced. In some cases, the mutation results in incorrectly spliced or partially spliced mRNA. For example, the mutation is located in at least one of a splice site, a silencer or enhancer sequence, an exon sequence, or an intron sequence in the protein-encoding gene. In some cases, the mutation results in a gene dysfunction. In some cases, the mutation results in a disease or disorder.

In some cases, diseases or disorders caused by incorrectly or partially spliced mrnas include, but are not limited to, neuromuscular diseases, genetic diseases, cancer, genetic diseases, or cardiovascular diseases.

In some cases, the genetic disease or disorder comprises an autosomal dominant disorder, an autosomal recessive disorder, an X-linked dominant disorder, an X-linked recessive disorder, a Y-linked disorder, a mitochondrial disease, or a multi-factorial or polygenic disorder.

In some cases, cardiovascular diseases such as hypercholesterolemia are caused by incorrectly or partially spliced mRNA. In hypercholesterolemia, a single nucleotide polymorphism in exon 12 of the Low Density Lipoprotein Receptor (LDLR) has been shown to promote exon skipping.

In some cases, incorrectly or partially spliced mRNA can lead to cancer. For example, incorrectly or partially spliced mRNA affects cellular processes associated with cancer, including but not limited to proliferation, motility, and drug response. In some cases, solid or hematologic cancer. In some cases, the cancer is bladder cancer, lung cancer, brain cancer, melanoma, breast cancer, non-hodgkin's lymphoma, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cancer, skin cancer, leukemia, thyroid cancer, liver cancer, or uterine cancer.

In some cases, incorrectly or partially spliced mRNA can lead to a neuromuscular disease or disorder. Exemplary neuromuscular diseases include muscular dystrophy, such as duchenne muscular dystrophy, becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some cases, the muscular dystrophy is genetic. In some cases, muscular dystrophy is caused by spontaneous mutations. Becker muscular dystrophy and duchenne muscular dystrophy have been shown to be associated with mutations in the DMD gene, which encodes dystrophin. Facioscapulohumeral muscular dystrophy has been shown to be associated with mutations in the double homeobox 4(DUX4) gene.

In some cases, incorrectly or partially spliced mRNA can lead to duchenne muscular dystrophy. Duchenne muscular dystrophy results in severe muscle weakness and is caused by mutations in the DMD gene that prevent the production of functional dystrophins. In some cases, duchenne muscular dystrophy is the result of an exon mutation in the DMD gene. In some cases, duchenne muscular dystrophy is the result of a mutation of at least one of exons 1, 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, and 79 in the DMD gene. In some cases, duchenne muscular dystrophy is the result of a mutation of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 in the DMD gene. In some cases, duchenne muscular dystrophy is the result of a mutation in at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, and 55 in the DMD gene. In some cases, multiple exons are mutated. For example, mutations in exons 48-50 are common in Duchenne muscular dystrophy patients. In some cases, duchenne muscular dystrophy is the result of a mutation in exon 51. In some cases, duchenne muscular dystrophy is the result of a mutation in exon 23. In some cases, the mutation involves the deletion of one or more exons. In some cases, the mutation involves the replication of one or more exons. In some cases, the mutation involves a point mutation in an exon. For example, some patients have been shown to have a nonsense point mutation in exon 51 of the DMD gene.

In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat muscular dystrophy. In some cases, the polynucleic acid molecule or pharmaceutical composition described herein is used to treat duchenne muscular dystrophy, becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy or myotonic dystrophy. In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat duchenne muscular dystrophy.

Polynucleic acid molecules

In some embodiments, the polynucleic acid molecules described herein induce insertions, deletions, duplications or alterations in mis-spliced mRNA transcripts to induce exon skipping or exon inclusion. In some cases, the polynucleic acid molecule restores the translational reading frame. In some cases, the polynucleic acid molecule produces a functional and truncated protein.

In some cases, the polynucleic acid molecule targets an mRNA sequence. In some cases, the polynucleic acid molecule targets a splice site. In some cases, the polynucleic acid molecule targets a cis regulatory element. In some cases, the polynucleic acid molecule targets a trans regulatory element. In some cases, the polynucleic acid molecule targets an exonic splicing enhancer or an intronic splicing enhancer. In some cases, the polynucleic acid molecule targets an exon splicing silencer or an intron splicing silencer.

In some cases, the polynucleic acid molecule targets a sequence found in an intron or exon. For example, a polynucleic acid molecule targets a sequence found in an exon that mediates splicing of the exon. In some cases, the polynucleic acid molecule targets an exon recognition sequence. In some cases, the polynucleic acid molecule targets a sequence upstream of the exon. In some cases, the polynucleic acid molecule targets a sequence downstream of the exon.

As described above, the polynucleic acid molecule targets a mis-processed mRNA transcript that results in a disease or disorder, not limited to a neuromuscular disease, a genetic disease, a cancer, a genetic disease, or a cardiovascular disease. In some cases, the polynucleic acid molecule targets an mRNA transcript that results in the misprocessing of a neuromuscular disease or disorder. In some cases, the neuromuscular disease or disorder is duchenne muscular dystrophy, becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some cases, the polynucleic acid molecule targets an mRNA transcript that results in misprocessing of duchenne muscular dystrophy, becker muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, or myotonic dystrophy. In some cases, the polynucleic acid molecule targets a mis-processed mRNA transcript that results in duchenne muscular dystrophy.

In some cases, the polynucleic acid molecule targets an exon mutated in the DMD gene that causes duchenne muscular dystrophy. Exemplary exons mutated in the DMD gene that cause duchenne muscular dystrophy include, but are not limited to, exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78. In some cases, the polynucleic acid molecule targets a sequence adjacent to the mutated exon. For example, if exon 50 is deleted, the polynucleic acid molecule targets a sequence in exon 51, skipping exon 51. In another case, if there is a mutation in exon 23, the polynucleic acid molecule targets a sequence in exon 22, skipping exon 23.

In some cases, the polynucleic acid molecules described herein target a region located at an exon-intron junction of exon 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at an exon-intron junction of exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at an exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region located at the exon-intron junction of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon junction or the 3' exon-intron junction of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at a 5 'intron-exon junction or a 3' exon-intron junction of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon junction or the 3' exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

In some cases, the polynucleic acid molecule binds to a target region located at a 5 'intron-exon junction (e.g., the 5' intron-exon junction of exon 3 is an intron 2-exon 3 junction hybrid) of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at a 5 'intron-exon junction (e.g., the 5' intron-exon junction of exon 3 is an intron 2-exon 3 junction) of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 44 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5' intron-exon junction of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule binds to a target region located at a 3 'exon-intron junction (e.g., the 3' exon-intron junction of exon 3 is an exon 3-intron 3 junction hybrid) of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at a 3 'exon-intron junction (e.g., the 3' exon-intron junction of exon 3 is the exon 3-intron 3 junction) of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 8 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 23 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 35 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 43 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 44 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 50 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 52 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 3' exon-intron junction of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecules described herein target splice sites of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice sites of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice sites of exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice site of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice sites of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice sites of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target the splice sites of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target splice sites of exon 55 of the DMD gene. As used herein, a splice site includes a canonical splice site, a cryptic splice site, or an alternative splice site that is capable of inducing an insertion, deletion, duplication, or alteration in a mis-spliced mRNA transcript to induce exon skipping or exon inclusion.

In some embodiments, the polynucleic acid molecules described herein target a partially spliced mRNA sequence comprising additional exons associated with duchenne muscular dystrophy, such as exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63.

In some cases, the polynucleic acid molecule hybridizes to the target region proximal to the exon-intron junction. In some cases, the polynucleic acid molecules described herein target a region at exon 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 upstream (or 5') of at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at exon 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 upstream (or 5') of at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000nt, 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt upstream (or 5') of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region at about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp upstream (or 5') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene.

In some cases, the polynucleic acid molecules described herein target a region at exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 downstream (or 3') of at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 10nt, or 5nt downstream (or 3') of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target a region at least 1000 nucleotides (nt), 500nt, 400nt, 300nt, 200nt, 100nt, 80nt, 60nt, 50nt, 40nt, 30nt, 20nt, 10nt, or 5nt downstream (or 3') of exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region at about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

In some cases, the polynucleic acid molecules described herein target an internal region within exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, or 78 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 8 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 23 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 35 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 43 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 44 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 45 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 48 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 49 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 50 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 51 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 52 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 53 of the DMD gene. In some cases, the polynucleic acid molecules described herein target an internal region within exon 55 of the DMD gene.

In some cases, the polynucleic acid molecule hybridizes to a target region within at least one of exons 2, 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, and 78 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region within at least one of exons 3, 4, 5, 6, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region within at least one of exons 8, 23, 35, 43, 44, 45, 50, 51, 52, 53, or 55 of the DMD gene.

In some embodiments, the polynucleic acid molecules described herein target partially spliced mRNA sequences comprising exon 44 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of exon 44. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp upstream (or 5') of exon 44. In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of exon 44. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of exon 44.

In some cases, the polynucleic acid molecule hybridizes to a target region within exon 44 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon 44 junction or the 3' exon 44-intron junction.

In some embodiments, the polynucleic acid molecules described herein target partially spliced mRNA sequences comprising exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of exon 45. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp upstream (or 5') of exon 45. In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of exon 45. In some cases, the polynucleic acid molecule hybridizes to a target region approximately 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of exon 45.

In some cases, the polynucleic acid molecule hybridizes to a target region within exon 45 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon 45 junction or the 3' exon 45-intron junction.

In some embodiments, the polynucleic acid molecules described herein target partially spliced mRNA sequences comprising exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of exon 51. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp upstream (or 5') of exon 51. In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of exon 51. In some cases, the polynucleic acid molecule hybridizes to a target region approximately 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of exon 51.

In some cases, the polynucleic acid molecule hybridizes to a target region within exon 51 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon 51 junction or the 3' exon 51-intron junction.

In some embodiments, the polynucleic acid molecules described herein target partially spliced mRNA sequences comprising exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region upstream (or 5') of exon 53. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp upstream (or 5') of exon 53. In some cases, the polynucleic acid molecule hybridizes to a target region downstream (or 3') of exon 53. In some cases, the polynucleic acid molecule hybridizes to a target region about 5, 10, 15, 20, 50, 100, 200, 300, 400, or 500bp downstream (or 3') of exon 53.

In some cases, the polynucleic acid molecule hybridizes to a target region within exon 53 of the DMD gene. In some cases, the polynucleic acid molecule hybridizes to a target region located at the 5 'intron-exon 53 junction or the 3' exon 53-intron junction.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to a target sequence of interest. In some embodiments, the polynucleic acid molecule consists of a target sequence of interest.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a target sequence of interest. In some cases, the second polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the target sequence of interest. In some cases, the polynucleic acid molecule comprises a first polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a target sequence of interest and a second polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a target sequence of interest.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 964-1285. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 964-1285.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1056-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1056-1094. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO: 1056-.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1147-1162. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 1147-1162.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1173-1211. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 1173-1211.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1056-1076. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 1056-1076.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1077-1094. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1077-. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 1077-.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1056-1058. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO: 1056-1058.

In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 50% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 60% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 70% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 75% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 80% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 85% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 90% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 95% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 96% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 97% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 98% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule comprises a sequence having at least 99% sequence identity to SEQ ID NO 1087-1089. In some embodiments, the polynucleic acid molecule consists of SEQ ID NO 1087-1089.

In some embodiments, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 964-1285. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-1094, 1147-1162 or 1173-1211. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-1076. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1077-. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1147-1162. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of a base sequence selected from the group consisting of SEQ ID NO 1173-1211. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1056-1058. In some cases, the polynucleic acid molecule comprises at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more consecutive bases of the base sequence selected from the group consisting of SEQ ID NO 1087-1089. In some cases, the polynucleic acid molecule further comprises 1, 2, 3 or 4 mismatches.

In some embodiments, the polynucleic acid molecule comprises a guide strand and a passenger strand. In some cases, the guide strand comprises a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO 964-1285. In some cases, the guide strand comprises a sequence selected from the group consisting of SEQ ID NO 964-1285.

In some embodiments, a polynucleic acid molecule described herein comprises RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some cases, the RNA comprises short interfering RNA (sirna), short hairpin RNA (shrna), microrna (mirna), double stranded RNA (dsrna), transfer RNA (trna), ribosomal RNA (rrna), or intranuclear heterogeneous RNA (hnrna). In some cases, the RNA comprises shRNA. In some cases, the RNA comprises miRNA. In some cases, the RNA comprises dsRNA. In some cases, the RNA comprises a tRNA. In some cases, the RNA comprises rRNA. In some cases, the RNA comprises hnRNA. In some cases, the RNA comprises siRNA. In some cases, the polynucleic acid molecule comprises an siRNA. In some cases, the polynucleic acid molecule is an antisense oligonucleotide (ASO).

In some embodiments, the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some cases, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some cases, the polynucleic acid molecule is about 45 nucleotides in length. In some cases, the polynucleic acid molecule is about 40 nucleotides in length. In some cases, the polynucleic acid molecule is about 35 nucleotides in length. In some cases, the polynucleic acid molecule is about 30 nucleotides in length. In some cases, the polynucleic acid molecule is about 25 nucleotides in length. In some cases, the polynucleic acid molecule is about 20 nucleotides in length. In some cases, the polynucleic acid molecule is about 19 nucleotides in length. In some cases, the polynucleic acid molecule is about 18 nucleotides in length. In some cases, the polynucleic acid molecule is about 17 nucleotides in length. In some cases, the polynucleic acid molecule is about 16 nucleotides in length. In some cases, the polynucleic acid molecule is about 15 nucleotides in length. In some cases, the polynucleic acid molecule is about 14 nucleotides in length. In some cases, the polynucleic acid molecule is about 13 nucleotides in length. In some cases, the polynucleic acid molecule is about 12 nucleotides in length. In some cases, the polynucleic acid molecule is about 11 nucleotides in length. In some cases, the polynucleic acid molecule is about 10 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 45 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 40 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 35 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 30 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 25 nucleotides in length. In some cases, the polynucleic acid molecule is from about 10 to about 20 nucleotides in length. In some cases, the polynucleic acid molecule is about 15 to about 25 nucleotides in length. In some cases, the polynucleic acid molecule is from about 15 to about 30 nucleotides in length. In some cases, the polynucleic acid molecule is from about 12 to about 30 nucleotides in length. In some cases, the polynucleic acid molecule is about 19 to about 30 nucleotides in length. In some cases, the polynucleic acid molecule is about 20 to about 30 nucleotides in length. In some cases, the polynucleic acid molecule is about 19 to about 25 nucleotides in length. In some cases, the polynucleic acid molecule is about 20 to about 25 nucleotides in length.

In some embodiments, the polynucleic acid molecule is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 nucleotides in length. In some cases, the polynucleic acid molecule is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some cases, the polynucleic acid molecule is at least 15 nucleotides in length. In some cases, the polynucleic acid molecule is at least 18 nucleotides in length. In some cases, the polynucleic acid molecule is at least 19 nucleotides in length. In some cases, the polynucleic acid molecule is at least 20 nucleotides in length. In some cases, the polynucleic acid molecule is at least 21 nucleotides in length. In some cases, the polynucleic acid molecule is at least 22 nucleotides in length. In some cases, the polynucleic acid molecule is at least 23 nucleotides in length. In some cases, the polynucleic acid molecule is at least 24 nucleotides in length. In some cases, the polynucleic acid molecule is at least 25 nucleotides in length. In some cases, the polynucleic acid molecule is at least 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50 nucleotides in length. In some cases, the polynucleic acid molecule is about 45 nucleotides in length. In some cases, the polynucleic acid molecule is about 40 nucleotides in length. In some cases, the polynucleic acid molecule is about 35 nucleotides in length. In some cases, the polynucleic acid molecule is about 30 nucleotides in length. In some cases, the polynucleic acid molecule is about 29 nucleotides in length. In some cases, the polynucleic acid molecule is about 28 nucleotides in length. In some cases, the polynucleic acid molecule is about 27 nucleotides in length. In some cases, the polynucleic acid molecule is about 26 nucleotides in length. In some cases, the polynucleic acid molecule is about 25 nucleotides in length. In some cases, the polynucleic acid molecule is about 24 nucleotides in length. In some cases, the polynucleic acid molecule is about 23 nucleotides in length. In some cases, the polynucleic acid molecule is about 22 nucleotides in length. In some cases, the polynucleic acid molecule is about 21 nucleotides in length. In some cases, the polynucleic acid molecule is about 20 nucleotides in length. In some cases, the polynucleic acid molecule is about 19 nucleotides in length. In some cases, the polynucleic acid molecule is about 18 nucleotides in length. In some cases, the polynucleic acid molecule is about 17 nucleotides in length. In some cases, the polynucleic acid molecule is about 16 nucleotides in length. In some cases, the polynucleic acid molecule is about 15 nucleotides in length. In some cases, the polynucleic acid molecule is about 14 nucleotides in length. In some cases, the polynucleic acid molecule is about 13 nucleotides in length. In some cases, the polynucleic acid molecule is about 12 nucleotides in length. In some cases, the polynucleic acid molecule is about 11 nucleotides in length. In some cases, the polynucleic acid molecule is about 10 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide. In some cases, the polynucleic acid molecule comprises a second polynucleotide. In some cases, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide is the sense strand or the passenger strand. In some cases, the second polynucleotide is an antisense strand or a guide strand.

In some embodiments, the polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is about 10 to about 50 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

In some cases, the first polynucleotide is about 50 nucleotides in length. In some cases, the first polynucleotide is about 45 nucleotides in length. In some cases, the first polynucleotide is about 40 nucleotides in length. In some cases, the first polynucleotide is about 35 nucleotides in length. In some cases, the first polynucleotide is about 30 nucleotides in length. In some cases, the first polynucleotide is about 25 nucleotides in length. In some cases, the first polynucleotide is about 20 nucleotides in length. In some cases, the first polynucleotide is about 19 nucleotides in length. In some cases, the first polynucleotide is about 18 nucleotides in length. In some cases, the first polynucleotide is about 17 nucleotides in length. In some cases, the first polynucleotide is about 16 nucleotides in length. In some cases, the first polynucleotide is about 15 nucleotides in length. In some cases, the first polynucleotide is about 14 nucleotides in length. In some cases, the first polynucleotide is about 13 nucleotides in length. In some cases, the first polynucleotide is about 12 nucleotides in length. In some cases, the first polynucleotide is about 11 nucleotides in length. In some cases, the first polynucleotide is about 10 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 50 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 45 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 40 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 35 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 30 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 25 nucleotides in length. In some cases, the first polynucleotide is about 10 to about 20 nucleotides in length. In some cases, the first polynucleotide is about 15 to about 25 nucleotides in length. In some cases, the first polynucleotide is about 15 to about 30 nucleotides in length. In some cases, the first polynucleotide is about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, about 19 to about 30, about 19 to about 25, about 19 to about 24, about 19 to about 23, about 20 to about 30, about 20 to about 25, about 20 to about 24, about 20 to about 23, or about 20 to about 22 nucleotides in length.

In some cases, the second polynucleotide is about 50 nucleotides in length. In some cases, the second polynucleotide is about 45 nucleotides in length. In some cases, the second polynucleotide is about 40 nucleotides in length. In some cases, the second polynucleotide is about 35 nucleotides in length. In some cases, the second polynucleotide is about 30 nucleotides in length. In some cases, the second polynucleotide is about 25 nucleotides in length. In some cases, the second polynucleotide is about 20 nucleotides in length. In some cases, the second polynucleotide is about 19 nucleotides in length. In some cases, the second polynucleotide is about 18 nucleotides in length. In some cases, the second polynucleotide is about 17 nucleotides in length. In some cases, the second polynucleotide is about 16 nucleotides in length. In some cases, the second polynucleotide is about 15 nucleotides in length. In some cases, the second polynucleotide is about 14 nucleotides in length. In some cases, the second polynucleotide is about 13 nucleotides in length. In some cases, the second polynucleotide is about 12 nucleotides in length. In some cases, the second polynucleotide is about 11 nucleotides in length. In some cases, the second polynucleotide is about 10 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 50 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 45 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 40 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 35 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 30 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 25 nucleotides in length. In some cases, the second polynucleotide is about 10 to about 20 nucleotides in length. In some cases, the second polynucleotide is about 15 to about 25 nucleotides in length. In some cases, the second polynucleotide is about 15 to about 30 nucleotides in length. In some cases, the second polynucleotide is about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the polynucleic acid molecule further comprises blunt ends, overhangs, or combinations thereof. In some cases, the blunt end is a 5 'blunt end, a 3' blunt end, or both. In some cases, the overhang is a 5 'overhang, a 3' overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base-pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, or 4 non-base-pairing nucleotides. In some cases, the overhang contains 1 non-base-pairing nucleotide. In some cases, the overhang contains 2 non-base-pairing nucleotides. In some cases, the overhang contains 3 non-base-pairing nucleotides. In some cases, the overhang contains 4 non-base-pairing nucleotides.

In some embodiments, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule is 100% complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5 or fewer mismatches with a target sequence described herein. In some embodiments, the sequence of the polynucleic acid molecule has 4 or fewer mismatches with a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule has 3 or fewer mismatches with a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule has 2 or fewer mismatches with a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule has 1 or fewer mismatches with a target sequence described herein.

In some embodiments, the specificity of a polynucleic acid molecule that hybridizes to a target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some cases, the hybridization is a highly stringent hybridization condition.

In some embodiments, the polynucleic acid molecule has a reduced off-target effect. In some cases, "off-target" or "off-target effect" refers to any situation in which a polynucleic acid polymer directed against a given target causes an unintended effect by interacting directly or indirectly with another mRNA sequence, DNA sequence, or cellular protein or other moiety. In some cases, an "off-target effect" occurs when other transcripts are simultaneously degraded due to partial homology or complementarity between the other transcripts and the sense and/or antisense strands of a polynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural or synthetic or artificial nucleotide analogues or bases. In some cases, the polynucleic acid molecule comprises a combination of DNA, RNA and/or nucleotide analogs. In some cases, the synthetic or artificial nucleotide analog or base comprises a modification at one or more of a ribose moiety, a phosphate moiety, a nucleoside moiety, or a combination thereof.

In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some cases, the modification comprises H, OR, R, halogen, SH, SR, NH2, NHR, NR2, OR CN, wherein R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary or tertiary), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification includes an azo group, a ketone group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazine, or hydroxyamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some cases, the alkyl moiety further comprises a hetero substitution. In some cases, the carbon of the heterocyclic group is replaced with nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino (pyrrolidino).

In some cases, the modification at the 2 'hydroxyl group is a 2' -O-methyl modification or a 2 '-O-methoxyethyl (2' -O-MOE) modification. In some cases, the 2 '-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, while the 2 'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety. Exemplary chemical structures of 2 '-O-methyl modifications of adenosine molecules and 2' -O-methoxyethyl modifications of uridine are shown below.

In some cases, the modification at the 2 ' hydroxyl group is a 2 ' -O-aminopropyl modification, wherein an extended amine group comprising a propyl linker binds an amine group to a 2 ' oxygen. In some cases, the modification neutralizes the overall negative phosphate-derived charge of the oligonucleotide molecule by introducing a positive charge from the amine group per sugar, thereby improving cellular uptake properties due to its zwitterionic nature. An exemplary chemical structure of the 2' -O-aminopropyl nucleoside phosphoramidite is shown below.

In some cases, the modification at the 2 ' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which an oxygen molecule bonded at the 2 ' carbon is linked to the 4 ' carbon through a methylene group, thereby forming a 2 ' -C,4 ' -C-oxy-methylene linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of the LNA is shown below. The representation shown on the left highlights the chemical connectivity of the LNA monomer. The right panel shows the 3' -endo highlighting the locking of the furanose ring of the LNA monomer: ( ³E) A conformation.

In some cases, the modification at the 2 ' hydroxyl group includes Ethylene Nucleic Acids (ENA), such as 2 ' -4 ' -ethylene bridged nucleic acids, which lock the sugar conformation to C_3’-an internal sugar folding (puckering) conformation. The ENA is part of a modified nucleic acid of the class of bridged nucleic acids, which modified nucleic acid also comprises LNA. Exemplary chemical structures of ENA and bridged nucleic acids are shown below.

In some embodiments, other modifications at the 2 'hydroxyl group include 2' -deoxy, T-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE), or 2 '-O-N-methylacetamido (2' -O-NMA).

In some embodiments, the nucleotide analogs comprise modified bases such as, but not limited to, 5-propynyl uridine, 5-propynyl cytidine, 6-methyladenine, 6-methylguanine, N, -dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5- (2-amino) propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methyluridine, 7-methyluridine, 2, 2-dimethyluridine, 5-methylaminoethyluridine, 5-methyloxyuridine, deaza nucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thiobases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, stevioside, gunoside, naphthyl and substituted naphthyl, any O-alkylated and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-hydroxyacetic acid, pyridin-4-one, pyridin-2-one, phenyl and modified phenyl such as aminophenol or 2,4, 6-trimethoxybenzene, Modified cytosine acting as a G-clamp (clamp) nucleotide, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyalkylalkylribonucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include nucleotides modified at the sugar moiety, as well as nucleotides having a non-ribosyl sugar or an analog thereof. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4' -thioribose, and other sugars, heterocyclic or carbocyclic rings. The term nucleotide also encompasses universal bases known in the art. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, the nucleotide analog further comprises morpholinos, Peptide Nucleic Acids (PNAs), methylphosphonate nucleotides, thiol phosphonate nucleotides, 2 '-fluoro N3-P5' -phosphoramidites, 1 ', 5' -anhydrohexitol nucleic acids (HNAs), or combinations thereof. Morpholino or Phosphorodiamidate Morpholino Oligonucleotides (PMOs) include synthetic molecules whose structure mimics the structure of natural nucleic acids but deviates from the normal sugar and phosphate structures. In some cases, the five-membered ribose ring is substituted with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked through a phosphorodiamidite group rather than a phosphate group. In such cases, the backbone change removes all positive and negative charges, enabling the morpholino neutral molecule to cross the cell membrane without the aid of a cell delivery agent, such as that used with charged oligonucleotides.

In some embodiments, the Peptide Nucleic Acids (PNAs) are free of sugar rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating backbone charges.

In some embodiments, one or more modifications optionally occur at the internucleotide linkage. In some cases, modified internucleotide linkages include, but are not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5 '-alkylenephosphonate, 5' -methylphosphonate, 3 '-alkylenephosphonate, boron trifluoride, 3' -5 'linked or 2' -5 'linked boranophosphate and selenophosphate, phosphotriester, thiocarbonylalkylphosphotriester, hydrophosphonate linkages, alkylphosphonate, alkylphosphonothioate, arylphosphorothioate, phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate, phosphinate, phosphoramidate, 3' -alkylaminophosphate, aminoalkylphosphoramidate, thiocarbonylaminophosphate, piperazine phosphate, aniline phosphorothioate (phosphoroanilothioate), aniline phosphate (phosphoranilidate), ketone, sulfone, sulfonamide, carbonate, carbamate, ketone, sulfone, and phosphate, Methylene hydrazine (methylene hydrazine), methylene dimethylhydrazine (methylene hydrazine), formacetal, thiometaldehyde, oxime, methylene imine, methylene methyl imine, thioamide, linkages with a ribose acetyl group, aminoethylglycine, silyl or siloxane linkages, alkyl or cycloalkyl linkages with or without heteroatoms, e.g., 1 to 10 saturated or unsaturated and/or substituted and/or heteroatom containing carbons, linkages with morpholino structures, amides or polyamides where the base is directly or indirectly attached to the nitrogen heteroatom of the backbone, and combinations thereof. Phosphorothioate antisense oligonucleotides (PS ASOs) are antisense oligonucleotides comprising phosphorothioate linkages. An exemplary PS ASO is shown below.

In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate or thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left) and methylphosphonate nucleotides (right) are shown below.

In some cases, modified nucleotides include, but are not limited to, 2 '-fluoro-N3-P5' -phosphoramidite, as shown below:

in some cases, modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1 ', 5' -anhydrohexitol nucleic acids (HNAs)) as shown below:

in some embodiments, the above-described nucleotide analogs or artificial nucleotide bases comprise a nucleotide nucleic acid having a modified 5 '-vinylphosphonate modification at the 5' hydroxyl of the ribose moiety. In some embodiments, the 5' -vinylphosphonate modified nucleotide is selected from the nucleotides provided below, wherein X is O or S; and B is a heterocyclic base moiety.

In some cases, the modification at the 2 ' hydroxyl group is a 2 ' -O-aminopropyl modification, wherein an extended amine group comprising a propyl linker binds an amine group to a 2 ' oxygen. In some cases, this modification neutralizes the overall negative phosphate-derived charge of the oligonucleotide molecule by introducing one positive charge per sugar from the amine group, and improves cellular uptake properties due to its zwitterionic nature.

In some cases, 5 '-vinylphosphonate modified nucleotides are further modified at the 2' hydroxyl group in a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), wherein the oxygen molecule bound to the 2 'carbon is linked to the 4' carbon through a methylene group, thereby forming a 2 '-C, 4' -C-oxy-methylene linked bicyclic ribonucleotide monomer. An exemplary representation of the chemical structure of the 5' -vinylphosphonate-modified LNA is shown below, wherein X is O or S; b is a heterocyclic base moiety; and J is an internucleotide linkage group attached to adjacent nucleotides of the polynucleotide.

In some embodiments, additional modifications at the 2 'hydroxyl group include 2' -deoxy, T-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE), or 2 '-O-N-methylacetamido (2' -O-NMA).

In some embodiments, the nucleotide analogs comprise modified bases such as, but not limited to, 5-propynyl uridine, 5-propynyl cytidine, 6-methyladenine, 6-methylguanine, N, -dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5- (2-amino) propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methyluridine, 7-methyluridine, 2, 2-dimethyluridine, 5-methylaminoethyluridine, 5-methyloxyuridine, deaza-nucleotides (such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine or 6-azothymidine), 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine, 4-thiouridine and 2-thiocytidine), dihydrouridine, pseudouridine, stevioside, gunoside, naphthyl and substituted naphthyl, any O-alkylated and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-hydroxyacetic acid, pyridin-4-one or pyridin-2-one), phenyl and modified phenyl such as aminophenol or 2,4, 6-trimethoxybenzene, modified cytosine acting as a G-clamp (clamp) nucleotide, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyalkylalkylribonucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. 5 '-vinylphosphonate modified nucleotides also include nucleotides modified at the sugar moiety, as well as 5' -vinylphosphonate modified nucleotides having a non-ribosyl sugar or an analog thereof. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4' -thioribose, and other sugars, heterocyclic or carbocyclic rings. The term nucleotide also encompasses universal bases known in the art. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

In some embodiments, the 5 ' -vinyl phosphonate modified nucleotide analog further comprises a morpholino, Peptide Nucleic Acid (PNA), methylphosphonate nucleotide, thiol phosphonate nucleotide, 2 ' -fluoro N3-P5 ' -phosphoramidite, or 1 ', 5 ' -anhydrohexitol nucleic acid (HNA). Morpholino or Phosphorodiamidate Morpholino Oligonucleotides (PMOs) include synthetic molecules whose structure mimics the structure of natural nucleic acids but deviates from the normal sugar and phosphate structures. In some cases, the five-membered ribose ring is substituted with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked through a phosphorodiamidite group rather than a phosphate group. In such cases, the backbone change removes all positive and negative charges, enabling the morpholino neutral molecule to cross the cell membrane without the aid of a cell delivery agent, such as that used with charged oligonucleotides. Non-limiting examples of 5' -vinylphosphonate modified morpholino oligonucleotides are shown below, wherein X is O or S; and B is a heterocyclic base moiety.

In some embodiments, the 5' -vinylphosphonate modified morpholino or PMO described above is a PMO comprising a positive or cationic charge. In some cases, the PMO is fmoplus (sarepta). FMOplus refers to phosphorodiamidate morpholino oligomers comprising any number of (1-piperazino) phosphinideneoxy, (1- (4- (ω -guanidino-alkanoyl)) -piperazino) phosphinideneoxy linkages (e.g., as described in PCT publication WO 2008/036127). In some cases, the PMO is the PMO described in us patent 7943762.

In some embodiments, the morpholino or PMO described above is PMO-x (sarepta). In some cases, PMO-X refers to phosphorodiamidate morpholino oligomers comprising at least one linkage or at least one disclosed end modification, such as those described in PCT publication WO2011/150408 and U.S. publication 2012/0065169.

In some embodiments, the morpholino or PMO described above is a PMO as described in table 5 of U.S. publication 2014/0296321.

An exemplary representation of the chemical structure of a 5' -vinylphosphonate-modified nucleic acid is shown below, wherein X is O or S; b is a heterocyclic base moiety; and J is an internucleotide linkage.

In some embodiments, the Peptide Nucleic Acids (PNAs) are free of sugar rings or phosphate linkages, and the bases are linked and appropriately spaced by oligoglycine-like molecules, thus eliminating backbone charges.

In some embodiments, one or more modifications of the 5' -vinylphosphonate modified oligonucleotide optionally occur at internucleotide linkages. In some cases, modified internucleotide linkages include, but are not limited to, phosphorothioates; a phosphorodithioate ester; methylphosphonate esters; 5' -alkylene phosphonates; 5' -methylphosphonate; 3' -alkylene phosphonates; boron trifluoride; 3 '-5' linked or 2 '-5' linked borane phosphates and selenophosphates; a phosphoric acid triester; thiocarbonylalkylphosphotriester; a hydrogen phosphonate bond; an alkyl phosphonate; an alkyl thiophosphate; an aryl thiophosphate; selenophosphoric acid esters (phosphoroselenoate); a phosphoric acid diselenide ester; a phosphinate; a phosphoramidate; 3' -alkyl phosphoramidates; aminoalkyl phosphoramidates; a thiocarbonylaminophosphoric acid ester; piperazine phosphate; aniline thiophosphate (phosphoroanilothioate); aniline phosphate (phosphoranilidate); a ketone; a sulfone; a sulfonamide; a carbonate ester; a carbamate; methylene hydrazine (methylene hydrazine); methylene dimethylhydrazono (methylene dimethylhydrazo); formacetal; a thiometal; an oxime; a methylene imine; a methylene methyl imine; thioamides; a linkage with a riboacetyl group; aminoethylglycine; a silyl or siloxane linkage; alkyl or cycloalkyl with or without heteroatoms linking, for example, 1 to 10 saturated or unsaturated and/or substituted and/or heteroatom containing carbons; a linkage having a morpholino structure, an amide, or a polyamide, wherein a base is directly or indirectly attached to the nitrogen aza nitrogen of the backbone; and combinations thereof.

In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate or thiol phosphonate modification. Exemplary thiol phosphonate nucleotides (left), phosphorodithioates (center), and methylphosphonate nucleotides (right) are shown below.

In some cases, 5' -vinylphosphonate modified nucleotides include, but are not limited to, phosphoramidites as shown below:

in some cases, the modified internucleotide linkage is a phosphorodiamidate linkage. Non-limiting examples of phosphorodiamidite linkages to morpholino systems are shown below.

In some cases, the modified internucleotide linkage is a methylphosphonate linkage. Non-limiting examples of methylphosphonate linkages are shown below.

In some cases, the modified internucleotide linkage is an amide linkage. Non-limiting examples of amide linkages are shown below.

In some cases, 5' -vinylphosphonate modified nucleotides include, but are not limited to, the modified nucleic acids shown below.

In some embodiments, the one or more modifications comprise a modified phosphate backbone, wherein the modification results in a neutral or uncharged backbone. In some cases, the phosphate backbone is modified by alkylation to produce an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, as used herein in the context of alkylation, alkyl refers to a straight or branched chain saturated hydrocarbon group containing 1 to 6 carbon atoms. In some cases, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1-dimethylbutyl, 2-dimethylbutyl, 3-dimethylbutyl, and 2-ethylbutyl. In some cases, the modified phosphate is a phosphate group as described in U.S. patent 9481905.

In some embodiments, the additional modified phosphate backbone comprises methylphosphonate, ethylphosphonate, methylphosphonothioate, or methoxyphosphonate. In some cases, the modified phosphate is methylphosphonate. In some cases, the modified phosphate is ethyl phosphonate. In some cases, the modified phosphate is a methyl thiophosphonate. In some cases, the modified phosphate is a methoxyphosphonate.

In some embodiments, the one or more modifications further optionally include modifications of the ribose moiety, the phosphate backbone, and the nucleoside, or modifications of the nucleotide analog at the 3 'or 5' terminus. For example, the 3 ' terminus optionally comprises a 3 ' cationic group, or the nucleoside is inverted with a 3 ' -3 ' linkage at the 3 ' terminus. In another alternative, the 3 'terminus is optionally conjugated to an aminoalkyl group, such as a 3' C5-aminoalkyl dT. In another alternative, the 3' terminus is optionally conjugated to an abasic site, for example to an apurinic or apyrimidinic site. In some cases, the 5 'terminus is conjugated to an aminoalkyl group, e.g., to a 5' -O-alkylamino substituent. In some cases, the 5' terminus is conjugated to an abasic site, e.g., to an apurinic or apyrimidinic site.

In some embodiments, the polynucleic acid molecule comprises one or more artificial nucleotide analogs described herein. In some cases, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more artificial nucleotide analogs described herein. In some embodiments, the artificial nucleotide analog comprises a 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy, T-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), or 2' -O-N-methylacetamido (2 '-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, N-methylacetamido (2' -O-NMA) modification, Methylphosphonate nucleotides, thiol phosphonate nucleotides, 2 '-fluoro N3-P5' -phosphoramidite, or combinations thereof. In some cases, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more artificial nucleotide analogs selected from the group consisting of 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy, T-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), or 2' -O-N-methylacetoacetyl Amino (2 ' -O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiol phosphonate nucleotide, 2 ' -fluoro N3-P5 ' -phosphoramidite, or a combination thereof. In some cases, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2' -O-methyl modified nucleotides. In some cases, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2 '-O-methoxyethyl (2' -O-MOE) modified nucleotides. In some cases, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiol phosphonate nucleotides.

In some embodiments, the polynucleic acid molecule comprises a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified non-natural nucleotides, and optionally comprises at least one inverted abasic moiety. In some cases, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20 or more phosphorodiamidate morpholino oligomer modified non-natural nucleotides. In some cases, the polynucleic acid molecule comprises 100% phosphorodiamidate morpholino oligomer modified non-natural nucleotides.

In some cases, the polynucleic acid molecule comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptide nucleic acid modified non-natural nucleotides. In some cases, the polynucleic acid molecule comprises 100% peptide nucleic acid modified non-natural nucleotides.

In some embodiments, the polynucleic acid molecule comprises one or more nucleotide analogs, wherein each nucleotide analog is in a stereochemically isomeric form. In such a case, the polynucleic acid molecule is a chiral molecule. In some cases, the nucleotide analog comprises a backbone stereochemistry. In other cases, the nucleotide analogs include chiral analogs described in U.S. Pat. nos. 9,982,257, 9,695,211, or 9,605,019.

In some cases, the polynucleic acid molecule comprises at least one of: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification.

In some cases, the polynucleic acid molecule comprises at least one of: about 10% to about 30% modification and about 20% to about 30% modification.

In some cases, the polynucleic acid molecule comprises from about 10% to about 20% modifications.

In some cases, the polynucleic acid molecule comprises from about 15% to about 90%, from about 20% to about 80%, from about 30% to about 70%, or from about 40% to about 60% of the modifications.

In further instances, the polynucleic acid molecule comprises at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modifications.

In some embodiments, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.

In some cases, the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.

In some cases, about 5% to about 100% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 5% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 10% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 15% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 20% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 25% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 30% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 35% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 40% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 45% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 50% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 55% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 60% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 65% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 70% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 75% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 80% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 85% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 90% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 95% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 96% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 97% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 98% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 99% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some cases, about 100% of the polynucleic acid molecules comprise an artificial nucleotide analog described herein. In some embodiments, the artificial nucleotide analog comprises a 2 '-O-methyl, 2' -O-methoxyethyl (2 '-O-MOE), 2' -O-aminopropyl, 2 '-deoxy, T-deoxy-2' -fluoro, 2 '-O-aminopropyl (2' -O-AP), 2 '-O-dimethylaminoethyl (2' -O-DMAOE), 2 '-O-dimethylaminopropyl (2' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 '-O-DMAEOE), or 2' -O-N-methylacetamido (2 '-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, N-methylacetamido (2' -O-NMA) modification, Methylphosphonate nucleotides, thiol phosphonate nucleotides, 2 '-fluoro N3-P5' -phosphoramidite, or combinations thereof.

In some embodiments, the polynucleic acid molecule comprises from about 1 to about 25 modifications, wherein the modifications comprise an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 1 modification, wherein the modification comprises an artificial nucleotide analog described herein. In some embodiments, the polynucleic acid molecule comprises about 2 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 3 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 4 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 5 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 6 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 7 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 8 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 9 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 10 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 11 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 12 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 13 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 14 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 15 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 16 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 17 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 18 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 19 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 20 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 21 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 22 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 23 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 24 modifications, wherein the modifications comprise artificial nucleotide analogs described herein. In some embodiments, the polynucleic acid molecule comprises about 25 modifications, wherein the modifications comprise artificial nucleotide analogs described herein.

In some embodiments, a polynucleic acid molecule is assembled from two separate polynucleotides, wherein one polynucleotide comprises the sense strand and the second polynucleotide comprises the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides in the sense strand comprise 2 '-O-methyl pyrimidine nucleotides and the purine nucleotides in the sense strand comprise 2' -deoxypurine nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotides present in the sense strand comprise 2' -deoxy-2 ' -fluoro pyrimidine nucleotides, and wherein the purine nucleotides present in the sense strand comprise 2' -deoxy purine nucleotides.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein a pyrimidine nucleotide is a 2' -deoxy-2 ' -fluoro pyrimidine nucleotide when present in the antisense strand, and a purine nucleotide is a 2' -O-methyl purine nucleotide when present in the antisense strand.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein a pyrimidine nucleotide, when present in the antisense strand, is a 2' -deoxy-2 ' -fluoro pyrimidine nucleotide, and wherein a purine nucleotide, when present in the antisense strand, comprises a 2' -deoxy-purine nucleotide.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a terminal cap moiety at the 5 'end, the 3' end, or both the 5 'and 3' ends of the sense strand. In other embodiments, the terminal headpiece is an inverted deoxyabasic moiety.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a phosphate backbone modification at the 3' end of the antisense strand. In some cases, the phosphate backbone modification is a phosphorothioate. In some cases, the passenger strand comprises more phosphorothioate modifications than the guide strand. In other cases, the guide strand contains more phosphorothioate modifications than the passenger strand. In further cases, the passenger strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications. In further instances, the guide strand comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate modifications.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises a glyceryl modification at the 3' end of the antisense strand.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, 5' end, or both 3 'and 5' ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense strand and/or antisense strand are chemically modified with 2 '-deoxy, 2' -O-methyl, and/or 2 '-deoxy-2' -fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or terminal cap molecules present in the same or different strand at the 3 ', 5' end or both the 3 'and 5' ends.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises from about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the sense strand; and wherein the antisense strand comprises from about 1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense strand and/or antisense strand are chemically modified with 2 '-deoxy, 2' -O-methyl, and/or 2 '-deoxy-2' -fluoro nucleotides, with or without about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages and/or terminal cap molecules at the 3 ', 5' end or both the 3 'and 5' ends present in the same or different strands.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 ', 5' end or both the 3 'and 5' ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, pyrimidine nucleotides of the sense strand and/or antisense strand are chemically modified with 2 '-deoxy, 2' -O-methyl, and/or 2 '-deoxy-2' -fluoro nucleotides, with or without one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages and/or terminal cap molecules at the 3 ', 5' end or both the 3 'and 5' ends present in the same or different strands.

In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises from about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the sense strand; and wherein the antisense strand comprises from about 1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages, and/or one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2 '-deoxy, 2' -O-methyl, 2 '-deoxy-2' -fluoro, and/or one or more (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and optionally a terminal cap molecule at the 3 'end, the 5' end, or both the 3 'and 5' ends of the antisense strand. In other embodiments, one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, pyrimidine nucleotides of the sense strand and/or antisense strand are chemically modified with 2 '-deoxy, 2' -O-methyl, and/or 2 '-deoxy-2' -fluoro nucleotides, with or without about 1 to about 5, e.g., about 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or terminal cap molecules present in the same or different strand, at the 3 'end, the 5' end, or both the 3 'and 5' ends.

In some embodiments, the polynucleic acid molecule is a duplex polynucleic acid molecule having one or more of the following properties: higher hepatocyte stability, reduced total charge, reduced hepatocyte uptake, or extended pharmacokinetics. In some embodiments, the duplex polynucleic acid molecule comprises a passenger strand (e.g., sense strand) and a guide strand (e.g., antisense strand) comprising a plurality of modifications.

In some embodiments, the duplex polynucleic acid molecule comprises a guide strand (e.g., antisense strand) having one or more of the above modifications and a passenger strand (e.g., sense strand) having a plurality of phosphorodiamidate morpholino oligomers or a plurality of peptide nucleic acid modified non-natural nucleotides.

In some embodiments, the polynucleic acid molecules described herein are chemically modified short interfering nucleic acid molecules having from about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotide linkages in each strand of the polynucleic acid molecule.

In another embodiment, a polynucleic acid molecule described herein comprises a 2 '-5' internucleotide linkage. In some cases, the 2 '-5' internucleotide linkage is at the 3 'end, the 5' end, or both the 3 'and 5' ends of one or both of the sequence strands. In other examples, the 2 '-5' internucleotide linkage is present at various other positions within one or both of the sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the pyrimidine nucleotides in one or both strands of the polynucleic acid molecule, including each internucleotide linkage, comprises a 2 '-5' internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the purine nucleotides in one or both strands of the polynucleic acid molecule, including each internucleotide linkage, comprises a 2 '-5' internucleotide linkage.

In some embodiments, the polynucleic acid molecule is a single stranded polynucleic acid molecule that mediates RNAi activity in a cell or reconstituted in vitro system, wherein the polynucleic acid molecule comprises a single stranded polynucleotide having complementarity to a target nucleic acid sequence, and wherein one or more of the pyrimidine nucleotides present in the polynucleic acid is a 2' -deoxy-2 ' -fluoropyrimidine nucleotide (e.g., wherein all of the pyrimidine nucleotides are 2' -deoxy-2 ' -fluoropyrimidine nucleotides, or a plurality of the pyrimidine nucleotides are 2' -deoxy-2 ' -fluoropyrimidine nucleotides), and wherein any purine nucleotides present in the polynucleic acid are 2' -deoxypurine nucleotides (e.g., wherein all of the purine nucleotides are 2' -deoxypurine nucleotides, or a plurality of purine nucleotides are 2' -deoxypurine nucleotides), and a terminal cap modification optionally present at the 3 ' end, the 5 ' end, or both the 3 ' and 5 ' ends of the antisense sequence, the polynucleic acid molecule optionally further comprising from about 1 to about 4 (e.g., about 1, 2, 3 or 4) terminal 2' -deoxynucleotides at the 3 ' end of the polynucleic acid molecule, wherein the terminal nucleotides further comprise one or more (e.g., 1, 2, 3 or 4) phosphorothioate internucleotide linkages, and wherein the polynucleic acid molecule optionally further comprises a terminal phosphate group, such as a 5 ' terminal phosphate group.

In some cases, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as rnase H, deoxyribonucleases such as dnase, or exonucleases such as 5 '-3' exonuclease and 3 '-5' exonuclease, as compared to the native polynucleic acid molecule. In some cases, 2 ' -O-methyl, 2 ' -O-methoxyethyl (2 ' -O-MOE), 2 ' -O-aminopropyl, 2 ' -deoxy, T-deoxy-2 ' -fluoro, 2 ' -O-aminopropyl (2 ' -O-AP), 2 ' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl (2 ' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), or 2 ' -O-N-methylacetamido (2 ' -O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, LNA, ENA, PNA, HNA, morpholino, N-methyl phosphonate, N-methyl, Artificial nucleotide analogs of thiol phosphonate nucleotides, 2 '-fluoro-N3-P5' -phosphoramidites, or combinations thereof are resistant to nucleases, e.g., ribonucleases such as RNase H, deoxyribonucleases such as DNase, or exonucleases such as 5 '-3' exonuclease and 3 '-5' exonuclease. In some cases, the 2 ' -O-methyl modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 ' -3 ' exonuclease, or 3 ' -5 ' exonuclease resistant). In some cases, the 2 'O-methoxyethyl (2' -O-MOE) modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, the 2 ' -O-aminopropyl modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 ' -3 ' exonuclease, or 3 ' -5 ' exonuclease resistant). In some cases, the 2 ' -deoxy modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 ' -3 ' exonuclease, or 3 ' -5 ' exonuclease resistant). In some cases, the T-deoxy-2 ' -fluoro modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 ' -3 ' exonuclease, or 3 ' -5 ' exonuclease resistant). In some cases, the 2 '-O-aminopropyl (2' -O-AP) modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, the 2 '-O-dimethylaminoethyl (2' -O-DMAOE) modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the 2 '-O-dimethylaminopropyl (2' -O-DMAP) -modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, the T-O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE) modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 ' -3 ' exonuclease or 3 ' -5 ' exonuclease resistant). In some cases, the 2 '-O-N-methylacetamide (2' -O-NMA) modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the LNA-modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, the ENA-modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the HNA-modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, the morpholino is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, PNA-modified polynucleic acid molecules are nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the methylphosphonate nucleotide modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the thiol phosphonate nucleotide modified polynucleic acid molecule is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease or 3 '-5' exonuclease resistant). In some cases, the polynucleic acid molecule comprising 2 '-fluoro-N3-P5' -phosphoramidite is nuclease resistant (e.g., rnase H, DNA enzyme, 5 '-3' exonuclease, or 3 '-5' exonuclease resistant). In some cases, a 5 ' conjugate described herein inhibits 5 ' -3 ' exonucleolytic cleavage. In some cases, a 3 ' conjugate described herein inhibits 3 ' -5 ' exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. Comprising 2 ' -O-methyl, 2 ' -O-methoxyethyl (2 ' -O-MOE), 2 ' -O-aminopropyl, 2 ' -deoxy, T-deoxy-2 ' -fluoro, 2 ' -O-aminopropyl (2 ' -O-AP), 2 ' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl (2 ' -O-DMAP), T-O-dimethylaminoethoxyethyl (2 ' -O-DMAE) or 2 ' -O-N-methylacetamido (2 ' -O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, amino acid esters, One or more artificial nucleotide analogs of a thiol phosphonate nucleotide or 2 '-fluoro N3-P5' -phosphoramidite have increased binding affinity for its mRNA target. In some cases, the 2' -O-methyl modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a 2 '-O-methoxyethyl (2' -O-MOE) modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, the 2' -O-aminopropyl modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, the 2' -deoxy modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a T-deoxy-2' -fluoro modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a 2 '-O-aminopropyl (2' -O-AP) modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a 2 '-O-dimethylaminoethyl (2' -O-DMAOE) modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a 2 '-O-dimethylaminopropyl (2' -O-DMAP) -modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE) -modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a 2 '-O-N-methylacetamide (2' -O-NMA) modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, the LNA-modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, the ENA modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a PNA-modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, the HNA-modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a morpholino modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, methylphosphonate nucleotide modified polynucleic acid molecules have increased binding affinity for their mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a thiol phosphonate nucleotide modified polynucleic acid molecule has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, a polynucleic acid molecule comprising 2 '-fluoro N3-P5' -phosphoramidite has increased binding affinity for its mRNA target relative to an equivalent native polynucleic acid molecule. In some cases, increased affinity is demonstrated by a lower Kd, a higher melting temperature (Tm), or a combination thereof.

In some embodiments, the polynucleic acid molecules described herein are chirally pure (or stereopure) polynucleic acid molecules, or polynucleic acid molecules comprising a single enantiomer. In some cases, the polynucleic acid molecule comprises L-nucleotides. In some cases, the polynucleic acid molecule comprises D-nucleotides. In some cases, the polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror image enantiomer. In some cases, the polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of the racemic mixture. In some cases, the polynucleic acid molecule is a polynucleic acid molecule described in U.S. patent publications 2014/194610 and 2015/211006 and PCT publication WO 2015107425.

In some embodiments, the polynucleic acid molecules described herein are further modified to comprise an aptamer conjugate moiety. In some cases, the aptamer conjugate moiety is a DNA aptamer conjugate moiety. In some cases, the aptamer-conjugated moiety is an alphamer (centauri therapeutics) that comprises an aptamer moiety that recognizes a particular cell surface target and a moiety that presents a particular epitope for attachment to circulating antibodies. In some cases, the polynucleic acid molecules described herein are further modified to include aptamer conjugate moieties as described in U.S. patents 8,604,184, 8,591,910 and 7,850,975.

In additional embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is an RNA (e.g., siRNA). In some cases, the polynucleic acid molecule is modified to increase its stability by one or more of the above-described modifications. In some cases, the polynucleic acid molecule is modified at the 2 'hydroxyl position, such as by 2' -O-methyl, 2 '-O-methoxyethyl (2' -O-MOE), 2 '-O-aminopropyl, 2' -deoxy, T-deoxy-2 '-fluoro, 2' -O-aminopropyl (2 '-O-AP), 2' -O-dimethylaminoethyl (2 '-O-DMAOE), 2' -O-dimethylaminopropyl (2 '-O-DMAP), T-O-dimethylaminoethoxyethyl (2' -O-DMAEOE), or 2 '-O-N-methylacetamido (2' -O-NMA), or by locking or bridging the ribose conformation (e.g., LNA or ENA) modified polynucleic acid molecules. In some cases, the polynucleic acid molecule is modified with 2 '-O-methyl and/or 2' -O-methoxyethyl ribose. In some cases, the polynucleic acid molecule further comprises morpholinos, PNAs, HNAs, methylphosphonate nucleotides, thiol phosphonate nucleotides and/or 2 '-fluoro N3-P5' -phosphoramidites to increase its stability. In some cases, the polynucleic acid molecule is a chirally pure (or stereopure) polynucleic acid molecule. In some cases, a chirally pure (or stereopure) polynucleic acid molecule is modified to increase its stability. Suitable modifications to the RNA to increase delivery stability will be apparent to the skilled person.

In some embodiments, the polynucleic acid molecules described herein have RNAi activity that modulates expression of an RNA encoded by a gene associated with muscular dystrophy, such as, but not limited to, DMD, DUX4, DYSF, EMD, or LMNA. In some cases, a polynucleic acid molecule described herein is a double stranded siRNA molecule that down-regulates expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein one strand of the double stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of at least one of DMD, DUX4, DYSF, EMD, or LMNA or an RNA encoded by at least one of DMD, DUX4, DYSF, EMD, or LMNA, or a portion thereof, and wherein a second strand of the double stranded siRNA molecule comprises a nucleotide sequence that is substantially similar to a nucleotide sequence of at least one of DMD, DUX4, DYSF, EMD, or LMNA or an RNA encoded by at least one of DMD, DUX4, DYSF, EMD, or LMNA, or a portion thereof. In some cases, a polynucleic acid molecule described herein is a double stranded siRNA molecule that down-regulates expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand. In some cases, a polynucleic acid molecule described herein is a double stranded siRNA molecule that down-regulates expression of at least one of DMD, DUX4, DYSF, EMD, or LMNA, wherein each strand of the siRNA molecule comprises from about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to nucleotides of the other strand. In some cases, the RNAi activity occurs in a cell. In other cases, the RNAi activity occurs in a reconstituted in vitro system.

In some embodiments, the polynucleic acid molecules described herein have RNAi activity that modulates expression of RNA encoded by the DMD gene. In some cases, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates expression of DMD, wherein the single-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of DMD or an RNA encoded by DMD, or a portion thereof. In some cases, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates DMD expression, wherein the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides. In some cases, a polynucleic acid molecule described herein is a single-stranded siRNA molecule that down-regulates DMD expression, wherein the siRNA molecule comprises from about 19 to about 23 nucleotides. In some cases, the RNAi activity occurs in a cell. In other cases, the RNAi activity occurs in a reconstituted in vitro system.

In some cases, the polynucleic acid molecule is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some cases, the polynucleic acid molecule is assembled from two separate polynucleotides, wherein one strand is the sense strand and the other strand is the antisense strand, wherein the antisense and sense strands are self-complementary (e.g., each strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the other strand; e.g., wherein the antisense and sense strands form a duplex or double-stranded structure, e.g., wherein the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, the polynucleic acid molecule is assembled from individual oligonucleotides, wherein the self-complementary sense and antisense regions of the polynucleic acid molecule are linked by a nucleic acid-based or non-nucleic acid-based linker.

In some cases, the polynucleic acid molecule is a polynucleotide having a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in the target nucleic acid molecule alone or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleic acid molecule is a circular single stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide is processed in vivo or in vitro to generate an active polynucleic acid molecule capable of mediating RNAi. In further instances, the polynucleic acid molecule further comprises a single-stranded polynucleotide having a nucleotide sequence that is complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof (e.g., where such a polynucleic acid molecule does not require the presence of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof within the polynucleic acid molecule), wherein the single-stranded polynucleotide further comprises a terminal phosphate group, such as a 5 ' -phosphate (see, e.g., Martinez et al, 2002, cell.,110,563-574 and Schwarz et al, 2002, molecular cell,10,537-568) or a 5 ', 3 ' -diphosphate.

In some cases, the asymmetry is a linear polynucleic acid molecule comprising an antisense region, a loop portion comprising a nucleotide or a non-nucleotide, and a sense region comprising fewer nucleotides than the antisense region, such that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a duplex with the loop. For example, an asymmetric hairpin polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in an in vitro system and a loop region comprising about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, the asymmetric hairpin polynucleic acid molecule further comprises a chemically modified 5' terminal phosphate group. In further instances, the loop portion of the asymmetric hairpin polynucleic acid molecule comprises a nucleotide, a non-nucleotide, a linker molecule or a conjugate molecule.

In some embodiments, the asymmetric duplex is a polynucleic acid molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region, such that the sense region has sufficient complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric double-stranded polynucleic acid molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cell or in an in vitro system and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

In some cases, universal bases refer to nucleotide base analogs that form a base pair with each natural DNA/RNA base with little distinction. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamide and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole and 6-nitroindole, as known in the art (see, e.g., Loakes,2001, Nucleic Acids Research,29, 2437-.

Synthesis of polynucleic acid molecules

In some embodiments, the polynucleic acid molecules described herein are constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the polynucleic acid molecule and the target nucleic acid. Exemplary methods include those described in U.S. patents 5,142,047, 5,185,444, 5,889,136, 6,008,400 and 6,111,086, PCT publication WO2009099942 or european publication 1579015. Additional exemplary methods include those described in the following documents: griffey et al, "2' -O-aminopropy ribotides A zwitterionic modification of the antibiotic resistance and biological activity of antisense," J.Med.chem.39(26):5100-5109 (1997)); obika et al, "Synthesis of2 '-O, 4' -C-methyleuridine and-cysteine, novel bicyclic nucleosides having attached F3, -end sun padding". Tetrahedron Letters 38(50):8735 (1997); koizumi, M. "ENA oligonucleotides as therapeutics". Current opinion in molecular therapeutics 8(2): 144-149 (2006); and Abramova et al, "Novel oligonucleotide peptides based on morpholino nucleic acids surfactants-antisense technologies: new chemical sites," Indian Journal of Chemistry 48B: 1721-. Alternatively, an expression vector is used to biologically produce a polynucleic acid molecule, wherein the polynucleic acid molecule has been subcloned into the expression vector in an antisense orientation (i.e., the RNA transcribed from the inserted polynucleic acid molecule will be in an antisense orientation to the target polynucleic acid molecule of interest).

In some embodiments, polynucleic acid molecules are synthesized by a tandem synthesis method in which two strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which is subsequently cleaved to provide individual fragments or strands, which hybridize and allow for purification of the duplex.

In some cases, the polynucleic acid molecule is further assembled from two different nucleic acid strands or fragments, wherein one fragment comprises the sense region and the second fragment comprises the antisense region of the molecule.

Other modification methods for incorporating, for example, sugar, base and phosphate modifications include: eckstein et al, International publication No. WO 92/07065; perrault et al Nature,1990,344, 565-; pieken et al Science,1991,253, 314-; usman and Cedergren, Trends in biochem. Sci.,1992,17, 334-; usman et al, International publication No. WO 93/15187; sproat, U.S. patent No. 5,334,711 and Beigelman et al, 1995, j.biol.chem.,270,25702; beigelman et al, international PCT publication No. wo 97/26270; beigelman et al, U.S. patent 5,716,824; usman et al, U.S. patent 5,627,053; woolf et al, International PCT publication No. WO98/13526; U.S. Ser. No.60/082,404, filed 4/20 of 1998 by Thompson et al; karpeisky et al, 1998, Tetrahedron Lett.,39,1131; earnshaw and Gait,1998, biopolymers (nucleic acids sciences),48, 39-55; verma and Eckstein,1998, Annu. Rev. biochem.,67, 99-134; and Burlina et al, 1997, bioorg.med.chem.,5, 1999-. These publications describe general methods and strategies for determining the location of incorporation of sugar, base and/or phosphate modifications, etc., into nucleic acid molecules without the need to modulate catalysis.

In some cases, while chemical modification of the internucleotide linkages of polynucleic acid molecules with phosphorothioate, phosphorodithioate and/or 5' -methylphosphonate linkages improves stability, excessive modification sometimes results in reduced toxicity or activity. Thus, when designing nucleic acid molecules, the amount of these internucleotide linkages is minimized in some cases. In such cases, a decrease in the concentration of these linkages results in decreased toxicity, increased efficacy, and greater specificity of these molecules.

Nucleic acid-polypeptide conjugates

In some embodiments, the polynucleic acid molecule is further conjugated to polypeptide a for delivery to a target site. In some cases, the polynucleic acid molecule is conjugated to polypeptide a and an optional polymeric moiety.

In some cases, at least one polypeptide a is conjugated to at least one B. In some cases, at least one polypeptide a is conjugated to at least one B to form an a-B conjugate. In some embodiments, at least one a is conjugated to the 5 'end of B, the 3' end of B, an internal site on B, or any combination thereof. In some cases, at least one polypeptide a is conjugated to at least two B. In some cases, at least one polypeptide a is conjugated to at least 2, 3, 4, 5, 6, 7, 8, or more B.

In some embodiments, at least one polypeptide a is conjugated at one terminus of at least one B and at least one C is conjugated at the opposite terminus of the at least one B to form an a-B-C conjugate. In some cases, at least one polypeptide a is conjugated at one terminus of at least one B, while at least one C is conjugated at an internal site on the at least one B. In some cases, at least one polypeptide a is directly conjugated to at least one C. In some cases, at least one B is indirectly conjugated to at least one polypeptide a through at least one C to form an a-C-B conjugate.

In some cases, at least one B and/or at least one C and optionally at least one D is conjugated to at least one polypeptide a. In some cases, at least one B is conjugated to at least one polypeptide a at a terminus (e.g., a 5 'terminus or a 3' terminus), or is conjugated to at least one polypeptide a through an internal site. In some cases, at least one C is directly conjugated to at least one polypeptide a, or indirectly conjugated to at least one polypeptide a through at least one B. If indirectly conjugated through at least one B, at least one C is conjugated to at least one polypeptide A at the same terminus to B, at the opposite terminus to at least one polypeptide A, or independently at an internal site. In some cases, at least one additional polypeptide a is further conjugated to at least one polypeptide A, B or C. In further instances, at least one D is optionally conjugated, directly or indirectly, to at least one polypeptide a, at least one B, or at least one C. If conjugated directly to at least one polypeptide a, at least one D is also optionally conjugated to at least one B to form an a-D-B conjugate, or optionally conjugated to at least one B and at least one C to form an a-D-B-C conjugate. In some cases, at least one D is directly conjugated to at least one polypeptide a, and indirectly conjugated to at least one B and at least one C, to form a D-a-B-C conjugate. If indirectly conjugated to at least one polypeptide a, at least one D is also optionally conjugated to at least one B to form an a-B-D conjugate, or optionally conjugated to at least one B and at least one C to form an a-B-D-C conjugate. In some cases, at least one additional D is further conjugated to at least one polypeptide A, B or C.

In some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

in some embodiments, the polynucleic acid molecule conjugate comprises a construct as shown below:

As shown aboveAre used for purposes of illustration only and include humanized antibodies or binding fragments thereof, chimeric antibodies or binding fragments thereof, monoclonal antibodies or binding fragments thereof, monovalent Fab', diabodiesA Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.

Binding moieties

In some embodiments, binding moiety a is a polypeptide. In some cases, the polypeptide is an antibody or fragment thereof. In some cases, the fragment is a binding fragment. In some cases, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a divalent Fab₂、F(ab)'₃Fragments, single-chain variable fragments (scFv), bis-scFv, (scFv)₂A diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

In some cases, a is an antibody or binding fragment thereof. In some cases, a is a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab ₂、F(ab)'₃Fragments, single-chain variable fragments (scFv), bis-scFv, (scFv)₂A diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein ("dsFv"), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some cases, a is a humanized antibody or binding fragment thereof. In some cases, a is a murine antibody or binding fragment thereof. In some cases, a is a chimeric antibody or binding fragment thereof. In some cases, a is a monoclonal antibody or binding fragment thereof. In some cases, a is a monovalent Fab'. In some cases, a is a bivalent Fab₂. In some cases, a is a single chain variable fragment (scFv).

In some embodiments, the binding moiety a is a bispecific antibody or binding fragment thereof. In some cases, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some cases, the bispecific antibody is a trifunctional antibody. In some cases, the trifunctional antibody is a full-length monoclonal antibody that comprises binding sites for two different antigens.

In some cases, the bispecific antibody is a bispecific miniantibody. In some cases, the bispecific miniantibody comprises a bivalent Fab₂、F(ab)'₃Fragment, bis-scFv, (scFv)₂A diabody, a minibody, a triabody, a tetrabody, or a bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single chain variable fragments (scfvs), wherein the two scfvs target epitopes of two different antigens.

In some embodiments, the binding moiety a is a bispecific miniantibody. In some cases, a is a bispecific Fab₂. In some cases, A is bispecific F (ab)'₃And (3) fragment. In some cases, a is a bispecific bis scFv. In some cases, a is bispecific (scFv)₂. In some embodiments, a is a bispecific diabody. In some embodiments, a is a bispecific miniantibody. In some embodiments, a is a bispecific triabody. In other embodiments, a is a bispecific tetrabody. In other embodiments, a is a bispecific T cell engager (BiTE).

In some embodiments, the binding moiety a is a trispecific antibody. In some cases, the trispecific antibody comprises f (ab)' ₃Fragments or triabodies. In some cases, A is trispecific F (ab)'₃And (3) fragment. In some cases, a is a triabody. In some embodiments, A is a trispecific antibody, as described by Dimas et al, "Development of antigenic designed to physiological and antigenic target antibodies on molecules," mol. pharmaceuticals, 12(9): 3490. sup. 3501 (2015).

In some embodiments, the binding moiety a is an antibody or binding fragment thereof that recognizes a cell surface protein. In some cases, the binding moiety a is an antibody or binding fragment thereof that recognizes a cell surface protein on a muscle cell. Exemplary cell surface proteins recognized by the antibody or binding fragment thereof include, but are not limited to, Sca-1, CD34, Myo-D, myogenin, MRF4, NCAM, CD43, and CD95 (Fas).

In some cases, the cell surface protein comprises a Cluster of Differentiation (CD) cell surface marker. Exemplary CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD11 9, CD, CD319(SLAMF7), CD326(EpCAM), etc.

In some cases, the binding moiety a is an antibody or binding fragment thereof that recognizes a CD cell surface marker. In some cases, binding moiety a is a binding moiety that recognizes CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11 8, CD15 8, CD, An antibody or binding fragment thereof to CD326(EpCAM) or a combination thereof.

In some embodiments, binding moiety a is an anti-myosin antibody, an anti-transferrin antibody, and an antibody that recognizes muscle-specific kinase (MuSK).

In some cases, binding moiety a is an anti-myosin antibody. In some cases, the anti-myosin antibody is a humanized antibody. In other cases, the anti-myosin antibody is a chimeric antibody. In other cases, the anti-myosin antibody is a monovalent, bivalent, or multivalent antibody.

In some cases, binding moiety a is an anti-transferrin (anti-CD 71) antibody. In some cases, the anti-transferrin antibody is a humanized antibody. In other cases, the anti-transferrin antibody is a chimeric antibody. In additional cases, the anti-transferrin antibody is a monovalent, bivalent, or multivalent antibody. In some embodiments, exemplary anti-transferrin antibodies include MAB5746 from R & D Systems, AHP858 from Bio-Rad Laboratories, A80-128A from Bethyl Laboratories, Inc., and T2027 from Millipore Sigma.

In some cases, binding moiety a is an antibody that recognizes MuSK. In some cases, the anti-MuSK antibody is a humanized antibody. In other cases, the anti-MuSK antibody is a chimeric antibody. In other cases, the anti-MuSK antibody is a monovalent, bivalent, or multivalent antibody.

In some embodiments, the binding moiety a is non-specifically conjugated to the polynucleic acid molecule (B). In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) in a non-site specific manner via a lysine residue or a cysteine residue. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) via a lysine residue in a non-site specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) in a non-site specific manner via a cysteine residue.

In some embodiments, the binding moiety a is conjugated to the polynucleotide molecule (B) in a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) via a lysine residue, a cysteine residue, at the 5 'terminus, at the 3' terminus, at a non-natural amino acid, or at an enzymatically modified or enzymatically catalyzed residue, in a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) via a lysine residue in a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) via a cysteine residue in a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) at the 5' end via a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) at the 3' end via a site-specific manner. In some cases, the binding moiety a is conjugated to the polynucleotide molecule (B) by an unnatural amino acid via site-specific means. In some cases, the binding moiety a is conjugated to the polynucleic acid molecule (B) via site-specific means by an enzymatically modified or enzymatically catalyzed residue.

In some embodiments, one or more polynucleic acid molecules (B) are conjugated to the binding moiety a. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 1 polynucleic acid molecule is conjugated to one binding moiety a. In some cases, about 2 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 3 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 4 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 5 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 6 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 7 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 8 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 9 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 10 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 11 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 12 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 13 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 14 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 15 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, about 16 polynucleic acid molecules are conjugated to one binding moiety a. In some cases, the one or more polynucleic acid molecules are identical. In other cases, the one or more polynucleic acid molecules are different.

In some embodiments, the number of polynucleic acid molecules (B) conjugated to a binding moiety a forms a ratio. In some cases, this ratio is referred to as a DAR (drug-antibody) ratio, where the drug as referred to herein is a polynucleic acid molecule (B). In some cases, the DAR ratio of the polynucleic acid molecule (B) to the binding moiety a is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 1 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 2 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 3 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 4 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 5 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 6 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 7 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 8 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 9 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 10 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 11 or greater. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 12 or greater.

In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 1. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 2. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 3. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 4. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 5. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 6. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 7. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 8. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 9. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 10. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 11. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 12. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 13. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 14. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 15. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is about 16.

In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 1. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 2. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 4. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 6. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 8. In some cases, the DAR ratio of polynucleic acid molecule (B) to binding moiety a is 12.

In some cases, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety a has improved activity compared to a conjugate comprising a polynucleic acid molecule (B) that does not comprise a binding moiety a. In some cases, the improved activity results in enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and efficacy in treating or preventing a disease state. In some cases, the disease state is the result of one or more mutated exons of the gene. In some cases, a conjugate comprising a polynucleic acid molecule (B) and a binding moiety a results in increased exon skipping of one or more mutated exons as compared to a conjugate comprising a polynucleic acid molecule (B) that does not comprise a binding moiety a. In some cases, exon skipping is increased by at least or about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% in a conjugate comprising a polynucleic acid molecule (B) and a binding moiety a, as compared to a conjugate comprising a polynucleic acid molecule (B) that does not comprise a binding moiety a.

In some embodiments, the antibody or binding fragment thereof is further modified using conventional techniques known in the art, e.g., by using amino acid deletions, insertions, substitutions, additions, either alone or in combination, and/or by recombination and/or any other modification known in the art (e.g., post-translational and chemical modifications, such as glycosylation and phosphorylation). In some cases, the modification further comprises a modification for modulating interaction with an Fc receptor. In some cases, the one or more modifications include, for example, those described in international publication WO97/34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications into the nucleic acid sequence of the amino acid sequence of an antibody or binding fragment thereof are well known to those skilled in the art.

In some cases, the antibody-binding fragment further includes derivatives thereof, and includes a polypeptide sequence comprising at least one CDR.

In some cases, the term "single-stranded" as used herein means that the first and second domains of the bispecific single-stranded construct are covalently linked, preferably in the form of a collinear amino acid sequence that can be encoded by a single nucleic acid molecule.

In some cases, a bispecific single chain antibody construct involves a construct comprising two antibody-derived binding domains. In such embodiments, the bispecific single chain antibody construct is a tandem bis scFv or diabody. In some cases, the scFv contains VH and VL domains connected by a linker peptide. In some cases, the length and sequence of the linker is sufficient to ensure that each of the first and second domains can retain their differential binding specificities independently of each other.

In some embodiments, binding or interaction with as used herein defines binding/interaction of at least two antigen-interaction-sites with each other. In some cases, an antigen-interaction-site defines a motif of a polypeptide that exhibits the ability to specifically interact with a particular antigen or a particular set of antigens. In some cases, binding/interaction is also understood to define specific recognition. In such cases, specific recognition means that the antibody or binding fragment thereof is capable of specifically interacting with and/or binding to at least two amino acids per target molecule. For example, specific recognition involves the specificity of an antibody molecule, or its ability to discriminate between specific regions of a target molecule. In other cases, the specific interaction of an antigen-interaction-site with its particular antigen results in signal initiation, e.g., due to induction of a change in antigen conformation, oligomerization of the antigen, etc. In a further embodiment, an example of binding is the specificity of the "key-lock principle". Thus, in some cases, the antigen-interaction-site and a particular motif in the amino acid sequence of the antigen bind to each other due to their primary, secondary or tertiary structure and secondary modifications of that structure. In such cases, the specific interaction of an antigen-interaction-site with its particular antigen also results in simple binding of the site to the antigen.

In some cases, a specific interaction further refers to a decrease in cross-reactivity or a decrease in off-target effects of the antibody or binding fragment thereof. For example, an antibody or binding fragment thereof that binds to a polypeptide/protein of interest but does not or does not substantially bind to any other polypeptide is considered to be specific for the polypeptide/protein of interest. Examples of specific interactions of an antigen-interaction-site with a particular antigen include the specificity of the ligand for its receptor, e.g., the interaction of an antigenic determinant (epitope) with the antigen-binding site of an antibody.

Additional binding moieties

In some embodiments, the binding moiety is a plasma protein. In some cases, the plasma protein comprises albumin. In some cases, the binding moiety a is albumin. In some cases, albumin is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some cases, albumin is conjugated to the polynucleic acid molecule by natural ligation chemistry. In some cases, albumin is conjugated to the polynucleic acid molecule by lysine conjugation.

In some cases, the binding moiety is a steroid. Exemplary steroids include cholesterol, phospholipids, diacyl and triacylglycerols, fatty acids, saturated, unsaturated, containing substituted hydrocarbons, or combinations thereof. In some cases, the steroid is cholesterol. In some cases, the binding moiety is cholesterol. In some cases, cholesterol is conjugated to the polynucleic acid molecule by one or more of the conjugation chemistries described herein. In some cases, cholesterol is conjugated to the polynucleic acid molecule by natural ligation chemistry. In some cases, cholesterol is conjugated to the polynucleic acid molecule by lysine conjugation.

In some cases, the binding moiety is a polymer, including but not limited to a polynucleic acid aptamer that binds to a specific surface marker on a cell. In this case, the binding moiety is a polynucleic acid that does not hybridize to a target gene or mRNA, but is capable of selectively binding to a cell surface marker similar to an antibody that binds to its specific epitope of the cell surface marker.

In some cases, the binding moiety is a peptide. In some cases, the peptide has about 1 to about 3 kDa. In some cases, the peptide has about 1.2 to about 2.8kDa, about 1.5 to about 2.5kDa, or about 1.5 to about 2 kDa. In some cases, the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some cases, the binding moiety is a bicyclic peptide (e.g., a bicyclic compound from Bicycle Therapeutics).

In other cases, the binding moiety is a small molecule. In some cases, the small molecule is a small molecule that recruits antibodies. In some cases, the small molecule that recruits an antibody comprises a target binding end and an antibody binding end, wherein the target binding end is capable of recognizing and interacting with a cell surface receptor. For example, in some cases, a target binding terminus comprising a glutamatereurea compound enables interaction with PSMA, thereby enhancing antibody interaction with PSMA-expressing cells. In some cases, the binding moiety is Zhang et al, "A remove arene-binding site on proteinaceous modified by antibody-detecting small molecules," JAm Chem Soc.132(36): 12711-; or McEnaney et al, "Antibody-detecting molecules: an interfering side for interfering with the immune function in the treating human disease," ACS Chem biol.7(7): 1139) -1151 (2012).

Conjugation chemistry

In some embodiments, the polynucleic acid molecule B is conjugated to a binding moiety. In some cases, the binding moieties include amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all of these classes of materials. Other examples of binding moieties also include steroids such as cholesterol, phospholipids, diacylglycerols and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or containing substitutions), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some cases, the binding moiety is an antibody or binding fragment thereof. In some cases, the polynucleic acid molecule is further conjugated to a polymer and optionally an endosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some cases, the polynucleic acid molecule is conjugated to the binding moiety through a natural linkage. In some cases, conjugation is as follows: "Synthesis of proteins by native chemical ligation," Science 1994,266, 776-779; dawson et al, "Modulation of Reactivity in Native Chemical Ligation of Chemical additions the use of Chemical additions," J.Am.chem.Soc.1997,119, 4325-4329; hackeng et al, "protein by natural chemical ligation," Expanded scope by using strain for manufacturing by means of, "Proc. Natl. Acad. Sci. USA 1999,96, 10068-; or Wu et al, "building complex polypeptides: Development of a cyclic-free chemical ligation protocol," Angew. chem. int. Ed.2006,45, 4116-. In some cases, conjugation is as described in us patent 8,936,910. In some embodiments, the polynucleic acid molecule is site-specifically or non-specifically conjugated to the binding moiety via native ligation chemistry.

In some cases, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method using "traceless" coupling technology (philichem). In some cases, this "traceless" coupling technique utilizes an N-terminal 1, 2-aminothiol group on a binding moiety, which is subsequently conjugated to a polynucleic acid molecule containing an aldehyde group (see Casi et al, "Site-specific cellular uptake coupling of pore cytotoxic drugs to recombinant antibodies for pharmacodary," JACS134(13): 5887. sup. 5892 (2012)).

In some cases, the polynucleic acid molecule is conjugated to the binding moiety by site-directed methods that utilize unnatural amino acids incorporated into the binding moiety. In some cases, the unnatural amino acid includes para-acetylphenylalanine (pAcPhe). In some cases, the keto group of pAcPPhe is selectively coupled to a conjugate moiety derived from an alkoxy-amine to form an oxime linkage (see Axup et al, "Synthesis of site-specific antibodies-drug conjugates using non-native amino acids," PNAS 109(40): 16101-.

In some cases, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method that utilizes an enzymatic process. In some cases, the fixed point method utilizes smart tag ^TMTechnique (Redwood). In some cases, smart ag^TMThe technique involves the oxidation of a formyl glycine generating enzyme (FGE) from a caspase in the presence of an aldehyde tagThe amino acid generates a formylglycine (FGly) residue, which is then conjugated to an alkylhydrazine functionalized polynucleic acid molecule via a hydrazino-Picture-Spengler (HIPS) linkage (see Wu et al, "Site-specific chemical modification of recombinant proteins in a macromolecular cell by using the genetic encoded aldehyde tag," PNAS 106(9):3000-3005 (2009); Agarwal et al, "A Picture-Spenglerlication for protein chemical modification," PNAS 110(1):46-51 (2013)).

In some cases, the enzyme-catalyzed process comprises microbial transglutaminase (mTG). In some cases, the polynucleic acid molecule is conjugated to the binding moiety using a microbial transglutaminase catalyzed process. In some cases, mTG catalyzes the formation of a covalent bond between the amide side chain of glutamine within the recognition sequence and a primary amine of the functionalized polynucleic acid molecule. In some cases, mTG is produced by Streptomyces mobarensis (Strop et al, "Location information: site of conjugation models status and pharmacologics of affinity drugs," Chemistry and Biology 20(2)161-167 (2013)).

In some cases, the polynucleic acid molecule is conjugated to the binding moiety by a method that utilizes a sequence-specific transpeptidase, as described in PCT publication WO 2014/140317.

In some cases, the polynucleic acid molecule is conjugated to a binding moiety by methods as described in U.S. patent publications 2015/0105539 and 2015/0105540.

Production of antibodies or binding fragments thereof

In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art that can be used to synthesize polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression techniques.

In some cases, the antibody or binding fragment thereof is expressed recombinantly, and nucleic acid encoding the antibody or binding fragment thereof is assembled from chemically synthesized oligonucleotides (e.g., as described by Kutmeier et al, 1994, BioTechniques 17: 242), which method comprises synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating the oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

Alternatively, nucleic acid molecules encoding the antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing immunoglobulins) by PCR amplification using synthetic primers that hybridize to the 3 'and 5' ends of the sequences, or by cloning using oligonucleotide probes specific for a particular gene sequence.

In some cases, polyclonal Antibodies are optionally generated by immunizing an animal such as a rabbit, or more preferably, Monoclonal Antibodies are generated to generate Antibodies or binding fragments thereof, for example, as described by Kohler and Milstein (1975, Nature 256: 495-. Alternatively, clones encoding at least the Fab portion of the antibody are optionally obtained by screening Fab expression libraries for clones that bind to a particular antigen (e.g., as described in Huse et al, 1989, Science 246: 1275-.

In some embodiments, techniques developed for generating "chimeric antibodies" by splicing genes from mouse antibody molecules with appropriate antigen specificity with genes from human antibody molecules with appropriate biological activity are used (Morrison et al, 1984, Proc. Natl.Acad.Sci.81:851 855; Neuberger et al, 1984, Nature312: 604-608; Takeda et al, 1985, Nature 314: 452-454). Chimeric antibodies are molecules in which different portions are derived from different animal species, such as antibodies having variable regions derived from murine monoclonal antibodies and human immunoglobulin constant regions, e.g., humanized antibodies.

In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird,1988, Science 242: 423-42; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-. Single chain antibodies are formed by connecting the heavy and light chain fragments of the Fv region via an amino acid bridge to produce a single chain polypeptide. The technique of assembling functional Fv fragments in E.coli (E.coli) is also optionally used (Skerra et al, 1988, Science 242: 1038-.

In some embodiments, the expression vector comprising the nucleotide sequence of the antibody or the nucleotide sequence of the antibody is transferred to a host cell by conventional techniques (e.g., electroporation, lipofection, and calcium phosphate precipitation), and the transfected cell is then cultured by conventional techniques to produce the antibody. In particular embodiments, expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

In some embodiments, the antibodies or binding fragments thereof described herein are expressed using a variety of host expression vector systems. Such host expression systems represent vectors through which the coding sequences for the antibodies are produced and subsequently purified, but also represent cells that express the antibodies or binding fragments thereof in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., escherichia coli and bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody or binding fragment-encoding sequences thereof; yeast (e.g., pichia pastoris) transformed with a recombinant yeast expression vector containing the coding sequence for the antibody or binding fragment thereof; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody or binding fragment thereof coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and Tobacco Mosaic Virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing antibody or binding fragment thereof-encoding sequences; or a mammalian cell system (e.g., COS, CHO, BH, 293T, 3T3 cells) with a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or a promoter derived from a mammalian virus (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

For long-term high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines that stably express the antibody are optionally engineered. Rather than using an expression vector containing a viral origin of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of the exogenous DNA, the engineered cells were grown in enrichment medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows the cell to stably integrate the plasmid into its chromosome and grow to form foci (focus) which are then cloned and expanded into cell lines. The method may advantageously be used to engineer cell lines expressing the antibody or binding fragment thereof.

In some cases, a variety of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Szybalski,192, Proc. Natl. Acad. Sci. USA 48:202) and adenine phosphoribosyl transferase (Lowy et al, 1980, Cell 22:817) genes used in tk-, hgprt-or aprt-cells, respectively. Furthermore, antimetabolite resistance was used as a basis for the selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, 1980, Proc. Natl. Acad. Sci. USA77: 357; O' Hare et al, 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg,1981, proc.Natl.Acad.Sci.USA 78: 2072); neo, which confers resistance to aminoglycoside G-418 (Clinical Pharmacy 12: 488-505; Wu and Wu,1991, Biotherapy 3: 87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol.32: 573-596; Mulligan,1993, Science260: 926-932; and Morgan and Anderson,1993, Ann. Rev. biochem.62: 191-217; May,1993, TIBTECH 11(5): 155-215); and hygro, conferring resistance to hygromycin (Santerre et al, 1984, Gene 30: 147). Methods well known in the art of recombinant DNA technology that can be used are described in Ausubel et al (eds.), 1993, Current protocols in Molecular Biology, John Wiley & Sons, NY; kriegler,1990, Gene transfer and Expression, A Laboratory Manual, Stockton Press, NY; and chapters 12 and 13, Dracopoli et al (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al, 1981, J.mol.biol.150: 1.

In some cases, The expression level of The antibody is increased by vector amplification (for review see Bebbington and Hentschel, The use of vector based on gene amplification for The expression of bound genes in mammalian cells in DNA binding, Vol.3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the antibody, the production of the antibody will also increase (Crouse et al, 1983, mol. cell biol.3: 257).

In some cases, any method known in the art for purifying or analyzing an antibody or antibody conjugate is used, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly for a particular antigen after protein a, and size column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and flash protein liquid chromatography.

Polymeric conjugate moieties

In some embodiments, the polymeric moiety C is further conjugated to a polynucleic acid molecule described herein, a binding moiety described herein, or a combination thereof. In some cases, the polymeric moiety C is conjugated to a polynucleic acid molecule. In some cases, the polymeric moiety C is conjugated to a binding moiety. In other cases, the polymeric moiety C is conjugated to a polynucleic acid molecule binding moiety molecule. In other cases, the polymeric moiety C is conjugated as shown above.

In some cases, polymer portion C is a natural or synthetic polymer consisting of a long chain of branched or unbranched monomers and/or a two-or three-dimensional cross-linked network of monomers. In some cases, the polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylene oxide (e.g., polyethylene glycol). In some cases, the at least one polymer moiety C includes, but is not limited to, alpha-, omega-dihydroxy polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactic acid (PLA), poly (glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound as well as in a block copolymer. In some cases, a block copolymer is a polymer in which at least a portion of the polymer is made up of monomers of another polymer. In some cases, polymer portion C includes a polyalkylene oxide. In some cases, polymer moiety C comprises PEG. In some cases, polymer moiety C comprises a Polyethyleneimine (PEI) or hydroxyethyl starch (HES).

In some cases, C is a PEG moiety. In some cases, the PEG moiety is conjugated at the 5 'terminus of the polynucleic acid molecule and the binding moiety is conjugated at the 3' terminus of the polynucleic acid molecule. In some cases, the PEG moiety is conjugated at the 3 'terminus of the polynucleic acid molecule and the binding moiety is conjugated at the 5' terminus of the polynucleic acid molecule. In some cases, the PEG moiety is conjugated to an internal site of the polynucleic acid molecule. In some cases, the PEG moiety, binding moiety, or combination thereof is conjugated to an internal site of the polynucleic acid molecule. In some cases, the conjugation is direct conjugation. In some cases, the conjugation is via a natural linkage.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydisperse or monodisperse compound. In some cases, polydisperse materials comprise a dispersion distribution of materials of different molecular weights, characterized by an average weight (weight average) magnitude and dispersibility. In some cases, monodisperse PEG comprises molecules of one size. In some embodiments, C is a polydisperse or monodisperse polyalkylene oxide (e.g., PEG), and the indicated molecular weights represent the average of the molecular weights of the polyalkylene oxide (e.g., PEG) molecules.

In some embodiments, the polyalkylene oxide (e.g., PEG) has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is a polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some cases, the molecular weight of C is about 200 Da. In some cases, C has a molecular weight of about 300 Da. In some cases, the molecular weight of C is about 400 Da. In some cases, the molecular weight of C is about 500 Da. In some cases, the molecular weight of C is about 600 Da. In some cases, the molecular weight of C is about 700 Da. In some cases, the molecular weight of C is about 800 Da. In some cases, C has a molecular weight of about 900 Da. In some cases, the molecular weight of C is about 1000 Da. In some cases, the molecular weight of C is about 1100 Da. In some cases, the molecular weight of C is about 1200 Da. In some cases, the molecular weight of C is about 1300 Da. In some cases, C has a molecular weight of about 1400 Da. In some cases, C has a molecular weight of about 1450 Da. In some cases, the molecular weight of C is about 1500 Da. In some cases, the molecular weight of C is about 1600 Da. In some cases, the molecular weight of C is about 1700 Da. In some cases, the molecular weight of C is about 1800 Da. In some cases, C has a molecular weight of about 1900 Da. In some cases, the molecular weight of C is about 2000 Da. In some cases, C has a molecular weight of about 2100 Da. In some cases, the molecular weight of C is about 2200 Da. In some cases, the molecular weight of C is about 2300 Da. In some cases, the molecular weight of C is about 2400 Da. In some cases, the molecular weight of C is about 2500 Da. In some cases, C has a molecular weight of about 2600 Da. In some cases, C has a molecular weight of about 2700 Da. In some cases, the molecular weight of C is about 2800 Da. In some cases, C has a molecular weight of about 2900 Da. In some cases, C has a molecular weight of about 3000 Da. In some cases, the molecular weight of C is about 3250 Da. In some cases, C has a molecular weight of about 3350 Da. In some cases, the molecular weight of C is about 3500 Da. In some cases, the molecular weight of C is about 3750 Da. In some cases, the molecular weight of C is about 4000 Da. In some cases, C has a molecular weight of about 4250 Da. In some cases, the molecular weight of C is about 4500 Da. In some cases, C has a molecular weight of about 4600 Da. In some cases, the molecular weight of C is about 4750 Da. In some cases, C has a molecular weight of about 5000 Da. In some cases, the molecular weight of C is about 5500 Da. In some cases, the molecular weight of C is about 6000 Da. In some cases, C has a molecular weight of about 6500 Da. In some cases, the molecular weight of C is about 7000 Da. In some cases, C has a molecular weight of about 7500 Da. In some cases, the molecular weight of C is about 8000 Da. In some cases, the molecular weight of C is about 10,000 Da. In some cases, the molecular weight of C is about 12,000 Da. In some cases, the molecular weight of C is about 20,000 Da. In some cases, the molecular weight of C is about 35,000 Da. In some cases, the molecular weight of C is about 40,000 Da. In some cases, the molecular weight of C is about 50,000 Da. In some cases, the molecular weight of C is about 60,000 Da. In some cases, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, wherein the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide unit. In some cases, the discrete peg (dpeg) comprises 2 to 60, 2 to 50, or 2 to 48 repeating ethylene oxide units. In some cases, the dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 2 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 3 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 4 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 5 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 6 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 7 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 8 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 9 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 10 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 11 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 12 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 13 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 14 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 15 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 16 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 17 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 18 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 19 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 20 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 22 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 24 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 26 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 28 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 30 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 35 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 40 or more repeating ethylene oxide units. In some cases, dPEG comprises about 42 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 48 or more repeating ethylene oxide units. In some cases, the dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, dPEG is synthesized in a stepwise manner from pure (e.g., about 95%, 98%, 99%, or 99.5%) starting material as a single molecular weight compound. In some cases, dPEG has a particular molecular weight, not an average molecular weight. In some cases, the dPEG described herein is dPEG from Quanta Biodesign, LMD.

In some embodiments, polymer moiety C comprises a cationic mucic acid-based polymer (cMAP). In some cases, the cMAP comprises one or more subunits of at least one repeating subunit, and the subunit structure is represented by formula (V):

wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 4 to 6 or 5; and n is independently at each occurrence 1, 2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

In some cases, the cMAP is further conjugated to a PEG moiety, resulting in a cMAP-PEG copolymer, mPEG-cMAP-PEGm triblock polymer, or cMAP-PEG-cMAP triblock polymer. In some cases, the PEG moiety ranges from about 500Da to about 50,000 Da. In some cases, the PEG moiety ranges from about 500Da to about 1000Da, greater than 1000Da to about 5000Da, greater than 5000Da to about 10,000Da, greater than 10,000 to about 25,000Da, greater than 25,000Da to about 50,000Da, or any combination of two or more of these ranges.

In some cases, polymer moiety C is a cMAP-PEG copolymer, mPEG-cMAP-PEGm triblock polymer, or cMAP-PEG-cMAP triblock polymer. In some cases, polymer moiety C is a cMAP-PEG copolymer. In other cases, polymer moiety C is a mPEG-cMAP-PEGm triblock polymer. In other cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

In some embodiments, the polymeric moiety C is conjugated to the polynucleic acid molecule, the binding moiety and optionally the endosomolytic moiety as shown above.

Endosomal lysis fraction

In some embodiments, the molecule of formula (I) -A-X-B-Y-C-further comprises an additional conjugation moiety. In some cases, the additional conjugate moiety is an endosomolytic moiety. In some cases, the endosomolytic moiety is a component released from a cellular compartment, such as a compound capable of being released from any cellular compartment known in the art, e.g., an endosome, lysosome, Endoplasmic Reticulum (ER), golgi apparatus, microtubule, peroxisome, or other vesicle within a cell. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide, an endosomolytic polymer, an endosomolytic lipid, or an endosomolytic small molecule. In some cases, the endosomolytic moiety comprises an endosomolytic polypeptide. In other cases, the endosomolytic moiety comprises an endosomolytic polymer.

Endosomolytic polypeptides

In some embodiments, the molecule of formula (I) -A-X-B-Y-C-is further conjugated to an endosomolytic polypeptide. In some embodiments, formula (V) -A- (X) ¹-B)_nOr formula (II) -A-X¹-(B-X²-C)_nThe molecule of (a) is further conjugated to an endosomolytic polypeptide. In some cases, the endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the endosomolytic polypeptide is an amphiphilic polypeptide. In other cases, theThe endosomolytic polypeptide is a peptidomimetic. In some cases, the endosomolytic polypeptide comprises INF, melittin, meucin, or a respective derivative thereof. In some cases, the endosomolytic polypeptide comprises INF or a derivative thereof. In other cases, the endosomolytic polypeptide comprises melittin or a derivative thereof. In further cases, the endosomolytic polypeptide comprises meucin or a derivative thereof.

In some cases, INF7 is a 24-residue polypeptide whose sequence comprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO:1) or GLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2). In some cases, INF7 or a derivative thereof comprises the sequence: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQ ID NO:3), GLFEAIEGFIENGWEGMIDGWYG- (PEG)6-NH2(SEQ ID NO:4) or GLFEAIEGFIENGWEGMIWDYG-SGSC-K (GalNAc)2(SEQ ID NO: 5).

In some cases, melittin is a 26-residue polypeptide whose sequence comprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO:6) or GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 7). In some cases, melittin comprises a polypeptide sequence as described in U.S. patent 8,501,930.

In some cases, meucin is an antimicrobial peptide (AMP) derived from the venom glands of the scorpions (Mesobuthus eupetus). In some cases, the meucin includes meucin-13 and meucin-18, and the sequence of meucin-13 includes IFGAIAGLLKNIF-NH₂(SEQ ID NO:8), the sequence of meucin-18 comprises FFGHLFKLATKIIPSLFQ (SEQ ID NO: 9).

In some cases, an endosomolytic polypeptide includes a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence identity to INF7 or a derivative thereof, melittin or a derivative thereof, or meucin or a derivative thereof. In some cases, the endosomolytic moiety comprises INF7 or a derivative thereof, melittin or a derivative thereof, or meucin or a derivative thereof.

In some cases, the endosomolytic moiety is INF7 or a derivative thereof. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs 1-5. In some cases, the endosomolytic moiety comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to seq id No. 1. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs 2-5. In some cases, the endosomolytic moiety comprises SEQ ID NO 1. In some cases, the endosomolytic moiety comprises SEQ ID NOS: 2-5. In some cases, the endosomolytic moiety consists of SEQ ID NO 1. In some cases, the endosomolytic portion consists of SEQ ID NOS: 2-5.

In some cases, the endosomolytic moiety is melittin or a derivative thereof. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 6 or 7. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 6. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ id No. 7. In some cases, the endosomolytic moiety comprises SEQ ID NO 6. In some cases, the endosomolytic moiety comprises SEQ ID NO 7. In some cases, the endosomolytic moiety consists of SEQ ID NO 6. In some cases, the endosomolytic portion consists of SEQ ID NO 7.

In some cases, the endosomolytic moiety is meucin or a derivative thereof. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 8 or 9. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 8. In some cases, the endosomolytic portion comprises a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ id No. 9. In some cases, the endosomolytic moiety comprises SEQ ID NO 8. In some cases, the endosomolytic moiety comprises SEQ ID NO 9. In some cases, the endosomolytic portion consists of SEQ ID NO 8. In some cases, the endosomolytic moiety consists of SEQ ID NO 9.

In some cases, the endosomolytic moiety comprises a sequence as shown in table 1 below.

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptide that antagonizes an inhibitor target, such as Bcl-2 and/or Bcl-x_LInducing apoptosis. In some cases, the endosomal solubilizing moiety comprises Albarran et al, "effective intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive&Bak BH3 polypeptide described by Functional Polymers 71:261-265 (2011).

In some cases, the endosomolytic moiety comprises a polypeptide described in PCT publication WO2013/166155 or WO2015/069587 (e.g., a cell penetrating polypeptide).

Endosomolytic polymers

In some embodiments, formula (V) -A- (X)¹-B)_n-or formula (VI) -A-X¹-(B-X²-C)_nThe molecule of (a) is further conjugated to an endosomolytic polymer. As used herein, endosomolytic polymers include linear, branched networks, star, comb, or ladder polymers. In thatIn some cases, the endosomolytic polymer is a homopolymer or copolymer comprising two or more different types of monomers. In some cases, the endosomolytic polymer is a polycationic polymer. In other cases, the endosomolytic polymer is a polyanionic polymer.

In some cases, the polycationic polymer comprises monomer units that are positively charged, neutral in charge, or negatively charged, and the net charge is positive. In other cases, the polycationic polymer comprises a non-polymeric molecule containing two or more positive charges. Exemplary cationic polymers include, but are not limited to, poly (L-lysine) (PLL), poly (L-arginine) (PLA), Polyethyleneimine (PEI), poly [ alpha- (4-aminobutyl) -L-glycolic acid ] (PAGA), 2- (dimethylamino) ethyl methacrylate (DMAEMA), or N, N-diethylaminoethyl methacrylate (DEAEMA).

In some cases, the polyanionic polymer comprises monomer units that are positively charged, neutral in charge, or negatively charged, and the net charge is negative. In other cases, the polyanionic polymer comprises a non-polymeric molecule comprising two or more negative charges. Exemplary anionic polymers include poly (alkyl acrylates) (e.g., poly (propyl acrylate) (PPAA)) or poly (N-isopropylacrylamide) (NIPAM). Additional examples include the L-phenylalanine-poly (L-lysine isophthalamide) polymers described in PP75, Khormain et al, "endoscopic analytical polymers for the cellular delivery of siRNAs amplified in vivo applications," Advanced Functional Materials 23: 565-.

In some embodiments, the endosomolytic polymer described herein is a pH-responsive endosomolytic polymer. pH-responsive polymers include polymers that increase (swell) or collapse depending on the pH of the environment. Polyacrylic acid and chitosan are examples of pH-responsive polymers.

In some cases, the endosomolytic moiety described herein is a membrane destructive polymer. In some cases, the membrane disruptive polymer comprises a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer. In some cases, the membrane disruptive polymer is a hydrophilic polymer.

In some cases, the endosomolytic moiety described herein is a pH-responsive membrane-disrupting polymer. Exemplary pH-responsive film-disrupting polymers include poly (alkylacrylic acid), poly (N-isopropylacrylamide) (NIPAM) copolymers, succinylated poly (glycidol), and poly (β -malic acid) polymers.

In some cases, the poly (alkylacrylic acid) includes poly (propylacrylic acid) (polyPAA), poly (methacrylic acid) (PMAA), poly (ethylacrylic acid) (PEAA), and poly (propylacrylic acid) (PPAA). In some cases, the poly (alkyl acrylic acid) includes poly (alkyl acrylic acid) as described in Jones et al, Biochemistry Journal 372:65-75 (2003).

In some embodiments, the pH-responsive membrane-disrupting polymer comprises poly (butyl acrylate-co-methacrylic acid). (see Bulmus et al, Journal of Controlled Release93:105-120(2003) and Yessing et al, Biochimica et Biophysica Acta 1613:28-38 (2003)).

In some embodiments, the pH-responsive film-disrupting polymer comprises a poly (styrene-maleic anhydride) alternating polymer. (see Henry et al, Biomacromolecules 7: 2407-.

In some embodiments, the pH-responsive film-disrupting polymer comprises a pyridyl disulfide acrylate (PDSA) polymer, such as a poly (MAA-co-PDSA), poly (EAA-co-PDSA), poly (PAA-co-PDSA), poly (MAA-co-BA-co-PDSA), poly (EAA-co-BA-co-PDSA), or poly (PAA-co-BA-co-PDSA) polymer. (see El-Sayed et al, "Rational design of composition and activity coatings for pH-responsive and glutathione-responsive polymer therapeutics," Journal of Controlled Release104: 417-.

In some embodiments, the pH-responsive film-disrupting polymer comprises a soluble polymer comprising the basic structure:

in some cases, the endosomolytic moieties described herein are further conjugated to additional conjugates, such as polymers (e.g., PEG) or modified polymers (e.g., cholesterol modified polymers).

In some cases, the additional conjugate comprises a detergent (e.g., Triton X-100). In some cases, an endosomolytic moiety described herein comprises a polymer (e.g., a poly (amidoamine)) conjugated to a detergent (e.g., Triton X-100). In some cases, the endosomal solubilizing moiety described herein comprises a poly (amidoamine) -Triton X-100conjugate (Duncan et al, "A polymer-Triton X-100conjugate capable of pH-dependent porous cell lysis: a model system irradiation the availability of Drug with acidic intracellular complexes," Journal of Drug Targeting2:341-347 (1994)).

Endosomolytic lipids

In some embodiments, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some embodiments, formula (V) -A- (X)¹-B)_nOr formula (VI) -A-X ¹-(B-X²-C)_nThe molecule of (a) is further conjugated to an endosomolytic lipid (e.g., a fusogenic lipid). Exemplary fusogenic lipids include 1, 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), Palmitoyl Oleoyl Phosphatidylcholine (POPC), (6Z,9Z,28Z,31Z) -thirty-seven-carbon-6, 9,28, 31-tetraen-19-ol (Di-Lin), N-methyl (2, 2-bis ((9Z,12Z) -octadeca-9, 12-dienyl) -1, 3-dioxolan-4-yl) methylamine (DLin-k-DMA) and N-methyl-2- (2, 2-bis ((9Z,12Z) -octadeca-9, 12-dienyl) -1, 3-dioxolan-4-yl) ethylamine (XTC).

In some cases, the endosomolytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT publication WO09/126,933.

Endosomolytic small molecules

In some embodiments, the endosomolytic moiety is a small molecule. In some embodiments, formula (I) -A- (X)¹-B)_nOr formula (II) -A-X¹-(B-X²-C)_nThe molecule of (a) is further conjugated to an endosomolytic small molecule. Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquine, amoprazine, primaquine, mefloquine, nivaquine, halofantrine, quinonimine, or combinations thereof. In some cases, the quinoline endosomolytic moiety includes, but is not limited to, 7-chloro-4- (4-diethylamino-1-methylbutyl-amino) quinoline (chloroquine); 7-chloro-4- (4-ethyl- (2-hydroxyethyl) -amino-1-methylbutyl-amino) quinoline (hydroxychloroquine); 7-fluoro-4- (4-diethylamino-1-methylbutyl-amino) quinoline; 4- (4-diethylamino-1-methylbutylamino) quinoline; 7-hydroxy-4- (4-diethyl-amino-1-methylbutylamino) quinoline; 7-chloro-4- (4-diethylamino-1-butylamino) quinoline (demethylchloroquine); 7-fluoro-4- (4-diethylamino-1-butylamino) quinoline); 4- (4-diethyl-amino-1-butylamino) quinoline; 7-hydroxy-4- (4-diethylamino-1-butylamino) quinoline; 7-chloro-4- (1-carboxy-4-diethylamino-1-butylamino) quinoline; 7-fluoro-4- (1-carboxy-4-diethyl-amino-1-butylamino) quinoline; 4- (1-carboxy-4-diethylamino-1-butylamino) quinoline; 7-hydroxy-4- (1-carboxy-4-diethylamino-1-butylamino) quinoline; 7-chloro-4- (1-carboxy-4-diethylamino-1-methylbutylamino) quinoline; 7-fluoro-4- (1-carboxy-4-diethyl-amino-1-methylbutylamino) quinoline; 4- (1-carboxy-4-diethylamino-1-methylbutylamino) quinoline; 7-hydroxy-4- (1-carboxy-4-diethylamino-1-methylbutylamino) quinoline; 7-fluoro-4- (4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; 4- (4-ethyl- (2-hydroxy-ethyl) -amino-1-methylbutylamino-) quinoline; 7-hydroxy-4- (4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; hydroxychloroquine phosphate; 7-chloro-4- (4-ethyl- (2-hydroxyethyl-1) -amino-1-butylamino) quinoline (demethylhydroxychloroquine); 7-fluoro-4- (4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 4- (4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 7-hydroxy-4- (4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 7-chloro-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quine (ii) an alkyl; 7-fluoro-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 7-hydroxy-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-butylamino) quinoline; 7-chloro-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; 7-fluoro-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; 4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; 7-hydroxy-4- (1-carboxy-4-ethyl- (2-hydroxyethyl) -amino-1-methylbutylamino) quinoline; 8- [ (4-aminopentyl) amino-6-methoxyquinoline dihydrochloride; 1-acetyl-1, 2,3, 4-tetrahydroquinoline; 8- [ (4-Aminopentyl) amino group]-6-methoxyquinoline dihydrochloride, 1-butyryl-1, 2,3, 4-tetrahydroquinoline, 3-chloro-4- (4-hydroxy- α' -bis (2-methyl-1-pyrrolidinyl) -2, 5-diphenylaminoquinoline, 4- [ (4-diethyl-amino) -1-methylbutyl-amino]-6-methoxyquinoline, 3-fluoro-4- (4-hydroxy- α' -bis (2-methyl-1-pyrrolidinyl) -2, 5-xylenylaminoquinoline, 4- [ (4-diethylamino) -1-methylbutyl-amino ]-6-methoxyquinoline, 4- (4-hydroxy- α' -bis (2-methyl-1-pyrrolidinyl) -2, 5-diphenylaminoquinoline, 4- [ (4-diethylamino) -1-methylbutyl-amino]-6-methoxyquinoline; 3, 4-dihydro-1- (2H) -quinolinecarboxaldehyde; 1, 1' -pentamethylenebisquinolinium diiodide; 8-hydroxyquinoline sulfate and its amino, aldehyde, carboxyl, hydroxyl, halogen, keto, mercapto and vinyl derivatives or analogs. In some cases, the endosomolytic moiety is a small molecule as described in Naisbitt et al (1997, J Pharmacol Exp Therapy280:884-893) and U.S. Pat. No. 5,736,557.

In some embodiments, the endosomolytic moiety is nigericin or a conjugate thereof, e.g., a folate-nigericin ester conjugate, a folate-nigericin amide conjugate, or a folate-nigericin carbamate conjugate. In some cases, the endosomolytic moiety is Nigericin as described in Rangasamy et al, "New mechanism for release of endogenous proteins"/biological lysine via niger-modified K +/H + exchange, "Bioconjugate chem.29:1047-1059 (2018).

Connecting body

In some embodiments, the linker described herein is a cleavable linker or a non-cleavable linker. In some cases, the linker is a cleavable linker. In other cases, the linker is a non-cleavable linker.

In some cases, the linker is a non-polymeric linker. By non-polymeric linker is meant a linker that does not contain monomeric repeat units generated by the polymerization process. Exemplary non-polymeric linkers include, but are not limited to, C₁-C₆Alkyl (e.g. C)₅、C₄、C₃、C₂Or C₁Alkyl), homobifunctional cross-linkers, heterobifunctional cross-linkers, peptide linkers, traceless linkers, self-immolative (self-immolative) linkers, maleimide-based linkers, or combinations thereof. In some cases, the non-polymeric linker comprises C₁-C₆Alkyl (e.g. C)₅、C₄、C₃、C₂Or C₁Alkyl), homobifunctional crosslinks, heterobifunctional crosslinks, peptide linkers, traceless linkers, self-immolative linkers, maleimide-based linkers, or combinations thereof. In other cases, the non-polymeric linker does not comprise more than two linkers of the same type, e.g., more than two homobifunctional cross-linkers, or more than two peptide linkers. In other cases, the non-polymeric linker optionally comprises one or more reactive functional groups.

In some cases, the non-polymeric linker does not comprise a polymer as described above. In some cases, the non-polymeric linker does not comprise the polymer comprised by polymer moiety C. In some cases, the non-polymeric linker does not comprise a polyalkylene oxide (e.g., PEG). In some cases, the non-polymeric linker does not comprise PEG.

In some cases, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include, but are not limited to, Lomant reagent dithiobis (succinimidyl propionate) DSP, 3 '3' -dithiobis (sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disuccinimidyl tartrate (sulfo DST), bis (succinimidyl succinate) ethylene glycol Ester (EGS), disuccinimidyl glutarate (DSG), N '-disuccinimidyl carbonate (DSC), dimethyl adipimide (DMA), dimethyl pimeimide (DMP), dimethyl suberoyl imide (DMS), dimethyl-3, 3' -Dithiodipropylimide (DTBP), 1, 4-bis-3 ' - (2 ' -pyridyldithio) propionamido) butane (DPDPDPB), Bismaleimidohexane (BMH), aryl halide-containing compounds (DFDNB) such as 1, 5-difluoro-2, 4-dinitrobenzene or 1, 3-difluoro-4, 6-dinitrobenzene, 4 ' -difluoro-3, 3 ' -dinitrophenylsulfone (DFDNPS), bis- [ beta- (4-azidosalicylamino) ethyl ] disulfide (BASED), formaldehyde, glutaraldehyde, 1, 4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3 ' -dimethylbenzidine, benzidine, alpha ' -p-diaminobiphenyl, diiodo-p-xylenesulfonic acid, N ' -ethylene-bis (iodoacetamide), or N, n' -hexamethylene-bis (iodoacetamide).

Exemplary heterobifunctional linkers include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3- (2-pyridyldithio) propionate (sPDP), long chain N-succinimidyl 3- (2-pyridyldithio) propionate (LC-sPDP), water-soluble long chain N-succinimidyl 3- (2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyl oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (sMPT), sulfosuccinimidyl-6- [ α -methyl- α - (2-pyridyldithio) toluidinoyl ] amide]Caproate (sulfo-LC-sMPT), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimidyl ester (sulfo-MBs), N-succinimidyl (4-iodoacetyl) aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl) Acetyl) aminobenzoate (sulfo-siaab), succinimidyl-4- (p-maleimidophenyl) butyrate (sMPB), sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate (sulfo-sMPB), N- (gamma-maleimidobutyryloxy) succinimidyl ester (GMBs), N- (gamma-maleimidobutyryloxy) sulfosuccinimidyl ester (sulfo-GMBs), succinimidyl 6- ((iodoacetyl) amino) hexanoate (siaax), succinimidyl 6- [6- (((iodosulfosuccinimidyl) amino) hexanoyl) amino]Caproate (sIAXX), succinimidyl 4- (((iodosulfosuccinimidyl) amino) methyl) cyclohexane-1-carboxylate (siaac), succinimidyl 6- ((((4-iodosulfosuccinimidyl) amino) methyl) cyclohexane-1-carbonyl) amino) caproate (sIACX), p-Nitrophenyliodoacetate (NPIA); carbonyl-reactive and thiol-reactive cross-linkers, such as 4- (4-N-maleimidophenyl) butyric acid hydrazide (MPBH), 4- (N-maleimidomethyl) cyclohexane-1-carboxy-hydrazide-8 (M)₂C₂H) 3- (2-pyridyldithio) propionyl hydrazide (PDPH); amine-reactive and photoreactive crosslinkers, such as N-hydroxysuccinimide-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl- (4-azidosalicylamido) hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2- (rho-azidosalicylamido) ethyl-1, 3 ' -dithiopropionate (sAsD), N-hydroxysuccinimide-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6- (4 ' -azido-2 ' -nitrophenyl) Alkylamino) hexanoate (sANPAH), sulfosuccinimidyl-6- (4 ' -azido-2 ' -nitrophenylamino) hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl-2- (m-azido-o-nitrobenzoylamino) -ethyl-1, 3 ' -dithiopropionate (sAND), n-succinimidyl-4 (4-azidophenyl) 1,3 '-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl) -1, 3' -dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4- (p-azidophenyl) butyrate (sulfo-sAPB). Sulfosuccinimidyl 2- (7-azido-4-methylcoumarin-3-acetamide) ethyl-1, 3' -dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), rho-nitrophenyldiazopyruvate (rho NPDP), rho-nitrophenyl-2-diazo-3, 3, 3-trifluoropropionate (PNP-DTP); thiol-reactive and photoreactive crosslinkers such as 1- (rho-azidosalicylamido) -4- (iodoacetamido) butane (AsIB), N- [4- (rho-azidosalicylamido) butyl]-3 '- (2' -pyridyldithio) propionamide (apd), benzophenone-4-iodoacetamide, benzophenone-4-maleimide; carbonyl-reactive and photoreactive crosslinks such as rho-azidobenzoyl hydrazide (ABH); carboxylate reactive and photoreactive crosslinkers, such as 4- (rho-azidosalicylamido) butylamine (AsBA); and arginine-reactive and photoreactive crosslinks such as rho-Azidophenylglyoxal (APG).

In some cases, the linker comprises a reactive functional group. In some cases, the reactive functional group comprises a nucleophilic group that is reactive with an electrophilic group present on the binding moiety. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, ketenes, acid halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazine.

In some embodiments, the linker comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer. In some cases, the maleimide group further comprises hexanoic acid, forming a maleimide hexanoyl group (mc). In some cases, the linker comprises a maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other instances, the maleimide group comprises a maleimide methyl group, such as succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (stmcc) or sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-stmcc) as described above.

In some embodiments, the maleimide group is a self-stabilized maleimide. In some cases, self-stabilized maleimides incorporate basic amino groups adjacent to the maleimide with diaminopropionic acid (DPR) to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby preventing the maleimide from undergoing elimination reactions through the reverse michael reaction. In some cases, the Self-stabilizing maleimide is a maleimide group as described by Lyon et al, "Self-hydrolyzing maleimides improving the stability and pharmacological properties of antibodies-drugs," Nat. Biotechnol.32(10):1059-1062 (2014). In some cases, the linker comprises a self-stabilized maleimide. In some cases, the linker is a self-stabilized maleimide.

In some embodiments, the linker comprises a peptide moiety. In some cases, the peptide moiety comprises at least 2, 3, 4, 5, or 6 or more amino acid residues. In some cases, the peptide moiety comprises up to 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some cases, the peptide moiety comprises about 2, about 3, about 4, about 5, or about 6 amino acid residues. In some cases, the peptide moiety is a cleavable peptide moiety (e.g., enzymatically or chemically). In some cases, the peptide moiety is a non-cleavable peptide moiety. In some cases, the peptide portion comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker comprises a peptide moiety, such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group or a derivative thereof. In some cases, the benzoic acid group or derivative thereof comprises para-aminobenzoic acid (PABA). In some cases, the benzoic acid group or derivative thereof comprises gamma aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and/or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of maleimide groups, peptide moieties, and/or benzoic acid groups. In some cases, the maleimide group is a maleimide hexanoyl group (mc). In some cases, the peptide group is val-cit. In some cases, the benzoic acid group is PABA. In some cases, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In further instances, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some cases, the linker includes a linker described in U.S. patent 9,089,614 or PCT publication WO 2015038426.

In some embodiments, the linker is a dendritic linker. In some cases, the dendritic linker comprises a branched multifunctional linker moiety. In some cases, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety a. In some cases, the dendritic linker comprises a PAMAM dendrimer.

In some embodiments, the linker is a traceless linker or a linker that does not leave a linker moiety (e.g., an atom or linker group) for binding moiety a, polynucleotide B, polymer C, or endosomolytic moiety D after cleavage. Exemplary traceless linkers include, but are not limited to, germanium linkers, silicon linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazine linkers. In some cases, the linker is a traceless aryl-triazene linker as described by Hejesen et al, "A tracess aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem11(15):2493-2497 (2013). In some cases, the linker is a Traceless linker as described by Blaney et al, "Traceless solid-phase organic Synthesis," chem. Rev.102:2607-2024 (2002). In some cases, the linker is a traceless linker as described in us patent 6,821,783.

In some cases, the linker is a linker described in the following documents: us patent 6,884,869; 7,498,298, respectively; 8,288,352, respectively; 8,609,105, respectively; or 8,697,688; U.S. patent publication 2014/0127239; 2013/028919, respectively; 2014/286970, respectively; 2013/0309256, respectively; 2015/037360, respectively; or 2014/0294851; or PCT publication WO 2015057699; WO 2014080251; WO 2014197854; WO 2014145090; or WO 2014177042.

In some embodiments, X, Y and L are independently a bond or a linker. In some cases, X, Y and L are independently a bond. In some cases, X, Y and L are independently linkers.

In some cases, X is a bond or a linker. In some cases, X is a bond. In some cases, X is a linker. In some cases, the linker is C₁-C₆An alkyl group. In some cases, X is C₁-C₆Alkyl radicals, e.g. C₅、C₄、C₃、C₂Or C₁An alkyl group. In some cases, this C₁-C₆Alkyl being unsubstituted C₁-C₆An alkyl group. As used in the context of a linker, particularly in the context of X, alkyl means a saturated straight or branched chain hydrocarbon group containing up to six carbon atoms. In some cases, X is a non-polymeric linker. In some cases, X comprises a homobifunctional or heterobifunctional linker as described above. In some cases, X comprises a heterobifunctional linker. In some cases, X comprises smacc. In other cases, X includes optionally conjugated to C ₁-C₆Heterobifunctional linkers for alkyl groups. In other cases, X includes optionally conjugated to C₁-C₆Alkyl sMCC. In other circumstancesIn this case, X does not include homobifunctional or heterobifunctional linkers as described above.

In some cases, Y is a bond or a linker. In some cases, Y is a bond. In other cases, Y is a linker. In some embodiments, Y is C₁-C₆An alkyl group. In some cases, Y is a homobifunctional or heterobifunctional linker as described above. In some cases, Y is a homobifunctional linker as described above. In some cases, Y is a heterobifunctional linker as described above. In some cases, Y comprises a maleimide group, such as the maleimidocaproyl (mc) group described above or a self-stabilizing maleimide group. In some cases, Y comprises a peptide moiety, such as Val-Cit. In some cases, Y comprises a benzoic acid group, such as PABA. In further cases, Y comprises a combination of maleimide groups, peptide moieties, and/or benzoic acid groups. In other cases, Y comprises a mc group. In other cases, Y comprises a mc-val-cit group. In other cases, Y comprises a val-cit-PABA group. In other cases, Y comprises a mc-val-cit-PABA group.

In some cases, L is a bond or a linker. In some cases, L is a bond. In other cases, L is a linker. In some embodiments, L is C₁-C₆An alkyl group. In some cases, L is a homobifunctional or heterobifunctional linker as described above. In some cases, L is a homobifunctional linker as described above. In some cases, L is a heterobifunctional linker as described above. In some cases, L comprises a maleimide group, such as the maleimide caproyl (mc) or a self-stabilizing maleimide group described above. In some cases, L comprises a peptide moiety, such as Val-Cit. In some cases, L comprises a benzoic acid group, such as PABA. In further cases, L comprises a combination of maleimide groups, peptide moieties, and/or benzoic acid groups. In other cases, L comprises a mc group. In other cases, L comprises a mc-val-cit group. In other cases, L comprises a val-cit-PABA group. In other cases, L comprises a mc-val-cit-PABA group.

In some embodiments, X¹And X²Each independently is a bond or a non-polymeric linker. In some cases, X¹And X²Each independently a bond. In some cases, X ¹And X²Each independently is a non-polymeric linker.

In some cases, X¹Is a bond or a non-polymeric linker. In some cases, X¹Is a bond. In some cases, X¹Is a non-polymeric linker. In some cases, the linker is C₁-C₆An alkyl group. In some cases, X¹Is C₁-C₆Alkyl radicals, e.g. C₅、C₄、C₃、C₂Or C₁An alkyl group. In some cases, this C₁-C₆Alkyl being unsubstituted C₁-C₆An alkyl group. E.g., in the context of a linker, particularly X¹Alkyl, as used in the context of (a), means a saturated straight or branched hydrocarbon group containing up to six carbon atoms. In some cases, X¹Including homobifunctional or heterobifunctional linkers as described above. In some cases, X¹Including heterobifunctional linkers. In some cases, X¹Including the smacc. In other cases, X¹Comprising optionally conjugated to C₁-C₆Heterobifunctional linkers for alkyl groups. In other cases, X¹Comprising optionally conjugated to C₁-C₆Alkyl sMCC. In other cases, X¹Homo-or heterobifunctional linkers as described above are not included.

In some cases, X²Is a bond or a linker. In some cases, X²Is a bond. In other cases, X²Is a connecting body. In other cases, X ²Is a non-polymeric linker. In some embodiments, X²Is C₁-C₆An alkyl group. In some cases, X²Are homobifunctional or heterobifunctional linkers as described above. In some cases, X²Are homobifunctional linkers as described above. In some cases, X²Is a heterobifunctional linker as described above. In some cases, X²Contain a maleimide group such as the maleimidocaproyl (mc) group described above or a self-stabilizing maleimide group. In some cases, X²Comprising a peptide moiety, such as Val-Cit. In some cases, X²Containing benzoic acid groups, such as PABA. In other cases, X²A combination comprising a maleimide group, a peptide moiety and/or a benzoic acid group. In other cases, X²Containing a mc group. In other cases, X²Containing the mc-val-cit group. In other cases, X²Comprising a val-cit-PABA group. In other cases, X²Containing a mc-val-cit-PABA group.

Pharmaceutical preparation

In some embodiments, the pharmaceutical formulations described herein are administered to a subject by a variety of routes of administration including, but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal routes of administration. In some cases, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, intracerebral, intracerebroventricular, or intracranial) administration. In other instances, the pharmaceutical compositions described herein are formulated for oral administration. In other instances, the pharmaceutical compositions described herein are formulated for intranasal administration.

In some embodiments, the pharmaceutical formulation includes, but is not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.

In some cases, the pharmaceutical formulation comprises a multiparticulate formulation. In some cases, the pharmaceutical formulation comprises a nanoparticle formulation. In some cases, the nanoparticle comprises cMAP, cyclodextrin, or a lipid. In some cases, the nanoparticle comprises a solid lipid nanoparticle, a polymeric nanoparticle, a self-emulsifying nanoparticle, a liposome, a microemulsion, or a micellar solution. Additional exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as metal chelates with covalent linkages), nanofibers, nanohorns, nano-onions, nanorods, nanoropes, and quantum dots. In some cases, the nanoparticles are metal nanoparticles, such as nanoparticles of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

In some cases, the nanoparticle comprises a core or a core and a shell, such as in a core-shell nanoparticle.

In some cases, the nanoparticle is further coated with a molecule for attaching a functional element (e.g., to one or more of the polynucleic acid molecules or binding moieties described herein). In some cases, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agar gum, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, alpha-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some cases, the nanoparticles comprise graphene-coated nanoparticles.

In some cases, the nanoparticle has at least one dimension less than about 500nm, 400nm, 300nm, 200nm, or 100 nm.

In some cases, the nanoparticle formulation comprises paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as metal chelates with covalent linkages), nanofibers, nanohorns, nano onions, nanorods, ropes, or quantum dots. In some cases, the polynucleic acid molecules or binding moieties described herein are conjugated to nanoparticles, either directly or indirectly. In some cases, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more of the polynucleic acid molecules or binding moieties described herein are conjugated, directly or indirectly, to a nanoparticle.

In some embodiments, the pharmaceutical formulation comprises a delivery vector, such as a recombinant vector, to deliver the polynucleic acid molecule into the cell. In some cases, the recombinant vector is a DNA plasmid. In other cases, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some cases, a recombinant vector capable of expressing a polynucleic acid molecule provides stable expression in a target cell. In other cases, viral vectors are used that provide for transient expression of the polynucleic acid molecules.

In some embodiments, the pharmaceutical formulation comprises a carrier or carrier material selected based on compatibility with the compositions disclosed herein and the release profile properties of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizing agents, stabilizing agents, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, cellulose and cellulose conjugates, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like. See, for example, Remington: The science and Practice of Pharmacy, nineteenth edition (Easton, Pa.: Mack Publishing Company, 1995); hoover, John e., Remington's Pharmaceutical Sciences, Mack Publishing co, Easton, Pennsylvania 1975; liberman, h.a. and Lachman, l. eds, Pharmaceutical DosageForms, Marcel Decker, New York, n.y., 1980; and Pharmaceutical document Forms and drug Delivery Systems, seventh edition (Lippincott Williams & Wilkins 1999).

In some cases, the pharmaceutical formulation further comprises a pH adjusting agent or buffer, which includes acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

In some cases, the pharmaceutical formulation includes one or more salts in an amount necessary to bring the osmolality of the composition within an acceptable range. Such salts include those having a sodium, potassium or ammonium cation and a chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anion; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

In some cases, the pharmaceutical formulations further comprise diluents to stabilize the compounds, as they provide a more stable environment. Salts dissolved in buffer solutions (which also provide pH control or maintenance) are utilized in the art as diluents, including but not limited to phosphate buffered saline solutions. In some cases, the diluent increases the volume of the composition to facilitate compression or to create sufficient volume for uniform blending for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Calcium hydrogen phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray dried lactose; pregelatinized starches, compressible sugars, e.g.(Amstar); mannitol, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, sugar fructose; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrate (dextrate); hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulation includes a disintegrant to facilitate the disintegration or disintegration of the substance. The term "disintegration" includes dissolution and dispersion of the dosage form when contacted with gastrointestinal fluids. Examples of disintegrants include starches, e.g. natural starches such as corn or potato starch, pregelatinized starches such as National 1551 or

Or sodium starch glycolate e.g.Or

Cellulose, e.g. wood products, methyl crystalline cellulose, e.g.PH101、

PH102、

PH105、

P100、

Andmethylcellulose, croscarmellose, or cross-linked cellulose such as croscarmellose sodium

Croscarmellose or cross-linked carmellose, cross-linked starches such as sodium starch glycolate, cross-linked polymers such as povidone, cross-linked polyvinylpyrrolidone, alginates such as alginic acid or salts of alginic acid such as sodium alginate, clays such as sodium alginate

HV (magnesium aluminum silicate), gums such as agar, guar gum, locust bean gum, karaya gum, pectin or tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactants, resins such as cation exchange resins, citrus pulp, sodium lauryl sulfate, combinations of sodium lauryl sulfate and starch, and the like.

In some cases, the pharmaceutical formulation comprises a filler, such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in the pharmaceutical formulations described herein to prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil

Higher fatty acids and their alkali metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc salts, stearic acid, sodium stearate, glycerin, talc, waxes,

boric acid, sodium benzoate, sodium acetate, sodium chloride, Leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax^TMSodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium lauryl sulfate or sodium lauryl sulfate, colloidal silica such as Syloid^TM，

Starches such as corn starch, silicone oils, surfactants, and the like.

Plasticizers include compounds that serve to soften the microencapsulated material or film coating to make it less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. Plasticizers also act as dispersing or wetting agents.

Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium lauryl sulfate, docusate sodium, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcyclodextrine, ethanol, N-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-.

Stabilizers include compounds such as any antioxidants, buffers, acids, preservatives, and the like.

Suspending agents include, for example, polyvinylpyrrolidone such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having a molecular weight of from about 300 to about 6000, or from about 3350 to about 4000, or from about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth and acacia, guar gum, xanthan gum (including xanthan gum), sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, carboxymethylcellulose sodium, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, Polyethoxylated sorbitan monolaurate, povidone, and the like.

Surfactants include, for example, sodium lauryl sulfate, docusate sodium, tween 60 or 80, triacetin, vitamin e tpgs, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomers (polaxomers), bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide such as (BASF) and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol (octoxynol)10, octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, gum arabic, chitosan, and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerol monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, docusate sodium, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, tween 80, vitamin E TPGS, ammonium salts, and the like.

Treatment regimens

In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic applications. In some embodiments, the pharmaceutical composition is administered once daily, twice daily, three times daily, or more. The pharmaceutical composition is administered daily, once daily, every other day, five days per week, once weekly, every other week, two weeks per month, three weeks per month, once monthly, twice monthly, three times monthly or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

In some embodiments, one or more pharmaceutical compositions are administered simultaneously, sequentially or at intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further instances, one or more pharmaceutical compositions are administered at intervals (e.g., a first administration of a first pharmaceutical composition on a first day and then at least 1, 2, 3, 4, 5 or more days before administration of at least a second pharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositions are co-administered. In some cases, two or more different pharmaceutical compositions are co-administered simultaneously. In some cases, two or more different pharmaceutical compositions are co-administered sequentially without a time interval between administrations. In other cases, two or more different pharmaceutical compositions are co-administered sequentially with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or more between administrations.

Continuing administration of the administration composition at the discretion of the physician in the event that the patient's condition is indeed improved; alternatively, the dose of the composition administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., the "drug holiday"). In some cases, the length of the drug holiday varies between 2 days and 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during the drug holiday is 10% -100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once the patient's condition has improved, if necessary, a maintenance dose is administered. Subsequently, depending on the change in symptoms, the dosage or frequency of administration, or both, can be reduced to a level at which improvement in the disease, disorder, or condition is maintained.

In some embodiments, the amount of a given agent corresponding to such amount varies depending on factors such as the particular compound, the severity of the disease, the characteristics (e.g., body weight) of the subject or host in need of treatment, etc., yet it is still routinely determined in a manner known in the art according to the specifics associated with the example, including, for example, the particular agent administered, the route of administration, and the subject or host being treated. In some cases, the required dose is conveniently presented in a single dose or in divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example 2, 3, 4 or more sub-doses per day.

The foregoing ranges are indicative only, as the number of variables relating to an individual treatment regimen is large, and it is not uncommon for significant deviations from these recommended values. Such dosages will vary depending upon a number of variables not limited to the activity of the compound employed, the disease or condition to be treated, the mode of administration, the requirements of the subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including but not limited to the determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio of LD50 to ED 50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of doses for use in humans. The dose of such compounds is preferably within a circulating concentration range that includes ED50 and has minimal toxicity. The dosage will vary within this range depending upon the dosage form employed and the route of administration utilized.

Kit/article of manufacture

In certain embodiments, disclosed herein are kits and articles of manufacture for use with one or more of the compositions and methods described herein. Such kits include a carrier, package, or container compartmentalized to receive one or more containers, e.g., vials, tubes, and the like, each container containing a separate element to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from various materials such as glass or plastic.

The articles provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

For example, the container comprises a target nucleic acid molecule as described herein. Such kits optionally comprise identifying descriptions or labels or instructions for their use in the methods described herein.

Kits typically include a label and/or instructions for use listing the contents, as well as a package insert with instructions for use. A set of instructions will also typically be included.

In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers or other characters comprising the label are attached, molded or etched onto the container itself; a label is associated with a container when the label is present within a receptacle or carrier that also holds the container (e.g., as a package insert). In one embodiment, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions regarding the use of the contents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are provided in a pack or dispenser device containing one or more unit dosage forms containing a compound provided herein. For example, the package comprises a metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the package or dispenser is accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects approval by the agency of the pharmaceutical form for human or veterinary administration. Such notice is, for example, a label approved by the U.S. food and drug administration for prescription drugs or an approved product insert. In one embodiment, a composition containing a compound provided herein formulated in a compatible pharmaceutical carrier is also prepared, placed in an appropriate container, and labeled for treatment of the indicated condition.

Certain terms

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 the claimed subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the terms "including" and other forms, such as "comprises," "comprising," and "having," are non-limiting.

As used herein, ranges and amounts can be expressed as "about" a particular value or range. "about" also includes the exact amount. Thus, "about 5 μ L" means "about 5 μ L", and also means "5 μ L". Generally, the term "about" includes amounts that are expected to be within experimental error.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

As used herein, the terms "individual," "subject," and "patient" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of these terms require or are limited to situations characterized by supervision (e.g., on a continuous or intermittent basis) by a healthcare worker (e.g., a doctor, a registered nurse, a practicing nurse, a physician's assistant, a caregiver, or a attending care worker).