Modified muscle targeting composition

Abstract

This invention provides an improved muscle-targeting site for delivering a cargo containing gene therapy drugs, such as antisense oligonucleotides, therapeutic polypeptides, or polynucleotides encoding peptides, to muscle cells. [Solution] A composition is provided comprising a targeting site effective for targeting muscle cells, wherein the targeting site comprises an n-mer motif; and a cargo, wherein the cargo is connected to or otherwise associated with the targeting site.

Claims

1. A targeting site effective for targeting muscle cells, wherein the targeting site includes an n-mer motif; and A cargo, wherein the cargo is connected to or otherwise associated with the targeted site. A composition containing the following:

2. The composition according to claim 1, wherein the n-mer motif comprises an RGD motif or a non-RGD n-mer motif.

3. The aforementioned RGD motif is X ｍ RGDX ｎ The composition according to claim 2, having the formula (wherein m is 0 to 4 amino acids, n is 0 to 15 amino acids, X is any amino acid, and each X amino acid present is independently selected from others from the group consisting of any amino acids).

4. The composition according to claim 3, wherein the RGD motif has the formula RGDXn (wherein n is 4 or 5, X is any amino acid, and each X amino acid present is independently selected from others from the group consisting of any amino acids).

5. The composition according to claim 2, wherein the n-mer motif is one of sequence numbers 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889.

6. The composition according to claim 1, wherein the targeting site comprises a polypeptide, polynucleotide, lipid, polymer, sugar, or a combination thereof.

7. The composition according to claim 1, wherein the targeting site comprises a viral protein.

8. The composition according to claim 7, wherein the viral protein is a capsid protein.

9. The composition according to claim 7, wherein the viral protein is an adeno-associated virus (AAV) protein.

10. The composition according to claim 7, wherein the n-mer motif is positioned between two amino acids of the viral protein such that the n-mer motif is outside the viral capsid, which is part of the viral capsid protein.

11. The composition according to claim 10, wherein the n-mer motif is inserted between amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 in the AAV9 capsid polypeptide, or any combination thereof, or between any two consecutive amino acids at similar positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

12. The composition according to claim 11, wherein the n-mer motif is inserted between amino acids 588 and 589 in the AAV9 capsid polypeptide or at a similar position in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

13. The composition according to claim 1, wherein the composition is a modified virus particle.

14. The composition according to claim 13, wherein the modified virus particles are modified AAV virus particles.

15. The composition according to claim 14, wherein the modified AAV virus particles are modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh. 74, or AAV rh. 10 virus particles.

16. The composition according to claim 1, wherein the cargo is capable of treating or preventing muscle diseases or disorders.

17. The aforementioned muscle disease or disorder is a. autoimmune diseases; b. Cancer; c. Muscular dystrophy; d. Neuromuscular diseases; e. Sugar or glycogen storage disorders; f. Elongation Repeat Syndrome; g. Dominant-negative disorders; h. Cardiomyopathy; i. Viral diseases; j. Progeria; or k. Any combination of them The composition according to claim 16.

18. The aforementioned cargo is a. Morpholino; b. Peptide-linked morpholino; c. Antisense oligonucleotides; d. PMO, therapeutic transgenes; e. Polynucleotides encoding therapeutic polypeptides or peptides; f. PPMO; g. One or more peptides or polypeptides; h. One or more polynucleotides encoding the CRISPR-Cas protein, guide RNA, or both; i. A ribonucleoprotein comprising a CRISPR-Cas system molecule; j. Therapeutic transgene RNA, or other genetically modified or therapeutic RNA and / or protein; or k. Any combination of them The composition according to claim 1.

19. The composition according to claim 1, wherein the cargo is capable of inducing exon skipping in genes.

20. The composition according to claim 1, wherein the cargo is capable of inducing exon skipping in the dystrophin gene.

21. The composition according to claim 1, wherein the cargo is a mini or microdystrophin gene.

22. The composition according to claim 21, wherein the mini or microdystrophin gene comprises spectrin-like repeats 1, 1', 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin-binding domain, one or more hinge regions, a dystroglycan-binding domain, or any combination thereof.

23. The composition according to claim 1, wherein the cargo is functionally linked to a muscle-specific promoter.

24. The composition according to claim 17, wherein the elongation repeat disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

25. The composition according to claim 17, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery Leifs muscular dystrophy, myotonic dystrophy, or FSHD.

26. The composition according to claim 25, wherein the myotonic dystrophy is type 1 or type 2 myotonic dystrophy.

27. The composition according to claim 17, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, Duchenne muscular dystrophy-associated cardiomyopathy, or Danon disease.

28. The composition according to claim 17, wherein the sugar or glycogen storage disorder is MPS type III disease or Pompe disease.

29. The composition according to claim 28, wherein the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID.

30. The composition according to claim 17, wherein the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

31. The composition according to claim 1, wherein the composition has increased muscle cell efficacy, muscle cell specificity, decreased immunogenicity, or any combination thereof.

32. One or more polynucleotides each encoding all or part of one or more targeting sites effective for targeting muscle cells, each targeting site comprising one or more n-mer motifs, each n-mer motif being an RGD motif or a non-RGD n-mer motif, and each polynucleotide encoding at least one or more of the one or more n-mer motifs; and Optionally, a control element functionally linked to one or more of the one or more polynucleotides. vectors containing A vector system including a vector system.

33. The aforementioned RGD motif is X ｍ RGDX ｎ The vector system according to claim 32, having the formula (wherein m is 0 to 4 amino acids, n is 0 to 15 amino acids, X is any amino acid, and each X amino acid present is independently selected from others from the group consisting of any amino acids).

34. The vector system according to claim 33, wherein the RGD motif has the formula RGDXn (wherein n is 4 or 5, X is any amino acid, and each X amino acid present is independently selected from others from the group consisting of any amino acids).

35. The vector system according to claim 33, wherein the n-mer motif is one of sequence numbers 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889.

36. The vector system according to claim 32, further comprising cargo.

37. The vector system according to claim 36, wherein the cargo is a cargo polynucleotide, and is optionally linked to one or more of the one or more polynucleotides encoding the targeting site, the control element, or both.

38. The vector system according to claim 36, wherein the cargo polynucleotide is present in the same vector as or in a different vector from the one or more polynucleotides encoding the targeting site.

39. The vector system according to claim 36, wherein the vector system is capable of generating virus particles containing the cargo.

40. The vector system according to claim 32, wherein the vector system is capable of generating a viral capsid polypeptide containing one or more of the targeting sites.

41. The vector system according to claim 32, wherein the vector system is capable of generating AAV virus particles.

42. The vector system according to any one of claims 41, wherein the AAV virus particles are modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh. 74, or AAV rh. 10 virus particles.

43. The vector system according to claim 40, wherein the capsid polypeptide is a modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

44. The vector system according to claim 39, wherein at least one of the one or more polynucleotides encoding the n-mer motif(s) is inserted between two codons corresponding to two amino acids of the viral protein such that at least one of the n-mer motifs is outside the viral capsid.

45. The vector system according to claim 44, wherein the two codons correspond to amino acids 262-269, 327-332, 382-386, 452-460, 488-505, 527-539, 545-558, 581-593, 704-714 in the AAV9 capsid polypeptide, or any combination thereof, or any two consecutive amino acids at similar positions in the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

46. The vector system according to claim 45, wherein the two codons correspond to amino acids 588 and 589 in the AAV9 capsid polynucleotide or to similar positions in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV rh. 74, or AAV rh. 10 capsid polypeptide.

47. The vector system according to any one of claims 32, wherein the vector comprises one or more polynucleotides each encoding all or part of the one or more targeting sites, and does not include a splice control element.

48. The vector system according to claim 32, further comprising a polynucleotide encoding a viral rep protein.

49. The vector system according to claim 48, wherein the viral rep protein is a rep protein of AAV.

50. The vector system according to claim 48, wherein the polynucleotide encoding the viral rep protein is in the same vector or a different vector as the one or more polynucleotides encoding all or part of the one or more targeting sites, respectively.

51. The vector system according to claim 48, wherein the viral rep protein is functionally linked to a regulatory element.

52. A polypeptide produced by expressing the vector system described in any one of claims 32 to 51.

53. The polypeptide according to claim 52, wherein the polypeptide is a viral polypeptide.

54. The polypeptide according to claim 53, wherein the viral polypeptide is an AAV polypeptide.

55. Particles generated by activating the vector system described in any one of claims 32 to 51.

56. The particle according to claim 55, wherein the particle is a virus particle.

57. The particle according to claim 56, wherein the virus particle is an adeno-associated virus (AAV) particle.

58. The virus particle is the particle according to claim 56, wherein the virus particle has muscle-specific targeting.

59. The cargo is a vector system according to any one of claims 36 to 51, a polypeptide according to any one of claims 52 to 54, or a particle according to any one of claims 55 to 58, which is capable of treating or preventing muscle diseases or disorders.

60. The aforementioned muscle disease or disorder is a. autoimmune diseases; b. Cancer; c. Muscular dystrophy; d. Neuromuscular diseases; e. Sugar or glycogen storage disorders; f. Elongation Repeat Syndrome; g. Dominant-negative disorders; h. Cardiomyopathy; i. Viral diseases; j. Progeria; or k. Any combination of them The vector system, polypeptide, or particle according to claim 59.

61. The aforementioned cargo is morpholino; a. Peptide-linked morpholino; b. Antisense oligonucleotides; c. PMO, therapeutic transgene; d. Polynucleotides encoding therapeutic polypeptides or peptides; e. PPMO; f. One or more peptides or polypeptides; g. One or more polynucleotides encoding the CRISPR-Cas protein, guide RNA, or both; h. A ribonucleoprotein comprising a CRISPR-Cas system molecule; i. Therapeutic transgene RNA, or other genetically modified or therapeutic RNA and / or protein; or j. Any combination of those The vector system, polypeptide, or particle according to claim 60.

62. The vector system, polypeptide, or particle according to claim 59, wherein the cargo is capable of inducing exon skipping in a gene.

63. The vector system, polypeptide, or particle according to claim 59, wherein the cargo is capable of inducing exon skipping in the dystrophin gene.

64. The vector system, polypeptide, or particle according to claim 59, wherein the cargo is a mini or microdystrophin gene.

65. The vector system, polypeptide, or particle according to claim 64, wherein the mini or microdystrophin gene comprises spectrin-like repeats 1, 1', 2, 3, 16, 17, 20, 21, 22, 23, 24, or any combination thereof, and optionally an nNOS domain, an actin-binding domain, one or more hinge regions, a dystroglycan-binding domain, or any combination thereof.

66. The vector system, polypeptide, or particle according to claim 60, wherein the elongation repeat disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

67. The vector system, polypeptide, or particle according to claim 60, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery Leifs muscular dystrophy, myotonic dystrophy, or FSHD.

68. The vector system, polypeptide, or particle according to claim 67, wherein the myotonic dystrophy is type 1 or type 2.

69. The vector system, polypeptide, or particle according to claim 60, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Danon disease.

70. The vector system, polypeptide, or particle according to claim 60, wherein the sugar or glycogen storage disorder is MPS type III disease or Pompe disease.

71. The vector system, polypeptide, or particle according to claim 70, wherein the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID.

72. The vector system, polypeptide, or particle according to claim 60, wherein the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

73. The polypeptide, the particles, or both thereof, having increased muscle cell efficacy, muscle cell specificity, decreased immunogenicity, or any combination thereof, according to any one of claims 52 to 58.

74. a. The composition according to any one of claims 1 to 31; b. The vector system according to any one of claims 32 to 51 or 59 to 72; c. The polypeptide according to any one of claims 52 to 54 or 59 to 73; d. Particles according to any one of claims 55 to 73; or e. Combinations of those Cells containing this substance.

75. The cell according to claim 74, wherein the cell is a prokaryotic cell.

76. The cell according to claim 74, wherein the cell is a eukaryote.

77. a. The composition according to any one of claims 1 to 31; b. The vector system according to any one of claims 32 to 51 or 59 to 72; c. The polypeptide according to any one of claims 52 to 54 or 59 to 73; d. Particles according to any one of claims 55 to 73; e. The cells according to any one of claims 74 to 76; or f. Combinations thereof; and Pharmacologically acceptable carriers A pharmaceutical preparation containing [the specified ingredient].

78. a. The composition according to any one of claims 1 to 31; b. The vector system according to any one of claims 32 to 51 or 59 to 72; c. The polypeptide according to any one of claims 52 to 54 or 59 to 73; d. Particles according to any one of claims 55 to 73; e. The cells according to any one of claims 74 to 76; f. The pharmaceutical preparation according to claim 77; or g. Combinations of those A method comprising administering it to a subject who needs it.

79. The method according to claim 78, wherein the subject requiring the same has a muscle disease or disorder.

80. The aforementioned muscle disease or disorder is a. autoimmune diseases; b. Cancer; c. Muscular dystrophy; d. Neuromuscular diseases; e. Sugar or glycogen storage disorders; f. Elongation Repeat Syndrome; g. Dominant-negative disorders; h. Cardiomyopathy; i. Viral diseases; j. Progeria; or k. Any combination of them The method according to claim 79.

81. The method according to claim 80, wherein the elongation repeat disease is Huntington's disease, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD).

82. The method according to claim 80, wherein the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery Leifs muscular dystrophy, myotonic dystrophy, or FSHD.

83. The method according to claim 82, wherein the myotonic dystrophy is type 1 or type 2.

84. The method according to claim 80, wherein the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, DMD-associated cardiomyopathy, or Danon disease.

85. The method according to claim 80, wherein the sugar or glycogen storage disorder is MPS type III disease or Pompe disease.

86. The method according to claim 85, wherein the MPS type III disease is MPS type IIIA, IIIB, IIIC, or IIID.

87. The method according to claim 80, wherein the neuromuscular disease is Charcot-Marie-Tooth disease or Friedreich's ataxia.

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