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1. (WO2018226602) ENHANCED MODIFIED VIRAL CAPSID PROTEINS
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What is claimed is:

1. A modified viral capsid protein comprising a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the interior of the viral capsid protein.

2. The modified viral capsid protein of claim 1, further comprising one or more linkers. 3. The modified viral capsid protein of claim 2, wherein the one or more linkers comprise SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 51, or an equivalent of each thereof.

4. The modified viral capsid protein of any one of claims 1-3, wherein the Cas9 protein or an equivalent thereof has been conjugated to the interior of the viral capsid protein via modular intein based assembly.

5. The modified viral capsid protein of claim 4, wherein the modular intein based assembly is fast intein based assembly.

6. The modified viral capsid protein of claim 4, wherein the modular intein based assembly is consensus fast (Cfa) based assembly.

7. The modified viral capsid protein of any one of the previous claims, wherein the viral capsid protein is an adeno-associated virus (AAV) capsid protein.

8. The modified viral capsid protein of claim 7, wherein the AAV capsid protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

9. The modified viral capsid protein of claim 8, wherein the AAV viral protein comprises VP2, or an equivalent thereof.

10. The modified viral capsid protein of claim 9, wherein the Cas9 protein or an equivalent thereof is conjugated to VP2 at amino acid position 228, 350, 419, 684, or 689 of SEQ ID NO: 59.

11. The modified viral capsid protein of claim 10, wherein the Cas9 protein or an equivalent thereof is conjugated to VP2 at amino acid position 228, 350, 419, 684, or 689 of SEQ ID NO: 59 via one or more linkers.

12. The modified viral capsid protein of any one of the previous claims, wherein the Cas9 protein or an equivalent thereof comprises or is derived from S. aureus Cas9, Cpf1, or GeoCas9.

13. The modified viral capsid protein of claim 12, wherein the Cas9 protein is S. aureus Cas9 or an equivalent thereof or a derivative thereof.

14. The modified viral capsid protein of claim 9, wherein the modified capsid protein comprises SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49, or an equivalent of each thereof.

15. A modified viral capsid protein comprising a viral capsid protein having a Cas9 protein or an equivalent thereof conjugated to the exterior of the viral capsid protein via modular intein based assembly.

16. The modified viral capsid protein of claim 15, further comprising one or more linkers. 17. The modified viral capsid protein of claim 16, wherein the one or more linkers comprise SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 51, or an equivalent of each thereof.

18. The modified viral capsid protein of claim 16, wherein the one or more linkers comprises SEQ ID NO: 31.

19. The modified viral capsid protein of claim 18, wherein the modular intein based assembly is fast intein based assembly.

20. The modified viral capsid protein of claim 18, wherein the modular intein based assembly is consensus fast (Cfa) based assembly.

21. The modified viral capsid protein of any one of claims 15-20, wherein the viral capsid protein is an adeno-associated virus (AAV) capsid protein.

22. The modified viral capsid protein of claim 21, wherein the AAV capsid protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

23. The modified viral capsid protein of claim 22, wherein the AAV viral protein comprises VP2, or an equivalent thereof.

24. The modified viral capsid protein of claim 23, wherein the Cas9 protein or an equivalent thereof is conjugated to the N-terminus of VP2.

25. The modified viral capsid protein of any one of claims 15-24, wherein the Cas9 protein or an equivalent thereof comprises or is derived from S. aureus Cas9, Cpf1, or GeoCas9.

26. The modified viral capsid protein of claim 25, wherein the Cas9 protein is S. aureus Cas9 or an equivalent thereof or a derivative thereof.

27. The modified viral capsid protein of claim 26, wherein the modified capsid protein comprises SEQ ID NO: 36, or an equivalent thereof.

28. An isolated polynucleotide encoding the modified viral capsid protein of any of claims 1-27.

29. A vector or host cell comprising the isolated polynucleotide of claim 28.

30. A method of preparing the modified viral capsid protein of claim 4 or 18, comprising coupling (i) a fusion protein comprising the Cas9 protein or an equivalent thereof and an N-terminal fragment of a split intein to (ii) a fusion protein comprising the viral capsid protein and a C-terminal fragment of a split intein under conditions suitable for modular intein based assembly.

31. The method of claim 30, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a fast intein.

32. The method of claim 30, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a Cfa intein.

33. A method of preparing a modified viral capsid protein comprising expressing the polynucleotide of claim 28.

34. A recombinant viral particle comprising a modified capsid, wherein the modified capsid comprises the modified viral capsid protein of any one of claims 1-27.

35. The recombinant viral particle of claim 34, wherein the modified capsid comprises between 1 to 5 modified viral capsid proteins per recombinant viral particle.

36. The recombinant viral particle of claim 34 or 35, further comprising a polynucleotide encapsidated within the modified capsid.

37. The recombinant viral particle of claim 36, wherein the polynucleotide comprises a polynucleotide encoding one or more guide RNAs (gRNAs).

38. The recombinant viral particle of claim 37, wherein the polynucleotide encoding the one or more gRNAs comprises:

a. a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or

b. a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).

39. The recombinant viral particle of any one of claims 34-38, further comprising a therapeutic polynucleotide.

40. The recombinant viral particle of claim 39, wherein the therapeutic polynucleotide comprises a repair template.

41. A recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the interior of the viral capsid, comprising:

(a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or an equivalent thereof and a viral capsid protein; and

(b) a helper plasmid.

42. A recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the interior of the viral capsid, comprising:

(a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or an equivalent thereof and an N-terminal fragment of a split intein;

(b) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising a viral capsid protein and a C-terminal fragment of a split intein; and (c) a helper plasmid.

43. The recombinant expression system of claim 42, wherein plasmid (a) and plasmid (b) are the same or different plasmids.

44. The recombinant expression system of claim 42 or 43, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a fast intein.

45. The recombinant expression system of claim 42 or 43, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a Cfa intein.

46. The recombinant expression system of any one of claims 41-45, wherein the viral capsid protein is an AAV capsid protein.

47. The recombinant expression system of claim 46, wherein the AAV capsid protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

48. The recombinant expression system of claim 47, wherein the AAV capsid protein comprises VP2, or an equivalent thereof.

49. The recombinant expression system of claim 48, wherein the Cas9 protein or an equivalent thereof is fused to VP2 at amino acid position 228, 350, 419, 684, or 689 of SEQ ID NO: 59.

50. The recombinant expression system of claim 49, wherein the Cas9 protein or an equivalent thereof is fused to VP2 at amino acid position 228, 350, 419, 684, or 689 of SEQ ID NO: 59 via one or more linkers.

51. A recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the exterior of the viral capsid, comprising:

(a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or an equivalent thereof and a viral capsid protein; and

(b) a helper plasmid.

52. A recombinant expression system for the generation of a modified viral particle expressing Cas9 or an equivalent thereof on the exterior of the viral capsid, comprising:

(a) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising the Cas9 or an equivalent thereof and an N-terminal fragment of a split intein;

(b) a plasmid comprising a DNA sequence encoding a fusion protein, the fusion protein comprising a viral capsid protein and a C-terminal fragment of a split intein; and (c) a helper plasmid.

53. The recombinant expression system of claim 52, wherein plasmid (a) and plasmid (b) are the same or different plasmids.

54. The recombinant expression system of claim 52 or 53, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a fast intein.

55. The recombinant expression system of claim 52 or 53, wherein at least one of the N-terminal fragment of a split intein and the C-terminal fragment of a split intein is derived from a Cfa intein.

56. The recombinant expression system of any one of claims 51-55, wherein the viral capsid protein is an AAV capsid protein.

57. The recombinant expression system of claim 56, wherein the AAV capsid protein comprises one or more of VP1, VP2, and VP3, or an equivalent of each thereof.

58. The recombinant expression system of claim 57, wherein the AAV capsid protein comprises VP2, or an equivalent thereof.

59. The recombinant expression system of any one of claims 51-58, wherein the Cas9 protein or an equivalent thereof is conjugated to the N-terminus of VP2.

60. The recombinant expression system of any one of claims 41-59, wherein plasmid (a) further comprises one or more polynucleotides encoding a linker.

61. The recombinant expression system of claim 42 or 52, wherein plasmid (b) further comprises one or more polynucleotides encoding a linker.

62. The recombinant expression system of claim 60 or 61, wherein the linker comprises SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 51, or an equivalent of each thereof.

63. The recombinant expression system of any one of claims 41-62, wherein the Cas9 protein or an equivalent thereof is S. aureus Cas9 or Cpf1.

64. The recombinant expression system of claim 63, wherein the Cas9 protein is S. aureus Cas9 or an equivalent thereof.

65. The recombinant expression system of claim 41, wherein the fusion protein comprises SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49, or an equivalent of each thereof.

66. The recombinant expression system of claim 51, wherein the fusion protein comprises SEQ ID NO: 36, or an equivalent thereof.

67. The recombinant expression system of claim 41 or 51, wherein plasmid (a) comprises a polynucleotide encoding VP2, a polynucleotide encoding Cas9, and one or more polynucleotides encoding a linker, or an equivalent of each thereof.

68. The recombinant expression system of any one of claims 41-67, wherein the helper plasmid comprises a DNA sequence selected from the group of a DNA sequence encoding VP1, a DNA sequence encoding VP3, or a DNA sequence encoding both VP1 and VP3, or an equivalent of each thereof.

69. The recombinant expression system of claim 68, wherein the helper plasmid comprises SEQ ID NO: 57 or an equivalent thereof.

70. The recombinant expression system of any one of claims 41-69, further comprising a polynucleotide encoding one or more gRNAs.

71. The recombinant expression system of any one of claims 41-70, further comprising a therapeutic polynucleotide.

72. A method of producing modified AAV expressing Cas9 on the interior of the viral capsid comprising transfecting one or more cells with the recombinant expression system of any one of claims 41-50 or 65.

73. A method of producing modified AAV expressing Cas9 on the exterior of the viral capsid comprising transfecting one or more cells with the recombinant expression system of any one of claims 51-59 or 66.

74. A modified AAV produced according to the method of claim 72 or 73.

75. An isolated tissue comprising the modified viral capsid protein of any one of claims 1-27.

76. A non-human transgenic animal comprising the modified viral capsid protein of any one of claims 1-27.

77. A method of gene editing or gene regulation comprising contacting a cell with the recombinant viral particle of any one of claims 34-40.

78. A method of gene editing or gene regulation comprising contacting a cell with the recombinant viral particle of claim 34 or 35 and a second viral particle comprising a polynucleotide.

79. The method of claim 78, wherein the polynucleotide comprises a polynucleotide encoding one or more guide RNAs (gRNAs).

80. The method of claim 79, wherein the polynucleotide encoding the one or more gRNAs comprises:

a. a fusion polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA); or

b. a polynucleotide comprising CRISPR RNA (crRNA) and trans-activating CRIPSPR RNA (tracrRNA).

81. The method of any one of claims 78-80, further comprising a therapeutic

polynucleotide.

82. The method of claim 81, wherein the therapeutic polynucleotide comprises a repair template.

83. The method of any of claims 77-82, wherein the contacting is in vitro, in vivo, or ex vivo.

84. A method of gene editing or gene regulation in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant viral particle of claim 34 or 35.

85. A method of gene editing or gene regulation in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant viral particle of any one of claims 36-38.

86. A method of gene editing or gene regulation in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant viral particle of claim 39 or 40.

87. The method of claim 85, wherein the polynucleotide is selected to treat a disease, disorder, or condition selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin, amyotrophic lateral sclerosis, Alzheimer’s, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson’s, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C.

88. The method of claim 86, wherein the therapeutic polynucleotide is selected to treat a disease, disorder, or condition selected from the group of hemophilia, muscular dystrophy, multiple sclerosis, alpha-1-antitrypsin, amyotrophic lateral sclerosis, Alzheimer’s, spinal muscular atrophy, cystic fibrosis, HIV, thalassemia, choroideremia, Parkinson’s, Leber congenital amaurosis, macular degeneration, aromatic amino acid decarboxylase deficiency, achromatopsia, Crigler Najjar syndrome, Pompe disease, X-linked retinoschisis, homozygous familial hypercholesteremia, Batten disease, retinal degeneration, ornithine transcarbamylase deficiency, mucopolysarccharidosis (I-IX), hepatitis B, and hepatitis C.

89. The method of claim 87 or 88, wherein the hemophilia is characterized by one or more of factor VIII or factor IX deficiency.

90. The method of claim 87 or 88, wherein the muscular dystrophy is selected from Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.