阅读说明：本技术 用于穿过维管结构的基因转移的方法和组合物 (For passing through the method and composition of the gene transfer of vasculature ) 是由 阿拉温德·阿索卡恩 吉里达尔·莫里达尔安 布莱克·奥尔布赖特 于 2018-02-15 设计创作，主要内容包括：本公开提供了在氨基酸序列中包含修饰的AAV衣壳蛋白,以及包含经修饰的AAV衣壳蛋白的病毒衣壳和病毒载体。本公开还提供了将本公开的病毒载体和病毒衣壳体内施用至细胞或受试者的方法。(Present disclose provides the AAV capsid proteins comprising modification in amino acid sequence, and viral capsid and viral vectors comprising modified AAV capsid protein.The disclosure, which additionally provides, will be applied to cell or the method for subject in the viral vectors of the disclosure and viral capsid body.)

Adeno-associated virus 1. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S262, A263, Modification at S264, T265, A267, S268 and H272, and the single amino acids residue insertion between residue G266 and A267, (VP1 number), wherein the number of each residue is based on the amino acid sequence or AAV2 (SEQ ID of AAV1 (SEQ ID NO:1) NO:2)、AAV3(SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO: 6), the equivalent amino acid residue in AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8).

2. AAV capsid protein as described in claim 1, be further contained in amino acid residue Q148, E152, S157, Between modification and residue E152 and P153 (VP1 number) at T162, T326, D328, V330, T331, V341 and S345 Single amino acids residue insertion, wherein amino acid sequence or SEQ ID NO of the number of each residue based on SEQ ID NO:1: 2, the equivalent amino acid residue in 3,4,5,6,7 or 8.

3. AAV capsid protein as claimed in claim 2, further includes amino acid residue L188, S205, N223 and A224 The modification of (VP1 number), wherein amino acid sequence or SEQ ID NO:2 of the number of each residue based on SEQ ID NO:1,3, 4, the equivalent amino acid residue in 5,6,7 or 8.

4. AAV capsid protein as claimed in any one of claims 1-3, wherein the modification be S262N, A263G, S264T, At least one of T265S, A267S, S268T and H272T.

5. such as AAV capsid of any of claims 1-2, wherein the modification be Q148P, E152R, S157T, At least one of T162K, T326Q, D328E, V330T, T331K, V341I and S345T.

6. AAV capsid as claimed in claim 3, wherein the modification be in L188I, S205A, N223S and A224S at least One.

7. AAV capsid protein as described in claim 1, it includes the amino acid sequences selected from the group being made of the following terms:

A) amino acid sequence (AAV1RX) of SEQ ID NO:9；

B) amino acid sequence (AAV2RX) of SEQ ID NO:10；

C) amino acid sequence (AAV3RX) of SEQ ID NO:11；

D) amino acid sequence (AAV6RX) of SEQ ID NO:12；

E) amino acid sequence (AAV7RX) of SEQ ID NO:13；

F) amino acid sequence (AAV8RX) of SEQ ID NO:14；With

G) amino acid sequence (AAV9RX) of SEQ ID NO:15.

8. AAV capsid as claimed in claim 2, it includes the amino acid sequences selected from the group being made of the following terms:

A) amino acid sequence (AAV1R6) of SEQ ID NO:16；

B) amino acid sequence (AAV2R6) of SEQ ID NO:17；

C) amino acid sequence (AAV3R6) of SEQ ID NO:18；

D) amino acid sequence (AAV6R6) of SEQ ID NO:19；

E) amino acid sequence (AAV7R6) of SEQ ID NO:20；

F) amino acid sequence (AAV8R6) of SEQ ID NO:21；With

G) amino acid sequence (AAV9R6) of SEQ ID NO:22.

9. AAV capsid as claimed in claim 3, it includes the amino acid sequences selected from the group being made of the following terms:

A) amino acid sequence (AAV1R7) of SEQ ID NO:23；

B) amino acid sequence (AAV2R7) of SEQ ID NO:24；

C) amino acid sequence (AAV3R7) of SEQ ID NO:25；

D) amino acid sequence (AAV6R7) of SEQ ID NO:26；

E) amino acid sequence (AAV7R7) of SEQ ID NO:27；

F) amino acid sequence (AAV8R7) of SEQ ID NO:28；With

G) amino acid sequence (AAV9R7) of SEQ ID NO:29.

10.AAV capsid, it includes capsid proteins as claimed in any one of claims 1-9 wherein.

11. viral vectors, it includes:

(a) AAV capsid described in any one of claim 10；With

(b) include at least one terminal repeat nucleic acid,

Wherein the nucleic acid is by AAV capsid involucrum.

12. composition, it includes viral vectors described in the claim 11 in pharmaceutically acceptable carrier.

13. the method that nucleic acid molecules are imported cell comprising make viral vectors described in the cell and claim 11 and/ Or the contact of composition described in claim 12.

14. the method that nucleic acid molecules are delivered to subject comprising Xiang Suoshu subject applies disease described in claim 11 Composition described in poisonous carrier and/or claim 12.

15. method as claimed in claim 14, wherein the viral vectors and/or composition are applied to the subject's Central nervous system.

16. method as claimed in claim 15, wherein the viral vectors and/or composition are delivered across blood-brain barrier.

17. the method as described in any one of claim 14,15 or 16, wherein the viral vectors includes interested nucleic acid Molecule.

18. the method for selectively delivering interested nucleic acid molecules to neuronal cell comprising make the neuronal cell with The contact of viral vectors described in claim 11, wherein the viral vectors includes the interested nucleic acid molecules.

19. method as claimed in claim 18 further includes that interested nucleic acid molecules are selectively delivered to cardiac muscle carefully Born of the same parents.

20. the method as described in any one of claim 17,18 or 19, wherein the interested nucleic acid molecule encoding treatment Property protein or therapeutic RNA.

21. the method as described in any one of claim 18,19 or 20, wherein the neuronal cell and cardiac muscle cell be In subject.

22. method as claimed in claim 21, wherein the viral vectors goes to target from splenocyte, liver cell or nephrocyte.

23. the method as described in any one of claim 14-17,21 or 22, wherein the subject is people experimenter.

24. the method for the neurological disorder or defect for the treatment of subject comprising Xiang Suoshu subject applies claim 11 The viral vectors, wherein the viral vectors includes that coding is effective therapeutic in treating the neurological disorder or defect The nucleic acid molecules of protein or therapeutic RNA.

25. the method for nerve and cardiovascular disorder or defect for treating subject comprising Xiang Suoshu subject applies power Benefit require 11 described in viral vectors, wherein the viral vectors include coding treat it is described nerve and cardiovascular disorder or lack The nucleic acid molecules of effective therapeutic protein or therapeutic RNA in falling into.

26. the method as described in any one of claim 14-17,21,22,23,24 or 25, wherein by the viral vectors or Composition is intravenous, is applied to the subject in intra-arterial or peritonaeum.

27. the method as described in any one of claim 14-17,21,22,23,24 or 25, wherein by the viral vectors or Composition by the ventricles of the brain, in brain pond, essence is interior, encephalic and/or intrathecal route are applied to the subject.

28. the method as described in any one of claim 24-27, wherein the viral vectors and/or composition to it is described by The systemic administration of examination person causes to miss the target the lower transduction in tissue.

Adeno-associated virus 29. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S262, A263, Modification at S264, T265 and A267 (VP1 number), and the single amino acids residue insertion between residue S268 and N269, Wherein the amino acid residue is based on the amino acid sequence or AAV2 (SEQ ID NO:2), AAV3 of AAV1 (SEQ ID NO:1) (SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9(SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8) in equivalent amino acid residue.

30. AAV capsid protein as claimed in claim 29, wherein the capsid protein is further contained in amino acid residue Modification at H272.

31. the AAV capsid protein as described in claim 29 or 30, wherein the AAV capsid protein is further contained in amino Modification at sour residue Q148, E152, S157, T162, H272, T326, D328, V330, T331, V341 and S345, Yi Ji Single amino acids residue insertion between residue E152 and P153.

32. the AAV capsid protein as described in any one of claim 29-31, wherein the AAV capsid protein further includes Modification at amino acid residue L188, S205, N223, A224 and H272.

33. the AAV capsid protein as described in any one of claim 29-32, wherein the modification be S262N, A263G, At least one of S264T, T265S and A267S.

34. the AAV capsid protein as described in any one of claim 31-33, wherein the modification be Q148P, E152R, At least one of S157T, T162K, H272T, T326Q, D328E, V330T, T331K, V341I and S345T.

35. the AAV capsid protein as described in any one of claim 32-24, wherein the modification be L188I, S205A, At least one of N223S, A224S and H272T.

36. the AAV capsid protein as described in any one of claim 29-35, wherein the institute between residue S268 and N269 State the insertion that amino acid residue insertion is single T residue.

37. the AAV capsid protein as described in any one of claim 31-36, wherein the institute between residue E152 and P153 State the insertion that amino acid residue insertion is single S residue.

38. AAV capsid protein as claimed in claim 29, wherein the amino acid sequence of the capsid protein is selected from by following The group of item composition:

A) amino acid sequence (AAV1RX) of SEQ ID NO:9；

B) amino acid sequence (AAV2RX) of SEQ ID NO:10；

C) amino acid sequence (AAV3RX) of SEQ ID NO:11；

D) amino acid sequence (AAV6RX) of SEQ ID NO:12；

E) amino acid sequence (AAV7RX) of SEQ ID NO:13；

F) amino acid sequence (AAV8RX) of SEQ ID NO:14；With

G) amino acid sequence (AAV9RX) of SEQ ID NO:15.

39. AAV capsid protein as claimed in claim 38, wherein the amino acid sequence of the capsid protein is SEQ ID NO: 9(AAV1RX)。

40. AAV capsid protein as claimed in claim 31, wherein the amino acid sequence of the capsid protein is selected from by following The group of item composition:

A) amino acid sequence (AAV1R6) of SEQ ID NO:16；

B) amino acid sequence (AAV2R6) of SEQ ID NO:17；

C) amino acid sequence (AAV3R6) of SEQ ID NO:18；

D) amino acid sequence (AAV6R6) of SEQ ID NO:19；

E) amino acid sequence (AAV7R6) of SEQ ID NO:20；

F) amino acid sequence (AAV8R6) of SEQ ID NO:21；With

G) amino acid sequence (AAV9R6) of SEQ ID NO:22.

41. AAV capsid protein as claimed in claim 40, wherein the amino acid sequence of the capsid protein is SEQ ID NO: 16(AAV1R6)。

42. AAV capsid protein as claimed in claim 32, wherein the amino acid sequence of the capsid protein is selected from by following The group of item composition:

A) amino acid sequence (AAV1R7) of SEQ ID NO:23；

B) amino acid sequence (AAV2R7) of SEQ ID NO:24；

C) amino acid sequence (AAV3R7) of SEQ ID NO:25；

D) amino acid sequence (AAV6R7) of SEQ ID NO:26；

E) amino acid sequence (AAV7R7) of SEQ ID NO:27；

F) amino acid sequence (AAV8R7) of SEQ ID NO:28；With

G) amino acid sequence (AAV9R7) of SEQ ID NO:29.

43. AAV capsid protein as claimed in claim 42, wherein the amino acid sequence of the capsid protein is SEQ ID NO: 23(AAV1R7)。

Adeno-associated virus 44. (AAV) capsid protein, wherein the AAV capsid protein includes to cause corresponding to natural A AV1 clothing Following amino acid sequence at the amino acid of the amino acid position 262 to 265 (VP1 number) of glutelin (SEQ ID NO:1) Modification:

X¹-X²-X³-X⁴

Wherein X¹It is the arbitrary amino acid in addition to S；

Wherein X²It is the arbitrary amino acid in addition to A；

Wherein X³It is the arbitrary amino acid in addition to S；With

Wherein X⁴It is the arbitrary amino acid in addition to T.

45. AAV capsid protein as claimed in claim 44, wherein the amino acid X¹It is N.

46. AAV capsid protein as claimed in claim 44, wherein the amino acid X²It is G.

47. AAV capsid protein as claimed in claim 44, wherein the amino acid X³It is T.

48. AAV capsid protein as claimed in claim 44, wherein the amino acid X⁴It is S.

49. AAV capsid protein as claimed in claim 44, wherein the amino acid X¹It is N, X²It is G, X³It is T, and X⁴It is S.

50. the AAV capsid protein as described in any one of claim 44-49, wherein the capsid protein is further contained in Modification at amino acid residue H272.

51. AAV capsid protein as claimed in claim 50, wherein the modification is H272T.

52. the AAV capsid protein as described in any one of claim 44-51, wherein the AAV capsid protein further includes Amino acid residue insertion between amino acid residue S268 and N269.

53. AAV capsid protein as claimed in claim 52, wherein amino acid residue insertion is the insertion of single T residue.

Adeno-associated virus 54. (AAV) carrier, it includes the AAV capsid proteins described in any one of claim 29-53.

55. AAV carrier as claimed in claim 54, further includes:

Nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is by the AAV capsid protein involucrum.

56. AAV viral vectors as claimed in claim 55, wherein the terminal repeat is AAV terminal repeat.

57. AAV viral vectors as claimed in claim 55, wherein the terminal repeat is non-AAV terminal repeat.

58. the AAV viral vectors as described in any one of claim 55-57, is controlled wherein the nucleic acid further includes coding The sequence of the property treated protein or therapeutic RNA.

59. pharmaceutical composition, it includes the viral vectors described in any one of claim 54-58.

60. pharmaceutical composition as claimed in claim 59, wherein the composition further includes pharmaceutically acceptable load Body.

61. the method that nucleic acid molecules are imported cell comprising make described in any one of the cell and claim 54-59 The contact of AAV viral vectors.

62. the method that nucleic acid molecules are imported cell comprising make pharmaceutical composition described in cell and claim 59 or 60 Contact.

63. the method as described in claim 61 or 62, wherein the viral vectors or the composition are applied to subject Central nervous system.

64. the method as described in claim 63, wherein the viral vectors or the composition are delivered across blood-brain barrier.

65. the method as described in claim 61 or 62, wherein the viral vectors or the composition go to target from liver.

66. the method as described in claim 61 or 62, wherein the viral vectors or the composition go to target from kidney.

67. the method as described in claim 61 or 62, wherein the viral vectors or the composition go to target from spleen.

68. selectively by the method for the neuronal cell of therapeutic protein or therapeutic RNA delivery to subject comprising Make group described in viral vectors described in any one of the neuronal cell and claim 54-58 or claim 59-60 Object contact is closed, wherein the viral vectors or the composition include the therapeutic protein or therapeutic RNA.

69. method as recited in claim 68, wherein the viral vectors or the composition go to target from liver.

70. method as recited in claim 68, wherein the viral vectors or the composition go to target from kidney.

71. method as recited in claim 68, wherein the viral vectors or the composition go to target from spleen.

72. the method for treating neural's obstacle or defect, wherein this method includes applying claim 54-58 to subject Any one of described in viral vectors or claim 59-60 described in composition, wherein the viral vectors or the combination Object includes coding nucleic acid molecules of effective therapeutic protein or therapeutic RNA in treatment neurological disorder.

73. the method as described in claim 72 further comprises the cardiovascular disorder or defect for treating subject.

74. the method as described in claim 72, wherein the viral vectors or the composition are selectively delivered to mind Through first cell.

75. the method as described in claim 72, wherein the viral vectors or the composition are selectively delivered to the heart Myocyte.

76. the method as described in claim 72, wherein by the viral vectors or the composition from liver, kidney and/or Spleen goes to target.

77. the method as described in any one of claim 61-76, wherein the subject is mammal.

78. method described in any one of claim 61-77, wherein the subject is people.

79. the method as described in any one of claim 61-78, wherein by the viral vectors or composition pass through selected from by The administration route of group of the following terms composition is delivered to the subject: in intravenous, intra-arterial, peritonaeum, in the ventricles of the brain, brain pond In interior, essence, encephalic and intrathecal.

80. the method as described in claim 79, wherein the viral vectors or composition are delivered to by intravenously application The subject.

Adeno-associated virus 81. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S262, A263, Modification at S264, T265, A267 and H272, and the single amino acids residue insertion between residue S268 and N269, (VP1 Number), wherein the number of each residue be based on AAV1 (SEQ ID NO:2) amino acid sequence or AAV1 (SEQ ID NO:1), AAV3(SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9 Equivalent amino acid residue in (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8).

82. the AAV capsid protein as described in claim 81, wherein the modification comprising S262N, A263G, S264T, T265S, At least one of A267G and H272T.

83. the AAV capsid protein as described in claim 81 or 82, wherein the single amino acids between residue S268 and N269 Residue insertion is single T residue insertion.

84. the AAV capsid protein as described in any one of claim 81 to 83, wherein the AAV capsid protein includes SEQ The sequence of ID NO:30 or SEQ ID NO.33.

Adeno-associated virus 85. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S262, Q263, Modification at S264, A266, S267 and H271, and at least one amino acid residue between residue S261 and S262 are inserted Enter, (VP1 number), wherein the number of each residue is based on the amino acid sequence or AAV1 (SEQ ID of AAV2 (SEQ ID NO:2) NO:1)、AAV3(SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO: 6), the equivalent amino acid residue in AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8).

86. the AAV capsid protein as described in claim 85, wherein the modification comprising S262T, Q263S, S264G, A266S, At least one of S267T and H271T.

87. the AAV capsid protein as described in claim 85 or 86, wherein the insertion between residue SS61 and S262 is single The insertion of amino acid residue.

88. the AAV capsid protein as described in claim 85 or 86, wherein the insertion between residue S251 and S252 is to be more than The insertion of one amino acid residue.

89. the AAV capsid protein as described in claim 88, wherein the insertion between residue S251 and S252 is N and G residue Insertion.

90. the AAV capsid protein as described in any one of claim 85 to 89, wherein the AAV capsid protein includes SEQ The sequence of ID NO:31.

Adeno-associated virus 91. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S262, Q263, Modification at S264, A266, A267, H271, and the single amino acids residue insertion between residue S261 and S262, wherein often The number of a residue is based on the amino acid sequence or AAV1 (SEQ ID NO:1), AAV2 (SEQ ID of AAV3 (SEQ ID NO:3) NO:2)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9(SEQ ID NO:7) Or the equivalent amino acid residue in AAVrh10 (SEQ ID NO:8).

92. the AAV capsid protein as described in claim 91, wherein the modification include S262T, Q263S, S264G, A266S, At least one of A267T, H271T.

93. the AAV capsid protein as described in claim 91 or 92, wherein the insertion between residue SS61 and S262 is single The insertion of amino acid residue.

94. the AAV capsid protein as described in claim 91 or 92, wherein the insertion between residue S251 and S252 is to be more than The insertion of one amino acid residue.

95. the AAV capsid protein as described in claim 94, wherein the insertion between residue S251 and S252 is N and G residue Insertion.

96. the AAV capsid protein as described in any one of claim 91 to 95, wherein the AAV capsid protein includes SEQ The sequence of ID NO:32.

Adeno-associated virus 97. (AAV) capsid protein, wherein the AAV capsid protein be included in amino acid residue S263, S269, Modification at A237 (VP1 number), wherein the number of each residue be based on AAV9 (SEQ ID NO:9) amino acid sequence or AAV1(SEQ ID NO:1)、AAV2(SEQ ID NO:2)、AAV3(SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7 Equivalent amino acid residue in (SEQ ID NO:5), AAV8 (SEQ ID NO:6) or AAVrh10 (SEQ ID NO:8).

98. the AAV capsid protein as described in claim 97, wherein the modification comprising in S263G, S269T and A273T extremely It is one few.

99. the AAV capsid protein as described in claim 97 or 98, wherein the AAV capsid protein includes SEQ ID NO:34 Sequence.

Adeno-associated virus 100. (AAV) capsid protein, wherein the AAV capsid protein includes SEQ ID NO:9 to SEQ ID The sequence of any of NO:34.

Adeno-associated virus 101. (AAV) carrier, it includes the AAV capsid proteins described in any one of claim 81-100.

102. the AAV carrier as described in claim 101, further includes:

Nucleic acid comprising at least one terminal repeat, wherein the nucleic acid is by the AAV capsid protein involucrum.

103. the AAV viral vectors as described in claim 102, wherein the terminal repeat is AAV terminal repeat.

104. the AAV viral vectors as described in claim 102, wherein the terminal repeat is that the non-end AAV repeats sequence Column.

105. the AAV viral vectors as described in any one of claim 101-104, wherein the nucleic acid further includes coding The sequence of therapeutic protein or therapeutic RNA.

106. pharmaceutical composition, it includes the viral vectors described in any one of claim 101-105.

107. the pharmaceutical composition as described in claim 106, wherein the composition further include it is pharmaceutically acceptable Carrier.

108. the method that nucleic acid molecules are imported cell comprising make any one of the cell and claim 101-105 institute The AAV viral vectors contact stated.

109. the method that nucleic acid molecules are imported cell, including making pharmaceutical composition described in cell and claim 106 or 107 Contact.

110. the method as described in claim 108 or 109, wherein the viral vectors or the composition are applied to tested The central nervous system of person.

111. the method as described in claim 110, wherein the viral vectors or the composition are passed across blood-brain barrier It send.

112. the method as described in claim 108 or 109, wherein the viral vectors or the composition remove target from liver To.

113. the method as described in claim 108 or 109, wherein the viral vectors or the composition remove target from kidney To.

114. the method as described in claim 108 or 109, wherein the viral vectors or the composition remove target from spleen To.

115. selectively by the method for the neuronal cell of therapeutic protein or therapeutic RNA delivery to subject, packet Including makes viral vectors described in any one of the neuronal cell and claim 101-105 or claim 106-107 institute The composition contact stated, wherein the viral vectors or the composition include the therapeutic protein or therapeutic RNA.

116. the method as described in claim 115, wherein the viral vectors or the composition go to target from liver.

117. the method as described in claim 115, wherein the viral vectors or the composition go to target from kidney.

118. the method as described in claim 115, wherein the viral vectors or the composition go to target from spleen.

119. the method for treating neural's obstacle or defect, wherein this method includes applying claim to the subject Composition described in viral vectors described in any one of 101-105 or claim 106-107, wherein the viral vectors or The composition includes coding nucleic acid molecules of effective therapeutic protein or therapeutic RNA in treatment neurological disorder.

120. the method as described in claim 119 further comprises the cardiovascular disorder or defect for treating subject.

121. the method as described in claim 119, wherein the viral vectors or the composition are selectively delivered to Neuronal cell.

122. the method as described in claim 119, wherein the viral vectors or the composition are selectively delivered to Cardiac muscle cell.

123. the method as described in claim 119, wherein by the viral vectors or the composition from liver, kidney and/ Or spleen goes to target.

124. the method as described in any one of claim 108-123, wherein the subject is mammal.

125. the method as described in any one of claim 108-124, wherein the subject is people.

126. the method as described in any one of claim 108-125, wherein by the viral vectors or composition by choosing The administration route of the group of free the following terms composition is delivered to the subject: in intravenous, intra-arterial, peritonaeum, in the ventricles of the brain, brain In pond, in essence, encephalic and intrathecal.

127. the method as described in claim 126, wherein the viral vectors or composition are delivered by intravenously application To the subject.

Invention field

The present invention relates to the modified capsid protein from adeno-associated virus (AAV) and include its viral capsid and disease Poisonous carrier.Particularly, the present invention relates to modified AAV capsid protein and comprising its capsid, viral vectors can be mixed In with assign the transduction needed for interested target tissue spectrum (profile).

Background technique

New adeno-associated virus (AAV) strain separated from animal tissue and adenoviral stocks, which extends, can be used for treating Property gene transfer application AAV vehicle group.Currently make great efforts to draw these tissues of AAV separation strains in animal model comprehensively Taxis.By AAV carrier go back to the nest be directed to selection sexual organ ability be useful for gene therapy and other treatment application 's.

The present invention solves demand of this field to the nucleic acid delivery vector with required targeting feature.

Summary of the invention

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white included in amino acid residue S262, A263, S264, T265, A267, S268 and H272 place modification, and residue G266 with Between A267 single amino acids residue insertion, (VP1 number), wherein the number of each residue based on AAV1 (SEQ ID NO: 1) amino acid sequence or AAV2 (SEQ ID NO:2), AAV3 (SEQ ID NO:3), AAV6 (SEQ ID NO:4), AAV7 In (SEQ ID NO:5), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8) etc. Same amino acid residue.In some embodiments, AAV capsid protein of the invention can be further contained in amino acid residue Modification and residue E152 and P153 at Q148, E152, S157, T162, T326, D328, V330, T331, V341 and S345 Single amino acids residue insertion between (VP1 number), wherein amino acid sequence of the number of each residue based on SEQ ID NO:1 Equivalent amino acid residue in column or SEQ ID NO:2,3,4,5,6,7 or 8.In a further embodiment, of the invention AAV capsid protein can further include the modification of amino acid residue L188, S205, N223 and A224 (VP1 number), wherein often Equivalent amino in amino acid sequence or SEQ ID NO:2,3,4,5,6,7 or 8 of the number of a residue based on SEQ ID NO:1 Sour residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg The white modification at amino acid residue S262, A263, S264, T265 and A267 (VP1 number), and residue S268 with Single amino acids residue insertion between N269, wherein amino acid residue is based on the amino acid sequence of AAV1 (SEQ ID NO:1) Or AAV2 (SEQ ID NO:2), AAV3 (SEQ ID NO:3), AAV6 (SEQ ID NO:4), AAV7 (SEQ ID NO:5), Equivalent amino acid residue in AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8). In some embodiments, capsid protein is further contained in the modification at amino acid residue H272.In some embodiments, AAV capsid protein be further contained in amino acid residue Q148, E152, S157, T162, H272, T326, D328, V330, Modification at T331, V341 and S345, and the single amino acids residue insertion between residue E152 and P153.Further Embodiment in, AAV capsid protein is further contained in repairing at amino acid residue L188, S205, N223, A224 and H272 Decorations.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein AAV capsid protein packet Containing the modification for generating following amino acid sequence: in the amino acid position for corresponding to natural A AV1 capsid protein (SEQ ID NO:1) X at the amino acid of 262 to 265 (VP1 numbers)¹-X²-X³-X⁴(SEQ ID NO:35), wherein X¹It is any ammonia in addition to S Base acid；Wherein X²It is the arbitrary amino acid in addition to A；Wherein X³It is the arbitrary amino acid other than S；Wherein X⁴It is any other than T Amino acid.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg The white modification at amino acid residue S262, A263, S264, T265, A267 and H272, and residue S268 and N269 it Between single amino acids residue insertion, (VP1 number), wherein the number of each residue based on AAV1 (SEQ ID NO:2) ammonia Base acid sequence or AAV1 (SEQ ID NO:1), AAV3 (SEQ ID NO:3), AAV6 (SEQ ID NO:4), AAV7 (SEQ ID NO:5), the equivalent amino acid in AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8) Residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg The white modification at amino acid residue S262, Q263, S264, A266, S267 and H271, and residue S261 and S262 it Between at least one amino acid residue insertion, (VP1 number), wherein the number of each residue be based on AAV2 (SEQ ID NO: 2) amino acid sequence or AAV1 (SEQ ID NO:1), AAV3 (SEQ ID NO:3), AAV6 (SEQ ID NO:4), AAV7 In (SEQ ID NO:5), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8) etc. Same amino acid residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg The white modification at amino acid residue S262, Q263, S264, A266, A267, H271, and residue S261 and S262 it Between single amino acids residue insertion, wherein the number of each residue be based on AAV3 (SEQ ID NO:3) amino acid sequence or AAV1(SEQ ID NO:1)、AAV2(SEQ ID NO:2)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8 Equivalent amino acid residue in (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8).

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein AAV capsid protein packet The modification being contained at amino acid residue S263, S269, A237 (VP1 number), wherein the number of each residue is based on AAV9 (SEQ ID NO:9) amino acid sequence or AAV1 (SEQ ID NO:1), AAV2 (SEQ ID NO:2), AAV3 (SEQ ID NO: 3), AAV6 (SEQ ID NO:4), AAV7 (SEQ ID NO:5), AAV8 (SEQ ID NO:6) or AAVrh10 (SEQ ID NO: 8) equivalent amino acid residue in.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg The white sequence comprising any of SEQ ID NO:9 to SEQ ID NO:34.

The disclosure is additionally provided with the AAV capsid of the capsid protein comprising the disclosure, and the AAV capsid comprising the disclosure Viral vectors.In some embodiments, viral vectors includes the AAV capsid of the disclosure and repeats comprising at least one end The nucleic acid of sequence, wherein the nucleic acid is by AAV capsid involucrum.

The disclosure is additionally provided with the pharmaceutical composition comprising AAV capsid disclosed herein and/or viral vectors.

The method that nucleic acid molecules are introduced into cell is also provided herein, including connecing the viral vectors of cell and the disclosure Touching, and the method that nucleic acid molecules are delivered to subject, including disclosed viral vectors is applied to subject.

In addition, the disclosure provides the method for selectively delivering interested nucleic acid molecules to neuronal cell comprising make The neuronal cell is contacted with viral vectors of the invention, wherein the viral vectors includes interested nucleic acid molecules.

In another embodiment again, the disclosure provides the method for treating the neurological disorder or defect of subject, It includes the viral vectors that the disclosure is applied to the subject, wherein the viral vectors includes coding in treatment neurological disorder Or the nucleic acid molecules of effective therapeutic protein or therapeutic RNA in defect.

Solve these and other aspects in more detail in the description being explained below.

Brief description

The transduction of Fig. 1 cortical neuron quantifies.It is 5 × 10 with dosage¹¹The packaging CBh-scGFP of vg (vector gene group) Carrier (AAV1, AAV1R6 or AAV1R7) or PBS as negative control by tail vein injection systemic administration to 6-8 week old Female BL6 mouse.21 days execution mouse after injection, and harvest tissue, be fixed and be sliced.Using DAB substrate to organize into Row GFP immunostaining observes GFP expression by glass slide scanning and ImageScope software.Spreading the multiple of every mouse In coronal brain section, artificial counting is carried out to the GFP positive neuron in cerebral cortex, quantifies and is averaged.For AAV1, N=2；For AAV1R6 and AAV1R7, n=3.

Fig. 2 passes through brain vasculature and neuron expression.To pack the AAV carrier of Cbh-scGFP transgenosis with 5 × 10¹¹The dosage of vg is applied to C57/B16 mouse by tail vein injection.21 days execution mouse after injection harvest tissue, by it Fixed, slice, DAB dyeing are imaged by Aperio Scanner and are carried out at image with ImageScope software to detect GFP Reason.Abbreviation: cortex (CTX), motor cortex (MCT), corpus straitum (STR), hippocampus (HC), dentate fascia (DG).N=3.

Fig. 3 liver goes to target.The AAV carrier of CBA- Luciferase transgenic will be packed with 1 × 10¹¹The dosage of vg passes through Tail vein injection is applied to C57/B16 mouse.14 days execution mouse after injection harvest tissue, are shredded, cracked and carried out Luciferase assay is horizontal to detect brain, heart, liver and the relative transduction of myeloid tissue.It is per gram of tissue by data normalization Relative light unit.N=3.

The Phylogenetic Analysis and structural analysis in Fig. 4 A-C.AAV1/rh.10 Domain swapping capsid library.Fig. 4 A. is from text The schematic diagram of the representative AAV1/rh.10 chimeric capsid group separated in library, which demonstrate obtained by reorganization (shuffling) Sequence polymorphism of the Domain swapping on amino acid levels.Single parent or chimeric capsid are vertically listed, and have difference C-terminal level of the VP1 capsid sequence of Domain swapping from the N-terminal in left side to right side is shown.Residue is derived from AAV1 with ash Color shows that AAVrh.10 residue is shown with dark green, and shared residue between the two is with black display.Fig. 4 B.AAV1/rh.10 The adjacent system of the VP1 capsid sequence of chimeric capsid occurs.Capsid amino acid sequence and ClustalW are compared, and use adjoining Method generates system.It is corrected using Poisson and calculates amino acid distance, be expressed as the quantity of the amino acid replacement in each site Unit.The tree is drawn to scale, and branch length is identical as the branch length of evolutionary distance, for inferring the tree.It uses 1000 times Duplication calculates Bootstrap value, and the percentages show for the replication tree that wherein relevant classification clustering gathers together is by branch Side.Parent of Fig. 4 C. selection for screening in vivo and capsid subunits tripolymer/3 times for representing sex-mosaicism AAV capsid mutants are right Claim the 3 d surface model in axis region.Amino acid residue from AAV1 with gray display, and from AAVrh.10 residue with Green-green display.Structural model is visualized and generated using PyMol.

Screening generates the AAV1/rh.10 chimera that blood-brain barrier can be passed through after intravenous application in Fig. 5 body.With 5×10¹¹By 3 weeks after tail vein injection application, the scanning of the brain section of immunostaining was shown by parent or packaging the dosage of vg The GFP table in cerebral cortex that the chimeric vector (or being PBS in the case where analogies are handled) of CBh-scGFP transgenosis mediates It reaches.Overall coronal brain section (above) indicates the cortex seen in the illustration shown for each parent or chimeric vector Rectangular body region represents their own transduction spectrum.Scale bar=100 μm.

The internal screening of Fig. 6 A-G.AAV1/rh.10 chimera passes through blood brain screen to obtain after they are applied in the blood vessels Hinder and the ability for various brain areas of transduceing.Mouse is 5 × 10 by tail vein injection acceptable dose¹¹Vg parent (AAV1 or AAVrh.10) or packaging CBh-scGFP transgenosis chimeric vector.After immunostaining and the display injection of the mouse brain slice of imaging GFP expression in 21 days, such as black/dark-brown dyeing.Scale bar=100 μm.The overall coronal brain section at figure top indicates to insert The various brain regions shown in figure: MCT, motor cortex (Fig. 6 A)；HC, hippocampus (Fig. 6 B)；DG, dentate fascia (Fig. 6 C)；TH, mound Brain (Fig. 6 D)；HY, hypothalamus (Fig. 6 E)；STR, corpus straitum (Fig. 6 F)；Amg, amygdaloid nucleus (Fig. 6 G).For every kind, n=3.

Compared with parent AAV1 and AAVrh.10, the CNS of AAV1R6 and AAV1R7 in brain transduces to be composed Fig. 7 A-H..For Parent serotypes A AV1 and AAVrh.10 (left column), and for AAV1R6 and AAV1R7 (right column), with 5 × 10¹¹The dosage of vg Three weeks after the AAV carrier of tail vein injection packaging CBh-scGFP transgenosis, it is shown that the transduction across various brain area domains is composed, institute Stating brain area domain includes motor cortex (Fig. 7 A), brain (body-sensing) cortex (Fig. 7 B), hippocampus (Fig. 7 C), dentate fascia (Fig. 7 D), thalamus (Fig. 7 E), hypothalamus (Fig. 7 F), corpus straitum (Fig. 7 G) and amygdaloid nucleus (Fig. 7 H).Scale bar=100 μm.

Fig. 8 A-H. is for the neuron of parent and chimeric capsid variant and the quantitative comparison of neuroglia levels of transduction.With 5x10¹¹The dosage of vg was by tail vein injection, by 3 weeks after the vector administration for packing CBh-scGFP transgenosis Relative neurons Levels of transduction is quantified by following: count for every 50 microns of coronal sections each brain area domain transduction neuron and Neuroglial quantity is based on Morphological Identification.Show parent serotypes A AV1 and AAVrh.10 and chimeric variant AAV1R6 and AAV1R7 passes through the transduction of each region of brain, and each region of the brain includes motor cortex (Fig. 8 A), brain (body-sensing) cortex (Fig. 8 B), hippocampus (Fig. 8 C), dentate fascia (Fig. 8 D), thalamus (Fig. 8 E), hypothalamus (Fig. 8 F), corpus straitum (Fig. 8 G) With amygdaloid nucleus (Fig. 8 H).Error bars indicate standard deviation (n > or=3).Use the not double tail T inspections in pairs corrected with Welch The significance,statistical that every group of neuron and neuroglia relative to AAV1 is transduceed is proved with unidirectional ANOVA, and is proved Difference between average value is respectively provided with significance,statistical.Ns, it is non-limiting；*, P < 0.05.

The structural analysis of Fig. 9 A-F.AAV1R6 chimeric capsid variant.The sequence of (Fig. 9 A) AAV1R6 and AAV1&AAVrh.10 Comparison is highlighted unique amino acid residue (AAV1R6 number) from AAVrh.10 and (is shown with black letters and prominent with blue Display).Conserved residues are shown with red letters, and are highlighted with yellow, and the non-conservative equivalent ones on AAV1 are with black Or green text is shown, and is highlighted with green or white.The three-dimensional structure mould of chimeric AAV1R6VP3 subunit (Fig. 9 B) monomer Type is generated using SWISS-MODEL, and wherein the crystal structure of AAV8 is used as the template of homologous modeling (PDB ID:2QAO). Tripolymer dimer/2 times AAV1R6VP3 subunit (Fig. 9 C) symmetry axis, (Fig. 9 D) tripolymer/three times symmetry axis, (Fig. 9 E) five are poly- The surface model that the surface of complete AAV1R6 capsid (60 aggressiveness) is presented in body/5 times symmetry axis and (Fig. 9 F) passes through VIPERdb oligomerization Body generator generates, and is visualized using PyMol.Residue from AAV1 is originated from the residual of AAVrh.10 with gray colored Base is gathered in the base portion of the protrusion of 3 times of symmetry axis, is with dark green chromatic colorant at the recess and 5 times of holes at 2 times of symmetry axis.

Figure 10 A-B. is compared with parent's capsid, the relative heart and liver transduction of AAV1R6 and AAV1R7.Pass through tail vein Injection is with 5 × 10¹¹The chimeric of CBh-scGFP transgenosis is packed in dosage application parent (AAV1 or AAVrh.10) of vg (AAV1R6 or AAV1R7) carrier.After injection 21 days, GFP immunostaining is carried out to heart and liver section and is imaged.Figure In the mouse that 10A. PBS (analogies), AAV1, AAVrh.10, AAV1R6 or AAV1R7 are handled, heart (above) and liver The GFP fluorescence of (following figure) tissue.Figure 10 B. handles every kind, by spreading the relatively glimmering of multiple images captured by quantization Light, the levels of transduction of measured heart (top) and liver (bottom).This indicates the range from minimum to peak, in Heart line indicates to spread the average value of sample.Relative fluorescence is normalized to the tissue of analogies processing.Error bars indicate standard deviation Poor (n=3).For every group, carries out unidirectional ANOVA and azygous double tail T with Welch correction are examined, and show opposite In the conspicuousness of AAVrh.10.Ns, it is non-limiting.*, P < 0.05.

Figure 11 A-H. is used for the rational design of the minimum AAVrh.10 footprint across BBB and function maps (functional mapping).The sequence alignment of Figure 11 A AAV1RX and AAV1 and AAVrh.10 show within neurophilic footprint and near Number amino acid residue.Conserved residues are highlighted with yellow, and the residue for constituting footprint is highlighted with cyan, non-conservative residual Base is highlighted with green or white.Use the structure of the engineered AAV1RX chimeric capsid of SWISS-MODEL Software Create Model is used for the modeling based on homology, and generates oligomer using VIPERdb.PyMol is sub- for generating AAV1RX VP3 Base monomer (Figure 11 B), tripolymer dimer/2 times symmetry axis (Figure 11 C), tripolymer/three times symmetry axis (Figure 11 D), pentamer/5 The model on the presentation surface of times symmetry axis (Figure 11 E) and complete capsids (60 aggressiveness) (Figure 11 F).Amino acid from AAV1 and Between AAV1 and AAVrh.10 homologous amino acid is indicated with grey, and include from AAVrh.10 for across BBB to The residue of neural footprint is indicated with green cyan.Figure 11 C. solid route map Projection Display is on AAV1RX capsid along triple right Claim that observes under axial direction to generate to neural footprint, and using RIVEM.The only amino acid residue of display surface exposure, respectively Between boundary described with black line.Green area depicts the topological protrusion on three-fold symmetry axis, and blue region representative is opened up Flutter recess.The critical amino acid residues of the footprint are restained as magenta (AAVrh.10VP1 number).Figure 11 H. packs CBh- The CNS of the AAV1RX of scGFP transgenosis, which transduces, to be composed, wherein the AAV1RX is with 5 × 10¹¹.vg dosage passes through tail vein injection Application.21 days after injection slices are subjected to immunostaining and are imaged.Scale bar=100 μm.

The peripheral tissues' transduction and bio distribution of AAV1R6, AAV1R7 and AAV1RX after Figure 12 A-H. is intravenously applied.With PBS or pack AAV1, AAVrh.10, AAV1R6, AAV1R7 or AAV1RX carrier of CBA-Luc reporter gene transgenosis with 1 × 10¹¹The dosage of vg passes through Tail Vein injection Mouse.2 weeks after injection, quantitative fluorescence element enzyme reporter gene transgene expression level The bio distribution (Figure 12 B, 12D, 12F, 12H) of (Figure 12 A, 12C, 12E, 12G) and viral genome copies in various tissues. Luciferase expression levels are normalized to the grams of Tissue Lysates, and are expressed as relative light unit.By vector gene group (vg) copy is normalized to the mouse lamin B (mLamB) as endogenous house-keeping gene, and is expressed as the vg of each cell Copy.Error bars represent standard deviation (for luciferase assay, n=3；For bio distribution, n=4).For every group, into The unidirectional ANOVA of row and the azygous double tail T corrected with Welch are examined, and show the conspicuousness relative to AAVrh.10. Ns, it is non-limiting.*, P < 0.05.

Figure 13 A-H. compared with parent and other chimeric capsid variants, transduce by the neuron and neuroglia that AAV1RX is mediated Horizontal quantifies.Mouse is 5 × 10 by tail vein injection acceptable dose¹¹The parent (AAV1 or AAVrh.10) of vg or packaging The chimeric vector of CBh-scGFP transgenosis.3 weeks after injection, brain is taken, is sliced, GFP immunostaining and is imaged.To opposite Neuron and neuroglia levels of transduction carry out GFP immunostaining and are imaged.Relative neuron and nerve are quantified by following Colloid levels of transduction: counting the transduction neuron for being directed to each region of every 50 μm of coronal sections and neuroglial quantity, Based on Morphological Identification.Show AAV1RX and parent's serotypes A AV1 and AAVrh.10 and chimeric variant AAV1R6 and The levels of transduction across each region of AAV1R7, the region include motor cortex (Figure 13 A), cerebral cortex (Figure 13 B), sea Horse (Figure 13 C), dentate fascia (Figure 13 D), thalamus (Figure 13 E), hypothalamus (Figure 13 F), corpus straitum (Figure 13 G) and amygdaloid nucleus (figure 13H).Error bars indicate standard deviation (n=3).Every group of phase is proved using examining with unpaired double tail T that Welch is corrected For the significance,statistical of AAV1.Ns: statistics is non-limiting；*, P < 0.05.Also average value is proved using unidirectional ANOVA Between difference be statistically significant.Neuron shown in this article across all brain area domains and neuroglia are turned It leads, *, P < 0.05.

Figure 14 is throughout the 1RX footprint of common AAV serotype and the sequence alignment of variable region I (VR-1).In variable region I (VR-1) it in relatively overall situation, is compared with common natural A AV serotype, the sequence of AAV1RX and common AAV serotype Compare the amino acid residue (VP1 number) for highlighting 1RX footprint.Above and below black line label 1RX footprint in it is specific Residue.Crucial: the identical residue throughout serotype is indicated with the red letters in yellow background；Conserved residues are carried on the back with cyan Blue letters on scape indicate；Similar residue is indicated with the black letters in green background；Weak Similar residues are carried on the back with white Green letter on scape indicates；Dissimilar residue, is indicated with the black letters in white background.The sequence alignment uses Vector NTI Advance 11.5.2 software generates.

The sequence alignment of Figure 15 .AAV1, AAV1R7 and AAVrh.10VP1 capsid protein.

Detailed description of the invention

Unless otherwise defined, otherwise all technical and scientific terms used herein have it is general with disclosure fields The logical identical meaning of the normally understood meaning of technical staff.Technology used herein is only used for the mesh of description specific embodiment , rather than it is restrictive.All publications, patent application, patent and other bibliography being mentioned above are with reference Mode is hereby incorporated by reference in its entirety.

Definition

Following term uses in the description herein and appended claims:

Unless the context is clearly stated, otherwise singular " one ", "one" and " described " are intended to also include plural number Form.

Further, when the amount for the length for referring to polynucleotides or polypeptide sequence, dosage, time, temperature etc. can be surveyed When magnitude, the term as used herein " about " mean to cover ± the 20% of specific quantity, ± 10%, ± 5%, ± 1%, ± 0.5% or Even ± 0.1% variation.

Same as used herein, "and/or" refers to and covers any and institute of one or more related listed items Possible combination, and what is combined when explaining in alternative ("or") lack.

As used herein, transition phrase " substantially by ... form " refers to that the scope of the claims should be interpreted to contain Documented designated substance or step in lid claim, " and those do not influence claimed invention substantiallySubstantially 'sWithIt is novelThose of feature ".Referring to 461,463 (CCPA of In re Herz, 537F.2d 549,551-52,190USPQ 1976) (in the Special attention will be given to of original text)；See also MPEP § 2111.03.Therefore, when in the claims herein in use, art Language " substantially by ... form " is not intended to be interpreted to be equal to "comprising".

Unless otherwise indicated by context, otherwise it especially refers to capsid protein, carrier, composition and side as described herein The various features of method can be in any combination.

In addition, the disclosure is it is also contemplated that (contemplate) in some embodiments, can be excluded or be omitted and is described in this paper Any feature or feature combination.

In order to further illustrate, for example, if specification points out that specific amino acids can be selected from A, G, I, L and/or V, This sentence also shows that amino acid can be selected from any subset of these amino acid, (such as A, G, I or L；A, G, I or V；A or G； Only L；Deng), as each such sub-portfolio is expressly recited herein.In addition, also show can be with the waiver of premium for such sentence Protect one or more specified amino acid.For example, in certain embodiments, amino acid is not A, G or I；It is not A；No It is G or V；Deng if possible as each abandon being expressly recited herein.

As used herein, term " reduction " and similar terms mean relative to control or referring at least about 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more decline.

As used herein, term " increase ", " enhancing " and similar terms indicate relative to control or referring at least about 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more increase or increasing By force.

Term " parvovirus " as used herein covers Parvoviridae, including the parvovirus independently replicated and dependence Virus.Autonomous parvovirus includes parvovirus category, red Tobamovirus, densovirus (Densovirus) category, Ai Tela virus (Iteravirus) belong to the member belonged to Kang Tela viral (Contravirus).Exemplary autonomous parvovirus includes but unlimited In piconavirus, bovine parvovirus, canine parvovirus, chicken parvovirus, Feline Panleukopenia Virus, the cat of mouse are thin Small virus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, B19 virus and times that be currently known or being later discovered that What his autonomous parvovirus.Other autonomous parvovirus are known to the skilled in the art.See, e.g., BERNARD N.FIELDS et al., VIROLOGY, volume 2, the 69th chapter (the 4th edition, Lippincott-Raven is published).

As used herein, term " adeno-associated virus " (AAV) includes but is not limited to 1 type of AAV, 2 type of AAV, 3 type of AAV (including 3A and 3B type), 4 type of AAV, 5 type of AAV, 6 type of AAV, 7 type of AAV, 8 type of AAV, 9 type of AAV, 10 type of AAV, AAV Rh10 type, 11 type of AAV, 12 type of AAV, fowl AAV, ox AAV, dog AAV, horse AAV, sheep AAV and currently known or quilt later It was found that any other AAV.See, e.g., BERNARD N.FIELDS et al., VIROLOGY, 2nd volume, the 69th chapter (the 4th edition, Lippincott-Raven is published).Many AAV serotypes and clade have been identified (see, e.g., Gao et al. (2004) J.Virology 78:6381-6388；Moris et al. (2004) Virology 33-:375-383；With table 1).

The genome sequence of the AAV of various serotypes and autonomous parvovirus, and natural terminal repeat (TR), The sequence of Rep albumen and capsid subunits is known in the art.Such sequence can be in the literature or such as It is found in the public database of database.See, e.g., GenBank accession number NC_044927, NC_002077, NC_ 001401、NC_001729、NC_001863、NC_001829、NC_001862、NC_000883、NC_001701、NC_001510、 NC_006152、NC_006261、AF063497、U89790、AF043303、AF028705、AF028704、J02275、J01901、 J02275、X01457、AF288061、AH009962、AY028226、AY028223、NC_001358、NC_001540、 AF513851,AF513852,AY530579；It, which is published in this article, is incorporated herein by the following way for instructing parvovirus and AAV Nucleic acid and amino acid sequence.It sees also, for example, Srivistava et al. (1983) J.Virology 45:555；Chiorini etc. People (1998) J.Virology 71:6823；Chiorini et al. (1999) J.Virology 73:1309；Bantel-Schaal Et al. (1999) J.Virology 73:939；Xiao et al. (1999) J.Virology 73:3994；Muramatsu et al. (1996)Virology 221:208；Shade et al. (1986) J.Virol.58:921；Gao et al. (2002) Proc.Nat.Acad.Sci.USA 99:11854；Moris et al. (2004) Virology 33-:375-383；International monopoly is public Open WO 00/28061, WO 99/61601, WO 98/11244；With United States Patent (USP) No.6,156,303；It is published in this article logical Cross the nucleic acid and amino acid sequence being incorporated by for instructing parvovirus and AAV.See also table 1.

The capsid structure of autonomous parvovirus and AAV are in BERNARD N.FIELDS et al., VIROLOGY, 2nd volume, and the 69th It is described in more detail in 70 chapters (the 4th edition, Lippincott-Raven is published).It sees also, AAV2 (Xie et al. (2002) ), Proc.Nat.Acad.Sci.99:10405-10 AAV4 (Padron et al. (2005) J.Virol.79:5047-58), AAV5 (Walters et al. (2004) J.Virol.78:3361-71) and CPV (Xie et al. (1996) J.Mol.Biol.6:497-520； Tsao et al. (1991) Science 251:1456-64；Drouin et al. (2013) Future Virol.8 (12): 1183- 1199) description of crystal structure.

Term " taxis " as used herein refers to that virus preferentially enters certain cell or tissues, optionally in table later The sequence carried up to (for example, transcription and optionally translation) by viral genome in cell, for example, for recombinant virus, expression Interested heterologous nucleic acids.It will be appreciated by those skilled in the art that in some embodiments, trans-acting factor is being not present In the case of, the transcription from virus genomic heterologous nucleic acid sequence not will start, for example, for inducible promoter or other The nucleic acid sequence being conditioned.In the case where recombinating AAV (rAAV) genome, can come from virus genomic gene expression Self-stabilization integration provirus and/or come from nonconformable episome, and virus can take in the cell it is any its His form.

As used herein, " systemic taxis " and " whole body F-duction " (and equivalent terms) shows the viral clothing of the disclosure Shell or viral vectors are shown respectively for body tissue's (for example, brain, lung, skeletal muscle, heart, liver, kidney and/or pancreas) Taxis and/or transduction.In embodiments, the whole body transduction of central nervous system (such as brain, nerve cell etc.) is observed It arrives.In other embodiments, the whole body transduction of cardiac muscular tissue is implemented.

Unless otherwise stated, " effective transduction " or " effective taxis " or similar terms, it can be by reference to closing Suitable control (for example, be respectively control at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more transduction or taxis) come It determines.In specific embodiments, viral vectors is effectively transduceed for neuronal cell and cardiac muscle cell or is had effective Taxis.Suitable control will depend on many factors, and the factor includes required taxis spectrum.

Similarly, by reference to suitably compareing, virus can be determined for target tissue " cannot effectively transduce " or " no With effective taxis " or similar terms.In certain embodiments, viral vectors for liver, kidney, sexual gland and/ Or reproduction cell cannot effectively transduce (that is, not having effective taxis).In specific embodiments, with desired target tissue (for example, the cell of central nervous system；Cardiac muscle cell) levels of transduction compare, tissue (such as liver) undesirable transduction be 20% or lower, 10% or lower, 5% or lower, 1% or lower, 0.1% or lower.

Unless otherwise indicated, the term as used herein " polypeptide " covers both peptide and protein.

" polynucleotides " are the sequence of nucleotide base, and can be RNA, DNA or DNA-RNA heterozygous sequence (including Naturally occurring and non-naturally occurring nucleotide), but in representative embodiment it is single-stranded or double-stranded DNA sequence dna.

As used herein, " separation " polynucleotides (for example, " isolated DNA " or " isolated RNA ") mean at least portion Point ground with naturally occurring organism or virus at least some other components (such as, generally it is found that it is related to polynucleotides The cell or virus structure component of connection or or other polypeptides or nucleic acid) separation polynucleotides.In representative embodiment, with Initial substance is compared, " separation " nucleic acid molecules enrichment at least about 10 times, 100 times, 1000 times, 10, and 000 times or more.

Equally, " separation " polypeptide mean at least partly with naturally occurring organism or virus it is at least some other Component (such as, generally it is found that cell associated with polypeptide or virus structure component or or other polypeptides or nucleic acid) separation Polypeptide.In representative embodiment, compared with initial substance, " separation " polypeptide be enriched to few about 10 times, 100 times, 1000 times, 10,000 times or more.

As used herein, pass through " separation " or " purifying " (or grammatical equivalents) viral vectors, it is intended that viral vectors is at least Partly separated at least some of initial substance other components.In representative embodiment, compared with initial substance, " separation " or " purifying " viral vectors is enriched to about 10 times, 100 times, 1000 times, 10,000 times or more few.

As used herein, " neuronal cell " includes sensory neuron, pseudounipolar neuron, motor neuron, multipole mind Through member, intrerneuron and/or be located at brain substructure (such as cortex, motor cortex, hippocampus, hypothalamus, corpus straitum, substrate mind Warp knuckle, amygdaloid nucleus, cerebellum, dorsal root ganglion and/or spinal cord) in bipolar neuron.

" therapeutic protein " is the egg that can reduce, reduce, prevent, postpone and/or stablize by cell or subject The protein of symptom caused by the shortage or defect of white matter, and/or be the other protein for assigning subject's benefit.

As used herein " therapeutic RNA molecule " or " functional RNA molecule " can be antisense nucleic acid, ribozyme (for example, Such as United States Patent (USP) No.5, described in 877,022), influence the trans-splicing that spliceosome mediates RNA (referring to, Puttaraju et al. (1999) Nature Biotech.17:246；United States Patent (USP) No.6,013,487；United States Patent (USP) No.6, 083,702), the RNA interfering including siRNA, shRNA or miRNA (RNAi) (its mediated gene silencing) is (referring to Sharp etc. People, (2000) Science 287:2431) and any other untranslatable rna (Gorman et al. for such as " instructing " RNA (1998)Proc.Nat.Acad.Sci.USA 95:4929；The United States Patent (USP) No.5 of Yuan et al., 869,248) etc., such as ability Known to domain.

Term " treatment " (and its grammatical variants) means that the severity of subject's patient's condition reduces, and is at least partially improved Or stablize, and/or realize some alleviations, mitigation, reduction or the stabilization of at least one clinical symptoms, and/or there are disease or diseases The delay of the progress of disease.

Term " prevention " (and its grammatical variants) refers to, will occur relative in the case where disclosed method is not present , prevent and/or postpone the breaking-out of the disease, illness and/or clinical symptoms of subject and/or reduces disease, illness and/or face The seriousness of the breaking-out of bed symptom.Prevention can be completely, such as absolutely not disease, illness and/or clinical symptoms.Prevention It is also possible to part, so that the severity of the disease of subject, the generation of illness and/or clinical symptoms and/or breaking-out is small In when there is no the disclosure the case where.

" treatment is effective " amount is to be enough to provide the amount of some improvement or benefit for subject as used herein.In other words, " treatment effective " amount is to provide some alleviate, mitigate, reducing and/or stable at least one clinical symptoms of subject Amount.It will be appreciated by those skilled in the art that therapeutic effect needs not be complete or healing, as long as providing some benefits to subject Place.

" prevention effective " used herein amount is, relative in the case where disclosed method is not present by generation, It is enough to prevent and/or postpone the disease, illness and/or onset of clinical symptoms of subject, and/or reduces and/or postpone disease, disease The amount of the severity of disease and/or onset of clinical symptoms.It will be appreciated by those skilled in the art that prevention level needs not be complete , as long as providing some benefits to subject.

Term " heterologous nucleotide sequence " and " exogenous nucleic acid molecule " are used interchangeably herein, and refer to non-natural The nucleic acid molecules and/or nucleotide sequence being present in virus.In general, exogenous nucleic acid molecule or nucleotide sequence include that opening is read Frame, the opening code-reading frame proteins of interest matter or untranslatable rna (for example, for delivery to cell or subject).

As used herein, term " viral vectors ", " carrier " or " gene delivery vector " refers to as nucleic acid delivery vector Virus (for example, AAV) particle to work, it includes be packaged in the intracorporal vector gene group of virus (for example, viral DNA [vDNA]).Alternatively, in some cases, term " carrier " can be used for referring to individual vector gene group/vDNA.

" rAAV vector gene group " or " rAAV genome " are the recombination AAV comprising one or more heterologous nucleic acid sequences Genome (i.e. vDNA).RAAV carrier is usually only necessary to (one or more) cis- terminal repeat (TR) to generate disease Poison.Every other virus sequence is all non-essential and can be with trans- offer (Muzyczka (1992) Curr.Topics Microbiol.Immunol.158:97).In general, rAAV vector gene group only retains one or more TR sequences, so as to most Bigization can be by the size for the transgenosis that carrier is effectively packed.Structure and non-structural protein coded sequence trans- can provide (example Such as, from carrier, such as plasmid, and/or pass through the sequence stable integration into incasing cells).In embodiments, rAAV is carried Body genome includes that at least one end repeats (TR) sequence (for example, AAV TR sequence), and optionally two TR are (for example, two AAV TR), would generally be located at and the vector gene end group 5' and 3' and be flanked with the heterologous nucleic acid sequence, but do not need and its It is adjacent.TR can be the same or different from each other.

Term " end repetition " or " TR " include forming hairpin structure and playing the role of inverted terminal repeat Any virus terminal of (that is, mediating required function, such as duplication, virus packaging, integration and/or provirus rescue etc.) repeats Sequence or composition sequence.TR can be AAV TR or non-AAV TR.For example, non-AAV TR sequence, such as other parvovirus (examples Such as, canine parvovirus (CPV), minute parvovirus of mice (MVM), human parvovirus B-19) sequence or any other suitable disease Malicious sequence (for example, the SV40 hair clip for being used as SV40 replication orgin), may be used as TR, and the TR can be by truncation, displacement, scarce It loses, be inserted into and/or add and further modify.In addition, TR can be it is partly or completely fully synthetic, such as Samulski's et al. " double D sequences " described in United States Patent (USP) No.5,478,745.

" AAV terminal repeat " or " AAV TR " can come from any AAV, including but not limited to serotype 1,2,3,4, 5,6,7,8,9,10,11,12 or currently known or any other AAV for being later discovered that (see, for example, table 1).The end AAV repeats Sequence does not need have natural terminal repeat (for example, natural A AV TR sequence can be by insertion, missing, displacement, truncation And/or missense mutation changes), as long as terminal repeat mediates required function, for example, duplication, virus packaging, integration and/ Or provirus rescue etc..

Viral vectors of the invention can also be " targeting " viral vectors (for example, have orientation taxis) and/or " miscellaneous Close " parvovirus (that is, wherein virus TR and viral capsid are from different parvovirus), such as International Patent Publication WO 00/ Described in 28004 and Chao et al. (2000) Molecular Therapy 2:619.

The viral vectors of the disclosure can also be as International Patent Publication WO 01/92551 (quote with it by its open passes through Be integrally incorporated herein) described in duplex parvovirus particle.Therefore, in some embodiments, double-strand (duplex) base Because group can be packaged into the viral capsid of the disclosure.

In addition, viral capsid or genomic elements may include other modifications, the modification includes insertion, lacks and/or set It changes.

As used herein, term " amino acid " covers any naturally occurring amino acid, modified forms and synthesizing amino Acid.

Naturally occurring left-handed (L-) amino acid is shown in table 3.

In addition, non-naturally occurring amino acid can be Wang et al. Annu Rev Biophys Biomol " non-natural " amino acid described in Struct.35:225-49 (2006).These unnatural amino acids be advantageously used for by Interested molecular chemistry is connected to AAV capsid protein.

Modified AAV capsid protein and viral capsid and viral vectors comprising it.

Present disclose provides include mutation (that is, modification, can be displacement or insertion or missing) in amino acid sequence AAV capsid protein, and viral capsid and viral vectors comprising modified AAV capsid protein.The AAV capsid egg of mutation It is white not find (that is, being non-natural) in nature with nucleic acid that is encoding them, and both do not have and find in nature Wild-type sequence sequence, also do not have these sequences function.On the contrary, the inventor has discovered that amino described herein Characteristic needed for the modification of sour position can assign the one or more of the viral vectors comprising modified AAV capsid protein, institute Need the characteristic to include but is not limited to: after (i) systemic injection after passing through blood-brain barrier neuronal cell selection F-duction；(ii) complete Heart tissue and nerve fiber while, transduce after body injection；(iii) from liver, spleen, kidney and other peripheral organs Targeting.

In specific embodiments, the modified AAV capsid protein of the disclosure includes natural A AV1 capsid protein One or more mutation in amino acid sequence or in the corresponding amino acid residue of the capsid protein from another AAV serotype (modifying), including but not limited to AAV2, AAV3, AAV6, AAV7, AAV8, AAV9 and AAVrh.10." corresponding to " natural A AV1 Other AAV serotypes of these positions in capsid protein or amino acid position in modified AAV capsid are for this field It is it will be apparent that and sequence alignment technology can be used (see, for example, the figure of WO 2006/066066 for technical staff 7) and/or crystal structure analysis (Padron et al. (2005) J.Virol.79:5047-58) is readily determined.

It will be appreciated by those skilled in the art that corresponding modification can be insertion and/or replace for some AAV capsid proteins, Whether this depends on whether corresponding amino acid position is partially or completely present in virus, or be completely absent.Similarly, When modified AAV rather than when AAV1, specific amino acids position can be different from the position in AAV1 (being numbered using VP1).As herein Discussed elsewhere, using well known technology, corresponding amino acid position be to those skilled in the art it is aobvious and It is clear to.

As used herein, " mutation " or " modification " in amino acid sequence includes displacement, insertion and/or missing, and every kind can It is related to one, two, three, four, five, six, seven, eight, nine, ten or more amino acid.In specific reality It applies in scheme, the modification is displacement.In other embodiments, the modification is (for example, two amino in amino acid sequence Single amino acids residue between sour residue) insertion.

Therefore, in one embodiment, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV clothing Glutelin include it is following, be substantially made up of or be made up of: amino acid residue S262, A263, S264, T265, Modification at A267 and S268, and the single amino acids residue insertion between residue 266 and 267 (VP1 number), wherein each The number of residue is based on the amino acid sequence or AAV2 (SEQ ID NO:2), AAV3 (SEQ ID of AAV1 (SEQ ID NO:1) NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9(SEQ ID NO:7) Or the equivalent amino acid residue in AAVrh10 (SEQ ID NO:8).In some embodiments, AAV capsid protein may include Modification at amino acid residue H272.In specific embodiments, modification be S262N, A263G, S264T, T265S, A267S, S268T, H272T and combinations thereof.In specific embodiments, the single amino acids residue between residue 266 and 267, which is inserted into, is G (is appointed as -267G).

In some embodiments, AVV capsid protein of the invention can further include it is following, substantially by with the following group At or be made up of: in amino acid residue Q148, E152, S157, T162, H272, T326, D328, V330, T331, V341 It is inserted into the modification at S345 and the single amino acids residue between residue 152 and 153 (VP1 number), wherein each residue Amino acid sequence or SEQ ID NO:2,3,4,5,6,7 or 8 of the number based on SEQ ID NO:1 in equivalent amino acid it is residual Base.In specific embodiments, modification be Q148P, E152R, S157T, T162K, H272T, T326Q, D328E, V330T, T331K, V341I and S345T.In specific embodiments, the single amino acids residue insertion between residue 152 and 153 is S (being appointed as -153S).

In some embodiments, above-described AAV capsid protein can further include it is following, substantially by following It forms or is made up of: the modification of amino acid residue L188, S205, N223, A224 and H272 (VP1 number), wherein each Equivalent amino acid in amino acid sequence or SEQ ID NO:2,3,4,5,6,7 or 8 of the number of residue based on SEQ ID NO:1 Residue.In specific embodiments, modification is L188I, S205A, N223S, A224S and H272T.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white comprising it is following, be substantially made up of or be made up of: in amino acid residue S262, A263, S264, T265 and A267 Modification at (VP1 number), and the single amino acids residue insertion between residue S268 and N269, wherein the volume of each residue Number be based on AAV1 (SEQ ID NO:1) amino acid sequence or AAV2 (SEQ ID NO:2), AAV3 (SEQ ID NO:3), AAV6 (SEQ ID NO:4), AAV7 (SEQ ID NO:5), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 Equivalent amino acid residue in (SEQ ID NO:8).In some embodiments, modification be S262N, A263G, S264T, At least one of T265S and A267S.In some embodiments, AAV capsid can further include in amino acid residue H272 The modification at place, it is consisting essentially of or be made from it.In some embodiments, AAV capsid protein can further include with Under, be substantially made up of or be made up of: amino acid residue Q148, E152, S157, T162, H272, T326, Modification at D328, V330, T331, V341 and S345, and the single amino acids residue between residue E152 and P153 are slotting Enter.In some embodiments, AAV capsid protein be further contained in amino acid residue L188, S205, N223, A224 and Modification at H272.In some embodiments, modification is at least one in L188I, S205A, N223S, A224S and H272T It is a.In some embodiments, the amino acid residue insertion between residue S268 and N269 is the insertion of single T residue.In In some embodiments, the amino acid residue insertion between residue E152 and P153 is the insertion of single S residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein AAV capsid protein packet Containing it is following, be substantially made up of or be made up of: generate the modification of following amino acid sequence: corresponding to natural A AV1 X at the amino acid of the amino acid position 262 to 265 (VP1 number) of capsid protein (SEQ ID NO:1)¹-X²-X³-X⁴(SEQ ID NO:35), wherein X¹It is the arbitrary amino acid in addition to S；Wherein X²It is the arbitrary amino acid in addition to A；Wherein X³S with Outer arbitrary amino acid；Wherein X⁴It is the arbitrary amino acid other than T.45. in some embodiments, amino acid X¹It is N, amino Sour X²It is G, amino acid X³It is T or amino acid X⁴It is S.In some embodiments, amino acid X¹It is N, amino acid X²It is G, amino Sour X³It is T, amino acid X⁴It is S.In some embodiments, AAV capsid protein further includes repairing for amino acid residue H272 Decorations.In some embodiments, modification is H272T.In some embodiments, AAV capsid protein further includes amino acid Amino acid residue insertion between residue S268 and N269.In some embodiments, amino acid residue insertion is single T residue Insertion.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white comprising it is following, be substantially made up of or be made up of: in amino acid residue S262, A263, S264, T265, A267 With the modification at H272, and the single amino acids residue insertion between residue S268 and N269, (VP1 number), wherein each The number of residue is based on the amino acid sequence or AAV1 (SEQ ID NO:1), AAV3 (SEQ ID of AAV1 (SEQ ID NO:2) NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9(SEQ ID NO:7) Or the equivalent amino acid residue in AAVrh10 (SEQ ID NO:8).In some embodiments, modification include S262N, At least one of A263G, S264T, T265S, A267G and H272T.In some embodiments, in residue S268 and N269 Between natural single amino acids residue insertion be single T residue insertion.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white comprising it is following, be substantially made up of or be made up of: in amino acid residue S262, Q263, S264, A266, S267 With the modification at H271, and the insertion of at least one amino acid residue between residue S261 and S262, (VP1 number), In each residue number be based on AAV2 (SEQ ID NO:2) amino acid sequence or AAV1 (SEQ ID NO:1), AAV3 (SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8(SEQ ID NO:6)、AAV9(SEQ ID ) or the equivalent amino acid residue in AAVrh10 (SEQ ID NO:8) NO:7.In some embodiments, modification include S262T, At least one of Q263S, S264G, A266S, S267T and H271T.In some embodiments, in residue SS61 and S262 Between insertion be single amino acids residue insertion.In some embodiments, the insertion between residue S251 and S252 It is the insertion of more than one amino acid residue.In some embodiments, the insertion between residue S251 and S252 is N and G The insertion of residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white comprising it is following, be substantially made up of or be made up of: amino acid residue S262, Q263, S264, A266, A267, Modification at H271, and the single amino acids residue insertion between residue S261 and S262, wherein the number base of each residue In amino acid sequence or AAV1 (SEQ ID NO:1), the AAV2 (SEQ ID NO:2), AAV6 (SEQ of AAV3 (SEQ ID NO:3) ID NO:4), AAV7 (SEQ ID NO:5), AAV8 (SEQ ID NO:6), AAV9 (SEQ ID NO:7) or AAVrh10 (SEQ ID NO:8) in equivalent amino acid residue.In some embodiments, modification include S262T, Q263S, S264G, A266S, At least one of A267T, H271T.In some embodiments, the insertion between residue SS61 and S262 is single amino The insertion of sour residue or more than one amino acid residue.In some embodiments, the insertion between residue S251 and S252 It is the insertion of N and G residue.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein AAV capsid protein packet Containing it is following, be substantially made up of or be made up of: repairing at amino acid residue S263, S269, A237 (VP1 number) Decorations, wherein the number of each residue be based on AAV9 (SEQ ID NO:9) amino acid sequence or AAV1 (SEQ ID NO:1), AAV2(SEQ ID NO:2)、AAV3(SEQ ID NO:3)、AAV6(SEQ ID NO:4)、AAV7(SEQ ID NO:5)、AAV8 Equivalent amino acid residue in (SEQ ID NO:6) or AAVrh10 (SEQ ID NO:8).In some embodiments, modification packet Include at least one of S263G, S269T and A273T.

In some embodiments, the disclosure provides adeno-associated virus (AAV) capsid protein, wherein the AAV capsid egg It is white comprising it is following, be substantially made up of or be made up of: the sequence of any of SEQ ID NO:9 to SEQ ID NO:34 Column.

The non-limiting reality of the modification of the capsid protein of the disclosure is generated in AAV serotype 1,2,3,6,7,8 and 9 respectively Example is shown in table 2, wherein the equivalent amino acid residue in each AAV serotype is identified.

In a further embodiment, the capsid protein of the disclosure may include following, it is substantially consisting of the following or by Following compositions: the amino acid sequence (AAV1RX) of SEQ ID NO:9；The amino acid sequence (AAV2RX) of SEQ ID NO:10；SEQ The amino acid sequence (AAV3RX) of ID NO:11；The amino acid sequence (AAV6RX) of SEQ ID NO:12；SEQ ID NO:13's Amino acid sequence (AAV7RX)；The amino acid sequence (AAV8RX) of SEQ ID NO:14；The amino acid sequence of SEQ ID NO:15 (AAV9RX)；The amino acid sequence (AAV1R6) of SEQ ID NO:16；The amino acid sequence (AAV2R6) of SEQ ID NO:17； The amino acid sequence (AAV3R6) of SEQ ID NO:18；The amino acid sequence (AAV6R6) of SEQ ID NO:19；SEQ ID NO: 20 amino acid sequence (AAV7R6)；The amino acid sequence (AAV8R6) of SEQ ID NO:21；The amino acid of SEQ ID NO:22 Sequence (AAV9R6)；The amino acid sequence (AAV1R7) of SEQ ID NO:23；The amino acid sequence of SEQ ID NO:24 (AAV2R7)；The amino acid sequence (AAV3R7) of SEQ ID NO:25；The amino acid sequence (AAV6R7) of SEQ ID NO:26； The amino acid sequence (AAV7R7) of SEQ ID NO:27；The amino acid sequence (AAV8R7) of SEQ ID NO:28；With SEQ ID The amino acid sequence (AAV9R7) of NO:29.In one embodiment, the capsid protein of the disclosure is with SEQ ID NO:9's Amino acid sequence (AAV1RX).In one embodiment, the capsid protein of the disclosure has the amino acid of SEQ ID NO:16 Sequence (AAV1R6).In one embodiment, the capsid protein of the disclosure has the amino acid sequence of SEQ ID NO:23 (AAV1R7)。

In some embodiments, present disclose provides AAV capsid protein, and have at least 80% below, at least 85%, at least 90%, at least 95%, at least 99%, including therebetween all ranges and subrange, sequence identity: SEQ The amino acid sequence (AAV1RX) of ID NO:9；The amino acid sequence (AAV2RX) of SEQ ID NO:10；The ammonia of SEQ ID NO:11 Base acid sequence (AAV3RX)；The amino acid sequence (AAV6RX) of SEQ ID NO:12；The amino acid sequence of SEQ ID NO:13 (AAV7RX)；The amino acid sequence (AAV8RX) of SEQ ID NO:14；With the amino acid sequence (AAV9RX) of SEQ ID NO:15.

In some embodiments, present disclose provides AAV capsid protein, and have at least 80% below, at least 85%, at least 90%, at least 95%, at least 99%, including therebetween all ranges and subrange, sequence identity: SEQ The amino acid sequence (AAV1R6) of ID NO:16；The amino acid sequence (AAV2R6) of SEQ ID NO:17；SEQ ID NO:18's Amino acid sequence (AAV3R6)；The amino acid sequence (AAV6R6) of SEQ ID NO:19；The amino acid sequence of SEQ ID NO:20 (AAV7R6)；The amino acid sequence (AAV8R6) of SEQ ID NO:21；The amino acid sequence (AAV9R6) of SEQ ID NO:22.

In some embodiments, present disclose provides AAV capsid proteins, have at least with amino acid sequence below 80%, at least 85%, at least 90%, at least 95%, at least 99%, including therebetween all ranges and subrange, sequence it is same One property: the amino acid sequence (AAV1R7) of SEQ ID NO:23；The amino acid sequence (AAV2R7) of SEQ ID NO:24；SEQ ID The amino acid sequence (AAV3R7) of NO:25；The amino acid sequence (AAV6R7) of SEQ ID NO:26；The amino of SEQ ID NO:27 Acid sequence (AAV7R7)；The amino acid sequence (AAV8R7) of SEQ ID NO:28；With the amino acid sequence of SEQ ID NO:29 (AAV9R7)。

The disclosure additionally provides adeno-associated virus (AAV) capsid protein, wherein AAV capsid protein in all positions or less than Include one or more displacements in any combination of all positions, generates amino acid sequence: X¹-X²-X³-X⁴(SEQ ID NO: 35).In some embodiments, the modification of amino acid sequence: X¹-X²-X³-X⁴It is positioned corresponding to natural A AV1 capsid protein At the amino acid of the amino acid position 262 to 265 (VP1 number) of (SEQ ID NO:1).In some embodiments, X¹It can be with It is any amino acid in addition to S.In some embodiments, X²It can be any amino acid in addition to A.In some implementations In scheme, X³It can be any amino acid in addition to S.In some embodiments, X⁴It can be any ammonia other than T Base acid.In one embodiment, amino acid X¹It is N.In one embodiment, amino acid X²It is G.In an embodiment In, amino acid X³It is T.In one embodiment, amino acid X⁴It is S.In another embodiment, X¹It is N, X²It is G, X³It is T and X⁴It is S.In some embodiments, X¹To X⁴One of be not replaced, and the amino acid residue at non-displacement position is wild Raw type amino acid residue.

The example that can be used for replacing the amino acid residue of the natural amino acid at each position described herein is listed in In table 3.

It should be understood that displacement and insertion described in the AAV capsid protein of the disclosure may include with conservative amino acid residues Displacement and/or insertion.This conservative substitution is well known in the art, and including such as nonpolar amino acid Gly, Ala, Val, Leu, Ile, Met, Phe, Trp and Pro can be replaced mutually；Polar amino acid Ser, Thr, Cys, Tyr, Asn and Gln can be set mutually It changes；Negatively charged amino acid Asp and Glu can be replaced mutually；Positively charged amino acid Lys, Arg and His can mutually be set It changes, with any combination.

The disclosure additionally provides the AAV capsid of the capsid protein comprising the disclosure, and the AAV capsid comprising the disclosure Viral vectors.In some embodiments, viral vectors includes following, substantially consisting of the following or consisting of the following: including The viral vectors of the AAV capsid of the disclosure；With the nucleic acid molecules comprising at least one terminal repeat, wherein the nucleic acid quilt AAV capsid involucrum.In some embodiments, terminal repeat is AAV terminal repeat.In some embodiments, Terminal repeat is non-AAV terminal repeat.

The capsid protein and/or viral vectors comprising the disclosure in pharmaceutically acceptable carrier is also provided herein Composition.

The disclosure additionally provides several method, the method including nucleic acid molecules are introduced cell, including makes cell and this public affairs Viral vectors and/or the composition contact opened, for example, being accommodated or being internalized by and carried by the virus by cell in thus viral vectors Under conditions of the nucleic acid molecules that body is introduced into are expressed in the cell.

The method that nucleic acid molecules are delivered to subject is also provided herein comprising the disease of the disclosure is applied to subject Poisonous carrier and/or composition.In specific embodiments, the viral vectors and/or composition are applied in subject Pivot nervous system.In certain embodiments, the viral vectors and/or composition are delivered across blood-brain barrier.

In some embodiments, the viral vectors of the disclosure may include interested nucleic acid molecules.In some embodiment party In case, interested nucleic acid molecules can encode therapeutic protein or therapeutic RNA molecule.

The disclosure also provides the method for selectively delivering interested nucleic acid molecules to neuronal cell comprising makes described Neuronal cell is contacted with the viral vectors of the disclosure, wherein the viral vectors includes interested nucleic acid molecules.Into one In the embodiment of step, this method, which can also comprise, is selectively delivered to cardiac muscle cell for interested nucleic acid molecules, for example, When this method carries out in subject's (for example, people experimenter).In some embodiments, composition is selectively delivered to Neuronal cell.In some embodiments, composition is selectively delivered to cardiac muscle cell.

In another embodiment, the disclosure provides the method for treating the neurological disorder or defect of subject, Including to the subject apply the disclosure viral vectors, wherein the viral vectors include coding treatment neurological disorder or The nucleic acid molecules of effective therapeutic protein or therapeutic RNA in defect.

It is additionally provided with the method for the nerve and cardiovascular disorder or defect for treating subject herein comprising to institute The viral vectors that subject applies the disclosure is stated, wherein the viral vectors includes coding in treatment nerve and cardiac disorder or lacks The nucleic acid molecules of effective therapeutic protein or therapeutic RNA in falling into.

In method described herein, the viral vectors and/or composition of the disclosure can be by systemic pathways (for example, quiet In arteries and veins, in intra-arterial, peritonaeum etc.) apply/be delivered to the subject of the disclosure.In some embodiments, viral vectors and/or Composition can be by (intracerebroventrical) in the ventricles of the brain, and in brain pond, in essence, encephalic and/or intrathecal route are applied With to subject.

In some embodiments, viral vectors and/or composition lead to tissue (example of missing the target to the systemic administration of subject Such as, in addition to central nervous system) lower transduction.In some embodiments of the present disclosure, viral vectors is from spleen, liver And/or targeting (detarget) is gone in kidney.In some embodiments, viral vectors is thin from splenocyte, liver cell and/or kidney It goes to target in born of the same parents.In some embodiments, compared with through the transduction of AAVrh.10, viral vectors to reduce at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% horizontal transduction liver, spleen and/or kidney, wherein viral transduction comes true optionally by luciferase or GFP expression It is fixed.Preferably, compared with the transduction by AAVrh.10, viral vectors is to reduce at least 50%-100%'s or 80%-90% Level transduction liver, spleen and/or kidney, wherein viral transduction is determined optionally by luciferase or GFP expression.

In some embodiments, compared with through the transduction of AAV1, viral vectors to increase at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% Horizontal transduction brain, wherein viral transduction is determined optionally by luciferase or GFP expression.Preferably, with pass through AAV1 Transduction compare, viral vectors with increase at least 2 times, 2.5 times, 3 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times of horizontal transduction brain, wherein viral transduction optionally by luciferase or GFP expresses to determine.In some embodiments, viral vectors is selectively transduceed neuron.

The modified capsid protein of the expected disclosure of the disclosure can currently known or what is be later discovered that appoints by modifying The capsid protein of what AAV generates.In addition, AAV capsid protein to be finished can be naturally occurring AAV capsid protein (example Such as, any AAV serotype shown in AAV2, AAV3a or 3b, AAV6, AAV7, AAV8, AAV9, AAVrh.10 or table 1), but It is not so limited.It will be appreciated by those skilled in the art that the various operations to AAV capsid protein are known in the art, and The present disclosure is not limited to the modifications of naturally occurring AAV capsid protein.For example, compared with naturally occurring AAV, capsid to be finished Albumen may have modification (for example, from naturally occurring AAV capsid protein, for example, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and/or AAV12 or currently known are later discovered that any Other AAV).Such AAV capsid protein is also within the scope of this disclosure.

Therefore, in specific embodiments, AAV capsid protein to be finished can derive from naturally occurring AAV, still One or more foreign sequences (for example, exogenous array of natural viral) can be further included, the foreign sequence is inserted Enter and/or replace and changes in the capsid protein and/or by the missing of one or more amino acid.

Therefore, when referenced herein specific AAV capsid protein (for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12 capsid protein or any AAV serotype shown in the table 1 Capsid protein etc.) when, it is intended to cover natural capsid protein and the capsid protein with the change in addition to the modification of the disclosure. It includes displacement, insertion and/or missing that these, which change,.In specific embodiments, compared with natural A AV capsid protein sequence, institute State capsid protein may include (other than the insertion of the invention) 1 being inserted, 2,3,4,5,6,7,8,9,10,11,12, 13,14,15,16,17,18,19 or 20,20 is less than, 30 is less than, is less than 40, is less than 50, is less than 60 or less than 70 amino Acid.In the embodiment of the disclosure, compared with natural A AV capsid protein sequence, the capsid protein may include (in addition to basis Except amino acid replacement of the invention) 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 It is a, 20 are less than, 30 is less than, is less than 40, is less than 50, is less than 60 or less than 70 amino acid replacements.In embodiments, with it is natural AAV capsid protein sequence is compared, the capsid protein may include (other than amino acid deletions of the invention) 1,2,3,4, 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20,20 is less than, 30 is less than, is less than 40, is less than 50, is few In the missing of 60 or less than 70 amino acid.

In specific embodiments, AAV capsid protein with natural A AV capsid protein sequence or has and natural A AV clothing Glutelin sequence at least about 90%, 95%, 97%, 98% or 99% similar or identity amino acid sequence.For example, specific In embodiment, the clothing such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 Glutelin covers natural A AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 Capsid protein sequence and with natural A AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 capsid protein sequence at least about 80%, 85%, 90%, 95%, 97%, 98% or 99% similar or identity Sequence.

The method for determining sequence similarity or identity between two or more amino acid sequences is known in the art 's.Standard technique known in the art can be used to determine in sequence similarity or identity, including but not limited to Smith and The local sequence identity algorithm of Waterman, Adv.Appl.Math.2,482 (1981), pass through Needleman and Wunsch J.Mol.Biol.48, the sequence identity alignment algorithm of 443 (1970), by Pearson and Lipman, The search for similarity method of Proc.Natl.Acad.Sci.USA 85,2444 (1988), it is real by the computerization of these algorithms Apply (Wisconsin Genetics software package, in Genetics Computer Group, 575Science Drive, Madison, WI GAP, BESTFIT, FASTA and TFASTA), by Devereux et al. Nucl.Acid Res.12,387-395 (1984) describe Best fit sequencer program, or pass through inspection.

Another suitable algorithm is BLAST algorithm, is described in Altschul et al. J.Mol.Biol.215,403- 410, (1990) and Karlin et al. Proc.Natl.Acad.Sci.USA 90,5873-5787 (1993).It is a kind of particularly useful Blast program be WU-BLAST-2 program, obtained from Altschul et al. Methods in Enzymology, 266, 460-480(1996)；http://blast.wustl/edu/blast/README.html.WU-BLAST-2 is searched using several Rope parameter, the parameter are optionally set to default value.

In addition, another useful algorithm be such as Altschul et al., (1997) Nucleic Acids Res.25, The BLAST jaggy of 3389-3402 report.

The viral capsid of modification may be used as " capsid carrier ", such as such as United States Patent (USP) No.5, described in 863,541.It can With by modification viral capsid pack and be transferred to the molecule in cell include allogeneic dna sequence DNA, RNA, polypeptide, small organic molecule, Metal or combinations thereof.

Heterologous molecule is defined as those of non-naturally-occurring in AAV infection, for example, not compiled by wild type AAV genome Those of code.In addition, the upper useful molecule for the treatment of can be associated with the outside of embedded virus capsid, for molecule to be transferred to In host target cells.Such associated molecule may include DNA, RNA, small organic molecule, metal, carbohydrate, lipid and/ Or polypeptide.In an embodiment of the disclosure, by the upper useful molecule covalent connection (i.e. conjugation or chemical coupling) for the treatment of To capsid protein.The method of covalent linker molecules is known to the skilled in the art.

The modified viral capsid of the disclosure also found any to escape for further modified antigen as template The purposes of neutralizing antibody in mammalian serum, such as described in PCT application sequence number PCT/2016/054143.

The modified viral capsid of the disclosure also found in terms of generation (raise) is for the antibody of novel capsid structure Purposes.It is used for an alternative it is possible to which exogenous amino acid sequences are inserted into modified viral capsid to thin as another kind The antigen presentation of born of the same parents, for example, being applied to subject to generate the immune response to exogenous amino acid sequences.

It in other embodiments, can be in the nucleic acid of the interested polypeptide of application delivering coding, peptide and/or functional r NA Before and/or at the same time of the viral vectors of molecule (for example, in mutual several minutes or a few hours), viral capsid is applied to block Certain cell sites.For example, capsid of the invention can be delivered to block the cell receptor on specific cells, and delivery vector It can subsequently or simultaneously apply, this can reduce the transduction for blocking cell, and enhance the transduction of other targets.

According to representative embodiment, modified viral capsid can be in the modified viral vectors according to the disclosure Before and/or at the same time of be applied to subject.In addition, present disclose provides the combinations comprising modified viral capsid of the invention Object and pharmaceutical preparation；Optionally, the composition also includes the modified viral vectors of the disclosure.

(optionally, the disclosure also provides the nucleic acid molecules of modified viral capsid and capsid protein disclosed in code book Isolated nucleic acid molecules).Additionally provide the carrier comprising nucleic acid molecules, and the nucleic acid molecules comprising the disclosure and/or carrier Cell (internal or culture).Suitable carrier includes but is not limited to viral vectors (for example, adenovirus, AAV, herpesviral, first Virus, cowpox, poxvirus, baculoviral etc.), plasmid, bacteriophage, YAC, BAC etc..Such nucleic acid molecules, carrier and cell can It is used as, for example, the reagent for generating modified viral capsid or viral vectors as described herein is (for example, packaging building The auxiliary of body or incasing cells).

Any method known in the art can be used (such as by from baculoviral table in viral capsid according to the present invention It is generated up to (Brown et al. (1994) Virology 198:477-488).

Modification according to the present invention to AAV capsid protein is " selectivity " modification.This method and using AAV serotype it Between entire subunit or big structure domain exchange Previous work be contrasted (see, for example, International Patent Publication WO 00/ 28004；Hauck et al. (2003) J.Virology 77:2768-2774；Shen et al. (2007) Mol Ther.15 (11): 1955-62；With Mays et al. (2013) J Virol.87 (17): 9473-85).According to the inventors knowledge, AAV capsid of the invention Albumen is first example made it possible across the specific amino acids variation of blood-brain barrier.

In some embodiments, AAV capsid protein, which contains, makes it possible that the specific amino acids across blood-brain barrier become Change.In some embodiments, AAV capsid protein include 8,9,10,11,12,13,14,15,16,17,18,19,20,21 or The displacement of 22 amino acid, wherein displaced amino acid source is from AAVrh.10, wherein the amino acid replacement makes across blood brain screen Barrier is possibly realized.In some embodiments, AAV capsid protein is originated from AAV1 capsid protein, and include 8,9,10,11,12, 13, the displacement of 14,15,16,17,18,19,20,21 or 22 amino acid, wherein displaced amino acid source is from AAVrh.10, Described in amino acid replacement make it possible across blood-brain barrier.In some embodiments, make across blood-brain barrier become can The specific amino acids of energy are 262N, 263G, 264T, 265S, 267G, 268S, 269T and 273T (VP1 numbers), wherein each residual The number of base is based on amino acid sequence SEQ ID NO:9 or SEQ ID NO:30.

In some embodiments, AAV capsid protein contains specific amino acid variation, the specific amino acid variation Make it possible to go targeting from liver, kidney and/or spleen.In some embodiments, AAV capsid protein include 8,9,10, 11, the displacement of 12,13,14,15,16,17,18,19,20,21 or 22 amino acid, wherein displaced amino acid source is certainly AAVrh.10, wherein the amino acid replacement makes it possible to go targeting from liver, kidney and/or spleen.In some embodiment party In case, AAV capsid protein is originated from AAV1 capsid protein, and include 8,9,10,11,12,13,14,15,16,17,18,19,20, 21 or 22 amino acid, wherein displaced amino acid source is from AAVrh.10, wherein the amino acid replacement makes from liver, kidney And/or spleen goes targeting to be possibly realized.

In some embodiments, displaced amino acid is located at the area VR-I of capsid.In some embodiments, displacement Amino acid is located on the surface of capsid.In some embodiments, displaced amino acid is positioned at the prominent of 3 times of symmetry axis in capsid The base portion risen.In some embodiments, displaced amino acid is located in the recess of 2 times of symmetry axis of capsid.In some implementations In scheme, displaced amino acid is located in the area VR-II of capsid protein, in DE- ring.In some embodiments, displaced ammonia Base acid is located in the β chain E of capsid.

In specific embodiments, " selectivity " modification cause be less than about 20,18,15,12,10,9,8,7,6,5,4,3 or The insertion and/or displacement of 2 adjacent amino acid and/or missing.

The modified capsid protein and capsid of the disclosure can further include it is currently known or identify later it is any its He modifies.

For example, the AAV capsid protein and viral capsid of the disclosure can be it is chimeric because they may include from another A kind of all or part of the capsid subunits of virus (optionally another parvovirus or others AAV serotype), for example, such as Described in International Patent Publication WO 00/28004.

Viral capsid may include targeting sequence (for example, in displacement and/or insertion viral capsid), the targeting sequence guidance Viral capsid interacts with the cell surface molecule being present on required target tissue (see, e.g., International Patent Publication WO 00/28004 and Hauck et al. (2003) J.Virology 77:2768-2774)；Shi et al. Human Gene Therapy [description integrin receptor binding motif RGD is at the position of AAV capsid subunits 520 and/or 584 by 17:353-361 (2006) Insertion]；United States Patent (USP) No.7,314,912 [describe AAV2 capsid subunits amino acid position 447,534,573 and 587 it The insertion of the P1 peptide containing RGD motif afterwards] and [the increased CNS of description display of International Patent Publication No.WO 2015038958 The selective recovery of the AAV carrier containing peptide sequence of transduction]).The other positions being resistant in the AAV capsid subunits of insertion are abilities (such as position 449 and 588 of Grifman et al. Molecular Therapy 3:964-975 (2001) description) known to domain.

For example, some viral capsids of the disclosure are for most of interested target tissues (for example, liver, skeletal muscle, the heart Dirty, diaphram, kidney, brain, stomach, intestines, skin, endothelial cell and/or lung) taxis with relative inefficiencies.Targeting sequence can have It is mixed in these low transduction vectors sharply, to assign taxis needed for viral capsid and optionally to the selection of specific organization Property taxis.AAV capsid protein, capsid and carrier vector comprising targeting sequence are described in such as International Patent Publication WO 00/ In 28004.As another possibility, can will as Wang et al. (Annu Rev Biophys Biomol Struct.35: 225-49 (2006)) description the orthogonal site of one or more non-naturally occurring amino acid incorporations AAV capsid subunits in, As the means that low transduction vector is redirected to required target tissue.These unnatural amino acids are advantageously used for will be interested Molecular chemistry be connected to AAV capsid protein, including but not limited to: glycan (mannose-dendritic cells targeting)；RGD, bombesin Or neuropeptide is for targeted delivery to specific cancer cell-types；Selected from targeting specific cells surface receptor (such as growth factor receptors, Integrin etc.) phage display RNA aptamer or peptide.The method of chemical modification amino acid be it is known in the art (referring to Such as Greg T.Hermanson, Bioconjugate Techniques, the first edition, Academic Press, 1996).

In representative embodiment, targeting sequence be can be the viral capsid sequence of infection guidance to particular cell types Column (for example, autonomous parvovirus capsid sequence, AAV capsid sequence or any other viral capsid sequence).

It, can be by heparin binding domain (for example, Respiratory Syncytial Virus(RSV) Heparin-binding as another non-limiting example Structural domain) it is inserted into or replaces to the capsid subunits (for example, AAV 4, AAV5) for not combining Heparan sulfate (HS) receptor usually In, so that heparin is in conjunction with gained mutant.

As another non-limiting example, amino acid footprint can make to be bound to glycan (such as galactolipin, saliva Acid, mannose, lactose, sulfo group-N- lactose amine, galactosamine, glucose, aminoglucose, fructose, fucose, gangliosides, shell Trisaccharide, chondroitin sulfate, keratin sulfate, dermatan sulfate) it is possibly realized, it can be grafted on capsid subunits, the capsid Subunit does not combine usually one or more of sugar listed above [for example, entitled Methods and compositions for The U.S. Patent Publication No.US20160017005 of dual glycan binding AAV vectors].

B19 infector is for red blood cell class progenitor cells using globoside as receptor (Brown et al. (1993) Science 262:114).The structure of B19 have determined thatResolution ratio (Agbandje-McKenna et al. (1994) Virology 203: 106).The region for being bound to the B19 capsid of globoside maps (Chapman et al. between amino acid 399-406 (1993) Virology 194:419), the annular section (Chipman et al. (1996) of β-between barrel structure E and F Proc.Nat.Acad.Sci.USA 93:7502).Therefore, the globoside receptor binding domain of B19 capsid can be by It replaces in AAV capsid protein, the viral capsid or viral vectors that will include it are targeted to class red blood cell.

In representative embodiment, external source targeting sequence can be any amino acid sequence of encoded peptide, and the peptide changes Become the taxis of the viral capsid comprising modified AAV capsid protein or viral vectors.In certain embodiments, it targets Peptide or protein matter can be naturally occurring, or alternatively can be and is wholly or partially synthetic.Exemplary target includes to sequence Ligand and other peptides for being bound to cell surface receptor and glycoprotein, such as RGD peptide sequence, bradykinin, hormone, peptide growth factor (such as epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet derived growth factor, Insulin-Like Growth factor I and II etc.), cell factor, melanocyte stimulates hormone (for example, α, β or γ), neuropeptide and endorphin etc., And retain their segment of the ability of cell-targeting to its homoreceptor.Other illustrative peptides and protein include substance P, keratinocyte growth factor, neuropeptide tyrosine, gastrin releasing peptide, interleukin 2, egg white lysozyme, promoting erythrocyte are raw Cheng Su, gonadoliberin, corticostatin, beta-endorphin, leucine enkephalin, dynorphin B (rimorphin), α- New enkephalins, angiotensins, pneumadin, vasoactive intestinal peptide, neurotensin, motilin and it is as described above its Segment.As another alternative, binding structural domain from toxin (such as tetanus toxin or ophiotoxin, such as α-silver Bungarotoxin etc.) can be used as targeting sequence be displaced in capsid protein.In another representative embodiment, AAV clothing Glutelin can by as described in Cleves (Current Biology 7:R318 (1997)) will it is " non-classical " input/it is defeated Signal peptide is (for example, fibroblast growth factor -1 and -2, interleukin 1, HIV-1Tat albumen, herpesviral VP22 out Deng) replace into AAV capsid protein and modify.Also cover the peptide motif for instructing specific cells to absorb, such as the triggering of FVFLP peptide motif By the intake of liver cell.

Display technique of bacteriophage and other technologies known in the art can be used for identifying any interested cell The peptide of type.

Targeting sequence can be encoded targeted to cell-surface binding sites (including receptor (such as protein, carbon hydrate Object, glycoprotein or proteoglycan)) any peptide.The example of cell-surface binding sites includes but is not limited to Heparan sulfate, Chondroitin sulfate and other glycosaminoglycans, sialic acid moities, poly sialic acid part, glycoprotein and gangliosides, MHC I sugar egg Carbohydrate ingredient that is white, being found on membrane glycoprotein, comprising: mannose, GalNAc, N- acetyl group-glucose Amine, fucose, galactolipin etc..

As another alternative, targeting sequence can be used for chemical coupling to another targeting and enter cell The peptide (can be by the arginine and/or lysine residue of its R group chemical coupling for example, may include) of molecule.

Above-mentioned modification can be combined with each other and/or combine with any other modification currently known or be later discovered that and be incorporated into In the capsid protein or capsid of the disclosure.

The disclosure also covers the viral vectors of modified capsid protein and capsid comprising the disclosure.Specifically implementing In scheme, viral vectors is parvovirus vectors (for example, comprising parvovirus capsid and/or vector gene group), such as AAV is carried Body (for example, including AAV capsid and/or vector gene group).In representative embodiment, viral vectors includes modified AAV capsid, the modified AAV capsid include the capsid subunits and vector gene group of the modification of the disclosure.

For example, viral vectors includes in representative embodiment: (a) modified viral capsid is (for example, through modifying AAV capsid), it includes the modified capsid proteins of the disclosure；(b) comprising terminal repeat (such as AAV TR) Nucleic acid, wherein the nucleic acid comprising terminal repeat is by modified viral capsid involucrum.The nucleic acid is optionally including two Terminal repeat (for example, two AAV TR).

In representative embodiment, viral vectors is recombinant viral vector, it includes encode interested protein or The exogenous nucleic acid molecule of functional r NA.It is described in more detail below recombinant viral vector.

It will be understood by those skilled in the art that the capsid protein of the modification of the disclosure, viral capsid and viral vectors exclude that Have the capsid protein, capsid and viral vectors of designated amino acid (that is, not being mutation in designated position under its native state a bit Body).

The method for generating viral vectors

The disclosure additionally provides the method for generating viral vectors of the present invention.In a representative embodiment, the disclosure The method for generating viral vectors is provided, this method includes providing to cell: (a) comprising at least one TR sequence (for example, AAV TR sequence) nucleic acid-templated, and (b) AAV sequence, be enough replicating nucleic acid template and be enough (example in capsidation to AAV capsid Such as, the AAV rep sequence of AAV capsid disclosed in code book and AAV cap sequence).Optionally, nucleic acid-templated also includes at least one A heterologous nucleic acid sequence.In specific embodiments, nucleic acid-templated includes two AAV ITR sequences, is located at heterologous nucleic acids sequence The 5' and 3' of column (if present), although they do not need directly to abut.

Nucleic acid-templated and AAV rep and cap sequence is provided under the following conditions: included in the nucleic acid of AAV capsid inner packing The viral vectors of template generates in cell.This method may further include the step of collecting viral vectors from cell.Virus Carrier can be collected from culture medium and/or through lytic cell.

Cell can be the cell for allowing AAV virus replication.Any suitable cell known in the art can be used.In In specific embodiment, cell is mammalian cell.Alternately, cell can be trans-complementation incasing cells System, the trans-complementation package cell line provide the function of lacking from replication defect type helper virus, for example, 293 cells or its His E1a trans-complementation cell.

AAV duplication and capsid sequence can be provided by any method known in the art.Current scheme is usually in list AAV rep/cap gene is expressed on a plasmid.AAV duplication and packaging sequence do not need to provide together, and even now, which is done, may be Easily.AAV rep and/or cap sequence can be provided by any virus or non-virus carrier.For example, rep/cap sequence can be with (for example, being inserted into the area E1a or E3 of the adenovirus vector of missing) is provided by hybrid adenoviral or herpesvirus vector.EBV Carrier can also be used for expression AAV cap and rep gene.It is additional that one advantage of this method, which is EBV carrier, but is existed always High copy number will be kept (that is, as extra-chromosomal element stable integration into cell, to be appointed as " base in continuous cell division In the core episome of EBV ", referring to Margolski (1992) Curr.Top.Microbiol.Immun.158:67).

As another alternative, rep/cap sequence can be stably integrated into cell.

In general, AAV rep/cap sequence will not be flanked by TR, to prevent the rescue and/or packaging of these sequences.

Any method known in the art can be used and be supplied to cell for nucleic acid-templated.For example, template can be by non-disease Malicious (such as plasmid) or viral vectors supply.In specific embodiments, nucleic acid-templated to be supplied by herpesviral or adenovirus vector Answer (for example, being inserted into the area E1a or E3 of the adenovirus of missing).As another illustration, Palombo et al. (1998) J.Virology 72:5025 describes the baculovirus vector for carrying the reporter gene for flanking AAV TR.EBV carrier can also answer For delivering template, as described in above for rep/cap gene.

In another representative embodiment, the nucleic acid-templated rAAV virus by replicating is provided.In again other implementation In scheme, comprising nucleic acid-templated AAV provirus stable integration into the chromosome of cell.

For enhanced virus titre, the helper viral function that generation property AAV can be promoted to infect to cell offer (for example, Adenovirus or herpesviral).Helper virus sequence necessary to AAV is replicated is known in the art.In general, these sequences will be by Helper adenovirus or herpesvirus vector provide.Alternatively, adenovirus or herpes viral sequences can by another non-viral or Viral vectors provides, for example, carrying all auxiliary bases for promoting effective AAV to generate as non-infectious adenovirus miniplasmids Because (such as by Ferrari et al., (1997) Nature Med.3:1295, and United States Patent (USP) No.6,040,183 and 6,093,570 It is described).

In addition, helper viral function can by with insertion chromosome in or remain stable extra-chromosomal element it is auxiliary Sequence incasing cells is helped to provide.In general, helper virus sequence cannot be packaged into AAV virion, for example, not flanking TR.

It will be appreciated by those skilled in the art that providing AAV duplication and the capsid sequence on single helper construct and auxiliary Virus sequence (for example, adenoviral sequence) is helped to may be advantageous.The helper construct can be non-viral or virus constructs. As a non-limitative illustration, helper construct can be hybrid adenoviral or heterozygosis blister comprising AAV rep/cap gene Exanthema virus.

In a specific embodiment, AAV rep/cap sequence and adenovirus auxiliary sequencel are auxiliary by single adenovirus Help carrier supplying.This carrier can also further include nucleic acid-templated.It can be by AAV rep/cap sequence and/or rAAV template It is inserted into the absent region (for example, the region E1a or E3) of adenovirus.

In a further embodiment, AAV rep/cap sequence and adenovirus auxiliary sequencel are assisted by single adenovirus Carrier supplying.According to the embodiment, rAAV template can be used as plasmid template offer.

In another illustrative embodiment, AAV rep/cap sequence and adenovirus auxiliary sequencel are by single adenovirus Assistant carrier provides, and rAAV template is integrated into cell as provirus.Alternatively, rAAV template is provided by EBV carrier, The EBV carrier maintains in the cell as extra-chromosomal element (for example, as core episome based on EBV).

In another exemplary embodiment, AAV rep/cap sequence and adenovirus auxiliary sequencel are by single adenovirus Auxiliary provides.The duplication viral vectors that rAAV template can be used as separation provides.For example, rAAV template can be by rAAV particle Or second recombined adhenovirus particle provide.

According to preceding method, hybrid adenoviral carrier generally comprises the adenovirus 5' for being sufficient to adenoviral replication and packaging With 3' cis sequence (i.e. adenoviral terminal repetitive sequence and PAC sequence).By AAV rep/cap sequence and rAAV template (if In the presence of if) it is embedded in adenoviral backbone and is flanked with 5' and 3' cis sequence, so that these sequences can be packaged into adenovirus In capsid.As described above, adenovirus auxiliary sequencel and AAV rep/cap sequence do not flank usually with TR, not so as to these sequences It is wrapped into AAV virion.

Zhang et al. ((2001) Gene Ther.18:704-12) is described comprising adenovirus and AAV rep and cap base Chimeric auxiliary of cause.

Herpesviral also is used as the helper virus in AAV packing method.Encode the heterozygosis herpesviral of AAV Rep albumen Expansible AAV carrier production decision can be advantageously facilitated.Express the heterozygosis herpes simplex virus I of AAV-2rep and cap gene Type (HSV-1) carrier has been described in (Conway et al. (1999) Gene Therapy 6:986 and WO 00/17377.

As another alternative, such as such as Urabe et al. (2002) Human Gene Therapy 13:1935-43 It is described, it can be used baculovirus vector to deliver rep/cap gene and rAAV template, the disclosure generated in insect cell Viral vectors.

The AAV carrier stoste for being free of pollution helper virus can be obtained by any method known in the art.For example, AAV and helper virus can be easily distinguished based on size.AAV can also be separated with helper virus based on affinity chromatography [for example, For heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973), or use affine resin (Wang et al. (2015), Mol Ther Methods Clin Dev 2:15040) or for example (summary is in Qu et al. (2015) for other methods Curr Pharm Biotechnol.16(8):684-95)].The replication defect type helper virus of deletion can be used, to appoint The helper virus of what pollution does not all have replication capacity.As another kind an alternative it is possible to using late gene expression is lacked Adenovirus auxiliary cell because needing adenovirus early gene expression only to mediate the packaging of AAV virus.For late gene The adenoviral mutants for expressing defect are (for example, ts100K and ts149 adenoviral mutants) known in the art.

Recombinant viral vector

The viral vectors of the disclosure can be used for delivery of nucleic acids to cell in vitro, in vitro and in vivo.Particularly, virus carries Body may be advantageously used with delivery of nucleic acids or be transferred to animal, including mammal, cell.

Any interested heterologous nucleic acid sequence can be delivered in the viral vectors of the disclosure.Interested nucleic acid includes The nucleic acid of polypeptide and/or functionality or therapeutic RNA molecule is encoded, the polypeptide includes therapeutic (for example, for medicine or beast Medical way) or immunogenicity (such as vaccine) protein.

Therapeutical peptide includes but is not limited to cystic fibrosis transmembrane regulatory protein (CFTR), dystrophin (including small flesh Albumen and micro- dystrophin are supported, see, for example, Vincent et al. (1993) Nature Genetics 5:130；United States Patent (USP) is public Open No.2003/017131；International Publication WO/2008/088895, Wang et al. Proc.Natl.Acad.Sci.USA 97: 13714-13719(2000)；With Gregorevic et al. Mol.Ther.16:657-64 (2008)), myostatin (myostatin) propetide, follistatin, activin type II soluble recepter, IGF-1, anti-inflammatory polypeptide such as Ikappa B are dominant prominent Variant, flesh is long (sarcospan), myotrophy GAP-associated protein GAP (utrophin) (Tinsley et al. (1996) Nature 384: 349), small uterus phosphatide (mini-utrophin), coagulation factor (for example, Factor IX, factors IX, factor X etc.) promote red thin Born of the same parents generate element, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, Leptin, LDL Receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globin, α-globin, spectrin, α₁Antitrypsin, Adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lyase are intimate Osamine enzyme A, branched keto acid dehydrogenase, RP65 albumen, cell factor is (for example, alpha-interferon, beta-interferon, interferon-γ are white Cytokine -2, interleukin 4, granulocyte-macrophage colony stimutaing factor, lymphotoxin etc.), peptide growth factor, mind Through trophic factors and hormone (such as growth hormone, insulin, type-1 insulin like growth factor and 2, platelet derived growth factor, Epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain source nerve battalion The factor is supported, bone morphogenetic protein (bone morphogenic proteins) [including RANKL and VEGF], neuroglia is spread out Raw growth factor, transforminggrowthfactor-α and-β etc.), lysosomal acid alpha-glucosidase, alpha-galactosidase A, receptor is (as swollen Tumor necrosis growth factor α soluble recepter), S100A1, parvalbumin, 6 type of adenylyl cyclase adjusts the molecule of Calcium treatment (for example, SERCA_2A, the inhibitor 1 and its segment [for example, WO 2006/029319 and WO 2007/100465] of PP1), influence G 2 type of receptor kinase of albumen coupling strikes low molecule, such as truncated constitutive activity bARKct, anti-inflammatory factors such as IRAP, anti-flesh Growth inhibition fibroin (anti-myostatin proteins), Aspartoacylase, monoclonal antibody (including single-stranded Dan Ke Grand antibody；Exemplary Mab isMab), neuropeptide and its segment (for example, neuromere peptide, neuropeptide tyrosine (referring to U.S.7,071,172), angiogenesis inhibitors such as angiostatin (Vasohibins) and other VEGF inhibitors (for example, Angiostatin 2 [referring to WO JP2006/073052]).Other illustrative heterologous nucleic acid sequence encoded suicide gene product (examples Such as, thymidine kinase, cytosine deaminase, diphtheria toxin and tumor necrosis factor), drug use for cancer treatment is assigned with resistance Protein, tumor suppressor gene products (for example, p53, Rb, Wt-1), TRAIL, FAS- ligand and having this need it is tested With any other polypeptide of therapeutic effect in person.AAV carrier can also be used for delivering monoclonal antibody and antibody fragment, such as needle Antibody or antibody fragment to myostatin is (see, for example, Fang et al. Nature Biotechnology 23:584- 590(2005))。

The heterologous nucleic acid sequence for encoding polypeptide includes those of encoding reporter polypeptides (such as enzyme).Report that polypeptide is this field It is known, including but not limited to green fluorescent protein (GFP), beta galactosidase, alkaline phosphatase, luciferase and chloramphenicol Acetyl transferase gene.

Optionally, exogenous nucleic acid molecule can encode the polypeptide of secretion (for example, being secrete polypeptide under its native state Polypeptide, or the polypeptide being engineered with secretion, for example, by with secretory signal sequence as known in the art operationally In conjunction with).

Alternatively, in the specific embodiment of the disclosure, heterologous nucleic acids can be with encoding antisense nucleic acid, ribozyme (for example, such as United States Patent (USP) No.5, described in 877,022), influence spliceosome mediate trans-splicing RNA (referring to Puttaraju Et al. (1999) Nature Biotech.17:246；United States Patent (USP) No.6,013,487；United States Patent (USP) No.6,083,702), packet The RNA interfering (RNAi) (its mediated gene silencing) of siRNA, shRNA or miRNA are included (referring to, Sharp et al., (2000) Science 287:2431) and other untranslatable rna (Gorman et al. (1998) for such as " instructing " RNA Proc.Nat.Acad.Sci.USA 95:4929；The United States Patent (USP) No.5,869,248 of Yuan et al.) etc..Exemplary untranslated RNA includes the RNAi for multi-drug resistant (MDR) gene product (for example, being used for treatment and/or pre- preventing tumor and/or being used for Heart is applied to prevent chemical damage), for myostatin RNAi (for example, be used for Du Shi muscular dystrophy Disease), for the RNAi (for example, for treat and/or pre- preventing tumor) of VEGF, for phospholamban RNAi (for example, for controlling Cardiovascular disease is treated, see, for example, Andino et al. J.Gene Med.10:132-142 (2008) and Li et al. people Acta Pharmacol Sin.26:51-55(2005))；Phospholamban inhibition or Dominant negative molecule such as phospholamban S16E (example Such as, for treating cardiovascular disease, see, for example, the Nat.Med.8:864-871 such as Hoshijima (2002)), swash for adenosine The RNAi (for example, being used for epilepsy) of enzyme, and for causal organism and virus (for example, hepatitis B and/or hepatitis C virus Poison, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus etc.) RNAi.

Furthermore, it is possible to deliver the nucleic acid sequence for instructing alternative splicing.In order to illustrate 5' with dystrophin exon 51 And/or the antisense sequences (or other inhibit sequence) of 3' splice site complementation can be with U1 or U7 small nut (sn) RNA promoter one Delivering is played to induce skipping for this exon.For example, comprising being located at antisense/inhibition sequence 5' U1 or U7snRNA promoter DNA sequence dna can be packed and be delivered in modification capsid of the invention.

Viral vectors can also include exogenous nucleic acid molecule, the locus on the exogenous nucleic acid molecule and host chromosome It recombinates with homology and therewith.This method can be used for, for example, the genetic defect in correction host cell.

The disclosure additionally provides the viral vectors of expression immunogenic polypeptide (such as vaccine inoculation).The nucleic acid molecules Codified any interested immunogene known in the art, the immunogene include but is not limited to come from human immunodeficiency Poison (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag albumen, tumour antigen, cancer antigen, bacterium The immunogene of antigen, viral antigen etc..

Immunogenic polypeptide, which can be, to be suitable for causing immune response and/or protects subject from infection and/or disease (packet Include but be not limited to the infection and disease of microorganism, bacterium, protozoan, helminth, fungi and/or virus) any polypeptide.Example Such as, immunogenic polypeptide can be orthomyxovirus immunogene (for example, influenza virus immunization is former, such as influenza virus hemagglutinin (HA) surface protein or influenza nucleoprotein or equine influenza virus immunogene) or slow virus immunogene (for example, equine infectious Anemia virus immunogene, simian immunodeficiency virus (SIV) immunogene or human immunodeficiency virus (HIV) immunogene, such as HIV or SIV coating GP160 albumen, HIV or SIV matrix/capsid protein and HIV or SIV gag, pol and env gene produce Object).Immunogenic polypeptide is also possible to arenavirus immunogene (for example, Lassa fever virus immunogene, such as Lassa fever virus core Capsid protein and Lassa fever envelope glycoprotein), poxvirus immunogene (such as vaccinia virus immunogene, such as cowpox L1 or L8 base Because of product), flavivirus immunogene (for example, flavivirus immunogene or japanese encephalitis virus immunogene), filamentous virus is immunized Former (for example, Ebola virus immunogene or Marburg virus immunogene, such as NP and GP gene product), bunyavirus is exempted from Epidemic focus (for example, RVFV, CCHF and/or SFS virus immunity are former) or coronavirus protein immunogens are (for example, infectious human coronary's disease Malicious immunogene, such as human corona virus's envelope glycoprotein or transmissible gastro-enteritis virus immunogene or birds infectiousness branch gas Pipe inflammation virus immunity is former).Immunogenic polypeptide can also be polio immunogene, bleb immunogene (for example, CMV, EBV, HSV immunogene), parotitis immunogene, measle immune is former, rubeola immunogene, diphtheria toxin or other diphtheria immunogenes, pertussis Antigen, hepatitis (for example, hepatitis A, hepatitis B, hepatitis C etc.) immunogene and/or this field it is currently known or with It is accredited as any other vaccine immunogens of immunogene afterwards.

Alternatively, immunogenic polypeptide can be any tumour or cancer cell antigen.Optionally, tumour or cancer antigen are thin in cancer It is expressed on the surface of born of the same parents.Exemplary cancers and tumor-cell antigen are described in S.A.Rosenberg (Immunity 10:281 (1991)) in.Other illustrative cancers and tumour antigen include but is not limited to: BRCA1 gene product, BRCA2 gene product, Gp100, tyrosinase, GAGE-1/2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, Guang day egg White enzyme -8, KIAA0205, HPVE, SART-1, PRAME, p15, Melanoma Tumor antigen (Kawakami et al. (1994) Proc.Natl.Acad.Sci.USA91:3515；Kawakami et al. (1994) J.Exp.Med., 180:347；Kawakami etc. People (1994) Cancer Res.54:3124), MART-1, gp100MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al. (1993) J.Exp.Med.178:489)；HER-2/neu gene product (United States Patent (USP) No.4,968,603), CA125, LK26, FB5 (endosialin), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialylated Tn antigen), c-erbB-2 albumen, PSA, L-CanAg, estrogen by Body, butter oil globulin, p53 tumor suppressor protein (Levine (1993) Ann.Rev.Biochem.62:623)；Mucoprotein is anti- Former (International Patent Publication No. WO 90/05142)；Telomerase；Nuclear matrix protein；Prostatic acid phosphatase；Papillomavirus Antigen；And/or antigen relevant to following cancer that is currently known or being later discovered that: melanoma, gland cancer, thymoma, lymph Tumor (such as non-Hodgkin lymphoma, Hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukaemia, uterine cancers, mammary gland Cancer, prostate cancer, oophoroma, cervical carcinoma, bladder cancer, kidney, cancer of pancreas, the cancer of the brain and it is currently known or identify later it is any its His cancer or the pernicious patient's condition (see, e.g., Rosenberg (1996) Ann.Rev.Med.47:481-91).

As another alternative, heterologous nucleic acids can encode expectation generation in cell in vitro, in vitro or in vivo Any polypeptide.For example, viral vectors can be introduced into in the cell of culture and therefrom be separated the gene product of expression.

In a further embodiment, heterologous nucleic acids can encode (a) be based on Zinc finger nuclease or meganuclease or The pointed decoration polypeptide of endonuclease or the nuclease based on TALEN or based on CRISPR/Cas system, contains and DNA target The RNA bound fraction to interact to RNA molecule (gRNA), or the mRNA of this polypeptide of coding；(b) one or more of guidances RNA molecule (gRNA), it includes the nucleotide sequences complementary with the sequence in target DNA, and interact with pointed decoration polypeptide The second section；And/or (c) one or more DNA donor template molecules, it includes dynamic to lactation designed for homologous recombination Single-stranded or double-stranded DNA sequence dna in object genome target site.For example, viral vectors can import in vitro stem cell and/or T leaching Bar cell, the cell of culture, in-vivo tissue and/or entire organism, and the gene product expressed can make the target base in host Editor, destruction, transcriptional activation and/or the inhibition of cause are possibly realized.

It will be understood by those skilled in the art that interested exogenous nucleic acid molecule can be with control sequence appropriate operationally Connection.For example, exogenous nucleic acid molecule can be operably connected with such as following expression control element: transcription/translation control letter Number, replication orgin, polyadenylation signal, internal ribosome entry site (IRES), promoter and/or enhancer etc..

Furthermore, it is possible to realize the expression of interested exogenous nucleic acid molecule adjusted in post-transcriptional level, such as pass through The existence or non-existence of the active oligonucleotides of montage, small molecule and/or other compounds is selectively blocked in specific site To adjust the alternative splicing (for example, as described in WO 2006/119137) of different intrones.

It will be understood by those skilled in the art that a variety of promoter/enhancer elements can be used, this depends on required level And tissue specific expression.Promoter/enhancer can be composing type or induction type, this depends on required expression pattern. Promoter/enhancer can be natural or external source, and can be natural or synthesis sequence.Pass through external source, it is intended that Do not find transcription initiation region in the wild-type host for being introduced into transcription initiation region.

In certain embodiments, promoter/enhancer element can be target cell or subject to be treated Natural.In representative embodiment, promoter/enhancer element can be heterologous nucleic acid sequence natural.It is logical Often selection promoter/enhancer element, makes it work in interested target cell.In addition, in specific embodiments, opening Mover/enhancer element can be mammalian promoter/enhancer element.Promoter/enhancer element can be composing type Or induction type.

Inducible expression control element is usually in the application that those need to provide the adjusting expressed heterologous nucleic acid sequence It is advantageous.Inducible promoter/enhancer element for gene delivery can be tissue specificity or preferred promoter/ Enhancer element, and including muscle specific or preferred (including heart, bone and/or smooth muscle specificity or preferably ), nerve fiber specificity or preferred (including brain-specificity or preferred), eye specificity or preferably (including retina Specificity and cornea specificity), liver specificity or preferred, marrow specificity or preferred, pancreas specificity or preferred, spleen Specificity or preferred and lung specificity or preferred promoter/enhancer element.Other inducible promoter/enhancers Element includes hormone inducible and metal inducible element.Exemplary inducible promoter/enhancer element includes but is not limited to Tet opening/closing member, RU486 inducible promoter, ecdysone inducible promoter, rapamycin inducible promoter and gold Belong to metallothoinein promoter.

It is transcribed in target cell in the embodiment then translated in wherein heterologous nucleic acid sequence, generally includes specificity Effective translation of the initial signal for the protein coding sequence of insertion.These Exogenous translational control sequences, may include ATG Initiation codon and flanking sequence can be a variety of sources, natural and synthesis.

Viral vectors according to the present invention provides the means being delivered to heterologous nucleic acids in extensive cell, including division And non-dividing cell.Viral vectors can be used in vitro by interested delivery of nucleic acids to cell, for example, more to generate in vitro Peptide is used for ex vivo gene therapy.Viral vectors can also be used in delivery of nucleic acids to its subject is needed, for example, being exempted from expression Epidemic focus or therapeutical peptide or functional r NA, method.In this way, polypeptide or function RNA can be in subject's bodies It generates.Subject may need polypeptide, because subject has the shortage of polypeptide.In addition it is possible to implement this method because by Polypeptide is generated in examination person or functional r NA can produce some beneficial effects.

Viral vectors can also be used in the cell of culture or subject generate interested polypeptide or functional r NA (example Such as, use subject as bioreactor, to generate the effect of polypeptide or overview function RNA to subject, for example, in conjunction with Screening technique).

In general, the viral vectors of the disclosure can be used for delivering coding polypeptide or the heterologous nucleic acids of functional r NA, with treatment And/or any delivering therapeutical peptide of prevention or the functional r NA morbid state beneficial to its.Illustrative morbid state include but It is not limited to: cystic fibrosis (cystic fibrosis transmembrane regulatory protein) and other tuberculosis, hemophilia A (Factor IX), hemophilia B (factors IX), thalassemia (beta-globin), anaemia (hematopoietin) and other hematologic diseases, Alzheimer disease (GDF；Enkephalinase), multiple sclerosis (beta-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington disease (RNAi is to remove repetition), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factor) and Other the nervous system diseases, cancer (endostatin, angiostatin, TRAIL, FAS ligand, the cell factor including interferon； RNAi comprising for VEGF or the RNAi of Multiresistant genes product, mir-26a [for example, being used for hepatocellular carcinoma]), glycosuria Sick (insulin), muscular dystrophy, including Du Shi muscular dystrophy (dystrophin, mini dystrophin, Insulin-like growth factor I, inose (sarcoglycan) [for example, α, beta, gamma], for myostatin, muscle growth Inhibin propetide, follistatin, activin II type soluble recepter, anti-inflammatory polypeptide such as Ikappa B dominant mutant, flesh are long (sarcospan), myotrophy GAP-associated protein GAP (utrophin), the RNAi of small myotrophy GAP-associated protein GAP (mini-utrophin), needle To the splice junction in dystrophin gene with the antisense or RNAi of inducing exon-skipping [see, e.g., WO/2003/ 095647], for U7snRNA with the antisense of inducing exon-skipping [see, for example, WO/2006/021724], and it is directed to muscle The antibody or antibody fragment of amicine or myostatin propetide) and the sick (glucocerebroside of Becker, Gaucher Enzyme), Hurler disease (α-L- iduronase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage disease (such as method cloth In sick [alpha-galactosidase] and pompe disease [lysosomal acid alpha-glucosidase]) and other metabolic disorders, congenital emphysema (alpha1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann- Pick disease (sphingomyelinase), Tay Sachs is sick (lysosome hexosaminidase A), maple syrup urine disease (branched keto acid dehydrogenase), retina Degenerative disease (and the other diseases of eyes and retina；Such as macular degeneration and/or angiostatin PDGF or Other of VEGF or other angiogenesis inhibitors inhibitor, with treatment/prevention retinal disease, such as type-1 diabetes mellitus), it is real (including Parkinson's disease [GDNF], astrocytoma [endostatin, angiostatin and/or are directed to VEGF to the disease of body organ such as brain RNAi], glioblastoma [endostatin, angiostatin and/or the RNAi for VEGF], liver, kidney, heart, packet Include congestive heart failure or peripheral arterial disease (PAD) (for example, by delivering protein phosphatase inhibitor) I (I-1) and its Segment (for example, I1C), serca2a adjust the zinc finger protein of phospholamban gene, Barkct, beta 2-adrenergic receptor, β 2- adrenergic receptor kinase 1 enzyme (BARK), PI-3 kinase (PI3 kinases), S100A1, parvalbumin, adenylate ring Change 6 type of enzyme, a kind of influences 2 type of g protein coupled receptor kinases strikes low molecule, such as truncated constitutive activity bARKct； Calsarcin, for the RNAi of phospholamban；Phospholamban inhibits or Dominant negative molecule, phospholamban S16E etc.), Arthritis (insulin-like growth factor), joint disease (type-1 insulin like growth factor and/or 2), endometrial hyperplasia (such as pass through Deliver enos, inos), improve the survival rate (superoxide dismutase) of heart transplant, AIDS (soluble CD4), muscle withers It contracts (insulin-like growth factor I), kidney deficiency (hematopoietin), anaemia (hematopoietin), arthritis is (anti-inflammatory The factor such as IRAP and TNF α soluble recepter), hepatitis (alpha-interferon), ldl receptor deficiency disease (ldl receptor), hyperammonemia (bird ammonia Sour transcarbamylase), Krabbe disease (galactocerebrosidase), spinal muscular atrophy (SMA), bar disease, myeloid brain is total Ji imbalance (spinal cerebral ataxias), including family ataxia, SCA1, SCA2 and SCA3, phenylpropyl alcohol Ketonuria (phenylalanine hydroxylase), autoimmune disease etc..The present invention can also use after organ transplantation, to increase transplanting Success and/or reduce the negative side-effects of organ transplant or adjuvant treatment (for example, by application immunosuppressor or inhibition Nucleic acid blocks the generation of cell factor).As another example, bone morphogenetic protein (including BMP 2,7 etc., RANKL And/or VEGF) can be applied together with bone allograft, it is cut for example, destroying (break) in cancer patient or performing the operation After removing.

The disclosure can also be used in the multipotential stem cell (iPS) for generating induction.For example, the viral vectors of the disclosure can be used for by Stem cell associated nucleic acid is delivered in non-pluripotent cell, such as adult fibroblast, Skin Cell, liver cell, nephrocyte, rouge Fat cell, cardiac muscle cell, nerve cell, epithelial cell, endothelial cell etc..The nucleic acid for encoding the factor relevant to stem cell is this Known to field.The non-limiting example of such factor relevant to stem cell and versatility includes Oct-3/4, SOX family (example Such as, SOX1, SOX2, SOX3 and/or SOX15), Klf family (for example, Klf1, Klf2, Klf4 and/or Klf5), Myc family (example Such as, C-myc, L-myc and/or N-myc), NANOG and/or LIN28.

The disclosure can also be practiced for treating and/or preventing epilepsy, apoplexy, traumatic brain injury, cognitive disorder, behavior Obstacle, phrenoblabia, Huntington's disease, Alzheimer disease, amyotrophic lateral sclerosis (ALS) and any other possibility Benefit from or need the nervus retrogression patient's condition of aixs cylinder/neuron regeneration or reparation.

In specific embodiments, the disclosure can be practiced to promote axon regeneration and neuron reparation, restore circuit and/ Or supplement lose neuron as corrective therapy, such as pass through targeting adjust or be overexpressed stem cell differentiation and reprogram the factor Such as FoxJ1, Fox2, NeuroD2, NG2 or Olig2 and/or the Microrna of such as miR-137, MiR124, and it is related to nerve Any other factor or miRNA of member development and differentiation.

Gene transfer has for understanding and providing the huge potential use of disease state treatments.There are many hereditary diseases Disease, dcc gene is known and has been cloned wherein.In general, above-mentioned morbid state is divided into two classes: defect state, The usually defect state of enzyme, usually with recessive manner heredity；And non-equilibrium state, it can be related to regulatory protein or structure egg It is white, and usually with dominant mode heredity.For defect state disease, gene transfer can be used for bringing into normal gene impacted Tissue in carry out replacement therapy, and the animal model of the disease is generated using anti-sense mutation.For unbalanced disease State, gene transfer can be used for generating morbid state in model system, and then it can be used for resisting the disease state.Therefore, root Allow to treat and/or prevent genetic disease according to the viral vectors of the disclosure.

Viral vectors according to the present invention can also be used for providing functional r NA to cell in vitro or in vivo.Functional r NA Expression in cell, such as, it is possible to reduce expression of the cell to specific target protein.Therefore, functional r NA can be applied to drop The expression of specific protein in low subject with this need.Functional r NA can also be applied in vitro cell to adjust base Because of expression and/or stechiology, for example, to optimize cell or tissue culture systems or screening technique.

In addition, can be used for diagnosis and screening technique according to the viral vectors of the disclosure, nucleic acid interested whereby is in cell In culture systems, or alternatively in transgenic animal model, instantaneous or stable expression.

Viral vectors of the invention can also be used in various non-treatment purposes, including but not limited to for assess gene target, In the scheme of removing, transcription, translation etc., as apparent to those skilled in the art.Viral vectors can also be used for assessment safety Property (diffusion, toxicity, immunogenicity etc.).These data, for example, by U.S.'s food and medication management before assessing clinical efficacy Office is considered as a part of regulatory approval process.

As on the other hand, the viral vectors of the disclosure can be used for generating immune response in subject.According to the implementation Scheme can apply the viral vectors of the heterologous nucleic acid sequence comprising encoding immunogenic polypeptide, and subject to subject Start active immunity response for immunogenic polypeptide.Immunogenic polypeptide is as described above.In some embodiments, protectiveness Immune response is initiated.

Alternatively, viral vectors can be applied to cell in vitro, and the cell of change is applied to subject.It will include different The viral vectors of source nucleic acid is introduced into cell, and the cell is applied to subject, and wherein the heterologous nucleic acids of encoding immunogens can It is expressed, and for the immune response in immunogene induction subject.In certain embodiments, cell is that antigen presentation is thin Born of the same parents' (for example, dendritic cells).

" active immunity response " or " active immunity " are characterized in that " ginseng of host tissue and cell after meeting with immunogene With.It is related to the differentiation and proliferation of immunocompetent cell in lymphoreticular tissue, leads to antibody synthesis or cell-mediated reaction The development of property, or both ".Herbert B.Herscowitz,Immunophysiology:Cell Function and Cellular Interactions in Antibody Formation,in IMMUNOLOGY:BASIC PROCESSES 117 (Joseph A.Bellanti ed.,1985).In other words, host is by infection or vaccine inoculation, to be exposed to immunogene laggard Row active immunity response.Active immunity can be opposite with passive immunity, and passive immunity is by " preformed substance is (anti- Body, transfer factor, thymic graft, interleukin 2) from the host of active immunity it is transferred to nonimmune host " and obtain 's.Id.

It is some to assign subject for " protectiveness " immune response or " protectiveness " immune instruction immune response as used herein Benefit, because it prevents or reduce the disease incidence of disease.Alternatively, protective immune response or protective immunity can be used for controlling Disease, especially cancer or tumour are treated and/or prevented (for example, being formed by pre- anti-cancer or tumour, by causing cancer or swelling Tumor subsides and/or by prevention transfer and/or the growth by preventing metastatic tubercle).Protective effect can be it is complete or Partial, only benefit to be treated is more than its any disadvantage.

In certain embodiments, the viral vectors comprising heterologous nucleic acids or cell can be applied with immunogenicity effective quantity With as described below.

It expresses one or more of cancer cell antigens (or molecule similar in immunology) by application or generates and be directed to cancer The viral vectors of the disclosure can be also used for cancer and exempted from by the viral vectors of any other immunogene of the immune response of cell Epidemic disease therapy.In order to illustrate immune response can be anti-comprising coding cancer cell by application for the cancer cell antigen in subject The viral vectors of former heterologous nucleic acids and generate, such as to treat patient with cancer and/or with pre- anti-cancer in subject Middle development.As described herein, viral vectors can be applied to subject in vivo or by using ex vivo approach.Alternatively, cancer is anti- Original can be expressed as a part or otherwise (for example, as described above) associated with viral capsid of viral capsid.

As another alternative, any other therapeutic nucleic acid (such as RNAi) known in the art or polypeptide (example Such as cell factor) it can apply with treatment and/or pre- anti-cancer.

As used herein, term " cancer " covers tumour formation cancer.Equally, term " cancerous tissue " covers tumour." cancer is thin Extracellular antigen " covers tumour antigen.

Term " cancer " is in the art with the meaning that it understands, for example, having the distant sites for being diffused into body The uncontrolled growth of the tissue of the potential of (that is, transfer).Exemplary cancers include but is not limited to melanoma, gland cancer, chest Adenoma, lymthoma (for example, non-Hodgkin lymphoma, Hodgkin lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukaemia, son Palace cancer, breast cancer, prostate cancer, oophoroma, cervical carcinoma, bladder cancer, kidney, cancer of pancreas, the cancer of the brain and it is currently known or later really Fixed any other cancer or the pernicious patient's condition.In representative embodiment, present disclose provides treatment and/or pre- preventing tumor shapes At the method for cancer.

Term " tumour " is also understood in the art, for example, as the different of the neoblast in multicellular organism body Chang Zhiliang.Tumour can be pernicious or benign.In representative embodiment, method disclosed herein is for preventing and controlling Treat malignant tumour.

By term " treating cancer ", " treatment of cancer " and equivalent terms, it is intended to reduce or at least partly eliminate cancer The seriousness of disease, and/or slow down and/or control the progress and/or stable disease of disease.In specific embodiments, these arts Language shows to prevent or reduce or at least partly eliminates cancer metastasis, and/or prevents or reduces or at least partly eliminate metastatic The growth of tubercle.

Pass through term " pre- anti-cancer " or " prevention of cancer " and equivalent terms, it is intended to this method at least partly eliminate or Reduce and/or postpone the incidence and/or seriousness of pathogenesis of cancer.In other words, the morbidity of cancer may be can in subject It reduces and/or postpones in energy property or probability.

In certain embodiments, cell can from cancer subject in take out, and be allowed to according to this public affairs The viral vectors contact for the expression cancer cell antigen opened.Then modified cell is applied to the subject, thus caused For the immune response of cancer cell antigen.This method may be advantageously used with the subject of immunocompromised host, cannot open in vivo Move enough immune responses (that is, enhancing antibody that sufficient amount cannot be generated).

Immune response known in the art can be enhanced by immunomodulating cytokines (for example, alpha-interferon, β-interference Element, gamma interferon, ω-interferon, τ-interferon, interleukin-1 alpha, interleukin-1 ' beta ', proleulzin, leucocyte are situated between Element -3, interleukin 4, t cell growth factor, interleukin-6, interleukin 7, interleukin 8, leucocyte are situated between Element -9, interleukin 10, interleukin 11, interleukin 12, interleukin-13, interleukin-14 are white Cytokine -18, Bcell growth factor, CD40 Ligand, tumor necrosis factor-alpha, tumor necrosis factor-β, monocyte chemotactic Albumen -1, granulocyte-macrophage colony stimutaing factor and lymphotoxin).Therefore, immunomodulating cytokines are (preferably, CTL inducing cytokine) subject can be applied to together with viral vectors.

Cell factor can be applied by any method known in the art.Can to subject apply foreign cell because Son, or suitable carrier alternatively can be used and generated by the delivery of nucleic acids of the Codocyte factor to subject, and in vivo Cell factor.

Subject, pharmaceutical preparation and administration mode

Viral vectors and capsid according to the present invention can be used for animal doctor and medical application.Suitable subject include birds and Mammal.The term as used herein " birds " includes but is not limited to chicken, duck, goose, quail, turkey, pheasant, parrot, long-tail parrot Nautilus etc..The term as used herein " mammal " includes but is not limited to people, non-human primate, ox, sheep, goat, horse, Cat, dog, Lagomorpha etc..Human experimenter includes newborn, baby, teenager, adult and aged subjects.

In representative embodiment, subject " needs " disclosed method.

In specific embodiments, the disclosure provides pharmaceutical composition, and it includes in pharmaceutically acceptable carrier The disclosure viral vectors and/or capsid, and other optional therapeutics, medicament, stabilizer, buffer, carrier, assistant Agent, diluent etc..For injection, carrier is usually liquid.For other method of administration, carrier can be solid or liquid.It is right It is applied in sucking, carrier will be inhalable, and optionally can be solid or liquid particles form.

" pharmaceutically acceptable " refers to and non-toxicity or otherwise undesirable substance, the i.e. substance can be applied With to subject without causing any undesirable biological effect.

An aspect of this disclosure is the method that nucleic acid is transferred to cell in vitro.It can be thin according to particular target is suitable for Viral vectors, is introduced into cell by the standard transduction method of born of the same parents with appropriate infection multiplicity.The titre of the viral vectors of application can be with Changed according to target cell type and quantity and specific viral vectors, and can be determined by those skilled in the art and nothing Need excessive experiment.It, will at least about 10 in representative embodiment³A infectious unit, optionally at least about 10⁵A infection Unit is introduced into cell.

The cell for introducing viral vectors can be any type, including but not limited to nerve cell (including periphery and maincenter The cell of nervous system, the especially brain cell of such as neuron and oligodendroglia), pneumonocyte, eye cell (including view Retinulae, retinal pigment epithelium and keratocyte), epithelial cell (such as enteron aisle and airway epithelial cell), muscle Cell (such as Skeletal Muscle Cell, cardiac muscle cell, smooth muscle cell and/or diaphragmatic muscle), dendritic cells, pancreatic cell (including pancreas Island cell), liver cell, cardiac muscle cell, osteocyte (such as stem cell), candidate stem cell, splenocyte, keratinocyte, Fibroblast, endothelial cell, prostatic cell, reproduction cell etc..In representative embodiment, cell can be any Progenitor cells.As another possibility, cell can be stem cell (for example, neural stem cell, liver stem cells).As another standby Scheme is selected, cell can be cancer or tumour cell.In addition, as noted, cell can come from any kind of next Source.

Viral vectors can be introduced into cell in vitro, for the cell of modification to be applied to the purpose of subject.In spy In fixed embodiment, cell is taken out from subject, by viral vectors import wherein, then cell is applied back again by Examination person.Cell is taken out from subject and is used for isolated operation, and the method for then leading back subject is known in the art (referring to example Such as United States Patent (USP) No.5,399,346).It is alternatively possible to which recombinant viral vector is introduced the cell from donor subject, draw Enter the cell of culture, or introduce the cell from any other suitable source, and cell is applied to and needs its subject (i.e. " receptor " subject).

Suitable cell in vitro delivery of nucleic acids is as described above.The dosage for being applied to the cell of subject will be according to tested Age, the patient's condition and the species of person, cell type, cell expression nucleic acid, method of application etc. and change.In general, every dosage is in medicine At least about 10 are applied in acceptable carrier²To about 10⁸A cell or at least about 10³To about 10⁶A cell.Specific In embodiment, with the cell of viral vector transduction with therapeutically effective amount or prevention effective dose be applied to together with pharmaceutical carriers by Examination person.

In some embodiments, viral vectors is introduced into cell, and cell is applied to subject and is directed to causing The immunogenic response of the polypeptide (for example, be expressed as transgenosis or express in capsid) delivered.In general, by a certain amount of table Cell up to a effective amount of polypeptide of immunogenicity is applied in combination with pharmaceutically acceptable carrier." immunogenicity effective quantity " is table Up to the amount of polypeptide, it is enough to cause the active immunity response for polypeptide in the subject of administration of pharmaceutical preparations.Specific In embodiment, the dosage is enough to generate protective immune response (as defined above).The degree of protection of imparting needs not be It is complete or permanent, as long as the benefit of application immunogenic polypeptide is more than its any disadvantage.

Another aspect of the present disclosure is the method for the application viral vectors and/or viral capsid to subject.It can lead to Cross the human subjects that viral vectors according to the present invention and/or capsid are applied to this needs by any method known in the art Person or animal.Optionally, viral vectors and/or capsid pharmaceutically have in acceptable carrier with treatment effective dose or prevention Imitate dose delivery.

Can further apply the disclosure viral vectors and/or capsid to cause immunogenic response (for example, as epidemic disease Seedling).In general, the immunogenic composition of the disclosure is a effective amount of comprising the immunogenicity combined with pharmaceutically acceptable carrier Viral vectors and/or capsid.Optionally, the dosage is enough to generate protective immune response (as defined above).The guarantor of imparting Shield degree needs not be complete or permanent, as long as the benefit of application immunogenic polypeptide is more than its any disadvantage.By Examination person and immunogene are as described above.

The dosage of viral vectors and/or capsid to be administered to subject depends on method of application, to be treated and/or prevention Disease or the patient's condition, the patient's condition of individual subjects, specific viral vectors or capsid and nucleic acid to be delivered etc., and It can determine in a usual manner.Exemplary dose for realizing therapeutic effect is at least about 10⁵、10⁶、10⁷、10⁸、10⁹、 10¹⁰、10¹¹、10¹²、10¹³、10¹⁴、10¹⁵The titre of a transduced unit, optionally about 10⁸-10¹³The titre of a transduced unit.

It in certain embodiments, can be using more than one application (for example, two, three, four or more times Application), to realize desired gene expression dose in different interim (for example, daily, weekly, monthly, every year etc.).

Exemplary method of application includes oral, rectum, and transmucosal intranasally sucks (for example, passing through aerosol), mouth Chamber (for example, sublingual), vagina is intrathecal, intraocularly, transdermal, intrauterine (or in ovum), parenteral (for example, it is intravenous, subcutaneously, skin Interior, intramuscular is intradermal, in pleura, intracerebral and intra-articular), part (for example, to skin and mucomembranous surface, including airway surface and Transdermal administration), in lymphatic vessel etc., and directly tissue or organ injection (for example, to liver, skeletal muscle is myocardium, diaphram or Brain).In some embodiments, intramuscular includes being applied to skeletal muscle, diaphram and/or cardiac muscle.Application is also possible to be applied to swollen Tumor (for example, in tumour or lymph node or near).In any given situation, most suitable approach will depend on treat with/ Or prevention the patient's condition property and severity and used specific support property.

The delivering to target tissue can also be realized by delivering the reservoir (depot) comprising viral vectors and/or capsid. In representative embodiment, skeletal muscle, cardiac muscle and/or diaphram tissue will be implanted into comprising the reservoir of viral vectors and/or capsid In, or tissue can be made to contact with the film comprising viral vectors and/or capsid or other matrix.This implantable base or bottom Object is described in United States Patent (USP) No.7,201,898.

In specific embodiments, viral vectors according to the present invention and/or viral capsid are applied to skeletal muscle, diaphragm Flesh and/or cardiac muscle (for example, to treat and/or prevent muscular dystrophy, heart disease).In some embodiments, basis is applied Viral vectors and/or viral capsid of the invention is to treat PAD or congestive heart failure.

It can also implement disclosed method to generate antisense RNA, RNAi or other function RNA (for example, ribozyme) and use In systemic delivery.

Injected material can be prepared with conventionally form, can be liquid solution or suspension, be suitble to dissolve or hang before the injection Floating solid form or lotion in a liquid.It is alternatively possible to apply the disease of the disclosure in the way of local and non-systemic Poisonous carrier and/or viral capsid, for example, in reservoir or sustained release preparation.In addition, viral vectors and/or viral capsid can be attached In the matrix for the implantation that can perform the operation deliver (for example, as described in U.S. Patent Publication No.US-2004-0013645-A1).

Viral vectors and viral capsid can be applied to the tissue (for example, brain, eye) of CNS, and can advantageously cause Viral vectors or capsid are more widely distributed than what is observed in the case where no disclosure.

In specific embodiments, the delivery vector of the disclosure can be applied to treat the disease of CNS, including hereditary disease Disease, neurodegenerative disease, mental disease and tumour.

The Exemplary diseases of CNS include but is not limited to Alzheimer disease, Parkinson's disease, Huntington disease, canavan's disease (Canavan disease), Leigh disease, Refsum disease, tourette syndrome, primary lateral sclerosis, amyotrophic side Rope sclerosis (ALS), progressive myatrophy disease, Pick disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Bin Si Prosperous lattice disease, wound caused by spinal cord injury and/or head injury (such as traumatic brain injury), Tay Sachs disease, thunder are had a rest disease (Lesch-Nyan disease), epilepsy, apoplexy, cerebral infarction, phrenoblabia include emotional handicap (for example, depression, two-phase feelings Feel obstacle, continue the disturbance of emotion, secondary emotional handicap), schizophrenia, pharmacological dependence is (for example, alcoholism and other substances Rely on), neurotica (for example, anxiety, obsessive-compulsive disorder, somatoform disorder, dissociative disorder is sad, post-natal depression), essence Refreshing disease (such as illusion and illusion), dull-witted, paranoea, attention deficit disorder, psychosexual disorder are any to benefit from or need axis Prominent/neuron regeneration and/or the neurodegenerative disease of reparation, cognitive disorder, behavior disorder, sleep disturbance, pain disorder, into The cancer and tumour (example of food or body weight disorders (such as obesity, cachexia, anorexia nervosa and baulimia (bulemia)) and CNS Such as, pituitary tumor).

The illness of CNS includes being related to retina, the eye disease of rear road and optic nerve (such as retinitis pigmentosa, sugar Urinate characteristic of disease retinopathy and other retina degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

One of most of (if not all) ophthalmology diseases and illness and three kinds of indications or more are related: (1) angiogenesis, (2) inflammation, and (3) denaturation.Delivery vector of the invention can be used for delivering anti-angiogenesis；It is anti-inflammatory because Son；Delay cell degeneration, promotes cell to retain (cell sparing) or promote the factor and combination above-mentioned of cell growth.

For example, diabetic retinopathy is characterized in that angiogenesis.Diabetic retinopathy can be by intraocular (for example, in vitreum) or eye circumference (for example, the region under fascia bulbi) deliver one or more of anti-angiogenesis To treat.One or more of neurotrophic factors can also intraocularly (for example, in vitreum) or eye circumference deliver jointly.

Uveitis is related to inflammation.The delivery vector of the disclosure can be applied by intraocular (for example, vitreum or anterior chamber) To apply one or more of anti-inflammatory factors.

In contrast, retinitis pigmentosa is characterized in that retinosis.In representative embodiment, pigment Property the retinitis can be applied by encoding the intraocular of delivery vector of one or more of neurotrophic factors (for example, glass Body application) it treats.

Age-related macular degeneration is related to angiogenesis and retinosis.It can be applied by intraocular (such as vitreum) With the delivery vector and/or intraocular or eye circumference of the one or more of neurotrophic factors of coding of the invention (for example, in eyeball muscle Region under film) delivery vector of the one or more of anti-angiogenesis of coding of the invention is applied to treat the illness.

Glaucoma is characterized in that intraocular pressure increases and retinal ganglial cells are lost.The treatment of glaucoma includes using this The delivery vector application one or more of invention protect cells from the neuroprotective agent of exitotoxicity damage.These medicament packets Include intraocular delivery, N-methyl-D-aspartate (NMDA) antagonist of optional intraocular delivery, cell factor and neurotrophy because Son.

In other embodiments, the disclosure can be used for treating epileptic attack, for example, with reduce the breaking-out of epileptic attack, Incidence or severity.The effect of therapeutic treatment of epileptic attack, can be by behavior (for example, eyes or oral cavity are shaken Dynamic, ticktack (ticks)) and/or electrograph means (most epilepsy breaking-out has characteristic electrograph abnormal) assess.Therefore, The present invention can also be used to treat epilepsy, over time characterized by multiple epileptic attack.

In a representative embodiment, growth hormone release inhibiting hormone (or its active fragment) is applied using the delivery vector of the disclosure With to brain to treat pituitary tumor.According to the embodiment, the delivery vector of encoding growth chalone (or its active fragment) passes through Micro infusion is administered in hypophysis.Equally, this treatment can be used for treating acromegalia (the misgrowth hormone of hypophysis point It secretes).Nucleic acid (such as GenBank accession number J00306) and amino acid (such as the GenBank accession number P01166 of growth hormone release inhibiting hormone； Active peptide somatostatin-28 and somatostatin-14 containing processing) sequence is known in the art.

In specific embodiments, carrier may include secretion signal, such as United States Patent (USP) No.7, described in 071,172.

In the representative embodiment of the disclosure, viral vectors and/or viral capsid are applied to CNS (for example, application To brain or eyes).Viral vectors and/or capsid can be introduced into spinal cord, brain stem (oblongata, pons), midbrain (hypothalamus, thalamus, Epithalamus, pituitary, black substance, pineal body), cerebellum, (corpus straitum, brain include occipital lobe, temporal lobe, top and frontal lobe, skin to akrencephalon Matter, basal ganglion, hippocampus and portal vein tongue (portaamygdala), limbic system, neopallium, corpus straitum, brain is under Mound.Viral vectors and/or capsid can also be applied to the different zones of eyes, such as retina, cornea and/or optic nerve.

Viral vectors and/or capsid can be delivered in celiolymph (for example, passing through lumbar puncture) for deliver load The more dispersion of body is applied.Viral vectors and/or capsid can further (such as the brains in the case where blood-brain barrier is disturbed Tumour or cerebral infarction) intravascular it is applied to CNS.

Viral vectors and/or capsid can be applied to the desired zone of CNS by any approach known in the art, including but It being not limited in the ventricles of the brain, in brain pond, in essence, encephalic is intrathecal, intraocularly, and intracerebral, intra-ventricle, intravenously (for example, in such as sweet dew In the presence of the sugar of alcohol), intranasally, in ear, intraocular (for example, in vitreum, under retina, anterior chamber) and eye circumference (for example, Under fascia bulbi) it conveys and is delivered to motor neuron with the intramuscular of retrograde delivering.

In specific embodiments, viral vectors and/or capsid pass through direct injection (for example, stereotactic injection) In The desired zone or compartment being applied in liquid preparation in CNS.In other embodiments, viral vectors and/or capsid can be with Desired zone is provided to by topical application or is provided by intranasal administration aerosol preparation.Eyes are applied to, can be passed through The local application of drop.As another alternative, viral vectors and/or capsid can be used as solid sustained-release preparation application (see, e.g., United States Patent (USP) No.7,201,898).

In a further embodiment, viral vectors, which can be used for driving in the wrong direction transporting, is related to kinesitherapy nerve to treat and/or prevent The disease and illness of member are (for example, amyotrophic lateral sclerosis (ALS)；Spinal muscular atrophy (SMA) etc.).For example, virus carries Body can be delivered to musculature, and therefrom it can be moved in neuron.

Following embodiment is for illustration purposes only, rather than restrictive.

Embodiment