Adeno-Associated Virus-Based Vectors for Delivery of Nucleic Acids to the Retina

Claims

1. An adeno-associated virus (AAV) vector comprising an AAV capsid polypeptide, wherein said capsid polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-5.

2. The vector according to claim 1, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that said amino acid sequence of any of SEQ ID NO: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO:

10.

3. The vector according to claim 1, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that said amino acid sequence of SEQ ID NO: 5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO:

10.

4. The vector according to claim 1, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of any of SEQ ID NO: 2-5.

5. The vector according to claim 1, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of SEQ ID NO:

5.

6. A vector according to any one of paragraphs 1-5, characterized in that said vector is a vector based on AAV2.

7. A vector according to any one of paragraphs 1-6, characterized in that said vector infects more than 2 percent of retinal cells in two or more areas of the retina when said vector is administered intravitrially into a human eye at a titer of at least 1×10 7 .

8. A vector according to any one of paragraphs 1-7, characterized in that said vector contains an exogenous nucleic acid encoding RNA or a polypeptide.

9. The vector according to claim 8, characterized in that said exogenous nucleic acid encodes RNA.

10. The vector according to claim 9, characterized in that said RNA is siRNA or microRNA.

11. The vector according to claim 8, characterized in that said exogenous nucleic acid encodes a polypeptide.

12. The vector according to claim 11, characterized in that said polypeptide is an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide.

13. The vector of any one of claims 1-12, wherein said vector expresses more nucleic acid in retinal cells in at least two regions of the retina than a comparable AAV-based vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, wherein said at least two regions of the retina are selected from the group consisting of the foveal region, the parafoveal region, the vascular arcade region, and the peripheral region.

14. The vector according to any one of paragraphs 1-13, characterized in that said capsid polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 11-42.

15. A composition comprising a vector according to any one of claims 1-14 and a pharmaceutically acceptable excipient.

16. The composition according to claim 15, characterized in that said composition contains from about 1×10 7 up to about 1×10 14 specified vectors.

17. The composition according to any one of claims 15, 16, characterized in that said pharmaceutically acceptable excipient contains one or more of: phosphate buffered saline, Hank's balanced salt solution and Pluronic F68.

18. A method for delivering an exogenous nucleic acid sequence to retinal cells in at least two different regions of the retina of a mammal, said method comprising contacting said retinal cells with an AAV-based vector comprising an AAV capsid polypeptide and said exogenous nucleic acid sequence, said capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5, said AAV-based vector infecting retinal cells within said at least two different regions of the retina, thereby delivering said exogenous nucleic acid sequence to said retinal cells, said at least two regions of the retina being selected from the group consisting of the foveal region, the parafoveal region, the vascular arcade region, and the peripheral region.

19. The method according to claim 18, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that said amino acid sequence of any of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO:

10.

20. The method according to claim 18, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that said amino acid sequence of SEQ ID NO: 5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO:

10.

21. The method according to claim 18, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of any of SEQ ID NO: 2-5.

22. The method according to claim 18, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of SEQ ID NO:

5.

23. The method according to any one of paragraphs 18-22, characterized in that said mammal is a human.

24. The method according to any one of paragraphs 18-23, characterized in that said vector is a vector based on AAV2.

25. The method according to any one of paragraphs 18-24, characterized in that said vector infects more than 2 percent of the retinal cells within said at least two areas of the retina when said vector is administered intravitrially into the human eye in a titer of at least 1×10 7 .

26. The method according to any one of paragraphs 18-25, characterized in that the said exogenous nucleic acid sequence encodes RNA or a polypeptide.

27. The method according to claim 26, characterized in that said exogenous nucleic acid encodes RNA.

28. The method according to claim 27, characterized in that said RNA is siRNA or microRNA.

29. The method according to claim 26, characterized in that said exogenous nucleic acid encodes a polypeptide.

30. The method according to claim 29, characterized in that said polypeptide is an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide.

31. The method according to any one of claims 18-30, characterized in that said vector expresses more of said exogenous nucleic acid sequence in said retinal cells of said at least two retinal regions than a comparable AAV-based vector containing a capsid polypeptide consisting of the amino acid sequence indicated in SEQ ID NO: 1 expresses in retinal cells.

32. The method according to any one of paragraphs 18-31, characterized in that said method comprises intravitreal administration of a composition containing said vector to said mammal, thereby bringing said retinal cells into contact with said vector.

33. The method according to claim 32, characterized in that said composition contains from about 1×10 7 up to about 1×10 14 specified vectors.

34. A method for treating a retinal disease in a mammal in need thereof, said method comprising contacting retinal cells of at least two retinal regions of a mammalian eye having said retinal pathology with AAV-based vectors comprising an AAV capsid polypeptide and an exogenous nucleic acid sequence, wherein said capsid polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, wherein said AAV-based vectors infect said retinal cells of said at least two retinal regions and provide expression of said exogenous nucleic acid sequence in said retinal cells of said at least two retinal regions, thereby treating said retinal pathology.

35. The method according to claim 34, characterized in that said mammal is a human.

36. The method according to any one of paragraphs 34, 35, characterized in that said retinal pathology is selected from the group consisting of LCA, OCA1, retinitis pigmentosa, rod-cone dystrophy, cone dystrophy, Stargardt disease, Usher syndrome, XLRP and XLRS.

37. The method according to any one of paragraphs 34-36, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1, except that said amino acid sequence of any of SEQ ID NO: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO:

1.

38. The method according to any one of paragraphs 34-36, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that said amino acid sequence of SEQ ID NO: 5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO:

10.

39. The method according to any one of claims 34-36, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of any one of SEQ ID NO: 2-5.

40. The method according to any one of paragraphs 34-36, characterized in that said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by said amino acid sequence of SEQ ID NO:

5.

41. The method according to any one of paragraphs 34-40, characterized in that said vectors are AAV2-based vectors.

42. The method according to any one of paragraphs 34-41, characterized in that said vectors infect more than 2 percent of retinal cells in said at least two areas of the retina when said vector is administered intravitrially into the human eye in a titer of at least 1×10 7 .

43. The method according to any one of paragraphs 34-42, characterized in that the said sequence of exogenous nucleic acid encodes RNA.

44. The method according to claim 43, characterized in that said RNA is siRNA or microRNA.

45. The method according to any one of paragraphs 34-42, characterized in that said exogenous nucleic acid encodes a polypeptide.

46. ​​The method according to claim 45, characterized in that said polypeptide is an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, and an NR2E3 polypeptide.

47. The method according to any one of claims 34-46, characterized in that said vectors express more of said exogenous nucleic acid sequence in said retinal cells of said at least two retinal regions than a comparable AAV-based vector containing a capsid polypeptide consisting of the amino acid sequence indicated in SEQ ID NO: 1 expresses in retinal cells of said at least two retinal regions.

48. The method according to any one of paragraphs 34-47, characterized in that said method comprises intravitreal administration of a composition containing said vectors to said mammal, thereby bringing said retinal cells of said at least two regions of the retina into contact with said vectors.

49. The method according to claim 48, characterized in that said composition contains from about 1×10 7 up to about 1×10 14 specified vectors.

50. The method according to any one of paragraphs 34-49, characterized in that said at least two regions of the retina are selected from the group consisting of the foveal region, the parafoveal region, the vascular arcade region, and the peripheral region.

51. A non-natural adeno-associated virus (AAV) vector comprising an AAV capsid polypeptide, wherein said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, containing an insert of the amino acid sequence of Formula A located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO: 10, wherein said Formula A is: -L1-EHQTRP (SEQ ID NO: 2)-L2-, wherein said L1 and said L2 each independently represent an optional amino acid linker comprising one, two or three amino acids.

52. The vector according to claim 51, characterized in that said L1 is one amino acid X1.

53. The vector according to claim 52, characterized in that said X1 is selected from the group of amino acid residues consisting of A, V, I and L.

54. The vector according to claim 52, characterized in that said X1 is A.

55. The vector according to claim 51, characterized in that said L1 represents two amino acids X2-X1.

56. The vector according to claim 55, characterized in that said X1 is selected from the group of amino acid residues consisting of A, V, I and L.

57. The vector according to claim 56, characterized in that said X1 is A.

58. A vector according to any one of paragraphs 55-57, characterized in that said X2 is selected from the group of amino acid residues consisting of A, V, I and L.

59. The vector according to claim 58, characterized in that said X2 is L.

60. The vector according to paragraph 55, characterized in that said X2-X1 is LA.

61. The vector according to claim 51, characterized in that said L1 represents three amino acids X3-X2-X1.

62. The vector according to claim 61, characterized in that said X1 is selected from the group of amino acid residues consisting of A, V, I and L.

63. The vector according to claim 62, characterized in that said X1 is A.

64. A vector according to any one of paragraphs 61-63, characterized in that said X2 is selected from the group of amino acid residues consisting of A, V, I and L.

65. The vector according to claim 64, characterized in that said X2 is L.

66. The vector according to claim 61, characterized in that said X2-X1 is LA.

67. A vector according to any one of paragraphs 61-66, characterized in that said X3 is selected from the group of amino acid residues consisting of A, V, I and L.

68. The vector according to paragraph 51, characterized in that said L1 is absent.

69. A vector according to any one of paragraphs 51-68, characterized in that said L2 is one amino acid of Z1.

70. The vector according to claim 69, characterized in that said Z1 is selected from the group of amino acid residues consisting of A, V, I and L.

71. The vector according to paragraph 69, characterized in that said Z1 is A.

72. A vector according to any one of paragraphs 51-68, characterized in that said L2 represents two amino acids Z1-Z2.

73. The vector according to claim 72, characterized in that said Z1 is selected from the group of amino acid residues consisting of A, V, I and L.

74. The vector according to claim 72, characterized in that said Z1 is A.

75. A vector according to any one of paragraphs 72-74, characterized in that said Z2 is selected from the group of amino acid residues consisting of A, V, I and L.

76. The vector according to claim 75, characterized in that said Z2 is L.

77. The vector according to item 76, characterized in that said Z1-Z2 represents AL.

78. A vector according to any one of paragraphs 51-68, characterized in that said L2 is three amino acids Z1-Z2-Z3.

79. The vector according to claim 78, characterized in that said Z1 is selected from the group of amino acid residues consisting of A, V, I and L.

80. The vector according to paragraph 79, characterized in that said Z1 is A.

81. A vector according to any one of paragraphs 78-80, characterized in that said Z2 is selected from the group of amino acid residues consisting of A, V, I and L.

82. The vector according to claim 81, characterized in that said Z2 is L.

83. The vector according to claim 78, characterized in that said Z1-Z2 represents AL.

84. A vector according to any one of paragraphs 78-83, characterized in that said Z3 is selected from the group of amino acid residues consisting of A, V, I and L.

85. A vector according to any of paragraphs 51-68, characterized in that said L2 is absent.

86. The vector according to claim 51, characterized in that said amino acid sequence insert contains any of SEQ ID NO: 2-5.

87. A non-naturally occurring AAV capsid polypeptide, wherein said capsid polypeptide comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, comprising an insertion of an amino acid sequence of Formula A located between amino acid positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO: 10, wherein said Formula A is: -L1-EHQTRP (SEQ ID NO: 2)-L2-, wherein said L1 and said L2 each independently represent an optional amino acid linker comprising one, two or three amino acids.

88. A method for administering an exogenous nucleic acid sequence to a mammal in need thereof, wherein said method comprises administering an effective amount of a vector according to any one of paragraphs 51-86 to said mammal, wherein said vector comprises said exogenous nucleic acid sequence.

89. The method according to claim 88, characterized in that said mammal is a human.

90. The method according to any one of paragraphs 88, 89, characterized in that said administration comprises administering said effective amount into the eye of said mammal.

91. The method according to any one of paragraphs 88-90, characterized in that said introduction is sufficient to ensure expression of said exogenous nucleic acid sequence in a cell of said mammal.

92. The method according to any one of paragraphs 88-91, characterized in that the said exogenous nucleic acid sequence encodes a therapeutic polypeptide.

93. A method for treating a retinal disease in a patient in need thereof, comprising administering to the eye of the patient an effective amount of an AAV-based vector, wherein the AAV-based vector comprises an AAV capsid polypeptide and an exogenous nucleic acid sequence, wherein the AAV capsid polypeptide is represented by formula A.