Adeno-associated virus virions with variant capsid and methods of use thereof
The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where the AAV virions exhibit greater infectivity of retinal cells compared to wild-type AAV. The present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating ocular disease.
1. A recombinant adeno-associated virus (rAAV) virion comprising:
a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an amino acid substitution at amino acid 451 of the AAV6 capsid sequence as set forth in SEQ ID NO:1, or the corresponding position in another AAV parental serotype, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising a wild-type AAV capsid protein, wherein the AAV capsid protein does not comprise an amino acid sequence present in a naturally occurring AAV capsid protein; and
b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
2. The rAAV virion of claim 1 , wherein the retinal cell is a Müller glial cell.
3. The rAAV virion of claim 1 , wherein the rAAV virion exhibits at least 5-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
4. The rAAV virion of claim 1 , wherein the rAAV virion exhibits at least 50-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
5. The rAAV virion of claim 1 , wherein gene product is a nucleic acid gene product.
6. The rAAV virion of claim 5 , wherein the nucleic acid gene product is an interfering RNA, a ribozyme, an antisense nucleic acid, or an aptamer.
7. The rAAV virion of claim 1 , wherein the gene product is a polypeptide.
8. The rAAV virion of claim 7 , wherein the polypeptide is a neuroprotective polypeptide.
9. The rAAV virion of claim 7 , wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, nurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, a soluble vascular endothelial growth factor (VEGF) receptor, an anti-VEGF antibody, or Sonic hedgehog.
10. The rAAV virion of claim 7 , wherein the polypeptide is an anti-angiogenic polypeptide.
11. The rAAV virion of claim 1 , wherein the parental AAV capsid protein is wild-type AAV6 capsid protein.
12. The rAAV virion of claim 6 , wherein the interfering RNA or the aptamer reduces the level of an angiogenic factor in the retinal cell.
13. The rAAV virion of claim 1 , wherein the variant capsid protein provides for selective infection of a Müller glial cell compared to other cells in the eye.
14. The rAAV virion of claim 1 , wherein the variant AAV capsid protein comprises an amino acid change at AAV6 position 532 or the corresponding position in another AAV parental serotype.
15. The rAAV virion of claim 14 , wherein the amino acid substitution at amino acid 532 of AAV6, or the corresponding position in another AAV parental serotype, is an asparagine.
16. The rAAV virion of claim 1 , wherein the amino acid substitution at amino acid 451 of AAV6, or the corresponding position in another AAV parental serotype, is an aspartic acid protein comprises.
17. The rAAV virion of claim 1 , wherein the variant AAV capsid protein comprises an asparagine to aspartic acid substitution at amino acid 451 of AAV6, or the corresponding position in another AAV parental serotype and an aspartic acid to asparagine substitution at amino acid 532 of AAV6, or the corresponding position in another AAV parental serotype.
18. The rAAV virion of claim 1 , wherein the variant capsid protein comprises from 1 to 10 amino acid differences compared to a wild-type AAV capsid protein.
19. The rAAV virion of claim 1 , wherein the retinal cell is a retinal glial cell.
20. The rAAV virion of claim 1 , wherein the retinal cell is an astrocyte.
21. A pharmaceutical composition comprising:
a) a recombinant adeno-associated virus (rAAV) virion according to claim 1 ; and
b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer.
22. A method of delivering a gene product to a retinal cell in an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
23. The method of claim 22 , wherein the gene product is a polypeptide.
24. The method of claim 22 , wherein the gene product is a nucleic acid.
25. The method of claim 23 , wherein the polypeptide is a neuroprotective factor or an anti-angiogenic factor.
26. The method of claim 25 , wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, nurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, a soluble vascular endothelial growth factor (VEGF) receptor, an anti-VEGF antibody, or Sonic hedgehog.
27. A method of treating a retinal disease, the method comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
28. The method of claim 27 , wherein said administering is by intraocular injection.
29. The method of claim 27 , wherein said administering is by intravitreal injection.
30. The method of claim 27 , wherein the ocular disease is glaucoma, retinitis pigmentosa, or macular degeneration.
31. An isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an amino acid substitution at amino acid 451 of the AAV6 capsid sequence as set forth in SEQ ID NO:1, or the corresponding position in another AAV parental serotype, and wherein the variant capsid protein, when present in an AAV virion, provides for increased infectivity of the AAV virion for a retinal cell.
32. An isolated, genetically modified host cell comprising the nucleic acid of claim 31 .
33. A variant AAV capsid protein, wherein the variant AAV capsid protein comprises an amino acid change at AAV6 position 532 or the corresponding position in another AAV parental serotype and wherein the variant capsid protein confers increased infectivity of a Müller glial cell compared to the infectivity of the Müller glial cell by an AAV virion comprising a wild-type AAV capsid protein, wherein the AAV capsid protein does not comprise an amino acid sequence present in a naturally occurring AAV capsid protein.
34. The capsid protein of claim 33 , where the amino acid change is D532N, alone or in combination with N451D.
35. A nucleic acid encoding a variant AAV capsid, wherein the nucleic acid encodes an amino acid change at AAV6 position 532 or the corresponding position in another AAV parental serotype and wherein the variant capsid protein confers increased transduction of a Müller glial cell compared to the transduction of the Müller glial cell by an AAV virion comprising a wild-type AAV capsid protein, wherein the AAV capsid protein does not comprise an amino acid sequence present in a naturally occurring AAV capsid protein.
36. The nucleic acid of claim 35 , where the amino acid change is D532N, alone or in combination with N451D.