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, when administered via intravitreal injection, 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 a peptide insertion relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence LGETTRP (SEQ ID NO:13), and wherein the insertion site is located between two adjacent amino acids at a position between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype; and
b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
2. The rAAV virion of claim 1 , wherein the peptide insertion contains the sequence TRP or TRPA at its C-terminus.
3. The rAAV virion of claim 1 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.
4. The rAAV virion of claim 1 , wherein the variant capsid protein confers increased infectivity of a photoreceptor cell by the rAAV virion compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein.
5. The rAAV virion of claim 1 , wherein the peptide insertion permits transduction of a photoreceptor cell by the rAAV when the rAAV is administered by intravitreal injection.
6. The rAAV virion of claim 1 , wherein the gene product is an interfering RNA.
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 or an anti-angiogenic polypeptide.
9. The rAAV virion of claim 3 , wherein the rAAV virion exhibits increased ability to cross the inner limiting membrane (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM.
10. A pharmaceutical composition comprising:
a) the recombinant adeno-associated virus virion of claim 1 ; and
b) a pharmaceutically acceptable excipient.
11. A method of delivering a gene product to a photoreceptor cell in an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
12. The method of claim 11 , wherein the gene product is an interfering RNA.
13. The method of claim 11 , wherein the gene product is a polypeptide.
14. The method of claim 13 , wherein the polypeptide is a neuroprotective factor, an anti-angiogenic polypeptide or an anti-apoptotic factor.
15. The method of claim 13 , wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, a rhodopsin, or Sonic hedgehog.
16. A method of treating an ocular 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 .
17. The method of claim 16 , wherein said administering is by intraocular injection.
18. The method of claim 16 , wherein said administering is by intravitreal injection.
19. The method of claim 16 , wherein the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, or diabetic retinopathy.
20. 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 a peptide insertion relative to a corresponding parental AAV capsid protein, wherein the peptide insertion has a length of from 7 amino acids to 10 amino acids and comprises the amino acid sequence LGETTRP (SEQ ID NO:13), wherein the insertion site is located between two adjacent amino acids at a position between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.
21. The isolated nucleic acid of claim 20 , wherein the peptide insertion contains the sequence TRP or TRPA at its C-terminus.
22. The isolated nucleic acid of claim 20 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.
23. The isolated nucleic acid of claim 20 , wherein the variant AAV capsid protein, when present in an AAV virion, provides for increased infectivity of a photoreceptor cell by the AAV virion compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein.
24. The isolated nucleic acid of claim 20 , wherein the variant AAV capsid protein, when present in an AAV virion, provides for increased ability to cross the inner limiting membrane (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM.
25. An isolated, genetically modified host cell comprising the nucleic acid of claim 20 .
26. A variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein the peptide insertion has a length of from 7 amino acids to 10 amino acids and comprises the amino acid sequence LGETTRP (SEQ ID NO:13), wherein the insertion site is located between two adjacent amino acids at a position between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.
27. The variant AAV capsid protein of claim 26 , wherein the peptide insertion contains the sequence TRP or TRPA at its C-terminus.
28. The variant AAV capsid protein of claim 26 , wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another AAV serotype.
29. The variant AAV capsid protein of claim 26 , wherein the variant capsid protein, when present in an AAV virion, confers increased infectivity of a photoreceptor cell by the AAV virion compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein.
30. The variant AAV capsid protein of claim 26 , wherein the variant AAV capsid protein, when present in an AAV virion, provides for increased ability to cross the inner limiting membrane (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM.
31. The rAAV virion of claim 3 , wherein the peptide insertion is inserted between amino acids 587 and 588 of VP1 of AAV2.
32. The rAAV virion of claim 31 , wherein the gene product is an anti-angiogenic polypeptide.
33. The method of claim 18 , wherein the peptide insertion is inserted between amino acids 587 and 588 of VP1 of AAV2.
34. The method of claim 33 , wherein the gene product is an anti-angiogenic polypeptide.
35. A recombinant adeno-associated virus 2 (rAAV2) virion comprising:
a) a variant AAV2 VP1 capsid protein, wherein the variant AAV2 VP1 capsid protein comprises a peptide insertion relative to a corresponding parental AAV2 VP1 capsid protein, wherein the peptide insertion has a length of from 7 amino acids to 10 amino acids and comprises the sequence of LGETTRP (SEQ ID NO:13), and wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2; and
b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
36. The rAAV2 virion of claim 35 , wherein the peptide insertion is inserted between amino acids 587 and 588 of VP1 of AAV2.
37. The rAAV2 virion of claim 36 , wherein the gene product is an anti-angiogenic polypeptide.
38. A pharmaceutical composition comprising:
a) the rAAV2 virion of claim 37 ; and
b) a pharmaceutically acceptable excipient.
39. A method of treating an ocular disease, the method comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus 2 (rAAV2) virion according to claim 37 .