Variant AAV capsids for intravitreal delivery
Provided are variant adeno-associated virus (AAV) capsid proteins and recombinant AAV virions having one or more variant AAV capsid proteins. Also provided are compositions and methods for the use of the recombinant AAV virions, such as for the treatment or prophylaxis of a disease or disorder.
1. A recombinant adeno-associated virus (AAV) virion comprising:
(a) a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parental AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to an AAV5 VP1 capsid protein; and
(b) a polynucleotide sequence encoding a therapeutic gene product.
2. The recombinant AAV virion of claim 1 , wherein the parental AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein.
3. The recombinant AAV virion of claim 1 , wherein the parental AAV capsid protein is an AAV2.5T capsid protein.
4. The recombinant AAV virion of claim 1 , wherein the parental AAV capsid protein is an AAV2.5T VP1 capsid protein.
5. The recombinant AAV virion of claim 1 , wherein the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3).
6. The recombinant AAV virion of claim 1 , wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO:5 or a capsid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.
7. The recombinant AAV virion of claim 1 , wherein the rAAV virion is a variant AAV5 or a variant AAV2 and AAV5 hybrid virion.
8. The recombinant AAV virion of claim 1 , wherein the rAAV virion is a variant AAV2.5T virion.
9. The recombinant AAV virion of claim 1 , wherein the recombinant AAV virion is capable of transducing cells of the retina when intravitreally injected into a mammal.
10. The recombinant AAV virion of claim 9 , wherein the recombinant AAV virion is capable of transducing one or more of: a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, and a retinal pigment epithelium cell when intravitreally injected into a mammal.
11. The recombinant AAV virion of claim 9 , wherein the recombinant AAV virion is capable of transducing retinal pigment epithelium cells when intravitreally injected into a mammal.
12. The recombinant AAV virion of claim 1 , wherein the therapeutic gene product is a siRNA, a miRNA, or a protein.
13. The recombinant AAV virion of claim 1 , wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product or an opsin.
14. The recombinant AAV virion of claim 1 , wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.
15. The recombinant AAV virion of claim 14 , wherein the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof.
16. The recombinant AAV virion of claim 14 , wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.
17. The recombinant AAV virion of claim 1 , wherein the recombinant AAV virion has an altered cellular tropism as compared to AAV2.5 T.
18. A pharmaceutical composition comprising the recombinant AAV virion of claim 1 .
19. A method for producing a rAAV virion comprising:
(a) culturing a host cell under a condition that rAAV virions are produced, wherein the host cell comprises:
(i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parental AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1);
(ii) a polynucleotide encoding a rep protein;
(iii) a polynucleotide cassette comprising a sequence that encodes a therapeutic gene product flanked by at least one AAV ITR; and
(iv) AAV helper functions; and
(b) recovering the rAAV virion produced by the host cell.
20. A method of providing a therapeutic gene product to a retina of a subject, comprising administering to the subject by intravitreal injection the recombinant AAV virion of claim 1 .
21. A method of treating a disease or disorder of the retina of a subject in need thereof, comprising administering to the subject by intravitreal injection the recombinant AAV virion of claim 1 .
22. The recombinant AAV virion of claim 1 for use in a method of treating a disease or disorder of the retina of a subject in need thereof, wherein the method comprises administering a pharmaceutical composition comprising the recombinant AAV virion to the subject by intravitreal injection.
23. The recombinant AAV virion of claim 1 for use in the preparation of a medicament for the treatment of a disease or disorder of the retina of a subject.
24. A variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parental AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to an AAV5 VP1 capsid protein.
25. The variant AAV capsid protein of claim 24 , wherein the parental AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein.
26. The variant AAV capsid protein of claim 24 , wherein the parental AAV capsid protein is an AAV2.5T capsid protein.
27. The variant AAV capsid protein of claim 24 , wherein the parental AAV capsid protein is an AAV2.5T VP1 capsid protein.
28. The variant AAV capsid protein of claim 24 , wherein the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO: 3) at amino acid residues 570-579 relative to the parental AAV capsid protein.
29. The variant AAV capsid protein of claim 24 , comprising a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO:5, or a capsid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.
30. A nucleic acid comprising a nucleic acid sequence encoding the variant AAV capsid protein of claim 24 .
31. An expression vector comprising the nucleic acid of claim 30 , wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence.
32. A cell comprising the expression vector of claim 31 .