ADENO-ASSOCIATED VIRUS VARIANT CAPSIDS AND METHODS OF USE THEREOF
Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer increased infectivity of one or more types of retinal cells as compared to the infectivity of the retinal cells by an AAV virion comprising the unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising a variant AAV capsid protein as described herein, methods of making these rAAV capsid proteins and virions, and methods for using these rAAV capsid proteins and virions in research and in clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more cells of the retina for the treatment of retinal disorders and diseases.
1 . A method for treating a human subject with wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization, the method comprising administering a therapeutically effective amount of an infectious recombinant adeno-associated virus (rAAV) particle, the rAAV particle comprising (i) a capsid comprising a capsid protein comprising a heterologous peptide with a length of 7-11 amino acids covalently inserted between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ ID NO:2) or the corresponding position in the capsid protein of another AAV serotype, the peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) and having from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the amino and/or carboxyl terminus of amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) a nucleic acid comprising a nucleotide sequence encoding a first anti-angiogenic polypeptide that inhibits the activity of a vascular endothelial growth factor (VEGF), said nucleotide sequence operably linked to an expression control sequence, or administering a pharmaceutical composition comprising said infectious rAAV particle and a pharmaceutically acceptable excipient.
2 . The method according to claim 1 , wherein the nucleic acid further comprises (a) a nucleotide sequence encoding a second anti-angiogenic polypeptide or (b) a nucleotide sequence encoding an interfering RNA that reduces expression of a pro-angiogenic gene, said nucleotide sequence operably linked to an expression control sequence.
3 . The method according to claim 1 , wherein the capsid protein comprises a P34A amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2) or the corresponding substitution in the capsid protein of another AAV serotype.
4 . The method according to claim 3 , wherein the capsid protein comprises an amino acid sequence at least 90% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:42.
5 . The method according to claim 4 , wherein the capsid protein comprises an amino acid sequence at least 95% identical to the entire length of the amino acid sequence set forth as SEQ ID NO:42.
6 . The method according to claim 5 , wherein the capsid protein comprises the amino acid sequence set forth as SEQ ID NO:42.
7 . The method according to claim 5 , wherein the capsid protein consists of the amino acid sequence set forth as SEQ ID NO:42.
8 . The method according to claim 2 , wherein the capsid protein comprises amino acid sequence set forth as SEQ ID NO:42 or comprises a sequence at least 95% identical thereto and comprises a P34A amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2).
9 . The method according to claim 8 , wherein the nucleotide sequence encoding the first anti-angiogenic polypeptide that inhibits the activity of a VEGF and/or the nucleotide sequence encoding the second anti-angiogenic polypeptide or interfering RNA that reduces expression of a pro-angiogenic gene is operably linked to an expression control sequence comprising a constitutively active promoter.
10 . The method according to claim 9 , wherein the nucleotide sequence encoding the first anti-angiogenic polypeptide that inhibits the activity of a VEGF and/or the nucleotide sequence encoding the second anti-angiogenic polypeptide or interfering RNA that reduces expression of a pro-angiogenic gene is operably linked to a CAG promoter.
11 . The 11. The method according to claim 1 , comprising administering to the human subject a pharmaceutical composition comprising from 10 8 to 10 15 particles of said rAAV and a pharmaceutically acceptable excipient.
12 . The method according to claim 10 , comprising administering to the human subject a pharmaceutical composition comprising from 10 8 to 10 15 particles of said rAAV and a pharmaceutically acceptable excipient.
13 . The method according to claim 1 , wherein the rAAV virion or the pharmaceutical composition is intravitreally administered to the subject.
14 . The method according to claim 10 , wherein the rAAV virion or the pharmaceutical composition is intravitreally administered to the subject.
15 . The method according to claim 1 , wherein the first anti-angiogenic polypeptide that inhibits the activity of a vascular endothelial growth factor (VEGF) is aflibercept.
16 . The method according to claim 10 , wherein the first anti-angiogenic polypeptide that inhibits the activity of a vascular endothelial growth factor (VEGF) is aflibercept.
17 . A method for treating a human subject with wet age-related macular degeneration, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization, the method comprising administering by intravitreal injection a therapeutically effective amount of an rAAV particle, said rAAV particle comprising (i) a capsid comprising a capsid protein comprising the amino acid sequence set forth as SEQ ID NO:42 and (ii) a heterologous nucleic acid comprising nucleotide sequence encoding aflibercept, said nucleotide sequence operably linked to a CAG promoter.
18 . The method according to claim 17 , wherein the heterologous nucleic acid further comprises a nucleotide sequence encoding an interfering RNA that reduces expression of a pro-angiogenic gene, said nucleotide sequence operably linked to an expression control sequence.
19 . The method according to claim 18 , wherein the nucleotide sequence encoding aflibercept and the nucleotide sequence encoding an interfering RNA that reduces expression of a pro-angiogenic gene are operably linked to the same CAG promoter.
20 . The method according to claim 19 , wherein the method is for treating a human subject with wet age-related macular degeneration.
21 . The method according to claim 17 , wherein the heterologous nucleic acid further comprises a nucleotide sequence encoding a second anti-angiogenic polypeptide, said nucleotide sequence operably linked to an expression control sequence.
22 . The method according to claim 21 , wherein the nucleotide sequence encoding aflibercept and the nucleotide sequence encoding the second anti-angiogenic polypeptide are operably linked to the same CAG promoter.
23 . The method according to claim 22 , wherein the method is for treating a human subject with wet age-related macular degeneration.