Treatments for retinal disease
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 clinical practice, for example in, e.g., the delivery of nucleic acid sequences to one or more ceils of the retina for the treatment of retinal disorders and diseases.
1. A recombinant adeno-associated virus (rAAV) comprising: (a) a variant AAV capsid protein comprising a heterologous peptide insertion with a length of 7, 8, 9, 10 or 11 amino acids covalently inserted in the GH-loop of the AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ NO:2) or between amino acids corresponding to amino acids 588 and 589 of VP1 of AAV2 (SEQ ID NO:2) or the corresponding position in the capsid protein of another AAV serotype and wherein if the insertion peptide is LAISDOTKHA (SEQ ID NO:28), the capsid protein does not comprise a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2).
2. The rAAV according to claim 1 , wherein the insertion peptide has 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).
3. The rAAV according to claim 2 , wherein the insertion peptide is LAISDQTKHA (SEQ ID NO:28).
4. The rAAV according to claim 2 , wherein the insertion peptide is LGISDQTKHA (SEQ ID NO:29).
5. The rAAV according to claim 1 , wherein the capsid protein comprises one or more amino acid substitution(s) relative to VP1 of AAV2 (SEQ ID NO:2) or one or more corresponding substitution(s) in the capsid protein of another AAV serotype.
6. The rAAV according to claim 5 , wherein the capsid protein comprises one or more of the following amino acid substitution(s) in the VP1 of AAV2 (SEQ ID NO:2) or one or more corresponding substitution(s) in the capsid protein of another AAV serotype: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, 1698V, V719M, S721L, and L735Q.
7. The rAAV according to claim 6 , wherein the variant capsid protein comprises a P34A amino acid substitution in the VP1 of AAV2 (SEQ ID NO:2) or the corresponding position in the capsid protein of another AAV serotype.
8. An rAAV comprising (i) a variant AAV capsid protein, said variant capsid protein comprising a P34A amino acid substitution relative to VP1 of AAV2 (SEQ ID NO:2) and a heterologous peptide insertion, wherein the insertion peptide is LAISDOTKHA (SEQ ID NO:28), wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 (SEQ NO:2) and, wherein the variant 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 and (ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
9. The rAAV according to claim 1 , wherein the gene product is a protein, a small interfering RNA, an antisense RNA, a microRNA, and/or a short hairpin RNA.
10. A pharmaceutical composition comprising the rAAV according to claim 9 and a pharmaceutically acceptable carrier.
11. A host cell comprising the rAAV according to claim 9 .
12. A pharmaceutical composition comprising the rAAV according to claim 1 and a pharmaceutically acceptable carrier.
13. A host cell comprising the rAAV according to claim 1 .
14. An in vitro method for delivering the heterologous nucleic acid comprising a nucleotide sequence encoding a gene product according to claim 1 to a retinal cell, the method comprising contacting the retinal cell with the rAAV according to claim 1 .
15. The method according to claim 14 , wherein the retinal cell is a retinal ganglion cell, an amacrine cell, a horizontal cell, a bipolar cell, a photoreceptor cell, a Müller glial cell, a microglial cell, or a retinal pigmented epithelium cell.
16. A method for delivering the heterologous nucleic acid comprising a nucleotide sequence encoding a gene product according to claim 1 to a retinal cell of a mammal, the method comprising subretinal, suprachoroidal and/or intravitreal administration of the rAAV according to claim 1 to the mammal.
17. The method according to claim 16 , wherein the rAAV is administered to the mammal by intravitreal injection.
18. The method according to claim 16 , wherein the retinal cell is a retinal ganglion cell, an amacrine cell, a horizontal cell, a bipolar cell, a photoreceptor cell, a Müller glial cell, a microglial cell, or a retinal pigmented epithelium cell.