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 muscle cells as compared to the infectivity of the muscle 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 muscle cells for the treatment of muscle disorders and diseases.
1. A method for delivering a heterologous nucleic acid encoding a gene product to a muscle cell, the method comprising contacting the muscle cell with a recombinant adeno-associated virus (rAAV), the rAAV comprising (i) the heterologous nucleic acid and (ii) a variant AAV capsid protein comprising a peptide insertion with a length of 7 to 13 amino acids covalently inserted in the GH-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14).
2. The method according to claim 1 , wherein the insertion peptide has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the amino terminus and/or has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the carboxyl terminus of amino acid sequence NKTTNKD (SEQ ID NO: 14), wherein each of Y 1 -Y 3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro.
3. The method according to claim 2 , wherein the insertion peptide is LANKTTNKDA (SEQ ID NO:28).
4. The method according to claim 3 , wherein the variant capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype and wherein the variant 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:48.
5. The method according to claim 4 , 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:48.
6. The method according to claim 1 , wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO:2), or the corresponding position in the capsid protein of another AAV serotype.
7. The method according to claim 6 , 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.
8. The method according to claim 1 , wherein the muscle cell is a cardiac or skeletal muscle cell.
9. The method 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. The method according to claim 1 , wherein the gene product is a protein selected from alpha galactosidase A (GLA), frataxin (FXN), dystrophin (DMD), acid alpha glucosidase (GAA), and muscle glycogen phosphorylase (PYGM).
11. The method according to claim 1 , wherein the rAAV is delivered to the muscle cell by intravenous and/or intramuscular injection.
12. A method for treating a disease selected from Fabry disease, Friedreich's ataxia, Duchenne's muscular dystrophy, Pompe disease, and myophosphorylase deficiency, the method comprising administering to a subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a recombinant adeno-associated virus (rAAV), the rAAV comprising (i) a heterologous nucleic acid comprising a nucleotide sequence encoding a protein selected from alpha galactosidase A (GLA), frataxin (FXN), dystrophin (DMD)), acid alpha glucosidase (GAA), and muscle glycogen phosphorylase (PYGM) and (ii) a variant AAV capsid protein comprising a peptide insertion with a length of 7 to 13 amino acids covalently inserted in the GH-loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14), wherein said nucleotide sequence encoding said protein is operably linked to an expression control sequence.
13. The method according to claim 12 , wherein the insertion peptide has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the amino terminus and/or has from 1 to 3 spacer amino acids (Y 1 -Y 3 ) at the carboxyl terminus of amino acid sequence NKTTNKD (SEQ ID NO: 14), wherein each of Y 1 -Y 3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro.
14. The method according to claim 13 , wherein the insertion peptide is LANKTINKDA (SEQ ID) NO: 28).
15. The method according to claim 14 , wherein the variant capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype and wherein the variant 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:48.
16. The method according to claim 15 , 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:48.
17. The method according to claim 12 , wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 (SEQ ID NO:2), or the corresponding position in the capsid protein of another AAV serotype.
18. The method according to claim 17 , 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.
19. The method according to claim 12 , wherein the disease is Fabry disease and wherein the heterologous nucleic acid comprises a nucleotide sequence encoding GLA.
20. The method according to claim 19 , wherein the rAAV or pharmaceutical composition comprising the rAAV is administered to the subject by intravenous and/or intramuscular injection.