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 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 an insertion of a heterologous peptide having a length of from 10 amino acids to 20 amino acids in the capsid protein GH loop relative to a corresponding parental AAV capsid protein, wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein, and
wherein the heterologous peptide comprises the amino acid sequence LATTSQNKPA (SEQ ID NO:50); and
b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product.
2. The rAAV virion of claim 1 , wherein the insertion site is 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.
3. The rAAV virion of claim 2 , 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 gene product is:
a) an interfering RNA or an aptamer;
b) a polypeptide; or
c) an RNA-guided endonuclease and a guide RNA.
5. The rAAV virion of claim 4 , wherein the gene product is a polypeptide, and wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances function of a retinal cell, or an RNA-guided endonuclease.
6. The rAAV virion of claim 4 , wherein the gene product is a polypeptide, and 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, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, a rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), Sonic hedgehog, or an RNA-guided endonuclease.
7. A pharmaceutical composition comprising:
a) a recombinant adeno-associated virus virion of claim 1 ; and
b) a pharmaceutically acceptable excipient.
8. A method of delivering a gene product to a retinal cell in an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to claim 1 .
9. 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 .
10. 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 an insertion of a heterologous peptide having a length of from 10 amino acids to 20 amino acids in the capsid protein GH loop relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein, when present in an AAV virion, provides for increased infectivity of the AAV virion of a retinal cell, wherein the insertion is in the GH loop of a native AAV capsid, and
wherein the heterologous peptide comprises the amino acid sequence LATTSQNKPA (SEQ ID NO:50).
11. The isolated nucleic acid of claim 10 , wherein the insertion site is 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.
12. The isolated nucleic acid of claim 10 , wherein the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.
13. A variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of a heterologous peptide having a length of from 10 amino acids to 20 amino acids wherein the insertion is in the GH loop of a native AAV capsid, and wherein the heterologous peptide comprises the amino acid sequence LATTSQNKPA (SEQ ID NO:50).
14. The variant AAV capsid protein of claim 13 , wherein the insertion site is 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.
15. The variant AAV capsid protein of claim 13 , wherein the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.