Adeno-associated virus virions with variant capsid and methods of use thereof

Abstract

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.

What is claimed is: 
     
         1 - 33 . (canceled) 
     
     
         34 . A recombinant adeno-associated virus (rAAV) virion comprising:
 a) a variant AAV capsid protein comprising an insertion of a heterologous peptide in the capsid protein GH loop relative to a corresponding parental AAV capsid protein,   wherein the heterologous peptide comprises the amino acid sequence:
 PDSTTRS, and 
 comprises a two amino acid long linker at the N-terminus prior to the amino acid P and one amino acid long linker at the C-terminus after the amino acid S; and 
   b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product.   
     
     
         35 . The rAAV virion of  claim 34 , wherein the rAAV virion exhibits at least 5-fold 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. 
     
     
         36 . The rAAV virion of  claim 34 , 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. 
     
     
         37 . The rAAV virion of  claim 36 , 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. 
     
     
         38 . The rAAV virion of  claim 34 , wherein gene product is:
 a) an interfering RNA or an aptamer;   b) a polypeptide, optionally wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, an anti-apoptotic polypeptide, or a polypeptide that enhances function of a retinal cell, or an RNA-guided endonuclease; or   c) an RNA-guided endonuclease and a guide RNA.   
     
     
         39 . The rAAV virion of  claim 34 , wherein the heterologous gene product 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), or Sonic hedgehog. 
     
     
         40 . The rAAV virion of  claim 34 , wherein the heterologous peptide comprises the amino acid sequence X 1 X 2 PDSTTRSX 10 , wherein X 1  is Leu, Ile, Pro, or Gln; X 2  is Ala, Pro, Ser, Asp, Gly, Thr, or Val; and X 10  is Ala, Phe, Asp, Thr, Val, or Met. 
     
     
         41 . The rAAV virion of  claim 34 , wherein the heterologous peptide has a length of 12-20 amino acids. 
     
     
         42 . A pharmaceutical composition, comprising:
 a) a recombinant adeno-associated virus (rAAV) virion of  claim 34 ; and   b) a pharmaceutically acceptable excipient.   
     
     
         43 . 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 34 . 
     
     
         44 . A method of treating an ocular diseases, the method comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to  claim 34 . 
     
     
         45 . The method of  claim 44 , wherein the rAAV virion is administered by intraocular or intravitreal injection. 
     
     
         46 . The method of  claim 44 , wherein the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber’s Congenital Amaurosis, diabetic retinopathy, achromotopsia, or color blindness. 
     
     
         47 . 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 in the capsid protein GH loop relative to a corresponding parental AAV capsid protein,
 wherein the heterologous peptide comprises the amino acid sequence PDSTTRS, and   comprises a two amino acid long linker at the N-terminus prior to the amino acid P and one amino acid long linker at the C-terminus after the amino acid S, and   wherein the variant capsid protein, when present in an AAV virion, provides for increased infectivity of the AAV virion of a retinal cell.   
     
     
         48 . The isolated nucleic acid of  claim 47 , wherein the insertion site is between amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. 
     
     
         49 . The isolated nucleic acid of  claim 47 , 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 AAV 10. 
     
     
         50 . The isolated nucleic acid of  claim 47 , wherein the heterologous peptide consists of the amino acid sequence X 1 X 2 PDSTTRSX 10 , wherein X 1  is Leu, Ile, Pro, or Gln; X 2  is Ala, Pro, Ser, Asp, Gly, Thr, or Val; and X 10  is Ala, Phe, Asp, Thr, Val, or Met. 
     
     
         51 . The isolated nucleic acid of claim  57 , wherein the heterologous peptide has a length of 12-20 amino acids. 
     
     
         52 . A host cell producing the rAAV virion of  claim 34 . 
     
     
         53 . A variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of a heterologous peptide in the capsid protein GH loop relative to a corresponding parental AAV capsid protein,
 wherein the heterologous peptide comprises the amino acid sequence PDSTTRS, and   comprises a two amino acid long linker at the N-terminus prior to the amino acid P and one amino acid long linker at the C-terminus after the amino acid S.