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, when administered via intravitreal injection, 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 - 27 . (canceled) 
     
     
         28 . A method of identifying a variant recombinant adeno-associated virus (rAAV) virion having greater infectivity of a cell as compared to the corresponding parental AAV virion, the method comprising:
 polymerase chain reaction (PCR) amplifying virion capsid DNA from cells contacted in vivo with a library of variant rAAV virions comprising variant AAV virion capsid protein and variant AAV virion capsid DNA encoding the variant AAV capsid protein.   
     
     
         29 . The method according to  claim 28 , wherein the variant AAV virion capsid protein comprises a peptide inserted into the GH loop of the corresponding parental AAV capsid protein. 
     
     
         30 . The method according to  claim 29 , wherein the peptide consists of five to eleven amino acids. 
     
     
         31 . The method according to  claim 29 , wherein the peptide is located between two adjacent amino acids within amino acids 570-614 of the corresponding parental AAV capsid protein. 
     
     
         32 . The method according to  claim 28 , wherein the variant AAV virion capsid protein comprises 1 to 25 amino acid substitutions. 
     
     
         33 . The method according to  claim 28 , wherein the variant AAV virion capsid protein is a chimeric capsid protein. 
     
     
         34 . The method according to  claim 28 , further comprising, following said PCR amplification, sequencing the variant AAV virion capsid DNA. 
     
     
         35 . The method according to  claim 28 , further comprising, following said PCR amplification,
 cloning the variant AAV virion capsid DNA, and   packaging variant AAV virions from the cloned variant AAV virion capsid DNA.   
     
     
         36 . The method according to  claim 35 , wherein the PCR amplification comprises error-prone PCR. 
     
     
         37 . The method according to  claim 28 , wherein the cell is a retinal cell. 
     
     
         38 . The method according to  claim 28 , wherein the increased infectivity is a greater infectivity of said cells following administration of the variant rAAV virion in vivo as compared to the infectivity of an AAV virion comprising the corresponding parental AAV capsid protein when administered in vivo. 
     
     
         39 . A method of screening a library of variant recombinant adeno-associated virus (rAAV) virions to identify a variant rAAV virion having greater infectivity of a cell as compared to a corresponding parental AAV virion, the method comprising:
 contacting cells in vivo with a library of variant rAAV virions comprising variant AAV capsid protein and variant AAV virion capsid DNA encoding the variant AAV capsid protein.   
     
     
         40 . The method according to  claim 39 , wherein the variant AAV virion capsid protein comprises a peptide inserted into the GH loop of the corresponding parental AAV capsid protein. 
     
     
         41 . The method according to  claim 40 , wherein the peptide consists of five to eleven amino acids. 
     
     
         42 . The method according to  claim 40 , wherein the peptide is located between two adjacent amino acids within amino acids 570-614 of the corresponding parental AAV capsid protein. 
     
     
         43 . The method according to  claim 40 , wherein the variant AAV virion capsid protein comprises 1 to 25 amino acid substitutions. 
     
     
         44 . The method according to  claim 40 , wherein the variant AAV virion capsid protein is a chimeric capsid protein. 
     
     
         45 . The method according to  claim 39 , further comprising:
 sequencing the variant AAV virion capsid DNA.   
     
     
         46 . The method according to  claim 39 , further comprising:
 polymerase chain reaction (PCR) amplifying the variant AAV virion capsid DNA from the cells.   
     
     
         47 . The method according to  claim 46 , further comprising, following said PCR amplification,
 cloning the variant rAAV virion capsid DNA, and   packaging variant AAV virions from the cloned variant rAAV virion capsid DNA.   
     
     
         48 . The method according to  claim 47 , wherein the PCR amplification comprises error-prone PCR. 
     
     
         49 . The method according to  claim 39 , wherein the cell is a retinal cell. 
     
     
         50 . The method according to  claim 39 , wherein the increased infectivity is a greater infectivity of said cells following administration of the variant rAAV virion in vivo as compared to the infectivity of an AAV virion comprising the corresponding parental AAV capsid protein when administered in vivo. 
     
     
         51 . A method of generating a library of variant AAV virions, each virion comprising a variant AAV capsid comprising at least one amino acid substitution, the method comprising:
 producing a library of AAV cap gene variants comprising a nucleic acid sequence that encodes for a peptide inserted into the GH loop of the encoded variant AAV capsid protein;   cloning said library of AAV cap gene variants; and   producing a library of variant rAAV virions comprising variant AAV capsid protein encoded by said AAV cap gene variants.   
     
     
         52 . The method according to  claim 51 , wherein the encoded peptide is located between two adjacent amino acids located between amino acids 570-614 of the corresponding parental AAV capsid protein. 
     
     
         53 . The method according to  claim 51 , wherein the encoded variant AAV capsid proteins further comprise 1 to 25 amino acid substitutions. 
     
     
         54 . The method according to  claim 51 , wherein the variant AAV virion capsid protein is a chimeric capsid protein. 
     
     
         55 . A library of rAAV virions generated by the method according to  claim 51 .