Adeno-associated virus variants and methods of use thereof

Abstract

The present disclosure provides recombinant adeno-associated virus (rAAV) virions comprising a variant AAV capsid protein, e.g., an AAV capsid protein derived from an ancestral AAV capsid protein amino acid sequence. An rAAV virion of the present disclosure can exhibit greater infectivity of a target cell. The present disclosure also provides methods of delivering a gene product to a target cell in an individual by administering to the individual an rAAV of the present disclosure. The present disclosure also provides methods of generating rAAV virions that have a variant AAV capsid protein derived from an ancestral AAV capsid protein amino acid sequence.

1 .- 33 . (canceled) 
     
     
         34 . A method of generating rAAV virions having variant AAV capsid proteins, comprising:
 subjecting an initial library of rAAV virions to a first round of selection in target cells,   wherein the rAAV virions in the initial library each comprise an initial AAV capsid protein having an AAV capsid protein amino acid sequence, and wherein the AAV capsid protein amino acid sequences comprise an ancestral AAV capsid protein amino acid sequence that differ among each other at one or more variable residues of the ancestral AAV capsid protein amino acid sequence, thereby generating a second library of rAAV virions having variant AAV capsid proteins.   
     
     
         35 . The method according to claim  33 , wherein the method comprises determining the ancestral AAV capsid protein amino acid sequence by:
 i) reconstructing a phylogenetic tree of a plurality of wild type AAV capsid protein amino acid sequences;   ii) selecting a node of the phylogenetic tree; and   iii) determining the most likely amino acid sequence at the node.   
     
     
         36 . The method according to  claim 34 , wherein the method further comprises estimating a confidence value at each node of the phylogenetic tree, and the selecting step comprises selecting a node of the phylogenetic tree based on the estimated confidence value at the node. 
     
     
         37 . The method according to  claim 34 , wherein the initial AAV capsid protein has an amino acid sequence at least 94% identical to the sequence set forth in SEQ ID NO: 7. 
     
     
         38 . The method according to  claim 34 , wherein the method comprises subjecting the second library of rAAV virions to a second round of selection. 
     
     
         39 . The method according to  claim 38 , wherein the second round of selection is performed in the same target cell type as the target cells used in the first round of selection. 
     
     
         40 . The method according to  claim 38 , wherein the second round of selection has increased stringency relative to the first round of selection. 
     
     
         41 . The method according to  claim 34 , wherein the target cells are selected from the group consisting of: muscle, epithelial, skin, glial, and human embryonic kidney cells. 
     
     
         42 . The method according to  claim 34 , wherein the subjecting step comprises:
 a) infecting target cells with the library of rAAV virions;   b) superinfecting the infected cells with a helper virus; and   c) harvesting rAAV virions released from superinfected cells.   
     
     
         43 . The method according to  claim 34 , wherein the method comprises isolating a rAAV virion comprising a variant AAV capsid protein, wherein the isolated rAAV virion has increased infectivity, enhanced tropism, or both, compared to an AAV virion comprising a wild type AAV serotype capsid protein.