Method of increasing the function of an aav vector

Abstract

A method of correcting singletons in a selected AAV sequence in order to increasing the packaging yield, transduction efficiency, and/or gene transfer efficiency of the selected AAV is provided. This method involves altering one or more singletons in the parental AAV capsid to conform the singleton to the amino acid in the corresponding position(s) of the aligned functional AAV capsid sequences.

1 . An adeno-associated virus (AAV) comprising an AAV capsid and a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in a host cell, wherein the AAV capsid comprises AAV vp1 proteins, AAV vp2 proteins, and AAV vp3 proteins, wherein the AAV vp1 proteins have i) the sequence of amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46) with a G135P modification, or ii) a sequence having at least 95% identity to the full length of amino acids 1 to 738 of SEQ ID NO:4, wherein the amino acid residue corresponding to position 135 in SEQ ID NO:4 is P when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4. 
     
     
         2 . The AAV according to  claim 1 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         3 . The AAV according to  claim 1 , wherein the heterologous gene encodes a dystrophin gene product. 
     
     
         4 . The AAV according to  claim 3 , wherein the dystrophin gene product is micro-dystrophin. 
     
     
         5 . The AAV according to  claim 1 , wherein the heterologous gene encodes Factor IX. 
     
     
         6 . A composition comprising the AAV according to  claim 1  and a physiologically compatible carrier. 
     
     
         7 . The AAV according to  claim 1 , wherein the AAV vp1 proteins further comprise i) the sequence of amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46) with a A136V modification, or ii) a sequence having at least 95% identity to the full length of amino acids 1 to 738 of SEQ ID NO:4 wherein the amino acid residue corresponding to position 136 is V when aligned along the full length of amino acids 1 to 738 of SEQ ID NO: 4. 
     
     
         8 . The AAV according to  claim 7 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         9 . The AAV according to  claim 7 , wherein the heterologous gene encodes a dystrophin gene product. 
     
     
         10 . The AAV according to  claim 9 , wherein the dystrophin gene product is micro-dystrophin. 
     
     
         11 . The AAV according to  claim 7 , wherein the heterologous gene encodes Factor IX. 
     
     
         12 . A composition comprising the AAV according to  claim 7  and a physiologically compatible carrier.