In vivo transduction with a chimeric aav capsid protein

Abstract

Recombinant adeno-associated viral (AAV) capsid proteins are provided. Methods for generating a library of recombinant adeno-associated viral capsid proteins are also provided.

What is claimed is: 
     
         1 . A method of generating a library of recombinant AAV plasmids, comprising:
 isolating AAV capsid nucleotide sequences from two or more serotypes of AAV;   digesting the AAV capsid nucleotide sequences into fragments;   reassembling the fragments using PCR to form re-assembled PCR products; and   cloning the re-assembled PCR products into plasmids to generate a library of recombinant AAV plasmids.   
     
     
         2 . The method of  claim 1 , wherein the reassembling the fragments using PCR is using primer-less PCR. 
     
     
         3 . The method of  claim 1 , wherein isolating includes isolating AAV capsid nucleotide sequences from human AAV serotypes and non-human AAV serotypes. 
     
     
         4 . The method of  claim 3 , wherein isolating includes isolating AAV capsid nucleotide sequences selected from the group consisting of AAV-2, AAV-8, and AAV-9. 
     
     
         5 . The method of  claim 1 , further comprising, after said cloning, transfecting cells with the plasmids to produce a viral library. 
     
     
         6 . The method of  claim 5 , wherein said transfecting comprises transfecting 293 kidney cells with a helper Adenovirus. 
     
     
         7 . The method of  claim 5 , further comprising, after said transfecting, passaging the viral library in a selected cell type in the presence of a stringent condition, and selecting AAV capsids that survive said passaging. 
     
     
         8 . The method of  claim 7 , wherein the stringent condition comprises the presence of human immune globulin. 
     
     
         9 . A recombinant AAV library prepared according to the method of  claim 1 .