AAV vectors for gene therapy

Abstract

The present invention is directed to methods for generating high titer, contaminant free, recombinant AAV vectors, methods and genetic constructs for producing AAV recombinant vectors conveniently and in large quantities, methods for the delivery of all essential viral proteins required in trans for high yields of recombinant AAV, recombinant AAV vectors for use in gene therapy, novel packaging cell lines which obviate the need for cotransfection of vector and helper plasmids, helper plasmids and vector plasmid backbone constructs, a reporter assay for determining AAV vector yield. Further provided are recombinant AAV vectors in a pharmaceutically acceptable carrier, methods of delivering a transgene of interest to a cell, compositions and methods for delivering a DNA sequence encoding a desired polypeptide to a cell, and transgenic non-human mammals that express a human chromosome 19 AAV integration locus.

What is claimed is:  
     
         1 . A composition for delivering a DNA sequence encoding a desired polypeptide or protein into a cell comprising: 
 an AAV rep protein or a nucleic acid sequence encoding an AAV rep protein; and a genetic construct including a DNA sequence encoding a protein or polypeptide or genetic transcript of interest; and a first AAV ITR or portion thereof, and a second AAV ITR or portion thereof, wherein said first and second AAV ITRs flank said DNA sequence encoding a polypeptide of interest and said promoter controlling said DNA sequence encoding a polypeptide.    
     
     
         2 . An expression vector for site-specific integration and cell-specific expression comprising a first and a second ITR of AAV and at least one cassette comprising a promoter capable of effecting cell-specific expression wherein said promoter is operably linked to a heterologous gene, and wherein said cassette resides between said interved terminal repeats.  
     
     
         3 . An AAV vector comprising the ITR sequences of AAV and a nucleic acid, wherein the ITR sequences promote expression of the nucleic acid in the absence of another promoter.  
     
     
         4 . A vector according to  claim 2  in a pharmaceutically acceptable carrier.  
     
     
         5 . A method of delivering a polypeptide of interest to a cell comprising infecting said cell with a vector according to  claim 2 .  
     
     
         6 . A packaging cell line 293-MT-DBP (ATCC CRL 12181).  
     
     
         7 . A packaging cell line 2C4 (ATCC CRL 12182).  
     
     
         8 . A packaging cell line 3B1 (ATCC CRL 12183).  
     
     
         9 . A non-human transgenic mammal expressing DNA encoding human chromosome 19 AAV integration locus.