Helper-free stocks of recombinant adeno-associated virus vectors

Abstract

The present invention relates to a method for producing helper-free stocks of recombinant adeno-associated virus (rAAV) which can be used to efficiently and stably transduce foreign genes into host cells or organisms. The method comprises the cotransfection of eukaryotic cells with rAAV and with helper AAV DNA in the presence of helper virus (e.g. adenovirus or herpesvirus) such that the helper AAV DNA is not associated with virion formation. The crux of the invention lies in the inability of the helper AAV DNA to recombine with rAAV vector, thereby preventing the generation of wild-type virus. In a specific embodiment of the invention, the vector comprises a recombinant AAV genome containing only the terminal regions of the AAV chromosome bracketing a non-viral gene, and the helper AAV DNA comprises a recombinant AAV genome containing that part of the AAV genome which is not present in the vector, and in which the AAV terminal regions are replaced by adenovirus sequences. In a further embodiment of the invention, cell lines are created which incorporate helper AAV DNA which can directly produce substantially pure recombinant AAV virus. The pure stocks of recombinant AAV produced according to the invention provide an AAV viral expression vector system with increased yield of recombinant virus, improved efficiency, higher definition, and greater safety than presently used systems.

1 - 57 . (canceled)  
   
   
       58 . A method for producing an adeno-associated virus packaging cell line, comprising: 
 a) transfecting cells permissive for adeno-associated virus replication with a helper adeno-associated virus DNA which provides viral functions sufficient for the replication and packaging into infectious virions of recombinant adeno-associated virus, and which comprises a nucleotide sequence not found in wild-type adeno-associated virus which promotes expression of adeno-associated virus genes which results in the viral functions provided, but which lacks adeno-associated virus terminal repeat sequence; and    b) selecting for stably transfected cells which express said helper adeno-associated virus DNA.    
   
   
       59 . The method of  claim 58 , wherein said helper adeno-associated virus DNA comprises adeno-associated virus derived cis-acting elements that control the expression of gene products.  
   
   
       60 . The method of  claim 58 , wherein said helper adeno-associated virus DNA comprises non-adeno-associated virus derived cis-acting elements that control the expression of gene products.  
   
   
       61 . The method of  claim 58 , wherein said helper adeno-associated virus DNA comprises inducible promoter/enhancer elements that control the expression of gene products.  
   
   
       62 . The method of  claim 58 , wherein said helper adeno-associated virus DNA comprises adeno-associated virus genes located between about nucleotide 195 and about nucleotide 4480.  
   
   
       63 . An adeno-associated virus packaging cell line produced using the method of  claim 58 .  
   
   
       64 . An adeno-associated virus packaging cell line produced using the method of  claim 59 .  
   
   
       65 . An adeno-associated virus packaging cell line produced using the method of  claim 60 .  
   
   
       66 . An adeno-associated virus packaging cell line produced using the method of  claim 61 .  
   
   
       67 . An adeno-associated virus packaging cell line produced using the method of  claim 62 .  
   
   
       68 . A method for producing a helper-free stock of recombinant adeno-associated virus comprising: 
 a) transfecting an adeno-associated virus packaging cell line produced by the method of  claim 58 , with a recombinant adeno-associated virus vector which contains a foreign DNA sequence and which can be incorporated into an infectious virion, in the presence of helper virus infection; and    b) collecting virions produced.    
   
   
       69 . The method of  claim 68 , wherein said helper virus is adenovirus or herpes virus.  
   
   
       70 . The method of  claim 68 , wherein said recombinant adeno-associated vector comprises up to 195 base pairs of the adeno-associated virus terminal repeated sequence, a foreign DNA sequence, and shares no adeno-associated virus sequence with the helper adeno-associated virus DNA.  
   
   
       71 . The method of  claim 68 , wherein the helper adeno-associated virus DNA comprises adeno-associated virus derived cis-acting elements that control the expression of gene products.  
   
   
       72 . The method of  claim 68 , wherein the helper adeno-associated virus DNA comprises non-adeno-associated virus derived cis-acting elements that control the expression of gene products.  
   
   
       73 . The method of  claim 68 , wherein the helper adeno-associated virus DNA comprises inducible promoter/enhancer elements that control the expression of gene products.  
   
   
       74 . The method of  claim 68 , wherein the helper adeno-associated virus DNA comprises adeno-associated virus genes located between about nucleotide 195 and about nucleotide 4480.