Production of rAAV in Vero Cells Using Particular Adenovirus Helpers

Abstract

The present invention relates to methods and materials for recombinant adeno-associated virus production. More particularly, in some embodiments the invention contemplates the use of an adenovirus known as Simian Adenovirus 13 (SAdV-13) and Vero cells for production of recombinant adeno-associated virus (rAAV).

1 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) infecting a Vero producer cell with simian adenovirus 13 (SAdV-13) helper virus and   b) culturing the cell to produce rAAV.   
     
     
         2 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) introducing a rAAV genome into a Vero packaging cell,   b) infecting the cell with simian adenovirus 13 (SAdV-13) helper virus and   c) culturing the cell to produce rAAV.   
     
     
         3 . The method of  claim 2  wherein the rAAV genome is introduced by infection with a rAd/AAV hybrid. 
     
     
         4 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) introducing a rAAV genome and AAV rep/cap genes into a Vero cell,   b) infecting the cell with simian adenovirus 13 (SAdV-13) helper virus and   c) culturing the cell to produce rAAV.   
     
     
         5 . The method of  claim 4  wherein steps a) and b) occur concurrently. 
     
     
         6 . The method of  claim 4  wherein the Vero cell is a packaging cell. 
     
     
         7 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) infecting a Vero producer cell with a SAdV-13-like adenovirus helper virus and   b) culturing the cell to produce rAAV.   
     
     
         8 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) introducing a rAAV genome into a Vero packaging cell,   b) infecting the cell with a SAdV-13-like adenovirus helper virus helper virus and   c) culturing the cell to produce rAAV.   
     
     
         9 . The method of  claim 8  wherein the rAAV genome is introduced by infection with a rAd/AAV hybrid. 
     
     
         10 . A method of producing infectious recombinant adeno-associated virus (rAAV) comprising the steps of:
 a) introducing a rAAV genome and AAV rep/cap genes into a Vero cell,   b) infecting the cell with a SAdV-13-like adenovirus helper virus and   c) culturing the cell to produce rAAV.   
     
     
         11 . The method of  claim 10  wherein steps a) and b) occur concurrently. 
     
     
         12 . The method of  claim 10  wherein the Vero cell is a packaging cell. 
     
     
         13 . The method of any of  claims 1 - 12  further comprising the step of isolating the rAAV produced by the cell. 
     
     
         14 . The method of  claim 3  or  9  wherein the Vero cell is infected with the rAd/AAV 16-24 hours after helper virus infection. 
     
     
         15 . In a method for producing infectious recombinant adeno-associated virus (rAAV), the improvement comprising infecting a Vero cell with SAdV-13 helper virus. 
     
     
         16 . In a method for producing infectious recombinant adeno-associated virus (rAAV), the improvement comprising infecting a Vero cell with a SAdV-13-like adenovirus helper virus. 
     
     
         17 . A method of producing infectious rAAV comprising culturing a Vero producer cell under conditions permissive for rAAV production, wherein the Vero producer cell comprises simian adenovirus 13 (SAdV-13) helper virus. 
     
     
         18 . A method of producing infectious rAAV comprising culturing a Vero producer cell under conditions permissive for rAAV production, wherein the Vero producer cell comprises simian adenovirus 13-like (SAdV-13-like) helper virus. 
     
     
         19 . The method of any of the preceding claims wherein the SAdV-13 helper virus is SAdV-13 PME-12.