Compositions and methods for producing recombinant adeno-associated virus

Abstract

The present invention provides compositions and methods of producing recombinant AAV (rAAV) virions in large amounts or high titers. Also provided are methods for producing stably transformed host cells capable of producing rAAV virions.

1 . A method of large-scale production of recombinant adeno-associated virus (rAAV) virions, comprising: 
 providing a population of cells capable of growth in suspension or in large-scale culture containers;    admixing a first polynucleotide comprising an AAV vector, a second polynucleotide comprising an AAV helper construct, and a third polynucleotide comprising an accessory function vector, with at least one transfection reagent to provide polynucleotide:transfection reagent complexes;    transfecting the cells with the polynucleotide:transfection reagent complexes to provide transfected cells;    culturing the transfected cells under conditions that permit the formation of rAAV virions; and    harvesting the rAAV virions.    
     
     
         2 . The method of  claim 1  wherein the population of cells are capable of growth in suspension culture and the transfected cells are cultured in suspension.  
     
     
         3 . The method of  claim 1  wherein the population of cells are capable of growth in suspension culture and further comprising the step of adhering the cells to a solid substrate prior to transfection.  
     
     
         4 . The method of  claim 1  wherein the cells are grown in roller bottles or expanded roller bottles.  
     
     
         5 . The method of  claim 1  wherein the cells are grown in T-225 flasks.  
     
     
         6 . The method of  claim 1  wherein the cells are grown in triple flasks.  
     
     
         7 . A method of large-scale production of recombinant adeno-associated virus (rAAV) virions, comprising: 
 providing a population of cells capable of growth in suspension or in large-scale culture containers;    admixing a first polynucleotide comprising an AAV vector and a second polynucleotide comprising an AAV helper construct with at least one transfection reagent to provide polynucleotide:transfection reagent complexes;    transfecting the cells with the polynucleotide:transfection reagent complexes;    infecting the cells with an AAV helper virus;    culturing the infected cells under conditions that permit the formation of rAAV virions; and    harvesting the rAAV virions.    
     
     
         8 . The method of  claim 7  wherein the population of cells are capable of growth in suspension culture and the transfected cells are cultured in suspension.  
     
     
         9 . The method of  claim 7  wherein the population of cells are capable of growth in suspension culture and further comprising the step of adhering the cells to a solid substrate prior to transfection.  
     
     
         10 . The method of  claim 7  wherein the cells are grown in roller bottles or expanded roller bottles.  
     
     
         11 . The method of  claim 7  wherein the cells are grown in T-225 flasks.  
     
     
         12 . The method of  claim 7  wherein the cells are grown in triple flasks.  
     
     
         13 . The method of  claim 7 , wherein the infecting is done concurrently with the transfecting.  
     
     
         14 . The method of  claim 7 , wherein the infecting is done subsequent to the transfecting.  
     
     
         15 . A method of preparing a stably transformed host cell capable of producing recombinant adeno-associated virus (rAAV) virions, comprising: 
 providing a population of cells capable of growth in suspension or in large-scale culture containers;    admixing a first polynucleotide comprising an AAV vector, a second polynucleotide comprising an AAV helper construct, and a third polynucleotide comprising an accessory function vector, with at least one transfection reagent to provide polynucleotide:transfection reagent complexes;    transfecting the cells with the polynucleotide:transfection reagent complexes to provide transfected cells;    culturing the transfected cells under conditions that permit the stable expression of the first polynucleotide, the second polynucleotide, and the third polynucleotide, or a combination thereof, by the host cell.    
     
     
         16 . The method of  claim 15  wherein the population of cells are capable of growth in suspension culture and the transfected cells are cultured in suspension.  
     
     
         17 . The method of  claim 15  wherein the population of cells are capable of growth in suspension culture and further comprising the step of adhering the cells to a solid substrate prior to transfection.  
     
     
         18 . The method of  claim 15  wherein the cells are grown in roller bottles or expanded roller bottles.  
     
     
         19 . The method of  claim 15  wherein the cells are grown in T-225 flasks.  
     
     
         20 . The method of  claim 15  wherein the cells are grown in triple flasks.  
     
     
         21 . The method of  claim 15 , wherein the infecting is done concurrently with the transfecting.  
     
     
         22 . The method of  claim 15 , wherein the infecting is done subsequent to the transfecting.  
     
     
         23 . A method of preparing a stably transformed host cell capable of producing recombinant adeno-associated virus (rAAV) virions, comprising: 
 providing a population of cells capable of growth in suspension culture or in large-scale culture containers;    admixing a first polynucleotide comprising an AAV vector and a second polynucleotide comprising an AAV helper construct with at least one transfection reagent to provide polynucleotide:transfection reagent complexes;    transfecting the cells with the polynucleotide:transfection reagent complexes;    infecting the cells with an AAV helper virus;    culturing the infected cells under conditions that permit the stable expression of the first polynucleotide, the second polynucleotides, the AAV helper virus, or a combination thereof, by the host cell.    
     
     
         24 . The method of  claim 23 , wherein the population of cells are capable of growth in suspension culture and the transfected cells are cultured in suspension.  
     
     
         25 . The method of  claim 23 , wherein the population of cells are capable of growth in suspension culture and further comprising the step of adhering the cells to a solid substrate prior to transfection.  
     
     
         26 . The method of  claim 23 , wherein the cells are grown in roller bottles or expanded roller bottles.  
     
     
         27 . The method of  claim 23 , wherein the cells are grown in T-225 flasks.  
     
     
         28 . The method of  claim 23 , wherein the cells are grown in triple flasks.  
     
     
         29 . The method of  claim 23 , wherein the infecting is done concurrently with the transfecting.  
     
     
         30 . The method of  claim 24 , wherein the infecting is done subsequent to the transfecting.