Use of ion concentrations to increase the packaging efficiency of recombinant adeno-associated virus

Abstract

The present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.

What is claimed is: 
     
         1 . A method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein said method comprises the steps:
 (A) culturing cells that have been transfected with said rAAV in an initial culture medium for an initial period under conditions sufficient to permit the production of rAAV, wherein said cells additionally contain an AAV helper function-providing polynucleotide and a non-AAV helper function-providing polynucleotide;   (B) changing the ionic strength of said culture medium after said initial period by adding one or more ions other than Na +  to said culture medium; and   (C) continuing said culturing of said cells to thereby produce a production titer of with said rAAV that is greater than a titer obtained in the absence of step (B).   
     
     
         2 . The method of  claim 1 , wherein each of said added ion(s) is provided in an amount sufficient to increase the concentration of such ion in said initial culture medium by from about 10 mM to about 80 mM. 
     
     
         3 . The method of any one of  claim 1  or  claim 2 , wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of said step (B). 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein said rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein said rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of said serotypes. 
     
     
         6 . The method of  claim 5 , wherein said rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of said serotypes. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein said added ions comprise one or more of K + , Ca ++ , or Mg ++ . 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein said added ions comprise one or more of CO 3   ═ , HCO 3   − , HPO 4   − , PO 4   ═ , SCN − , SO 4   ═ , HSO 4   − , and Cl − . 
     
     
         9 . The method of any one of  claims 1 - 7 , wherein said added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate. 
     
     
         10 . The method of any one of  claims 1 - 8 , wherein said added ions comprise K +  and CO 3   ═ . 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein said cells are human embryonic kidney cells. 
     
     
         12 . The method of  claim 11 , wherein said cells are HEK293 cells. 
     
     
         13 . The method of any one of  claims 1 - 10 , wherein said cells are baby hamster kidney cells. 
     
     
         14 . The method of  claim 13 , wherein said cells are BHK21 cells. 
     
     
         15 . The method of any one of  claims 1 - 10 , wherein said cells are sf9 insect cells. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein said initial culture medium is Dulbecco's Modified Eagle's Medium. 
     
     
         17 . The method of  claim 16 , wherein said initial culture medium is supplemented with serum. 
     
     
         18 . A pharmaceutical composition that comprises:
 (A) a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of  claims 1 - 17 , wherein said rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and wherein said pharmaceutical composition contains an effective amount of said rAAV preparation; and   (B) a pharmaceutically acceptable carrier.   
     
     
         19 . The preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of  claims 1 - 17 , wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the pharmaceutical composition of  claim 18 , for use in the treatment of a genetic or heritable disease or condition.