Aav production using suspension adapted cells

Abstract

Provided herein are methods of improving rAAV production in cells, the method comprising increasing the salt concentration in the media in which the cells are infected or transfected, cultured, or in which they produce AAV. Aspects of the disclosure relate to improved methods of rAAV production by co-infecting suspension adapted cells (e.g., suspension adapted HEK293 cells) with viruses that encode one or more AAV components for producing rAAV particles within the suspension adapted cells.

1 . A method of improving rAAV production in cells, the method comprising supplementing with salt the media in which the cells are infected or transfected to introduce AAV rep and cap genes (transfection or infection media), and/or supplementing with salt the media in which the cells produce rAAV (producer media). 
     
     
         2 - 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the supplementing with salt is carried out before, at or after the time of transfection or infection in the infection media. 
     
     
         5 . The method of  claim 1 , wherein the producer media is the same as the transfection or infection media. 
     
     
         6 . The method of  claim 1 , wherein the culture media is the same as the transfection or infection media. 
     
     
         7 . The method of  claim 1 , further comprising diluting or changing the producer media such that the concentration of salt is decreased. 
     
     
         8 . The method of  claim 7 , wherein the diluting or changing the producer media occurs 0.5-8 h after supplementing the producer media or the transfection or infection media with salt. 
     
     
         9 . The method of  claim 1 , wherein the salt is an inorganic salt. 
     
     
         10 . The method of  claim 9 , wherein the salt is an alkali halide, wherein the alkali halide is comprised of an alkali metal and a halogen. 
     
     
         11 . The method of  claim 10 , wherein the alkali metal is lithium (Li), sodium (Na), potassium (K), rubidium (Rb), or caesium (Cs). 
     
     
         12 . The method of  claim 10 , wherein the halogen is fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). 
     
     
         13 . The method of  claim 1 , wherein the concentration of supplemented salt is selected from the following group of ranges: between 5 mM and 200 mM, between 30 mM and 150 mM, and 60 mM to 90 mM. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the concentration of salt is supplemented such that the total concentration of salt is selected from the following group of ranges: between 125 and 250 mM, between 130 mM and 200 mM, and 135 mM to 180 mM. 
     
     
         17 - 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the AAV rep and cap genes are introduced to the cells by transfection of one or more plasmid vectors, or by one or more viral vectors. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 19 , wherein the one or more viral vectors are one or more rHSV vectors. 
     
     
         22 . The method of  claim 1 , wherein the cells are cultured in adherent format or suspension format. 
     
     
         23 - 28 . (canceled) 
     
     
         29 . The method of  claim 1 , wherein the cells are mammalian or insect cells. 
     
     
         30 . (canceled) 
     
     
         31 . A method of producing rAAV, the method comprising co-infecting suspension-adapted HEK293 cells with:
 a first rHSV encoding a gene of interest flanked by AAV ITRs, and,   a second rHSV encoding AAV rep and cap genes.   
     
     
         32 . The method of  claim 31 , further comprising isolating rAAV from the co-infected suspension-adapted HEK293 cells. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 31 , wherein the suspension-adapted HEK293 cells are derived from adherent HEK293 cells. 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The method of  claim 31 , wherein one or more of the AAV rep or cap genes are delivered to the suspension-adapted cells using more than one second rHSV. 
     
     
         38 - 47 . (canceled)