Scalable production method for aav

Abstract

A method for producing AAV, without requiring cell lysis, is described. The method involves harvesting AAV from the supernatant. For AAV having capsids with a heparin binding site, the method involves modifying the AAV capsids and/or the culture conditions to ablate the binding between the AAV heparin binding site and the cells, thereby allowing the AAV to pass into the supernatant, i.e., media. Thus, the method of the invention provides supernatant containing high yields of AAV which have a higher degree of purity from cell membranes and intracellular materials, as compared to AAV produced using methods using a cell lysis step.

1 . A method for scalable production of DNase resistant AAV particles comprising
 (a) culturing an AAV packaging cell in media which maintains the cell and allows production of AAV particles which secrete into the media without disruption of the packaging cell, wherein the AAV particles comprise an AAV capsid and a nucleic acid molecule which comprises an AAV 5′ inverted terminal repeat (ITR), a nucleic acid sequence encoding a gene product, and a 3′ ITR, and wherein the cell comprises (i) the nucleic acid molecule to be packaged into the AAV capsid, (ii) a coding sequence for the AAV capsid under control of sequences which direct its expression in the packaging cell, (iii) an AAV rep coding sequence which expresses a rep protein in the cell to permit packaging of the nucleic acid molecule into the AAV capsid, and (iv) one or more helper functions required for packaging the nucleic acid molecule into the AAV capsid,   (b) collecting the AAV particles from spent media from the cell culture of (a) in the substantial absence of cell lysis or cell disruption;   (c) isolating the AAV particles from the collected media of the cell culture.   
     
     
         2 . The method according to  claim 1 , wherein the isolating step further comprises concentrating AAV particles through chromatography, filtration and/or precipitation. 
     
     
         3 . The method according to  claim 2 , wherein the chromatography is column based. 
     
     
         4 . The method according to  claim 3 , wherein the column-based chromatography is affinity chromatography. 
     
     
         5 . The method according to  claim 2 , wherein the chromatography is membrane based. 
     
     
         6 . The method according to  claim 1 , wherein the isolating is performed by eluting the collected media containing the AAV particles over an anion exchange resin in a buffer having a pH in the range of about 6 to about 9. 
     
     
         7 . The method according to  claim 6 , wherein the pH is about 8 to about 9. 
     
     
         8 . The method according to  claim 6 , wherein the elution is performed using a 0 to 500 mM salt gradient. 
     
     
         9 . The method according to  claim 8 , wherein the elution is performed using a 100 to 150 mM salt gradient. 
     
     
         10 . The method according to  claim 1 , wherein the cell is stably transformed with one or more sequences encoding adenovirus helper functions. 
     
     
         11 . The method according to  claim 10 , wherein the adenovirus helper functions are expressed under an activatable or inducible promoter. 
     
     
         12 . The method according to  claim 1 , wherein the cell is stably transformed with a sequence encoding the AAV rep and/or a sequence encoding the AAV capsid protein. 
     
     
         13 . The method according to  claim 12 , wherein the AAV rep protein and/or the AAV capsid protein are expressed under direction of an activatable or inducible promoter. 
     
     
         14 . The method according to  claim 1 , wherein the cell is stably transformed with the nucleic acid molecule to be packaged. 
     
     
         15 . The method according to  claim 1 , further comprising a step of adding fresh media during or following collection of the spent media to provide a continuous production process. 
     
     
         16 . The method according to  claim 1 , wherein the method steps are repeated at least two to 100 times. 
     
     
         17 . The method according to  claim 1 , wherein the culturing step is performed in serum-free medium. 
     
     
         18 . The method according to  claim 1 , wherein the AAV capsid is AAV8 
     
     
         19 . The method according to  claim 1 , wherein the AAV capsid has been modified to ablate a native heparin binding site. 
     
     
         20 . The method according to  claim 19 , wherein the heparin binding site is characterized by the amino acid sequence RxxR (SEQ ID NO: 12), where x is any amino acid. 
     
     
         21 . The method according to  claim 1 , wherein the AAV capsid is selected from AAV2, hu. 51, hu.34, hu.35, hu.45, or hu.47. 
     
     
         22 . The method according to  claim 20 , wherein the heparin binding site is modified at the first amino acid of the RxxR (SEQ ID NO:12) sequence from Arg to Ser or Glu, and/or at the last amino acid of the RxxR (SEQ ID NO:12) sequence from Arg to Thr. 
     
     
         23 . The method according to  claim 1 , wherein the AAV particles are produced in a HEK 293 cell. 
     
     
         24 . The method according to  claim 1 , wherein the isolated AAV particles provide a yield of at least 60%. 
     
     
         25 . A cell culture media collected from the cell culture according to  claim 1 , which comprises the AAV particles in a yield of at least 60%. 
     
     
         26 . A kit for production of an AAV particle lacking a heparin binding site from the supernatant, said kit comprising one or more:
 (a) a production cell capable of directing the packaging of an AAV viral particle, whereby the AAV particle is secreted into the media without disruption of the production cell, wherein the AAV particles comprise an AAV capsid and a nucleic acid molecule which comprises an AAV 5′ inverted terminal repeat (ITR), a nucleic acid sequence encoding a gene product, and a 3′ ITR, wherein the cell comprises (i) a coding sequence for the AAV capsid under control of sequences which direct its expression in the packaging cell, (ii) an AAV rep coding sequence which expresses a rep protein in the cell to permit packaging of the nucleic acid molecule into the AAV capsid, and (iii) one or more helper functions required for packaging the nucleic acid molecule into the AAV capsid, wherein the AAV capsid lacks a heparin binding site, and wherein the kit further comprises one or more of:   (b) a transfection reagent;   (c) a plasmid component for construction of a vector;   (d) a component necessary for collection, purification, concentration or harvest of the assembled AAV particle;   (e) a reagent for negative or positive selection of viral particle in order to purify;   (f) a reagent for concentration of viral preparation; and   (g) a reagent for enzymatic digestion of contaminants in viral preparation.