Scalable production method for aav
AssigneeThe Trustees of the University of Pennsylvania
InventorsMartin Lock, Luc H. Vandenberghe, James M. Wilson
Life of this patent
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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.
Claims26
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.
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.
Dependencies are inferred from the claim text.