Scalable production method for AAV
OwnerMartin Lock, Luc H. Vandenberghe +1inventors
InventorsMartin Lock, Luc H. Vandenberghe, James M. Wilson
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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.
Claims17
A method for scalable production of DNA resistant AAV particles, said method comprising the steps of:
- (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 cell comprises one or more adenovirus helper functions necessary for packaging into an AAV capsid, an AAV rep protein sufficient for packaging, a nucleic acid sequence which expresses an AAV capsid in the cell, a nucleic acid molecule comprising an expression cassette to be packaged, said expression cassette comprising AAV inverted terminal repeats and a heterologous nucleic acid, and one or more helper functions for packaging the nucleic acid molecule into the AAV capsid;
- (b) collecting the AAV from supernatant by removing spent media from the cell culture of (a) without collection of cell pellet or cell disruption, wherein the AAV particle comprises the expression cassette packaged in the AAV capsid;
- and
- (c) isolating the AAV particles from the collected media of the cell culture of (b).
Dependencies are inferred from the claim text.