Optimised helper virus-free production of high-quality adeno-associated virus vectors

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Abstract
Background Clinical development of adeno‐associated virus (AAV) requires standardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several strategies have been reported for the approach to the manufacturing problem. We report a helper virus‐free process that produces high quality rAAV stocks. Methods rAAV were produced by triple transfection, a helper virus‐free process. After lysis of the cells in the presence of nuclease, the rAAV produced were purified by HPLC through two ion‐exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological activity and for the presence of residual contaminants. The titer of infectious particles and of rep+ particles was determined by dRA assay. Contaminating DNA and RNA were determined by fluorescent dye binding and real‐time PCR. The protein content of the rAAV stocks was characterised by SDS‐PAGE, ELISA test, Western blot and specific enzymatic assays for putative residual contaminating protein. The in vivo biological activity of the stocks was evaluated in mouse muscle. Results rAAV stocks obtained following this procedure elicit: 2–5×1012 pp/ml; 3–6×1010 ip/ml; 3 rep+ particles/ml; in vivo expression in mouse muscle without any immune reaction detected. Conclusions This work demonstrates the possibility of producing purified high‐quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large‐scale production of clinical rAAV vectors. Copyright © 2001 John Wiley & Sons, Ltd.