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

Background Clinical development of adeno-associated virus (AAV) requires st andardised, safe, efficient and scalable procedures for the manufacture of the rAAV vector, including production, purification and testing. Several st rategies have been reported for the approach to the manufacturing problem. We report a helper virus-free process that produces high quality rAAV stock s. Methods rAAV were produced by triple transfection, a helper virus-free proc ess. After lysis of the cells in the presence of nuclease, the rAAV produce d were purified by HPLC through two ion-exchange columns in tandem followed by dialysis. rAAV stocks were thoroughly characterised for biological acti vity and for the presence of residual contaminants. The titer of infectious particles and of rep + particles was determined by dRA assay. Contaminatin g 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, ELIS A test, Western blot and specific enzymatic assays for putative residual co ntaminating protein. The in vivo biological activity of the stocks was eval uated in mouse muscle. Results rAAV stocks obtained following this procedure elicit: 2-5 x 10(12) pp/ml; 3-6 x 10(10) ip/ml; < 10(3) rep + particles/ml; <0.3 mUeq/ mi of res idual benzonase activity; non-detectable Ad or beta -galactosidase proteins ; <35 pg/ml of cellular genomic DNA; in vivo expression in mouse muscle wit hout any immune reaction detected. Conclusions This work demonstrates the possibility of producing purified hi gh-quality rAAV free of helper virus. The procedure described in this paper is easily adaptable for large-scale production of clinical rAAV vectors. C opyright (C) 2000 John Wiley & Sons, Ltd.