Premium
Optimised helper virus‐free production of high‐quality adeno‐associated virus vectors
Author(s) -
Drittanti Lila,
Jenny Christine,
Poulard Karine,
Samba Anne,
Manceau Peggy,
Soria Nestor,
Vincent Nathalie,
Danos Olivier,
Vega Manuel
Publication year - 2001
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/1521-2254(2000)9999:9999<::aid-jgm152>3.0.co;2-u
Subject(s) - virus , in vivo , helper virus , western blot , biology , microbiology and biotechnology , adeno associated virus , virology , dna , chemistry , vector (molecular biology) , recombinant dna , biochemistry , gene , viral replication
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: 2–5×10 12 pp/ml; 3–6×10 10 ip/ml; <10 3 rep+ particles/ml; <0.3 mUeq/ml of residual benzonase activity; non‐detectable Ad or β‐galactosidase proteins; <35 pg/ml of cellular genomic DNA; 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.