Optimised helper virus-free production of high-quality adeno-associated virus vectors
| Autor: | Nestor Soria, Karine Poulard, Peggy Manceau, Anne Samba, Manuel Vega, Lila Drittanti, Nathalie Vincent, Christine Jenny, Olivier Danos |
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| Rok vydání: | 2001 |
| Předmět: |
viruses
Genetic Vectors Biology medicine.disease_cause Virus Cell Line Mice Western blot In vivo Drug Discovery Genetics medicine Animals Humans Molecular Biology Adeno-associated virus Genetics (clinical) DNA Primers Recombination Genetic Nuclease Mice Inbred BALB C medicine.diagnostic_test Base Sequence Transfection Dependovirus Virology Molecular biology Titer Helper virus biology.protein Molecular Medicine Helper Viruses |
| Zdroj: | The journal of gene medicine. 3(1) |
| ISSN: | 1099-498X |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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