Optimization of recombinant adeno-associated virus production using an herpes simplex virus amplicon system.
A major limitation of adeno-associated virus (AAV) based vectors for clinical applications to date is the production of high-titer recombinant AAV vector stocks. Despite recent improvements, the amount of recombinant adeno-associated virus vectors (rAAV) particles produced per cell continues to be significantly lower than that of wild-type AAV. In this study, an HSV-based system for rAAV production was used to examine the influence of different parameters including transfection conditions (vector-to-packaging plasmid ratio, amount of total transfected DNA, cell confluency) and multiplicity of infection of herpes helper virus on the resulting titre of rAAV stocks. For herpes helper virus, time-course experiments were carried out to analyse the effect on rAAV yields up to 72 h postinfection and to determine the ideal harvesting time. Taken together, the optimized production scheme consistently yields more than 3x10(3) transducing units per producer cell.
Feudner
E
E
University Eye Hospital, Röntgenweg 11, 72076, Tübingen, Germany.
de Alwis
M
M
Thrasher
A J
AJ
Ali
R R
RR
Fauser
S
S
D000818:Animals; D002460:Cell Line; D004279:DNA, Viral; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006367:HeLa Cells; D006378:Helper Viruses / Q000235:genetics* / Q000502:physiology; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics* / Q000472:pathogenicity / Q000502:physiology; D014161:Transduction, Genetic; D014162:Transfection; D014776:Virus Cultivation; D014779:Virus Replication
D004279:DNA, Viral