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.