Abstract
Abstract The production of recombinant adeno-associated virus (rAAV) commonly requires plasmid co-transfection, which hinders its mass production. Herein we developed a novel process for rAAV production by combining the advantages of baculovirus-mediated gene delivery and BelloCell bioreactor (a novel packed-bed reactor for animal cell culture). We constructed 3 baculovirus vectors: Bac-LacZ carried the lacZ gene flanked by AAV inverted terminal repeats (ITR), Bac-RC harbored AAV rep and cap genes and Bac-Helper carried helper genes derived from adenovirus. Co-transduction of HEK293 cells with these 3 baculoviruses resulted in successful production of rAAV, and the protein and rAAV yield did not decrease with Bac-RC passage for up to passage 4. By adjusting the dosage ratio of Bac-LacZ to Bac-RC, addition of sodium butyrate, and transferring the production process to BelloCell500-AP (500 ml) which allowed for high density culture and effective baculovirus transduction of HEK293 cells, the maximum specific rAAV yield reached □3.8□104 vector genome (VG) or 247 infectious viral particles (IVP) per cell, which corresponded to □1×1014 VG or 8.5×1011 IVP per reactor run. The yield was comparable or superior to those obtained using other production systems. The baculovirus transduction is simple and cost-effective and the BelloCell500-AP offers high density culture of HEK293 cells. Altogether the combination of baculovirus transduction and BelloCell reactor culture provides a novel and economically viable approach for rAAV production.