rAAV vector production in suspension adapted HEK293 cells
Proprietary ITR, Helper and Rep/Cap plasmid vectors used for rAAV8 and rAAV9 production were supplied by REGENXBIO using GFP as a reporter/transgene. Plasmids were transfected using polyethylenimine (PEI) as a gene delivery vehicle at optimised PEI:DNA and DNA:cell ratios. For rAAV production, plasmids and PEI were separately diluted in a serum free medium before being combined and incubated at RT. After incubation, the DNA:PEI polyplex transfection mixture was added to cells. For analysis, 450 µL of total cell culture was added to 50 µL 10x cell lysis buffer containing 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche) and incubated for 1 h at 37 °C with gentle agitation. Samples were briefly centrifuged at 12,000 RPM to remove cell debris and the resulting supernatant used to determine viral genome and capsid titre.