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.