rAAV production and purification
Sf9 cells were infected at a density of 106 cells/ml
with the combination of BEV rep2/cap8 or BEV rep2/capAnc80 and BEV
ITR-GFP at MOI 1 IU per baculovirus. Four days after infection, cells
were lysed by the addition of 0.5% Triton X-100 (Sigma-Aldrich, MO) for
2.5 hours at 27°C with agitation. The crude bulk was clarified by
centrifugation for 15 min at 500·g and 20°C, the supernatant is then
filtrated through a polyethersulfone (PES) membrane with 0.2 µm (Merck
Millipore, Billerica, MA). rAAV vectors were purified by immune affinity
chromatography with a single POROS™ CaptureSelect™ AAV8 column
(ThermoFisher Scientific, MA) and formulated in Dulbecco’s phosphate
buffered saline (Lonza, Verviers, Belgium) containing 0.001% Poloxamer
188 (Merck KGaA, Darmstadt, Germany).