Figure 7

Representative scheme of the rAAV production procedure. Recombinant AAV vectors were generated using a three-plasmid transfection protocol. Briefly, HEK293 cells were tri-transfected using polyethylenimine (PEI 25 kDa; Aldrich) with the helper plasmid carrying adenovirus helper functions, the packaging plasmid expressing the rep and cap genes and the rAAV vector plasmid containing the vector genome sequence. Three days after, cells were harvested and lysed to release the recombinant vectors from the producer cells. Samples were centrifuged at 1500 g for 15 minutes at 4°C and supernatants were treated with 25 U/ml benzonase for 30 minutes at 37°C, centrifuged at 10000 g for 20 minutes at 4°C. Then, one volume of cold saturated ammonium sulfate was added to supernatants and samples were incubated 1 h on ice and centrifuged at 12 000 g for 30 minutes at 4°C to precipitate the recombinant viral particles. rAAVs, resuspended in PBS with Ca²⁺ and Mg²⁺ were then purified by 2 rounds of ultracentrifugation on isopycnic CsCl₂ gradients. The vector-containing fractions were then pooled and desalted by dialysis against sterile PBS supplemented with Ca²⁺ and Mg²⁺. Purified recombinant vectors were aliquoted and stored at -80°C. For the titration of the physical particles, an aliquot was treated with DNAse I for 10 minutes at 37°C to eliminate residual DNA. Then, the sample was treated with proteinase K to degrade the capsids and to release the viral DNA. Viral genome titer of the sample was then determined by real-time qPCR.