AAV vector production and administration

Recombinant AAV vectors were produced in HEK 293 cells using a triple (AAV5) or double (AAV8) plasmid-based transfection method, and purified via Iodixanol density centrifugation as described previously (Zolotukhin et al. 1999). For AAV5 preps, pHelper (Agilent cat no. 240071-52) was used to supply the adenoviral helper genes and pACG2R5C (Zolotukhin et al. 2002) was used to supply the AAV2 rep and AAV5 cap genes. For AAV8 production, a single helper plasmid (pDG8) containing both the adenoviral genes as well as the AAV2 rep and AAV8 cap genes was used (Grimm et al. 1998). Table 2 shows the plasmids used in the transfection to produce each recombinant AAV vector. Vectors were titered using a PicoGreen-based assay described by Piedra et al. (2015), and were sterile filtered before administration to animals.

Plasmid constructs used in the production of recombinant AAV vectors

*Contains both Adenovirus helper genes and AAV rep/cap genes.