Figure 3.

Serial passaging of the SGMO Helper

(A) Western blot analysis of cell lysates collected 72 h post-infection for the indicated passage of each individual helper (Rep78-VP2/3 or Rep52-VP1). Cell lysates were analyzed using an antibody specific to either Rep or Cap. Naive Sf9 cells were run as a negative control for antibody recognition. The individual Rep (Rep78 or Rep52) and Cap (VP1, VP2, or VP3) bands are indicated in each panel. An additional band resulting from internal translation initiation from the VP1 ORF is indicated by an asterisk. (B) In vitro potency of AAV6 produced using 293 cells or Sf9 cells (Bac-RepCap and SGMO Helper) on HepG2 cells. SGMO Helper baculovirus from passages zero, three, and six were used to generate baculovirus-infected insect cells (BIICs) and the BIICs were used to initiate AAV production in naive Sf9 cells. HepG2 cells were transduced using the indicated MOI and, after 5 days, cell supernatants were assayed for α-Gal A activity. The nmol/h/mL α-Gal A activity was calculated based on fluorescent activity using a standard curve. Each virus sample was assessed for potency in triplicate, with each of the replicates being assayed in duplicate, and the standard deviation of the six data points was used to plot error bars.