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. 2024 Mar 4;32(2):101228. doi: 10.1016/j.omtm.2024.101228

Figure 2.

Figure 2

Testing of AAV6 produced using either Bac-RepCap or the SGMO Helper

(A) Western blot analysis of cell lysates collected at day 6 were harvested 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 and Rep52) and Cap (VP1, VP2, and VP3) proteins are indicated in their respective panels. (B) SDS-PAGE and Coomassie blue stained gel of purified AAV6 from four independent production runs. The individual Cap (VP1, VP2, and VP3) proteins are indicated on the panel. The proteolytic cleavage fragment observed with AAV6 produced using Bac-RepCap is indicated by an asterisk. AAV8 produced using the 293 production system (293 Reference) was also included for comparison. The capsid ratio for each production run is reported in Table 1. (C) In vitro potency of AAV6 produced using 293 cells or Sf9 cells (Bac-RepCap and SGMO Helper) on HepG2 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. (D) In vitro potency of AAV6 produced using 293 cells or Sf9 cells (Bac-RepCap and SGMO Helper) on HuH7 cells. HuH7 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. (E) In vitro potency of AAV6 produced using 293 cells or Sf9 cells (Bac-RepCap and SGMO Helper) on HepG2 cells. HepG2 cells were transduced using an MOI of 1E+06 and, after 5 days, cell supernatants were assayed for huFIX expression. The ng/mL huFIX quantity was calculated 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 three data points was used to plot error bars.