Figure 5.

Testing of AAV6 produced using Bac-RepCap and the SGMO Helper for in vivo potency

(A) SDS-PAGE and Coomassie blue stained gel of purified AAV6. 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.

(B) In vitro potency of AAV6 produced using 293 cells or Sf9 cells (Bac-RepCap and SGMO Helper) on Hepa1-6 cells. Hepa1-6 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. (C) Mouse plasma levels of α-Gal A activity from mice dosed with 5E+12 vg/kg or 2E+13 vg/kg of 293 AAV6 or Sf9 AAV6 produced using Bac-RepCap or the SGMO Helper. Twenty-eight days post-administration, mice were euthanized and plasma samples were collected to assay for α-Gal A activity. The nmol/h/mL α-Gal A activity was calculated based on fluorescent activity using a standard curve. p values were calculated using a two-way ANOVA with Tukey's multiple comparisons test (∗∗∗∗ = p < 0.0001).