Abstract
Presentation Description :
Recombinant adeno-associated (rAAV) vectors could be manufactured by plasmid transfection into human HEK293 cells or infection of Spodoptera frugiperda (Sf9) insect cells using baculoviruses. However, systemic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. The authors constructed an rAAV construct using a novel engineered AAV capsid, AAV.N54, and Aflibercept transgene. rAAV products from suspension cultures of Sf9 cells at 2-50 L (Sf9-rAAV) and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized via yields, purity, aggregation, VP1:2:3 ratio, genome contents, post-translational modifications (PTM), next generation sequencing (NGS), infectivity, in vitro and in vivo transgene expression and efficacy. Dynamic light scattering (DLS) and HPLC analysis showed that HEK-rAAV had more aggregation than Sf9-rAAV when rAAV concentration was higher concentrations, >5E+12 vg/mL, while NGS showed that HEK-rAAV had ~10-fold more host cell DNA. LC-MS/MS identified different PTM profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher TCID50/vg than HEK-rAAV using RC32 indicator cells, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.