Gain And Loss-Of-Function Screens Identify Targets That Ramp Up AAV Production

By Christopher A. Reid, Francis Grafton, Lily Leveque-Eichhorn, Jinna Brim, Isha Ukani, Kaylin Fisher, Markus Hoerer, Mohammad A. Mandegar

Recombinant AAV (rAAV) is a promising gene therapy vector, but current manufacturing methods yield relatively low productivity and quality. This study explores three strategies to enhance rAAV production:

• Small Molecule Modulation: A high-throughput screen of over 3,000 bioactive small molecules using the ATLAS platform identified SM-016, a novel compound that increases rAAV9 production up to threefold in a robust, dose-dependent manner.
• HEK293 Cell Engineering: A proprietary clonal suspension-adapted HEK293 cell line (AC001.230) demonstrated improved productivity across 7 of 10 tested serotypes compared to HEK293F and parental polyclonal cells. A CRISPR/Cas9 screen further identified three classes of gene targets that significantly enhanced AAV9 production, leading to the development of knockout cell lines (AC003 and AC010) with over twofold increased rAAV9 yield.

These strategies, now being validated in small-scale bioreactors for yield and quality impact, have the potential to significantly lower rAAV production costs, increasing accessibility for patients.