ATLAS: A High-Throughput Gain And Loss-Of-Function Screening Platform For Optimizing AAV Production

By Christopher A. Reid, Francis Grafton, Lily Leveque-Eichhorn, JinnaBrim, IshaUkani, Kaylin Fisher, and Mohammad A. Mandegar

Recombinant AAV (rAAV) is one of the most widely used vectors for in vivo gene delivery, with over 900 ongoing preclinical and clinical programs requiring substantial manufacturing capacity. However, inefficiencies in current production methods have led to prohibitively high gene therapy costs, limiting the accessibility of AAV-based treatments for both rare and common diseases. In this study, we introduce ATLAS (Arrayed Targeted Library for AAV Screening), a modality-agnostic, high-throughput platform designed to identify targets for enhancing rAAV production.

To enable miniaturized screening, we developed a highly sensitive cell-based reporter gene assay capable of detecting functional AAV9 at vector genome levels as low as 2.5E8 vg/mL. Using this assay, we performed high-throughput screening of small molecules and an ORF overexpression library, identifying a novel combination of factors that increased rAAV9 production by over 30-fold in our clonal suspension HEK 293 cells compared to commercially available HEK 293F cells. Ongoing efforts are focused on scaling these findings to a 50L bioreactor, assessing potential impacts on key quality attributes, and expanding the analysis to a broader panel of capsids.