Small Molecule Screen Identifies Targets That Increase AAV9 Production In Suspension HEK293 Cells

By Francesca Ispaso, Kaylin Fisher, Francis Grafton, Markus Hoerer, Mohammad A. Mandegar, Christopher A. Reid

The use of recombinant AAV as a gene delivery vector is widespread, with over 900 preclinical and clinical programs in progress. However, inefficient manufacturing methods drive up costs, limiting the accessibility of gene therapies. In this study, we present a high-throughput small molecule screening strategy to identify compounds that enhance cellular capacity for AAV9 production. Previously, we identified a novel small molecule (SM-016) from a screen of 3,000 bioactive compounds, which we validated in shake flasks and Ambr15 bioreactors.

Building on this work, we aimed to expand the diversity of biological targets screened. To achieve this, we developed ATLAS (Arrayed Targeted Library for AAV Screening), a miniaturized, suspension-adapted high-throughput screening platform. Optimization studies demonstrated that AAV9 yields were reproducible, scalable, and translatable from a 96-well format to 125 mL shake flasks. We then screened a curated library of over 700 small molecules, identifying key targets including epigenetic modulators, DNA damage response pathways, GPCR and transmembrane transporters, cell cycle regulators, anti-infective agents, and metabolic modulators. Evaluation of the top hits is currently in progress.