A Genome-Wide High-Throughput Gain-Of-Function Screen Identifies Novel Targets For Improved AAV9 Production

By Chad Wang, Muskan Sheth, Francesca Ispaso, Isha Ukani, Markus Hoerer, Mohammad A. Mandegar, and Christopher A. Reid

Recombinant adeno-associated virus (AAV), a widely used vector for in vivo gene delivery, plays a crucial role in ongoing preclinical and clinical gene therapy programs. However, current manufacturing processes for AAV are plagued by significant inefficiencies, leading to high production costs, low yields, and compromised product quality.

These challenges create barriers to the broader application of gene therapies. High costs limit accessibility, while quality issues—such as reduced potency, increased immunogenicity, and compromised overall safety—necessitate higher therapeutic dosages. This increases the risk of severe adverse events in patients following administration.

To address these issues, our study leveraged the ATLAS (Arrayed Targeted Library for AAV Screening) miniaturized screening platform. This innovative system systematically over-expressed approximately 18,000 open reading frames (ORFs) to identify targets capable of enhancing AAV9 production.

Through this screen, we identified several classes of targets that significantly boosted AAV9 production, including transcription factors, epigenetic regulators, DNA replication factors, RNA regulators, protein ubiquitination pathways, and metabolic targets. Follow-up validation studies revealed a dose-dependent increase in AAV9 yields, achieving up to a threefold enhancement compared to baseline levels. Importantly, we confirmed that these enhancements did not compromise the potency of the AAV vector.

Extending our findings, we demonstrated that the identified targets also improved production across other AAV serotypes, including AAV2, AAV5, and AAV8.