A human genome wide screen identifies miRNAs to increase recombinant Adeno-associated virus production in HEK293 cells.

Adeno-associated virus vectors have become increasingly important vehicles for gene therapy applications. However, the inability to produce vector with high yields and quality is a bottleneck hindering applicability to larger indications. microRNAs have successfully been utilized as cell engineering tools to optimize therapeutic protein production cell lines. The present study aimed at identifying miRNAs capable of increasing rAAV production in HEK293 cells by screening a miRNA mimic library. Out of 2428 miRNAs examined, 144 miRNAs increased functional rAAV titer. A secondary screen confirmed almost 50 % of miRNAs displaying the strongest positive effect. Further validation of six miRNA lead candidates hsa-miR-125a-5p, hsa-miR-135b-5p, hsa-miR-519c-5p, hsa-miR-563, hsa-miR-6857-3p and hsa-miR-3074-3p using plasmid-based miRNA expression and scale-up rAAV production in the Ambr®15 bioreactor system, confirmed their positive impact on functional rAAV titer without substantially compromising critical quality attributes. Bioinformatic analysis showed the miRNA-directed regulation of distinct viral sensing and signalling pathways, as well as apoptosis, which are hypothesized to be the underlying cause of the increased productivity. Our results emphasize the applicability of miRNAs as a powerful tool for cell line engineering capable of optimizing rAAV production in HEK293 cells.