Unveiling small non-coding RNA dynamics during recombinant Adeno-associated virus production.

Recombinant adeno-associated viruses (rAAVs) are a cornerstone of modern gene therapy, offering precise delivery and stable transgene expression with minimal immunogenicity. Despite clinical advancements, large-scale rAAV production faces significant challenges such as low yield, high costs, and variability, hindering its scalability. Recent research suggests that miRNAs and snoRNAs can modulate viral replication, transcriptional regulation, and host defense mechanisms, making them attractive targets for improving rAAV production. This study examines the expression profiles of microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) during rAAV plasmid transfection and production in HEK293F cells. We used microarrays to detail differential expression during rAAV production versus mock transfection and basal expression and identified significantly up- and down-regulated small non-coding RNAs (ncRNAs). HEK293F cells were either mock- or rAAV transfected and cultivated over 72 h, each in biological triplicates. During cultivation and production, samples were taken at 0 h, as well as 6, 12, 24, 48 and 72 h post-transfection to analyse cellular parameters and rAAV titers. Ultimately, RNA specimens of each sampling time point were isolated and analysed via microarray expression analysis. Differential expression was considered significant regarding difference in fold change ≥1.5, p-value <0.05 and FDR <0.1.