Unveiling Small Non‐Coding RNA Dynamics During Recombinant Adeno‐Associated Virus Production

TL;DR

This study explores how non-coding RNAs influence the production of gene therapy vectors, revealing new regulatory roles in the process.

Contribution

The study identifies novel microRNA and snoRNA dynamics during rAAV production, highlighting their regulatory roles in host-virus interactions.

Findings

142 microRNAs were differentially expressed during peak rAAV production, linked to viral processes and cellular pathways.

Five snoRNAs showed significant expression changes, suggesting a possible role in viral replication.

The findings reveal new regulatory layers in rAAV biogenesis that could improve vector manufacturing.

Abstract

Recombinant adeno‐associated viruses (rAAVs) play a pivotal role in gene therapy, yet the molecular interactions underlying rAAV production in host cells remain incompletely understood. Non‐coding RNAs (ncRNAs), particularly microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs), are increasingly recognized as key regulators of viral and cellular processes. This study investigates the dynamic expression profiles of miRNAs and snoRNAs during rAAV plasmid transfection and vector production in HEK293F cells. A total of 142 miRNAs were differentially expressed during the peak phase of rAAV production, with 128 associated with the Gene Ontology term “viral process”, indicating broad involvement in host‐virus interactions. Target gene analysis linked these miRNAs to biological pathways such as nucleocytoplasmic transport, innate immunity, apoptosis, and transcriptional regulation, highlighting potential roles of miRNAs in shaping the cellular environment during viral vector assembly. In contrast, snoRNAs exhibited more modest changes in expression, yet five were significantly differentially expressed during active production, suggesting a possible, underexplored involvement in viral replication. These findings illuminate the underexplored contributions of ncRNAs to the host response during rAAV biogenesis and provide a valuable resource for understanding how cellular regulatory networks are engaged throughout vector production.

In this study, we explored the largely underexamined role of non‐coding RNAs in recombinant adeno‐associated virus (rAAV) production. Using a time‐course, multi‐layered analytical approach in HEK293F cells, we identified 142 differentially expressed microRNAs and five snoRNAs involved in key cellular processes, including nucleocytoplasmic transport, immune response, and transcriptional regulation. These findings provide new insights into the molecular landscape of rAAV biogenesis and highlight previously unrecognized regulatory layers that may suggest potential strategies for improving vector manufacturing.

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