Unveiling small non-coding RNA dynamics during recombinant Adeno-associated virus production.
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PROVIDER: PRJNA1225130 | ENA |
REPOSITORIES: ENA
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Project description: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).
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Project description:Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.
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