Integrative transcriptomic and proteomic analysis unveils dynamic molecular responses to single, dual, and triple plasmid transfection of HEK293 cells for recombinant adeno-associated virus vector production

Despite clinical success, scalable and high-titer manufacturing of recombinant adeno-associated virus (rAAV) remains challenging, partially due to a limited understanding of the underlying cellular mechanisms and regulatory factors that govern production. We systemically investigate single, dual and the prevailing triple plasmid transfection process for rAAV production in human embryonic kidney (HEK293) cells and subsequently elucidate cellular molecular impacts of each approach through integrative transcriptomics, proteomics and chemometrics. Using a ‘high concentration, H’ single plasmid transfection process yielded increased rAAV titer (3-12-fold) compared to dual or triple transfection with 1556 differentially expressed genes (DEGs) identified. Single plasmid transfection activated fundamental processes intrinsic to transient transfection, including immune and defense response of the host cells, in addition to regulation of viral processes. Key pathways correlating with improved rAAV productivity were identified, including MYC targets, interferon responses, E2F targets, oxidative phosphorylation, G2M checkpoint, mitotic spindle, and WNT beta catenin signalling. The top 25 signature genes correlating with rAAV productivity and top 30 hub genes were also identified, which collectively serve as targeted genome and pathway engineering points for improved rAAV production. The data provides a rich repository of knowledge for the rational optimization of transient transfection strategies for tangible improvements in rAAV manufacturing.