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

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.