Unraveling Cytotoxicity in HEK293 Cells During Recombinant AAV Production for Gene Therapy Applications

ABSTRACT Transient transfection of HEK293 cells represents the dominant technique for the production of recombinant adeno‐associated virus (AAV) vectors. However, recombinant AAV (rAAV) production is cytotoxic, potentially impacting process performance, product yields, and quality, complicating downstream processing. This study characterizes cell death response for rAAV producing HEK293 cells and explores the potential to control cytotoxicity. Initial analysis of triple transfected cells revealed caspase‐mediated apoptosis as a likely mechanism of cellular death. Next, the causes of this cytotoxicity were investigated by dissecting transfection steps. Exposing cells to polyethyleneimine (PEI) alone or complexed with a blank plasmid at typical concentrations had a limited impact on cell growth. However, the inclusion of plasmid constructs containing genes to produce rAAVs triggered significant cell death, with the helper plasmid being the most toxic both independently and in combination with packaging and transgene plasmids. Additionally, apoptosis in transfected cultures could be inhibited using the pan‐caspase inhibitor, N ‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (Z‐VAD.fmk), leading to a 65% increase in peak viable cell density (VCD). Although the rAAV genome titer remained relatively unaltered, capsid levels declined upon cell death inhibition. Consequently, the ratio of full to empty capsids, an important product quality attribute (PQA) for rAAVs increased following caspase inhibition. This study provides insights into apoptosis activation in rAAVs and uncovers avenues for its modulation to alter PQAs.