Inhibition of Caspases Improves Non-Viral T Cell Receptor Editing

SIMPLE SUMMARY: Chimeric antigen receptor (CAR) T cells have shown promising results in cancer treatment. They can be generated with T cells from patients for personalized therapy or donors for off-the-shelf products. A critical step in producing the allogeneic CAR T cells is to knock out the endogenous T cell receptor (TCR). The objective of this study is to determine if inhibition of caspase activities in T cells could increase the cell viability after the TCR knockout mediated by the electrotransfer technology. We observed that inhibition of caspases, especially caspase 3, could significantly improve the cell viability and electrotransfer efficiency. Treatment of cells with caspase inhibitors post electrotransfer could also improve the efficiency of TCR knockout in primary human T cells using electrotransferred ribonucleoprotein. Our data suggest that inhibition of caspases is a promising strategy for improving CAR T cell production. ABSTRACT: T cell receptor (TCR) knockout is a critical step in producing universal chimeric antigen receptor T cells for cancer immunotherapy. A promising approach to achieving the knockout is to deliver the CRISPR/Cas9 system into cells using electrotransfer technology. However, clinical applications of the technology are currently limited by the low cell viability. In this study, we attempt to solve the problem by screening small molecule drugs with an immortalized human T cell line, Jurkat clone E6-1, for inhibition of apoptosis. The study identifies a few caspase inhibitors that could be used to simultaneously enhance the cell viability and the efficiency of plasmid DNA electrotransfer. Additionally, we show that the enhancement could be achieved through knockdown of caspase 3 expression in siRNA treated cells, suggesting that the cell death in electrotransfer experiments was caused mainly by caspase 3-dependent apoptosis. Finally, we investigated if the caspase inhibitors could improve TCR gene-editing with electrotransferred ribonucleoprotein, a complex of Cas9 protein and a T cell receptor-α constant (TRAC)-targeting single guide RNA (sgRNA). Our data showed that inhibition of caspases post electrotransfer could significantly increase cell viability without compromising the TCR disruption efficiency. These new findings can be used to improve non-viral T cell engineering.