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Published on: January 22, 2019
Inhibition of Caspases Improves Non-Viral T Cell Receptor Editing
Chunxi Wang1, Chun-Chi Chang1, Liangli Wang1
1Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.
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.
Insights
Small molecule drugs, specifically caspase inhibitors, enhance T cell viability and CRISPR/Cas9 gene-editing efficiency after electrotransfer. This approach improves non-viral T cell engineering for cancer immunotherapy.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- T cell receptor (TCR) knockout is essential for developing universal chimeric antigen receptor (CAR) T cells for cancer immunotherapy.
- Electrotransfer of CRISPR/Cas9 systems is a key method for TCR knockout, but limited by low cell viability.
Purpose of the Study:
- To improve cell viability and electrotransfer efficiency using small molecule drugs.
- To investigate the role of apoptosis in cell death during electrotransfer.
- To assess the impact of caspase inhibitors on TCR gene editing efficiency.
Main Methods:
- Screening of small molecule drugs for apoptosis inhibition in Jurkat T cells.
- siRNA-mediated knockdown of caspase 3 expression.
- Evaluating cell viability and TCR knockout efficiency after plasmid DNA and ribonucleoprotein electrotransfer.
Main Results:
- Caspase inhibitors were identified that enhance both cell viability and plasmid DNA electrotransfer efficiency.
- Caspase 3-dependent apoptosis was confirmed as the primary cause of cell death.
- Inhibition of caspases post-electrotransfer increased cell viability without reducing TCR gene-editing efficiency.
Conclusions:
- Caspase inhibitors can significantly improve non-viral T cell engineering by enhancing cell viability and gene-editing efficiency.
- Targeting caspase-dependent apoptosis is a viable strategy to overcome limitations in T cell engineering for immunotherapy.
Related Concept Videos
01:28CRISPR/Cas9 Genome Editing
01:24Caspases
01:59CRISPR
01:17The Extrinsic Apoptotic Pathway