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Updated: Mar 26, 2026
Production of Human CRISPR-Engineered CAR-T Cells
Published on: March 15, 2021
CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients
1The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
Abstract:
Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.
Insights
Disrupting the programmed death-1 (PD-1) gene in human T cells via electroporation enhances their anti-tumor activity. This non-viral method boosts T-cell function for improved immunotherapy strategies.
Area of Science:
- Immunology
- Cell Biology
- Gene Editing
Background:
- T-cell function is crucial for effective immunotherapy.
- Programmed death-1 ligand (PD-L1) negatively regulates T-cell activity by binding to the programmed death-1 (PD-1) receptor on T cells.
- Targeting this inhibitory pathway is a key strategy in cancer treatment.
Purpose of the Study:
- To investigate a non-viral method for reprogramming primary human T cells by disrupting the PD-1 gene.
- To assess the feasibility and impact of PD-1 gene knockout on T-cell function and viability.
Main Methods:
- Utilized CRISPR-Cas9 technology with electroporation to deliver plasmids encoding sgRNA and Cas9 into primary human T cells.
- Performed gene knockout of the PD-1 gene.
- Cultured gene-modified T cells in vitro for extended periods.
- Assessed T-cell viability, PD-1 expression, IFN-γ production, and cytotoxicity.
Main Results:
- Demonstrated the technical feasibility of PD-1 gene knockout in primary human T cells using electroporation.
- Achieved significant reduction in PD-1 expression without compromising T-cell viability during prolonged in vitro culture.
- Observed up-regulated interferon-gamma (IFN-γ) production and enhanced cytotoxic activity in gene-modified T cells.
Conclusions:
- Developed an efficient non-viral approach for checkpoint inhibitor disruption in T cells.
- This strategy offers a potential method to enhance the efficacy of T-cell based adoptive immunotherapies.
- Highlights a promising new avenue for targeting immune checkpoints to improve cancer treatment outcomes.
