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Sci Rep. 2016 Jan 28;6:20070. doi: 10.1038/srep20070.

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.

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The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing 210061, China.
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai 201210, China.


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.

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