title: CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients.
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.
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Shu
authors.initials: S
authors.lastname: Su
authors.affiliation: 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.
authors.forename: Bian
authors.initials: B
authors.lastname: Hu
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Shao
authors.affiliation: State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Shen
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Du
authors.affiliation: 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.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Du
authors.affiliation: 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.
authors.forename: Jiankui
authors.initials: J
authors.lastname: Zhou
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Yu
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Lianru
authors.initials: L
authors.lastname: Zhang
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Fangjun
authors.initials: F
authors.lastname: Chen
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Huizi
authors.initials: H
authors.lastname: Sha
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cheng
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Fanyan
authors.initials: F
authors.lastname: Meng
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Zhengyun
authors.initials: Z
authors.lastname: Zou
authors.affiliation: 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.|School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai 201210, China.
authors.forename: Xingxu
authors.initials: X
authors.lastname: Huang
authors.affiliation: The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University &Clinical Cancer Institute of Nanjing University, Nanjing 210008, China.
authors.forename: Baorui
authors.initials: B
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D003602:Cytotoxicity, Immunologic; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008213:Lymphocyte Activation; D009369:Neoplasms / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D061026:Programmed Cell Death 1 Receptor / Q000172:deficiency / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
chemical_list: D016207:Cytokines; D061026:Programmed Cell Death 1 Receptor; D000094704:RNA, Guide, CRISPR-Cas Systems
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