Author:
Liu Ming,Zhang Linlin,Zhong Mingtian,Long Yihao,Yang Wenhui,Liu Ting,Huang Xingxu,Ma Xiaodong
Abstract
AbstractCAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects. Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells. We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines. We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF-α, IL-2 and IFN-γ in vitro. Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy.
Funder
National Natural Science Foundation of China
Publisher
Springer Science and Business Media LLC
Subject
Cancer Research,Oncology,Hematology
Cited by
9 articles.
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