Oncotarget

Research Papers:

A versatile system for rapid multiplex genome-edited CAR T cell generation

Jiangtao Ren, Xuhua Zhang, Xiaojun Liu, Chongyun Fang, Shuguang Jiang, Carl H. June and Yangbing Zhao _

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Oncotarget. 2017; 8:17002-17011. https://doi.org/10.18632/oncotarget.15218

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Abstract

Jiangtao Ren1, Xuhua Zhang1, Xiaojun Liu1, Chongyun Fang1, Shuguang Jiang1, Carl H. June1,2, Yangbing Zhao1,2

1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

Correspondence to:

Yangbing Zhao, email: yangbing@upenn.edu

Keywords: CRISPR/CAS9, T lymphocytes, chimeric antigen receptors, PD-1, CD95

Received: November 18, 2016     Accepted: January 27, 2017     Published: February 09, 2017

ABSTRACT

The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.


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