A Versatile System for Rapid Multiplex Genome-Edited CAR T Cell Generation

Oncotarget. 2017 Mar 7;8(10):17002-17011. doi: 10.18632/oncotarget.15218.

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.

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

MeSH terms

  • Animals
  • Apoptosis / genetics
  • CRISPR-Cas Systems
  • CTLA-4 Antigen / genetics
  • Cell Line, Tumor
  • Cells, Cultured
  • Gene Editing / methods*
  • Genes, MHC Class I / genetics
  • Humans
  • Interleukin Receptor Common gamma Subunit / deficiency
  • Interleukin Receptor Common gamma Subunit / genetics
  • K562 Cells
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, SCID
  • Neoplasms, Experimental / genetics
  • Neoplasms, Experimental / pathology
  • Programmed Cell Death 1 Receptor / genetics
  • Receptors, Antigen, T-Cell / genetics*
  • Recombinant Fusion Proteins / genetics*
  • Reproducibility of Results
  • T-Lymphocytes / metabolism*
  • Transplantation, Heterologous

Substances

  • CTLA-4 Antigen
  • CTLA4 protein, human
  • Il2rg protein, mouse
  • Interleukin Receptor Common gamma Subunit
  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor
  • Receptors, Antigen, T-Cell
  • Recombinant Fusion Proteins