Human gene therapyMay 1, 2018

Using gene editing to disable a cell death protein in targeted cancer-fighting immune cells

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Apr 2, 2026

Abstract

On average, 91.5% of the PD-1 gene sites were disrupted in engineered CAR T cells.

CRISPR/Cas9 technology was used to disrupt PD-1 in chimeric antigen receptor T cells.
Transient expression of Cas9 via plasmid transfection may simplify the process compared to traditional methods.
The resulting PD-1-deficient CAR T cells exhibited similar cytokine secretion levels to conventional CAR T cells.
These PD-1-deficient CAR T cells demonstrated improved proliferation and cytotoxicity in vitro.
In an orthotopic mouse model of glioma, the engineered CAR T cells showed enhanced tumor growth inhibition.

Simplified

CRISPR/Cas9-mediated programmed cell death protein 1 (PD-1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD-1 gene and the piggyBac transposon system for expressing CD133-specific CAR in one reaction. Based on drug selection, CD133-specific CAR T cells were obtained in which, on average, 91.5% of the PD-1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD-1-deficient CD133-specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133-CAR T cells. The described method could be useful for the production of PD-1-deficient CAR T cells for cancer immunotherapy.

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Using gene editing to disable a cell death protein in targeted cancer-fighting immune cells

Abstract

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