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Published on: July 22, 2019
Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in
Bian Hu1, Yan Zou2, Linlin Zhang1
11 MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University , National Resource Center for Mutant Mice, Nanjing, China .
Abstract:
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.
Insights
Disrupting programmed cell death protein 1 (PD-1) in chimeric antigen receptor (CAR) T cells enhances their anti-tumor activity. This study developed a simpler method for creating PD-1-deficient CAR T cells for improved cancer immunotherapy.
Area of Science:
- Immunology
- Gene Editing
- Cancer Therapy
Background:
- The tumor microenvironment often suppresses T cell function.
- Chimeric antigen receptor (CAR) T cells show promise but can be hindered by immunosuppression.
- Programmed cell death protein 1 (PD-1) is a key immune checkpoint target.
Purpose of the Study:
- To develop an efficient method for generating PD-1-deficient CAR T cells.
- To assess the therapeutic potential of these engineered T cells in a glioma model.
Main Methods:
- Utilized CRISPR/Cas9 and piggyBac transposon system via nucleofection in human primary T cells.
- Co-delivered plasmids for PD-1 gene disruption and CD133-specific CAR expression in a single reaction.
- Employed drug selection to isolate engineered CAR T cells.
Main Results:
- Achieved high efficiency (91.5%) of PD-1 gene disruption in CD133-specific CAR T cells.
- Engineered CAR T cells exhibited enhanced proliferation, cytotoxicity, and tumor growth inhibition in vitro and in vivo.
- Minimal Cas9 expression was detected in the final CAR T cell product.
Conclusions:
- A simplified, single-reaction method for producing PD-1-deficient CAR T cells was established.
- PD-1 disruption enhances CAR T cell efficacy against glioma in preclinical models.
- This approach offers a promising strategy for advancing cancer immunotherapy.
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