Nucleofection with Plasmid DNA for CRISPR/Cas9-Mediated Inactivation of Programmed Cell Death Protein 1 in CD133-Specific CAR T Cells

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.

The Potential of CAR T Cell Therapy in Pancreatic Cancer

“…Another clinical trial is recruiting patients with non-hematologic tumors to evaluate the novel combination of autologous MUC1-redirected CAR T cells engineered to express antibodies targeting CTLA-4 and PD-1 (NCT03179007). Finally, sophisticated gene editing technologies such as CRISPR/Cas9 are being utilized to further engineer CAR T cells that lack genes encoding factors such as PD-1 that mediate an exhausted phenotype and function ( 71 , 72 ) or alternatively aid in the design of universal, “off-the-shelf” CAR T cell approaches that lack self-recognition factors ( 83 , 84 ). Other gene engineering strategies are also in development which incorporate suicide genes to improve the safety of these cell therapy strategies so that the therapeutic effect can be turned on or off, on demand ( 85 ).…”

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

“…Unlike the previously mentioned studies that employed electroporation for CRISPR-mediated ablation of troublesome receptors of T cells, Hu et al evaluated nucleofection as a simpler and more robust alternative technique to disrupt PD-1 receptor and PiggyBac transposon in CD133 CAR T-cells. This method yielded more than 90 percent disruption of PD-1 and the recombinant CARs showed better anti-glioma responses both in vitro and in mice models compared to conventional CD133 CAR T-cell immunotherapy [ 110 ].…”

CAR T Cells in Solid Tumors: Blueprints for Building Effective Therapies

“…The dominant negative receptor enhanced the functionality of CAR T cells and survival of mice treated with meso-CAR against mesothelioma compared with control CAR with the ability to signal PD-1 ( 178 ). Recent advances in genome editing using CRISPR/Cas9 technology have permitted removal of PD-1 entirely from T cells, and in two solid tumor models (prostate and glioma) have shown benefits of this intervention for tumor regression ( 179 , 180 ). While important for efficacy of transferred cells, and likely to be incorporated into more T cell therapies in the near future, removal of PD-1 would not benefit endogenous exhausted cells specific for potentially unknown antigens.…”