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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. [ABSTRACT FROM AUTHOR]

Genetically engineered T cell therapy is emerging as a potent strategy for treating hematological and solid malignancies. Although lentivirus is the most common vector for T cell gene modification, its transduction efficacy remains unsatisfied especially during the manufacturing process. Herein, glycometabolic bioorthogonal chemistry is utilized to establish a highly efficient viral transduction system for human primary T lymphocytes. Azide motifs are anchored on the T cell surface via the intrinsic glycometabolism of exogenous azide–glucose, serving as an artificial ligand for viral binding. The complementary functional moiety dibenzocyclooctyl (DBCO)‐conjugated PEI1.8K (PEI‐DBCO) is then coated on lentiviral surface, which strengthens the virus–T cell interaction through DBCO/azide bioorthogonal chemistry. The results show that the PEI‐DBCO/azide–glucose system effectively facilitates viral binding to T cells and elevates the transduction efficiency of the lentivirus from 20% to 80% without any effect on T cell proliferation and activity. More importantly, the PEI‐DBCO/azide–glucose system significantly doubles the yield of anti‐CD19 chimeric antigen receptor T (CAR‐T) cells and robustly boosts their antitumor capability compared to polybrene‐assisted lentiviral transduction both in vitro and in vivo. Overall, the bioorthogonal PEI‐DBCO/azide–glucose system significantly boosts viral transduction efficacy and exhibits a powerful gene‐manipulating capability in human primary T cells, thereby showing a great potential for clinical‐engineered T lymphocytes manufacture. [ABSTRACT FROM AUTHOR]