MTORas a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

Abstract

ABSTRACTChimeric antigen receptors (CARs) reprogram T cells to recognize and target cancer cells. Despite remarkable responses observed with CAR-T cell therapy in patients with hematological malignancies, CAR-T cell engineering still relies mostly on randomly integrating vectors, limiting the possibilities of fine-tuning T cell function. Here, we designed a CRISPR-based marker-free selection strategy to simultaneously target a therapeutic transgene and a gain-of-function mutation to theMTORlocus to enrich cells resistant to rapamycin, a clinically used immunosuppressant. We readily engineered rapamycin-resistant (RapaR) CAR-T cells by targeting CAR expression cassettes to theMTORlocus. Usingin vitrocytotoxicity assays, and a humanized mouse model of acute lymphoblastic leukemia, we show that RapaR-CAR-T cells can efficiently target CD19+leukemia cells in presence of rapamycin. Furthermore, our strategy allows multiplexed targeting of rapamycin-regulated immunoreceptors complexes (DARICs) to theMTORandTRACloci to pharmacologically control CAR-T cells’ activity. We foresee that our approach could both facilitate the enrichment of CRISPR-engineered CAR-T cellsex vivoandin vivowhile improving tumor eradication.