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Pharmacological control of CAR T cells through CRISPR-driven rapamycin resistance

Levesque, S.; Carleton, G.; Duque, V.; Goupil, C.; Fiset, J.-P.; Villeneuve, S.; Normandeau, E.; Morin, G.; Dumont, N.; Nelson, B. H.; Laganiere, J.; Boyle, B.; Lum, J. J.; Doyon, Y.

2024-02-05 cancer biology
10.1101/2023.09.14.557485 bioRxiv
Show abstract

Chimeric 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 the MTOR locus 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 the MTOR locus. Using in vitro cytotoxicity 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 immunosuppressing doses of rapamycin. Furthermore, our strategy allows multiplexed targeting of rapamycin-regulated immunoreceptors complexes (DARICs) to the MTOR and TRAC loci to pharmacologically control CAR-T cells activity. We foresee that our approach could both facilitate the enrichment of CRISPR-engineered CAR-T cells ex vivo and in vivo while improving tumor eradication.

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