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Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

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