Gough, O. J.; Georgiadis, C.; Preece, R.; Kadirkamanathan, R.; QASIM, W.

Chimeric Antigen Receptor (CAR) T cells are now established as therapies for some haematological malignancies. While lentiviral or {gamma}-retroviral vectors are commonly used for CAR delivery due to their efficiency and stable integration, supply constraints have created bottlenecks to wider applications and access. Alternatively, genome editing tools such as CRISPR-Cas9 can insert CAR genes by homology-directed repair (HDR) into specific genomic loci. 'Universal' donor CAR-T cells devoid of endogenous TCR{beta} after CRISPR-Cas9-mediated editing of the T cell receptor alpha (TRAC) locus are being investigated for more cost-effective, 'off-the-shelf' therapies. Targeting insertion of CARs into the TRAC locus places transcription under the control of native regulatory machinery while simultaneously disrupting endogenous TCR{beta}, and this has been reported to reduce exhaustion and extend persistence in modelling studies using humanised mice. We compared anti-CD20 CAR-T cells, generated with CAR inserts at either TRAC or CD3{zeta} loci using entirely virus-free manufacture, and universal CAR20-T cells generated using existing lentiviral procedures and CRISPR/Cas9 knockout. While non-viral cell yields were lower than lentiviral products cytotoxic function in vitro was comparable between groups. Studies in humanised murine models of leukaemia inhibition found non-viral CAR20-T cells were generally less efficacious than LV-CAR20 and exhibited more exhausted phenotypes. Non-viral approaches offer the prospect of sophisticated editing and precise CAR insertion but careful preclinical evaluation and well-designed clinical trials benchmarked against lentiviral approaches are recommended.