Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells

Chimeric Antigen Receptor (CAR) redirected T-cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T-cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome editing method for efficient CAR insertion into the TRAC locus of primary human T-cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knock-in rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knock-in rates and CAR T-cell yield. Resulting TRAC-replaced CD19-CAR T-cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon-sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair modulating small molecules. With TRAC-integrated CAR+ T-cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T-cells.