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

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

Blog Article

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 Acrylic Keychain (custom charms) 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 NO-CREASE CLIPS relative CAR knockin rates, respectively.While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin 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.