Off-the-shelf car t cells production using crispr/cas9 and mRNA technologies

Abstract

Autologous chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in hematologic malignancies but are limited by complex manufacturing, high costs, and variability due to patient-derived T cells. Allogeneic, off-the-shelf CAR T cells derived from healthy donors offer a promising solution. We will produce allogeneic, off-the-shelf CAR T cells by transiently expressing CAR molecules via mRNA in TCR-negative T cells. We will also optimize cryopreservation protocols to enable flexible CAR expression on demand. CRISPR/Cas9 was used to knock out the TRAC locus in healthy donor T cells, yielding TCR-negative cells that were cryopreserved and later transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, T cell phenotype, viability, and cytotoxicity were evaluated across different cryopreservation-transfection strategies. CRISPR/Cas9 editing achieved high TCRα/β- and CD3-knockout efficiency with 95% post-thaw viability. mRNA transfection yielded CAR expression of 90.03% (CD117), 85.65% (BCMA), and 63.97% (CD19), with tumor killing efficiencies of 76.63%, 94.09%, and 55.57%, respectively, comparable to fresh cells. When mRNA transfection was performed after thawing, the resulting CAR T cells had higher CAR-T levels and reduced the expression of activation (CD69) and exhaustion (TIM3) markers compared to pre-transfection cryopreservation. This study presents an optimized workflow for producing allogeneic CAR T cells using cryopreservable, TCR-deficient T cells and mRNA transfection performed after thawing.