Lipid-based mRNA chimeric antigen receptor T cells for enhanced in vitro antitumor activity

Summary: Chimeric antigen receptor (CAR) T cell therapy provides substantial clinical benefits in hematologic malignancies. However, cell engineering using conventional viral vectors is costly, labor intensive, and prone to safety risks, while electroporation-induced cellular damage limits manufacturing scalability. This study reports a safe, high-efficiency, nonviral platform for generating mRNA-engineered CAR T cells. Herein, eight ionizable lipids are synthesized and formulated into lipid nanoparticles. Systematic screening and structural optimization identified lipid 6 as the lead candidate. Transfection efficiency is validated by delivering enhanced green fluorescent protein ( eGFP ) mRNA into DC2.4, peripheral blood mononuclear, dendritic, and natural killer cells. Murine models confirm robust in vivo expression and a favorable safety profile. Lipid 6-mediated delivery of MUC1 mRNA generates MUC1 -CAR T cells characterized by high surface receptor expression and potent cytotoxicity against MUC1-positive tumor cells. These findings establish lipid 6 as a feasible strategy for primary human T cell engineering, facilitating functional CAR expression with minimal toxicity.