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

Abstract

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.