Cationic mRNA Lipid Nanoparticles for Ex Vivo NanoCAR‐T Cell Engineering

Lipid nanoparticles (LNPs) are proposed as an attractive non-viral alternative for mRNA-based CAR-T cell engineering. However, the impact of the LNP charge and the composition of the transfection medium remain to be fully explored. Here, charge-neutral C12-200 LNPs and cationic DOTAP(C12-200) LNPs were compared for ex vivo transfection of activated primary human T cells. C12-200 LNPs achieved efficient transfection with high cell viability in serum-free medium supplemented with apolipoprotein E. Transfection efficiency correlated with low-density lipoprotein receptor expression following T cell activation, suggesting receptor-mediated uptake. In contrast, cationic LNPs demonstrated superior mRNA expression independent of medium composition or T cell activation state, albeit with reduced cytocompatibility. Both LNPs efficiently delivered anti-CD20 nanoCAR mRNA into primary T cells, enabling potent cytotoxicity against CD20⁺ Raji cells in vitro. Together, our findings demonstrate that LNP charge, selected proteins in the culture medium, and T cell activation collectively play a critical role in ex vivo T cell engineering. Here, cationic LNPs offer the advantage of unspecific charge-dependent transfection, inducing high mRNA expression independent on T cell activation status or complex cell culture media. These traits offer opportunities to extend expression kinetics, transfect less activated T cell phenotypes, and simplify CAR-T cell manufacturing.