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

ABSTRACT 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.