Rapid receptor internalization potentiates CD7-targeted lipid nanoparticles for efficient mRNA delivery to T cells and <i>in vivo</i> CAR T-cell engineering

Targeted lipid nanoparticles (tLNPs) enable efficient mRNA delivery to T cells, allowing for <i>in situ</i> generation of chimeric antigen receptor (CAR) T cells without <i>ex vivo</i> manipulation. This strategy has shown promising therapeutical efficacy in preclinical studies of cardiac fibrosis, cancer, and autoimmune diseases. While multiple T-cell surface receptors have been targeted across studies for tLNP-mediated <i>in vivo</i> CAR T-cell generation and exhibit diverse efficiencies, their comparative performance and the mechanisms underlying these differences remain unclear. Here, we systematically compared tLNPs with antibody-based moieties targeting T-cell receptors including CD2, CD4, CD5, CD7, CD8, or a CD4/8 dual-targeting combination under identical conditions, assessing their mRNA delivery efficiency in human T cells and PBMCs <i>in vitro</i>, and subsequently validating the best performer <i>in vivo</i> in humanized mice. Among all moieties tested, CD7-targeting tLNPs achieved the highest mRNA delivery to T cells and efficiently generated functional CAR T cells <i>in vivo</i>. Mechanistic analysis revealed that receptor internalization, rather than the receptor abundance, is the primary determinant of delivery efficiency, a property intrinsic to each receptor and largely independent of antibody clone. These findings provide a rational framework for selecting optimal targeting moiety to enable highly efficient <i>in vivo</i> CAR T-cell engineering.