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

Abstract Targeted lipid nanoparticles (tLNPs) enable efficient mRNA delivery to T cells, allowing for in situ generation of chimeric antigen receptor (CAR) T cells without ex vivo 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 in vivo 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 in vitro, and subsequently validating the best performer in vivo 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 in vivo. 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 in vivo CAR T-cell engineering. Highlights Targeting CD7 outperforms other receptors for tLNP-mRNA delivery to T cells Receptor abundance does not predict tLNP-mRNA delivery efficiency Receptor internalization kinetics governs tLNP-mRNA delivery efficiency CD7-targeting LNP-mRNA enables efficient in vivo CAR T-cell engineering Competing Interest Statement Hamideh Parhiz received research support from BioNTech. Hamideh Parhiz and Tyler Ellis Papp are inventors (University of Pennsylvania) on patents describing some of the work presented here. These interests have been fully disclosed to the University of Pennsylvania.