Cytokine co-presentation on targeted lipid nanoparticles enhances in vivo T cell engineering.

T lymphocytes are attractive cellular targets for gene therapy and cell engineering, due to their critical role in adaptive immunity; however, efficient in vivo T cell transfection remains challenging. Antibody-conjugated lipid nanoparticles (LNPs) have emerged as a non-viral approach to engage T cells in vivo, but existing platforms primarily deliver activation signals 1 and 2 and lack cytokine-mediated signal 3 support. Here, we introduce a signal 3-augmented LNP design in which interleukin-7 (IL-7) is co-conjugated to the LNP surface with anti-CD3 and anti-CD28 antibodies, enabling localized tri-signal presentation at the T cell-nanoparticle interface. IL-7 co-presentation by these anti-CD3/anti-CD28/IL-7 LNPs significantly enhances particle uptake and transgene expression in primary human T cells compared to antibody-only LNPs or equivalent supplementation with soluble IL-7, demonstrating the importance of particle-bound signal 3 delivery. In naïve T cells, IL-7-conjugated LNPs promote coupled proliferation and transgene expression, increasing the fraction of transfected cells within both proliferating and non-proliferating populations. In vivo, IL-7-conjugated LNPs bias functional mRNA transfection toward splenic T cells while reducing hepatic T cell transfection, without altering overall organ-level biodistribution. As a proof of concept, IL-7-conjugated LNPs deliver chimeric antigen receptor (CAR) mRNA to generate functional CAR-expressing T cells in vivo, enabling antigen-specific target cell depletion. Together, these results establish cytokine signal 3 co-presentation as an effective design approach for T cell-targeted mRNA LNPs, resulting in enhanced engagement, uptake, and transfection in T cells in vivo.