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Systemic delivery of messenger RNA (mRNA) for tissue-specific targeting using lipid nanoparticles (LNPs) holds great therapeutic potential. Nevertheless, how the structural characteristics of ionizable lipids (lipidoids) impact their capability to target cells and organs remains unclear. Here we engineered a class of siloxane-based ionizable lipids with varying structures and formulated siloxane-incorporated LNPs (SiLNPs) to control in vivo mRNA delivery to the liver, lung and spleen in mice. The siloxane moieties enhance cellular internalization of mRNA-LNPs and improve their endosomal escape capacity, augmenting their mRNA delivery efficacy. Using organ-specific SiLNPs to deliver gene editing machinery, we achieve robust gene knockout in the liver of wild-type mice and in the lungs of both transgenic GFP and Lewis lung carcinoma (LLC) tumour-bearing mice. Moreover, we showed effective recovery from viral infection-induced lung damage by delivering angiogenic factors with lung-targeted Si5-N14 LNPs. We envision that our SiLNPs will aid in the clinical translation of mRNA therapeutics for next-generation tissue-specific protein replacement therapies, regenerative medicine and gene editing.

Ionizable lipid nanoparticles (LNPs) are the most clinically advanced nonviral platform for mRNA delivery. While they have been explored for applications including vaccines and gene editing, LNPs have not been investigated for placental insufficiency during pregnancy. Placental insufficiency is caused by inadequate blood flow in the placenta, which results in increased maternal blood pressure and restricted fetal growth. Therefore, improving vasodilation in the placenta can benefit both maternal and fetal health. Here, we engineered ionizable LNPs for mRNA delivery to the placenta with applications in mediating placental vasodilation. We designed a library of ionizable lipids to formulate LNPs for mRNA delivery to placental cells and identified a lead LNP that enables in vivomRNA delivery to trophoblasts, endothelial cells, and immune cells in the placenta. Delivery of this top LNP formulation encapsulated with VEGF-A mRNA engendered placental vasodilation, demonstrating the potential of mRNA LNPs for protein replacement therapy during pregnancy to treat placental disorders.

Immune modulation through the intracellular delivery of nucleoside-modified mRNA to immune cells is an attractive approach for in vivoimmunoengineering, with applications in infectious disease, cancer immunotherapy, and beyond. Lipid nanoparticles (LNPs) have come to the fore as a promising nucleic acid delivery platform, but LNP design criteria remain poorly defined, making the rate-limiting step for LNP discovery the screening process. In this study, we employed high-throughput in vivoLNP screening based on molecular barcoding to investigate the influence of LNP composition on immune tropism with applications in vaccines and systemic immunotherapies. Screening a large LNP library under both intramuscular (i.m.) and intravenous (i.v.) injection, we observed differential influences on LNP uptake by immune populations across the two administration routes, gleaning insight into LNP design criteria for in vivoimmunoengineering. In validation studies, the lead LNP formulation for i.m.administration demonstrated substantial mRNA translation in the spleen and draining lymph nodes with a more favorable biodistribution profile than LNPs formulated with the clinical standard ionizable lipid DLin-MC3-DMA (MC3). The lead LNP formulations for i.v.administration displayed potent immune transfection in the spleen and peripheral blood, with one lead LNP demonstrating substantial transfection of splenic dendritic cells and another inducing substantial transfection of circulating monocytes. Altogether, the immunotropic LNPs identified by high-throughput in vivoscreening demonstrated significant promise for both locally- and systemically-delivered mRNA and confirmed the value of the LNP design criteria gleaned from our screening process, which could potentially inform future endeavors in mRNA vaccine and immunotherapy applications.

Cell-based therapies for autoimmune diseases have gained significant traction, with several approaches centered around the regulatory T (Treg) cell─a well-known immunosuppressive cell characterized by its expression of the transcription factor Foxp3. Unfortunately, due to low numbers of Tregcells available in circulation, harvesting and culturing Tregcells remains a challenge. It has been reported that engineering Foxp3 expression in CD4+T cells can result in a Treg-like phenotype; however, current methods result in the inefficient engineering of these cells. Here, we develop an ionizable lipid nanoparticle (LNP) platform to effectively deliver Foxp3 mRNA to CD4+T cells. We successfully engineer CD4+T cells into Foxp3-T (FP3T) cells that transiently exhibit an immunosuppressive phenotype and functionally suppress the proliferation of effector T cells. These results demonstrate the promise of an LNP platform for engineering immunosuppressive T cells with potential applications in autoimmunity therapies.