Rational Design of Unsaturated, Thioether Ionizable Lipids for Enhanced In vivo mRNA Delivery

Therapies based on mRNA technology have offered hope to millions of patients worldwide by disrupting the way we treat diseases. The safe and functional delivery of the delicate mRNA molecules to the target tissue is a crucial step in the development of effective vaccines and therapeutic interventions. Lipid nanoparticles (LNP) are the most clinically advanced delivery vehicles for mRNA drugs. Key to the success of LNP is the inclusion of an ionizable cationic lipid. However, the structure-function relationships between ionizable lipids and efficient in-vivo mRNA delivery are still poorly understood. In this work, we focused on the rational design and sequential structural optimization of previously identified ionizable lipids that performed well in vitro , but not in vivo . Through two distinct iterative optimization cycles — one targeting the lipid tail and the other the headgroup — we aimed to understand how the fusogenicity and apparent p K a of the ionizable lipid contribute to LNP delivery performance in vivo . By engineering unsaturated lipids with more hydrophobic, less protonatable amino headgroups with longer alkyl group at the tertiary nitrogen, we achieved both significant improvement of protein expression in vitro , reduced hemolysis risk, and more than 200-fold improvement of in vivo mRNA delivery. When compared head-to-head to a market-approved LNP benchmark, the newly developed ionizable lipids/LNP resulted in equally highly efficient in vivo mRNA delivery, with strong liver and spleen tropism upon intravenous injection, while matching the safety of the approved platform. Our findings are pivotal for the development of next-generation mRNA-LNP therapies and vaccines.