Comprehensive Engineering of Ionizable Lipid Nanoparticles and mRNA Elements for Next-Generation Vaccines.

Messenger RNA (mRNA) vaccines in lipid nanoparticles (LNPs) are effective, yet immunity can wane, and reactogenicity increases with dose. We integrate ionizable lipid design, LNP formulation, and untranslated region (UTR) tuning to improve potency at low doses. From a 96-member biodegradable ionizable lipid library, stereodefined H9T6 emerged as the lead ionizable lipid. Design of experiments (DoE) identified an H9T6:DOPG:cholesterol:PEG composition (40:16:43.5:0.5 mol %) that achieved more than 3.5-fold higher dendritic-cell transfection than SM-102, increased endosomal escape, and localized expression predominantly to the injection site. For UTRs, mechanism-linked pooled screens of synthetic, barcoded randomized libraries ranked more than 630,000 3' UTR variants by half-life in primary human nasal epithelial cells and more than 68,000 5' UTR sequences by ribosome loading via polysome profiling, followed by predefined sequence filters consistent with efficient scanning and start codon recognition. The resulting 5B-8 scaffold outperformed benchmark UTRs from authorized mRNA vaccines in peak and cumulative antigen expression in vitro and in vivo. Encapsulation of 5B-8 mRNA in H9T6 LNPs enhanced lymph node delivery, and add-back plus receptor-blocking assays support an albumin-mediated transport mechanism. In mice, the formulation elicited strong T helper 1 (Th1)-biased humoral and cellular responses at low-microgram doses. Antibody magnitude depended primarily on UTR architecture, whereas T-cell responses reflected contributions from both the lipid and the UTR. Repeat-dose studies in rats up to 100 μg showed only transient, reversible findings without additional safety signals. These results outline a general framework for low-dose, lymph node-targeted mRNA vaccines that couples rational lipid design with mechanism-informed UTR selection.