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.