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

Area of Science:

• Vaccinology
• Biotechnology
• Immunology

Background:

• Messenger RNA (mRNA) vaccines delivered via lipid nanoparticles (LNPs) are effective but face challenges with waning immunity and dose-dependent reactogenicity.
• Optimizing mRNA vaccine formulations is crucial for enhancing potency, reducing side effects, and improving immune responses.

Purpose of the Study:

• To develop a novel mRNA vaccine platform that improves potency at low doses by integrating ionizable lipid design, LNP formulation, and untranslated region (UTR) optimization.
• To enhance vaccine efficacy, targeted delivery to lymph nodes, and immune responses, particularly T helper 1 (Th1)-biased responses.

Main Methods:

• Screened a library of 96 biodegradable ionizable lipids, identifying H9T6 as a lead candidate.
• Employed Design of Experiments (DoE) to optimize LNP composition for enhanced dendritic-cell transfection and endosomal escape.
• Utilized mechanism-linked pooled screens to identify optimal 5' and 3' UTR sequences for mRNA stability and translation.
• Evaluated mRNA-LNP formulations in vitro and in vivo for antigen expression, lymph node delivery, and immune responses in mice and safety in rats.

Main Results:

• The optimized H9T6-based LNPs demonstrated over 3.5-fold higher dendritic-cell transfection and improved expression localization compared to SM-102.
• The developed 5B-8 UTR scaffold significantly outperformed benchmark UTRs in both peak and cumulative antigen expression.
• Formulations enhanced lymph node delivery, mediated by an albumin-transport mechanism, and elicited strong Th1-biased humoral and cellular responses at low microgram doses.
• Repeat-dose studies in rats showed no additional safety signals, with only transient and reversible findings.

Conclusions:

• A general framework for low-dose, lymph node-targeted mRNA vaccines was established by combining rational lipid design with mechanism-informed UTR selection.
• This approach holds promise for developing more potent and safer mRNA vaccines with improved efficacy and reduced reactogenicity.
• The findings pave the way for next-generation mRNA vaccines with tailored immune responses and enhanced delivery characteristics.