Rational design of ionizable lipids for intramuscular messenger ribonucleic acid vaccines

Messenger ribonucleic acid (mRNA) technology holds immense potential to revolutionize vaccine development. However, to expand its application beyond COVID-19, improvements in both immunogenicity and reactogenicity are needed. Lipid nanoparticle (LNP) chemistry plays a critical role in overcoming these limitations by modulating delivery pathways and immune responses. This thesis focuses on the rational design of LNPs for intramuscular mRNA vaccines in mice, providing insights into their structure- activity relationships. First, we showed that the chemical structure of ionizable lipids, the key component of LNPs, can be systematically optimized to improve protein yield after intramuscular administration of mRNA-LNPs. A direct relationship between the pH-driven ionization behavior of LNPs and protein yield was identified, with an optimal pKa near 6.5. Using these insights, we designed an ionizable lipid that doubled the protein yield achieved with commercial lipids. Next, we investigated the adjuvant activity of LNPs. We showed that LNPs trigger acute inflammation, characterized by elevated IL-6 levels and recruitment of inflammatory monocytes–the primary immune cells translating the mRNA cargo and expressing antigen. Using a prime-boost influenza mRNA vaccine model, we found that current LNPs elicited protective humoral responses but weak antigen-specific CD8+ T cell responses. Importantly, we discovered that modifying the ionizable lipid to enhance both antigen production and monocyte activation improved antigen-specific CD8+ T cell responses tenfold, marking a significant advance in mRNA development. Finally, we addressed the challenge of slow ionizable lipid elimination from organs, which can hinder vaccine tolerability. We developed a cell-free platform to assess lipid biodegradation kinetics and identified steric factors as key determinants of organ clearance. These insights enabled the design an ionizable lipid that was rapidly degraded in vivo, reducing systemic exposure by approximately 70% over 7 days compared to a slow-degrading analog. Together, this thesis advances our understanding of the role of ionizable lipid chemistry in mRNA vaccines. These findings demonstrate that the rational design of ionizable lipids can enhance both immunogenicity and tolerability, providing a framework to design next-generation mRNA vaccines.