Design and Synthesis of Ionizable Lipids for mRNA Delivery

Messenger ribonucleic acids (mRNA) fulfill the role of relaying genetic information from the nucleus to the protein synthesis machinery of the cell. Once there, mRNA is translated into one of the myriad proteins that form living things. Now, with the ability to design and synthesize any mRNA at the lab bench, we too have gained the ability to instruct cells on which proteins to synthesize. However, mRNA made in a lab is unable to cross the cell membrane on its own and is recognized by the body’s immune system as foreign material to be degraded. We must improve our understanding of these biological barriers and develop methods to overcome them to achieve widespread adoption of mRNA therapies. The most clinically advanced systems for mRNA delivery are lipid nanoparticles (LNPs), which protect mRNA and carry it into the cell. The main components of LNPs that drive their efficacy are known as ionizable lipids; these special lipids are designed with pH sensitive motifs that help encapsulate mRNA and overcome cellular barriers. The chemistry of these ionizable lipids can be tuned in many ways to elicit different biological responses. As a result, the chemical space available to ionizable lipids is vast: even subtle changes in chemical structure may have large effects on biological outcomes. Therefore, this Thesis focuses on the design, synthesis, and experimental validation of ionizable lipids for mRNA delivery to cells. A design process for ionizable lipids is developed in the context of intramuscular mRNA delivery – a route of administration that is relevant for clinical translation of vaccines. Multiple families of ionizable lipids are designed using this process and optimized for properties such as assembly into high-quality LNPs, overall safety, and efficient cellular delivery of mRNA. Notable findings include: a clear relationship between ionization behaviour of LNPs and mRNA delivery efficacy; the influence of lipid biodegradability on inflammatory responses; and mRNA uptake into cells and escape from endosomes as key barriers for effective delivery. Altogether, this body of work advances the field by providing clear structure-function relationships for the development of ionizable lipids, and insights into the mechanisms of cellular delivery and immune responses.