Systematic Design, Synthesis, and Evaluation of Ionizable Lipids for Lipid Nanoparticles Delivering mRNA

Nucleic acids offer immense potential to act as targeted therapeutics and overcome limitations of traditional small molecule drugs. Diseases that are conventionally deemed untreatable and "untargetable" by small molecules are being addressed by alternative approaches, including nucleic acid therapeutics. However, to enable the feasibility of nucleic acid therapeutics, the challenge of delivery must be answered. To deliver RNA, both extracellular and intracellular barriers must be overcome. Due to the immune system's ability to safeguard against foreign materials and RNA's propensity for degradation, the delivery of RNA requires a carrier to protect it. Lipid nanoparticles (LNPs) have been developed to provide a modality enabling delivery of genetic cargos and represent the most promising vehicle to date for its reproducibility, safety, and efficacy. The works within this dissertation provide an insight into how chemical structures of ionizable amino lipids can control the functional mRNA delivery. Ionizable amino lipids were synthesized to study the fundamental structure-activity relationship (SAR) between the lipids and the in vitro and in vivo delivery of mRNA. Identification of potent ionizable amino lipids led to analysis of its respective physiochemical properties as it relates to in vivo LNP potency, revealing a range of physiochemical properties that expanded beyond the previously established ranges. Additionally, a systematic design, synthesis, and evaluation of ionizable amino lipids containing unsaturated motifs was conducted, inspired by the importance of unsaturated lipids in nature. These studies revealed that increased number of unsaturation does not necessarily result in improved mRNA-LNP potency. The inclusion of unsaturated motifs derived from natural products were most successful. Mechanistic studies highlighted how unsaturation can improve fusogenicity and affect cellular trafficking of LNPs. Combining unsaturated lipids with saturated lipids resulted in the best performing LNP formulations, suggesting there needs to be a fine balance of both components for successful mRNA delivery. These studies reiterate the importance of the design of chemical structure of the ionizable lipid used in LNPs, but also highlights the complexity of delivering mRNA.