Ionizable Triazine Lipids for RNA Delivery

PROJECT SUMMARY There are many potential applications of RNA-based therapies, including silencing gene expression, gene editing, and developing mRNA vaccines. RNA must get inside cells to serve its purpose, but its size, charge, immunogenicity, and instability lead to its degradation and do not allow it to pass through the plasma membrane. One of the most effective ways to protect RNA and transport it into the cytoplasm is by packaging it in a lipid nanoparticle (LNP). A key component of LNP formulations is a cationic or ionizable lipid that electrostatically interacts with the phosphate backbone of RNA and helps to encapsulate it. It also interacts with the cell membrane and disrupts the endosomal membrane, causing it to rupture and release the RNA into the cytoplasm. The structure of the cationic or ionizable lipid plays an important role in determining its ability to form LNPs with RNA and deliver it into cells. Many cationic and ionizable lipids have been prepared, and with so many potential applications across different diseases, types of RNA, and cell types, it is important to have a diverse toolbox of lipids available so the optimal formulation can be developed for each application. The major goal of the proposed work is to develop a library of triazine-based, ionizable lipids that can be used to form lipid nanoparticles (LNPs) to deliver RNA into cells. A structure-activity relationship (SAR) correlating lipid structure, LNP physical properties, RNA delivery in vitro, and cytotoxicity will also be developed and used to guide the preparation of new lipids. Specifically, modifications to the structures of the amine-based hydrophilic head groups (Aim 1) and the hydrophobic tails (Aim 2) will be done to determine how targeted structural changes affect LNP physical properties and in vitro biological activity. The synthetic approach will make it easy to prepare lipids with symmetrical and unsymmetrical head groups and tails. Additionally, a protecting group-free synthesis will be developed to streamline lipid library preparation (Aim 3). Successful completion of these aims will result in a novel class of triazine-based lipids for RNA delivery and an SAR that will be used to develop more active lipids.