ABSTRACT
Purpose/Objectives: The delivery of nucleic acids to cells has revolutionized medicine and enabled new technologies such as mRNA vaccines and stem cell therapies. These recent advances rely on delivery vehicles to stabilize the genetic payload and increase cellular transfection. While engineered viruses are efficient vectors for ex vivo cellular reprogramming, they are not ideal for in vivo gene therapies as repeated dosing leads to anti-vector immunity. Lipid nanoparticles have thus emerged as the best alternative to viral vectors for in vivo nucleic acid delivery. However, all FDA-approved lipid nanoparticles have been linked to inflammatory responses, undesirable for regenerative medicine applications that require precise immunomodulation. Thus, non-immunogenic delivery materials must be developed to fulfill the immense potential of gene therapy in regenerative medicine. Lipid nanoparticles typically comprise 4 different lipids, with the ionizable amino lipid being the main driver of potency and immunogenicity. A way to reduce immunogenicity is to develop lipid nanoparticles that minimize the amount of lipids per gram of nucleic acids. To do so, we developed a novel class of ionizable amino lipids with high charge density. Our primary objective is to design a lipid nanoparticle that maximizes RNA delivery and minimizes immunogenicity. Methodology: We designed a library of proprietary ionizable lipids based on the structure of a poly(amido amine) dendron. The structure is modular, which allowed us to systematically vary molecular motifs to optimize important physiochemical parameters: Lipid-to-RNA ratio;apparent pKa;surface zeta potential;size distribution;and RNA encapsulation These structures are also designed to include a higher number of amines compared to current ionizable lipids. This improves ionization charge density of the lipid and lowers the amount of lipid required to encapsulate RNA. In this study, lipid nanoparticles contain an ionizable lipid selected from our library, cholesterol, a phospholipid, and a PEG-lipid. The lipids and formulation conditions were selected to mimic Moderna's COVID-19 vaccine (SpikeVax), albeit with different lipid-to-RNA ratios. C57BL/6 mice were injected intramuscularly with nanoparticles co-formulated with a firefly luciferase mRNA and ovalbumin mRNA to simultaneously study transfection efficiency and antigen-specific immune responses. Nanoparticles that comprise SM-102, the ionizable lipid used in SpikeVax, were used as a comparative control due to their high potency and immunogenicity. Luciferase activity was detected using an IVIS Spectrum, and key organs were harvested for immune phenotyping. Results: We have so far determined the effect of hydrophobic motifs on apparent pKa and RNA encapsulation. Our best lipids with optimized tails did not induce IFN-I responses in vitro and demonstrated comparable in vivo efficacy to SM-102. We are currently in the process of collecting immunogenicity data which we expect to complete prior to the conference. Conclusion/Significance: We have produced a novel set of lipid nanoparticles that efficiently transfect cells in vivo. These new particles deliver RNA with half of the lipid mass used in SpikeVax, which can reduce the amount of material-induced immunogenicity. This result opens the door to developing mRNA vaccines with fewer side effects and equitable gene therapies for untreatable diseases such as inflammatory and autoimmune disorders.