Friday, May 9, 2025
5/9/2025
Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids - PROJECT SUMMARY RNA therapeutics hold great promise for the treatment of a number of diseases significantly impacting human health, such as chronic infections, genetic disorders, certain cancers, and presently COVID-19. The leading RNA delivery vehicles approved by the FDA, as well as being considered in several clinical trials, are non-viral lipid- based nanoparticles (LNPs). State-of-the art LNPs comprise standard phospholipids, cholesterol, and ionizable lipids (ILs) that get protonated in acidic conditions. Analogous to enveloped virus, LNPs hijack the endocytic pathway to enter cells. The efficacy of RNA delivery hinges on the ability of LNPs to escape the endosome by fusing with its membrane. However, the factors that control LNPs–endosome fusion remain largely unknown. Enveloped viruses contain proteins that promote fusion by stabilizing the formation of highly curved membrane pores. In LNPs, alternative strategies to bolster fusion include using lipids with non-zero spontaneous curvature that are elusively deemed “fusogenic”. However, understanding membrane fusion requires the consideration of membrane elasticity beyond spontaneous curvature. Specifically, the formation of a fusion pore between two bilayers is dictated by an interplay between the bending modulus and the Gaussian curvature modulus. However, the Gaussian modulus is rarely considered when designing “fusogenic” LNPs, even though bilayer fusion is an occasion for which its value matters the most. The central hypothesis of this work is that raising the Gaussian modulus of LNPs by inclusion of a new class of lipids termed Gaussian curvature lipids (GCLs) has a dramatic effect on the ability of LNPs to fuse with endosomal membranes. Furthermore, we conjecture that membrane fusion, as boosted by GCL integration, is synergistically favored in living systems during active proton pumping and endosome acidification. We combine a team of experts in RNA delivery to cells, membrane protein purification as well as experimental, computational, and theoretical membrane elasticity to test the central hypotheses via two aims. In Aim 1 we will establish the biophysical elastic properties of LNPs to maximize fusion with endosomes. We investigate how fusion takes place at a microscopic level, namely deciphering if the dominant effect is the formation of fusion pores and/or if LNPs feed lipids to endosomal membranes remodeling them and making them more prone to rupture. In Aim 2 we investigate the impact of membrane activity and endosome acidification by measuring in live cells RNA delivery and endosomal fusion of LNPs comprising increasing amounts of GCLs. We will also develop endosome-mimetic vesicular systems reconstituted with endosomal membrane proton pumps (V- ATPase) to elucidate the mechanism of LNP-endosomal membrane fusion during active proton pumping. Our work will raise new physical insights on LNP endosomal escape and establish the desired LNP membrane properties to boost fusion in living systems, resulting in substantially more effective RNA delivery vehicles.
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