Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids

项目摘要 RNA治疗剂对于治疗显著影响人类的许多疾病具有很大的希望， 健康，如慢性感染，遗传性疾病，某些癌症，目前COVID-19。主要RNA FDA批准的以及在几项临床试验中考虑的递送载体是非病毒脂质， 基于纳米颗粒（LNP）的纳米颗粒。现有技术的LNP包含标准磷脂、胆固醇和可电离的磷脂。 脂质（IL）在酸性条件下质子化。类似于包膜病毒，LNP劫持内吞作用， 进入细胞的途径。RNA递送的功效取决于LNP通过脂质体释放而逃离内体的能力。 与细胞膜融合然而，控制LNP-内体融合的因素在很大程度上仍然未知。 包膜病毒含有通过稳定高度弯曲膜的形成来促进融合的蛋白质 毛孔在LNP中，支持融合的替代策略包括使用具有非零自发曲率的脂质 被认为是“融合的”然而，理解膜融合需要考虑 膜弹性超过自发弯曲。具体地说，两个细胞之间融合孔的形成是通过 双层由弯曲模量和高斯曲率模量之间的相互作用决定。然而，在这方面， 当设计“融合”LNP时，很少考虑高斯模量，即使双层融合是一种 它的价值最重要的场合。 这项工作的中心假设是，通过包含一类新的 被称为高斯曲率脂质（GCL）的脂质对LNP与LNP融合的能力具有显著影响。 内体膜此外，我们推测，膜融合，作为推动GCL整合， 在活性质子泵送和内体酸化期间，在生命系统中协同有利。 我们联合收割机的专家团队在RNA输送到细胞，膜蛋白纯化以及实验， 计算，和理论膜弹性，通过两个目标来测试中心假设。在目标1中， 建立LNP的生物物理弹性特性以最大化与内体的融合。我们将探讨如何 融合发生在一个微观的层面上，也就是说，如果主导效应是融合的形成， 孔和/或如果LNP将脂质供给到内体膜，则重塑内体膜并使其更容易 破裂在目标2中，我们通过测量细胞内的蛋白质来研究膜活性和内体酸化的影响。 活细胞RNA递送和包含增加量的GCL的LNP的内体融合。我们还将 开发用内体膜质子泵（V-）重建的内体模拟囊泡系统。 ATP酶），以阐明主动质子泵期间LNP-内体膜融合的机制。 我们的工作将提出新的物理见解LNP内体逃逸和建立所需的LNP膜 这些特性可以促进生命系统中的融合，从而产生更有效的RNA递送载体。

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.