Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids

项目总结 RNA疗法有望用于治疗一些严重影响人类健康的疾病 健康，如慢性感染、遗传疾病、某些癌症，目前为新冠肺炎。领先的核糖核酸 FDA批准的给药载体，以及正在进行的几项临床试验，都是非病毒性脂质-- 基于纳米颗粒(LNPs)。最新的LNPs包括标准的磷脂、胆固醇和可电离的 在酸性条件下质子化的类脂(ILs)。类似于包膜病毒，LNPs劫持内吞 进入细胞的途径。RNA传递的有效性取决于LNPs通过以下途径逃脱内体的能力 与它的膜融合。然而，控制LNPs-内小体融合的因素仍然很大程度上是未知的。 被包裹的病毒含有通过稳定高度弯曲的膜的形成来促进融合的蛋白质 毛孔。在LNPs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 难以捉摸的被认为是“融合基因”的基因。然而，理解膜融合需要考虑 超过自发曲率的膜弹性。具体地说，在两个物体之间形成一个融合孔 双层膜由弯曲模数和高斯曲率模数之间的相互作用决定。然而， 在设计“融合”LNPs时，很少考虑高斯模，尽管双层融合是一种 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LNPs来提高LNPs的高斯模 被称为高斯曲率脂质(GCL)的脂质对LNPs与LNPs的融合能力有很大的影响 内体膜。此外，我们推测在GCL整合的促进下，膜融合是 在主动质子泵送和内体酸化过程中，在生命系统中协同有利。 我们结合了一组专家团队，包括向细胞递送RNA、膜蛋白纯化以及实验、 通过两个目标来验证中心假设的计算性和理论性的膜弹性。在目标1中，我们将 确定LNPs的生物物理弹性性质，以最大限度地与内小体融合。我们调查了 核聚变发生在微观层面上，即破译是否主导效应是核聚变的形成。 毛孔和/或如果LNPs向内体膜提供脂类，重塑它们并使它们更容易 破裂。在目标2中，我们调查了膜活性和内体酸化的影响，通过测量 活细胞、RNA递送和含有越来越多的GCL的LNPs的内体融合。我们还将 开发用内吞体膜质子泵(V-V)重组的内体模拟囊泡系统 ATPase)，以阐明主动质子泵过程中LNP-内膜融合的机制。 我们的工作将对LNP内体逃逸提出新的物理见解，并建立所需的LNP膜 具有促进生物系统融合的特性，从而产生更有效的RNA输送载体。

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.