Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids

包含高斯曲率脂质的基于脂质的纳米粒子的内体逃逸

基本信息

批准号：
10446400
负责人：
Cecilia Leal
金额：
$ 39.77万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-10 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10446400
关键词：
Affect Biological Assay Biophysics COVID-19 COVID-19 vaccine Cell Line Cell Membrane Proteins Cells Cholesterol Clinical Clinical Trials Communication Confocal Microscopy Cryoelectron Microscopy Cytosol DNA delivery Data Diffuse Disease Drug Delivery Systems Elasticity Endocytosis Pathway Endosomes Eukaryotic Cell Evaluation Event Evolution FDA approved Flow Cytometry Fluorescence Resonance Energy Transfer Formulation Gaussian model Gene Delivery Generations Genetic Diseases Goals Health Human Label Lipids Malignant Neoplasms Measures Membrane Membrane Fusion Messenger RNA Microfluidics Microscopic Modeling Modulus Molecular Nucleic Acids Organism Pathway interactions Phase Phospholipids Process Property Proteins Proton Pump RNA delivery Research Support Roentgen Rays Role Rupture Saccharomyces cerevisiae Series Spectrum Analysis Stress Swelling System Testing Therapeutic Work Yeasts base cell type chronic infection delivery vehicle design experimental study fluorophore innovation insight interdisciplinary approach lipid nanoparticle mRNA delivery membrane activity microscopic imaging mimetics models and simulation molecular modeling nanoparticle delivery novel strategies protein expression protein purification reconstitution simulation therapeutic RNA unilamellar vesicle vacuolar H+-ATPase virus envelope

项目摘要

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.

项目摘要 RNA疗法对多种疾病的治疗具有很大的希望健康，例如慢性感染，遗传疾病，某些癌症和目前的癌症。领先的RNA FDA批准的运输车辆以及在几项临床试验中被考虑的是非病毒脂质的 - 基于纳米颗粒（LNP）。最先进的LNP包括标准磷脂，胆固醇和可离子化在酸性条件下质子化的脂质（IL）。类似于包裹的病毒，LNP劫持了内吞进入细胞的途径。 RNA递送的效率取决于LNP的能力逃脱内体的能力与膜融合。然而，控制LNP-粘体融合的因素在很大程度上仍然未知。包围病毒含有蛋白质，可通过稳定高度弯曲的膜的形成来促进融合毛孔。在LNP中，提高融合的替代策略包括使用具有非零赞助商自发曲率的脂质被认为是“融合的”。但是，了解膜融合需要考虑膜弹性超出了赞助曲率。具体而言，两个之间的融合孔的形成双层由弯曲模量与高斯曲率模量之间的相互作用决定。然而，在设计“ Fusogen ogen” 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。这项工作的核心假设是，通过包括一类新的类别来提高LNP的高斯模量称为高斯曲率脂质（GCLS）的脂质对LNP与融合的能力具有巨大影响内体膜。此外，我们猜想GCL集成所增强的膜融合是在活性质子泵送和内体酸化过程中，在生物系统中有协同偏爱。我们结合了一个在RNA向细胞，膜蛋白纯化以及实验性的专家组合组合的团队，计算和理论膜弹性通过两个目的测试中心假设。在目标1中，我们将建立LNP的生物物理弹性特性，以最大程度地与内体融合。我们研究了如何融合发生在显微镜水平上，即如果主要效应是融合的形成，则解密毛孔和/或LNP是否将脂质喂给内体膜进行重塑，使它们更容易容易破裂。在AIM 2中，我们通过测量膜活性和内体酸化的影响活细胞的RNA递送和LNP的内体融合，完成了增加数量的GCL。我们也会开发与内体膜质子泵重构的内体模拟囊泡系统（V- ATPase）阐明活性质子泵送过程中LNP-粘膜融合的机理。我们的工作将提出有关LNP内体逃生的新物理见解，并建立所需的LNP膜属性以增强生活系统的融合，从而产生更有效的RNA输送车辆。