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.

项目概要 RNA疗法对于治疗许多对人类有重大影响的疾病有着巨大的希望健康，例如慢性感染、遗传性疾病、某些癌症以及目前的 COVID-19。经 FDA 批准并在多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) 的最先进的 LNP 包含标准磷脂、胆固醇和可电离的物质。与包膜病毒类似，LNP 会劫持内吞细胞。 RNA 进入细胞的途径取决于 LNP 逃逸内体的能力。然而，控制 LNPs 与内体融合的因素仍然很大程度上未知。有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在 LNP 中，促进融合的替代策略包括使用具有非零自发曲率的脂质。然而，理解膜融合需要考虑以下因素：具体来说，膜弹性超出自发曲率，在两者之间形成融合孔。双层由弯曲模量和高斯曲率模量之间的相互作用决定。在设计“融合”LNP 时很少考虑高斯模量，尽管双层融合是一种其价值最重要的场合。这项工作的中心假设是通过包含一类新的 LNP 来提高 LNP 的高斯模量称为高斯曲率脂质 (GCL) 的脂质对 LNP 融合的能力具有显着影响此外，我们推测，GCL 整合促进了膜融合。在活性质子泵和内体酸化过程中，在生命系统中具有协同作用。我们拥有一支由 RNA 递送至细胞、膜蛋白纯化以及实验、在目标 1 中，我们将通过计算和理论膜弹性来测试中心假设。建立 LNP 的生物物理弹性特性，以最大限度地与内涵体融合。融合发生在微观层面，即破译主导效应是否是融合的形成毛孔和/或如果 LNP 将脂质供给内体膜，重塑它们并使它们更容易发生在目标 2 中，我们通过测量来研究膜活性和内体酸化的影响。活细胞 RNA 递送和 LNP 的内体融合，包含越来越多的 GCL。开发用内体膜质子泵（V- ATPase）阐明活性质子泵期间 LNP-内体膜融合的机制。我们的工作将提出关于 LNP 内体逃逸的新物理见解并建立所需的 LNP 膜促进生命系统融合的特性，从而产生更有效的 RNA 递送载体。