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

• 批准号: 10798629
• 负责人: Cecilia Leal
• 金额: $ 16.79万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 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/10798629
• 关键词: Acoustics; COVID-19 pandemic; COVID-19 vaccine; Cell Therapy; Cells; Cholesterol; Clinical Trials; Computer software; Cytosol; Development; Disease; Drug Delivery Systems; Endosomes; Ensure; Equipment; Event; FDA approved; Flow Cytometry; Formulation; Gene Delivery; Genetic Diseases; Goals; Grant; Health; Human; Image; Image Enhancement; Individual; Intake; Lipids; Location; Malignant Neoplasms; Membrane; Messenger RNA; Morphology; Nanostructures; Organism; Pathway interactions; Phospholipids; Population; Property; RNA; RNA delivery; Records; Research; Research Support; Resolution; Speed; System; Testing; Therapeutic Studies; Virus; Work; chronic infection; delivery vehicle; design; endosome membrane; experimental study; gene therapy; insight; instrument; lipid nanoparticle; monoolein; novel; particle; success; therapeutic RNA

PROJECT SUMMARY We propose the acquisition of a new image-enhanced flow cytometer (Attune CytPix) to support the research of 1R01GM143723-01A1: Endosomal escape of lipid-based nanoparticles comprising Gaussian curvature lipids. RNA-based therapies offer significant potential for treating a variety of diseases that have a major impact on human health. These include chronic infections, genetic disorders, specific cancers, and the current COVID-19 pandemic. Non-viral lipid-based nanoparticles (LNPs) are the primary RNA delivery vehicles approved by the FDA and are also being evaluated in numerous clinical trials. LNPs are composed of standard phospholipids, cholesterol, and ionizable lipids (ILs) that become protonated in acidic conditions. Similar to enveloped viruses, LNPs exploit the endocytic pathway to gain entry into cells. The success of RNA delivery depends on the ability of LNPs to fuse with the endosomal membrane and escape the endosome. However, the mechanisms that govern LNP-endosome fusion remain largely unknown. The central goal of 1R01GM143723-01A1 is to test the hypothesis that the inclusion of a new class of structural lipids with single chains and small headgroups like glycerol monooleate onto state-of-the art LNP formulations allows us to prescribe well-defined internal nanostructures of LNPs directly impacting their ability to fuse with endosomal membranes and releasing RNA cargo into the cytosol. Acquiring the new Attune CytPix equipment is vital to the progression of our project for two main reasons. Firstly, the only flow cytometry instrument in close proximity to our primary research location is currently non-operational due to frequent breakdowns and high user demand, resulting in prolonged periods of downtime. Secondly, the Attune CytPix offers simultaneous high throughput flow cytometry and high resolution brightfield imaging through acoustic focusing, which is not available with our current equipment. This feature is particularly important for quantifying LNP cargo loading and delivery efficiency. The high-speed brightfield camera records individual events as they pass through the flow cell, and the Attune Cytometric Software ensures that analyzed events originate from single cells and particles rather than doublets, clumps, or debris. This capability is critical in cell and gene therapy research, as well as other flow cytometry experiments that aim to understand the morphology of each cell population. Through our research, we aim to uncover novel physical insights into the endosomal escape of LNPs and determine the optimal membrane properties of LNPs to enhance fusion in living systems. This will lead to the development of RNA delivery vehicles that are significantly more effective in delivering their cargo.

项目概要 我们建议购买新型图像增强流式细胞仪 (Attune CytPix) 以支持以下研究 1R01GM143723-01A1：包含高斯曲率脂质的基于脂质的纳米颗粒的内体逃逸。 基于 RNA 的疗法为治疗多种对人类健康产生重大影响的疾病提供了巨大的潜力。 人类健康。其中包括慢性感染、遗传性疾病、特定癌症和当前的 COVID-19 大流行。非病毒脂质纳米颗粒 (LNP) 是 FDA 批准的主要 RNA 递送载体 FDA 也在众多临床试验中对其进行评估。 LNP 由标准磷脂组成， 胆固醇和在酸性条件下质子化的可电离脂质 (IL)。与有包膜病毒类似， LNP 利用内吞途径进入细胞。 RNA 递送的成功取决于能力 LNP 与内体膜融合并逃离内体。然而，这些机制 控制 LNP-内体融合的机制仍然很大程度上未知。 1R01GM143723-01A1 的中心目标是检验以下假设：包含一类新的结构 将具有单链和小头基的脂质（例如单油酸甘油酯）添加到最先进的 LNP 配方中 允许我们规定明确的 LNP 内部纳米结构，直接影响它们与 内体膜并将 RNA 货物释放到细胞质中。 购买新的 Attune CytPix 设备对于我们项目的进展至关重要，主要原因有两个。首先， 靠近我们主要研究地点的唯一流式细胞仪目前无法运行 由于频繁的故障和高用户需求，导致长时间的停机。其次， Attune CytPix 通过以下方式同时提供高通量流式细胞术和高分辨率明场成像 声学聚焦，这是我们现有设备所不具备的。这个功能对于 量化 LNP 货物装载和交付效率。高速明场相机记录个体 当事件通过流动池时，Attune 细胞计数软件可确保分析的事件 源自单个细胞和颗粒，而不是双联体、团块或碎片。这种能力在细胞中至关重要 和基因治疗研究，以及其他旨在了解形态学的流式细胞术实验 每个细胞群。 通过我们的研究，我们的目标是揭示 LNP 内体逃逸的新物理见解 确定 LNP 的最佳膜特性，以增强生命系统中的融合。这将导致 开发RNA递送载体，其递送货物的效率显着提高。