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.

项目摘要 我们建议获得新的图像增强流式细胞仪（Attune Cytpix），以支持 1R01GM143723-01A1：基于高斯曲率脂质的基于脂质的纳米颗粒的内体逃逸。 基于RNA的疗法为治疗各种疾病提供了巨大潜力 人类健康。这些包括慢性感染，遗传疾病，特定的癌症和当前的Covid-19 大流行。非病毒脂质的纳米颗粒（LNP）是由批准的主要RNA输送车 FDA，也在许多临床试验中进行评估。 LNP由标准磷脂组成， 胆固醇和可离子脂质（ILS）在酸性条件下变成质子。类似于包裹的病毒， LNP利用内吞途径进入细胞。 RNA传递的成功取决于能力 LNP与内体膜融合并逃脱内体。但是，机制 负责LNP - 粘体融合仍然很大。 1R01GM143723-01A1的核心目标是检验以下假设：包括一类新的结构性 带有单链和小型头组（如甘油单引血）的脂质上最先进的LNP配方 允许我们开处方定义明确的LNP的内部纳米结构，直接影响其融合的能力 内体膜并将RNA货物释放到细胞质中。 获得新的Attune Cytpix设备对于我们项目的发展至关重要，这是两个主要原因。首先， 唯一靠近我们主要研究位置的流式细胞仪仪器目前是非运营的 由于经常发生故障和用户需求较高，导致停机时间延长。其次， Attune Cytpix提供同时通过高通量流式细胞术和高分辨率Brightfield成像 声学焦点，我们当前的设备无法使用。此功能对于 量化LNP货物加载和交付效率。高速Brightfield相机记录个人 事件在通过流动池时，Attune细胞仪软件可确保分析事件 起源于单细胞和颗粒，而不是双重，团块或碎屑。这种能力在细胞中至关重要 和基因疗法研究以及其他旨在理解形态的流式细胞仪实验 每个细胞种群。 通过我们的研究，我们旨在发现对LNP和LNP和 确定LNP的最佳膜特性，以增强生活系统中的融合。这将导致 开发RNA输送车辆在交付货物方面的有效性更大。