Enabling isolation and characterization of single extracellular vesicles and their molecular contents using multi-marker surface signatures

使用多标记表面特征来分离和表征单个细胞外囊泡及其分子内容物

• 批准号: 10000244
• 负责人: David Aaron Routenberg
• 金额: $ 40.76万
• 依托单位: MESO SCALE DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-21 至 2021-09-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000244
• 关键词: Address; Antibodies; Bar Codes; Biological; Biological Assay; Biological Process; Blood; Blood Circulation; Body Fluids; Caliber; Cell Culture Techniques; Cells; Communication; Consumption; Custom; Disease; Ensure; Flow Cytometry; Goals; Human; Human body; Individual; Label; Measurement; Measures; Methods; MicroRNAs; Modification; Molecular; Molecular Target; Oligonucleotides; Optical Instrument; Parents; Particle Size; Phase; Physiological; Plasma; Population; Population Distributions; Process; Proteins; Protocols documentation; Publishing; RNA; RNA Transport; Recovery; Reference Values; Reproducibility; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Running; Sampling; Sensitivity and Specificity; Signaling Molecule; Specificity; Surface; Techniques; Time; Urine; analytical method; base; cell type; combinatorial; cost effective; design; digital; experience; extracellular vesicles; flexibility; human subject; improved; instrument; instrumentation; interest; next generation sequencing; novel; novel strategies; particle; screening; single molecule; transcriptome sequencing

Abstract Extracellular vesicles (EVs) are believed to be an important means of transporting RNA and other signaling molecules between cells. Most types of cells in the human body secrete EVs which are each likely to have distinct biological functions. Studying different types of EVs and their role in normal physiologic function and disease-related processes requires a reliable means of capturing these particles from readily accessible body fluids, like blood or urine. Our goal is to develop a new way to isolate specific types of EVs based on the molecules present on their surface. Our hypothesis is that we can use the presence of two or more specific molecules on the surface of select EVs secreted by a particular cell type. Once we isolate highly purified populations we can more easily identify and measure their molecular contents. We will develop a new scalable approach to identifying combinations of surface molecules present on EVs from particular cell types and use this to identify multi-marker “surface signatures” for several types of cells known to secrete EVs directly into the bloodstream. We will develop a new method to isolate the EVs with each of these surface signatures. This approach, which will only capture the EVs having all of the targeted surface markers, should greatly improve the capture specificity, thus, addressing one of the main shortcomings of the existing EV isolation methods. We will verify the purity of the isolated EVs by measuring the presence of each of the surface-signature markers on every EV using recently developed multi-marker assays and a new high-sensitivity single-EV characterization instrument. Generating highly purified EVs from specific cell types will enable more targeted studies of EVs than those that are presently performed. For example, we will identify specific regulatory RNA molecules in the purified EVs by next generation sequencing and measure the distribution of these molecules at the single EV level using the new instrumentation and assay techniques. Lastly we will refine the isolation methods to enable high-throughput isolation of EVs from several commonly used biofluids and automate the single EV characterization instrumentation to enable large studies that presently would be either impossible or too time-consuming to be cost-effective.

抽象的 细胞外囊泡（EV）被认为是运输 RNA 的重要手段 以及细胞间的其他信号分子。人体中大多数类型的细胞都会分泌 每个电动汽车都可能具有不同的生物学功能。研究不同类型的电动汽车 它们在正常生理功能和疾病相关过程中的作用需要可靠的 从血液或尿液等容易接触到的体液中捕获这些颗粒的方法。我们的 目标是开发一种新方法，根据存在的分子来分离特定类型的电动汽车 他们的表面。我们的假设是我们可以利用两个或多个特定分子的存在 位于特定细胞类型分泌的选定 EV 的表面。一旦我们分离出高度纯化的 我们可以更轻松地识别和测量其分子含量。 我们将开发一种新的可扩展方法来识别表面组合 来自特定细胞类型的 EV 上存在的分子，并用它来识别多标记 已知将 EV 直接分泌到细胞中的几种类型细胞的“表面特征” 血流。我们将开发一种新方法，将电动汽车与这些表面隔离 签名。这种方法只会捕获具有所有目标表面的电动汽车 标记物，应该大大提高捕获特异性，从而解决主要问题之一 现有EV隔离方法的缺点。 我们将通过测量每个的存在来验证隔离电动汽车的纯度 使用最近开发的多标记检测和一个 新型高灵敏度单电动汽车表征仪器。生成高度纯化的电动汽车 特定的细胞类型将使电动汽车的研究比目前的研究更有针对性 执行。例如，我们将鉴定纯化的 EV 中的特定调节 RNA 分子 通过下一代测序并测量这些分子在单个 EV 上的分布 使用新的仪器和检测技术来提高水平。最后我们将完善隔离 能够从几种常用的生物流体中高通量分离 EV 的方法， 自动化单个 EV 表征仪器，以实现目前的大型研究 要么不可能，要么太耗时，不具有成本效益。