Development and application of a scalable workflow for immunomagnetic separation of exRNA carrier subclasses and molecular analysis of their cargo

开发和应用可扩展的工作流程，用于 exRNA 载体亚类的免疫磁性分离及其货物的分子分析

• 批准号: 9812007
• 负责人: LOUISE CHANG LAURENT
• 金额: $ 56.73万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9812007
• 关键词: Age; Antibodies; Apolipoproteins; Apolipoproteins A; Atlases; Benchmarking; Biogenesis; Biological Assay; Biological Markers; Biological Process; Biology; Biophysics; Blood Platelets; Cardiac Myocytes; Cell Culture Techniques; Cells; Centrifugation; Clinical; Complex; Coupled; Data; Density Gradient Centrifugation; Deposition; Development; Diagnostic; Disease; Effectiveness; Epithelial ovarian cancer; FBXW7 gene; Female; Flow Cytometry; Fractionation; Funding; Future; Goals; Hepatocyte; High Density Lipoproteins; Human; Immunomagnetic Separation; Information Dissemination; Interferometry; Knowledge; Laboratories; Lead; Lipids; Lipoproteins; Literature; Malignant neoplasm of ovary; Measurement; Mediator of activation protein; Metadata; Methods; MicroRNAs; Modeling; Molecular Analysis; Molecular Sieve Chromatography; Myocardial Infarction; Phase; Physiological; Plasma; Preparation; Process; Proteins; Proteomics; Public Health; Quantitative Reverse Transcriptase PCR; RNA; RNA marker; Reagent; Reference Values; Reproducibility; Ribonucleoproteins; Risk; Sampling; Sorting - Cell Movement; Specificity; Technology; Testing; Western Blotting; Work; base; biomarker discovery; biophysical properties; cancer cell; carrier testing; case control; cell type; cohort; cost effective; experimental study; extracellular; extracellular vesicles; human tissue; innovative technologies; intercellular communication; magnetic beads; microscopic imaging; novel; particle; pregnant; prognostic; protein biomarkers; scale up; screening; sex; technology development; theranostics; therapeutic RNA; transcriptome sequencing; trophoblast; uptake

SUMMARY Extracellular RNAs (exRNAs) have been found in all tested human biofluids, and there is increasing evidence that they can serve as mediators of intercellular communication, as well as diagnostic, prognostic, and theranostic biomarkers for a wide range of disease and physiological conditions. ExRNAs are associated with a variety of carriers subclasses (CSs), including extracellular vesicles (EVs), ribonucleoprotein complexes (RNPs), and lipoproteins (LPP), many of which are as-yet unknown or poorly characterized. This transdisciplinary team, with expertise in exRNA and lipoprotein biology, exRNA biomarker discovery, exRNA therapeutics, exRNA sequencing, low-input proteomics and lipidomics, and flow cytometry, will work together to develop and apply a rigorous, reproducible, efficient, scalable, and cost-effective immunomagnetic separation (IMS) workflow for preparative isolation of CSs for downstream omic analysis. In addition, the potential for multiplex bead-based flow sorting for even more efficient separation of CSs will be explored. Aim 1A will focus on development of reagents for identification and separation of known and suspected CSs using appropriate cell culture models and healthy human plasma and serum samples. This work will include screening of available antibodies against markers for known general CSs (e.g. tetraspanins, AGO proteins, apolipoproteins) and a variety of cell type- specific markers to identify antibodies that perform well for Western Blot and IMS, and dissemination of results for both successful and unsuccessful antibodies. In Aim 1B, the results from Aim 1A will be applied to build and test an IMS workflow for separation and small and long RNAseq, proteomic, and lipidomic analysis of general CSs and cell type-specific EVs. In addition to building a comprehensive knowledge set encompassing the RNA, protein, and lipid cargo of known and suspected CSs, profiling the material that is not captured by the IMS workflow will reveal novel CSs. Aim 2A will encompass refinement of the IMS workflow and application to three clinical cohorts: Pregnant and non-pregnant female controls; Post-myocardial infarction and age- and sex- matched at-risk controls; and Epithelial ovarian cancer and age-matched healthy female controls. Analysis of the exRNAs associated with general CSs and cell type-specific EVs by small and long RNAseq will reveal whether the relative abundance and exRNA cargo of of these CSs differs between cases and controls in these cohorts. Aim 2B will consist of development of a flow cytometry-based strategy for multiplexed simultaneous separation of multiple CSs from human plasma and serum. If successful, this project will result in development of a rigorous workflow for separation of exRNA CSs that reproducibly and rapidly produces fractions that are highly enriched for desired CSs with minimal contamination by other CSs in a cost-effective manner on clinically feasible volumes of input material, and yields sufficient material for downstream molecular analysis. In addition, the comprehensive omic data generated on during the course of this project will yield valuable reference profiling data on the RNA, protein, and lipid cargo carried by previously known and novel CSs.

概括 在所有测试的人类生物体液中都发现了细胞外 RNA (exRNA)，并且越来越多的证据 它们可以作为细胞间通讯的中介，以及诊断、预后和 适用于多种疾病和生理状况的治疗诊断生物标志物。 ExRNA 与 多种载体亚类 (CS)，包括细胞外囊泡 (EV)、核糖核蛋白复合物 (RNP)、 和脂蛋白（LPP），其中许多仍然未知或特征不明确。这个跨学科的团队， 拥有 exRNA 和脂蛋白生物学、exRNA 生物标志物发现、exRNA 疗法、exRNA 方面的专业知识 测序、低输入蛋白质组学和脂质组学以及流式细胞术将共同开发和应用 严格、可重复、高效、可扩展且经济高效的免疫磁分离 (IMS) 工作流程 用于下游组学分析的 CS 的制备分离。此外，基于多重珠的潜力 将探索用于更有效地分离 CS 的流式分选。目标 1A 将重点发展 使用适当的细胞培养模型识别和分离已知和可疑的 CS 的试剂 以及健康人血浆和血清样本。这项工作将包括筛选可用的抗体 已知一般 CS 的标记（例如四跨膜蛋白、AGO 蛋白、载脂蛋白）和各种细胞类型 - 用于识别在蛋白质印迹和 IMS 中表现良好的抗体的特异性标记物以及结果传播 对于成功和不成功的抗体。在目标 1B 中，目标 1A 的结果将应用于构建和 测试 IMS 工作流程，以进行一般性分析的分离以及小和长 RNAseq、蛋白质组学和脂质组学分析 CS 和特定于细胞类型的 EV。除了构建涵盖 RNA 的综合知识集之外， 已知和可疑 CS 的蛋白质和脂质货物，分析 IMS 未捕获的材料 工作流程将揭示新颖的 CS。目标 2A 将包括将 IMS 工作流程和应用程序细化为三个 临床队列：怀孕和未怀孕的女性对照；心肌梗塞后与年龄和性别 匹配的风险控制；上皮性卵巢癌和年龄匹配的健康女性对照。分析 通过小和长 RNAseq 与一般 CS 和细胞类型特异性 EV 相关的 exRNA 将揭示 这些 CS 的相对丰度和 exRNA 货物在这些病例和对照之间是否存在差异 队列。目标 2B 将包括开发基于流式细胞术的多重同步策略 从人血浆和血清中分离多种CS。如果成功，该项目将带来发展 严格的 exRNA CS 分离工作流程，可重复且快速地产生以下组分： 高度富集所需的 CS，同时以具有成本效益的方式在临床上将其他 CS 的污染降至最低 可行量的输入材料，并产生足够的材料用于下游分子分析。此外， 该项目过程中生成的全面组学数据将产生有价值的参考分析 有关先前已知和新型 CS 携带的 RNA、蛋白质和脂质货物的数据。