Molecular dissection and imaging of extracellular vesicles to define their origin and targets

细胞外囊泡的分子解剖和成像以确定其起源和目标

基本信息

批准号：
10350010
负责人：
Saumya Das
金额：
$ 110.01万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-16 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10350010
关键词：
Acute Disease Address Affinity Antibodies Biological Markers Blood Blood Plasma Volume Brain CD81 gene Cardiac Cell Separation Cells Characteristics Clinic Clinical Collaborations Communication Computer Analysis Coupled Development Disease Disease Progression Disease regression Dissection Exercise Fluorescence Future Genetic Goals Health Heart Hematopoietic Heterogeneity Human Image Individual Lead Maps Mediating Mediator of activation protein Membrane Proteins Methodology MicroRNAs Microscopy Molecular Mus Myocardial Infarction Myocardial Ischemia Organ Partner in relationship Patients Peripheral Blood Mononuclear Cell Phase Physiological Processes Plasma Population Prognostic Marker Proteins Proteomics RNA Reporter Research Personnel Resolution Specificity Stress Stroke Surface Techniques Technology Testing Time Tissue Microarray Tissues Validation Vesicle Work base calmodulin-dependent protein kinase II cell type cerebrovascular data mining data repository diagnostic biomarker disease stressor experimental study extracellular extracellular vesicles human disease human model imaging modality improved innovation insight intercellular communication mouse model nano nanoflow cytometry novel novel diagnostics physiologic stressor population based predictive marker protein biomarkers response single cell sequencing single molecule tool transcriptome sequencing transcriptomics translational impact

项目摘要

Extracellular vesicles (EVs) and the RNAs contained within them (EV-RNAs) are secreted into biofluids by every cell type. EV-RNAs have emerged as potential prognostic or predictive biomarkers of a wide range of diseases, providing a targetable, accurate real-time representation of the disease state. However, advancement of EV- RNAs in the clinic as biomarkers of disease has been impeded by challenges in their isolation and characterization. Most notably, there is a lack of tools and techniques to i) isolate and precisely characterize tissue-specific EV-RNA populations; ii) define heterogeneity in surface markers and RNA content in tissue- specific EV populations; and iii) determine changes in EV-RNAs associated with disease state. In this multi-PI proposal, we will use a collaborative and innovative approach to advance technology that would allow isolation and granular characterization of EV populations from hematopoietic cells, brain, and heart. Our objective in the UG3 phase is to identify cell/tissue specific markers for isolation of EVs using computational analysis, transcriptomics, and EV-tracking in genetic mouse models; this approach would allow for fluorescence/antibody-based identification of EVs in a cell-specific manner. Information will also be obtained on individual EVs with a novel quantitative single molecule localization microscopy (qSMLM) approach. qSMLM, a sensitive fluorescence-based imaging method, will be used to quantify the number of affinity isolated EVs, their size, and key RNA content using molecular beacons. The identified tissue-specific EV-markers and EV-RNAs will be used in the UH3 phase for validation in a variety of human models. Our objective in the UH3 phase is to determine cellular/tissue contribution to EV-RNAs from Tissue-Chip effluents; and assess dynamic changes in EVs from human plasma from subjects with acute disease (coronary ischemia or cerebrovascular accident) or physiological processes (exercise). We will validate key EV-RNAs (using nano-flow cytometry and molecular beacons) and use qSMLM with molecular beacons to provide a quantitative profile of EVs at a single vesicle resolution. Notably, proposed experiments will help determine contribution of different tissues to the plasma biofluid RNA landscape at baseline, and in response to physiological or disease stressors. Together, the tools and techniques developed in the UG3 phase, and validated in the UH3 phase would serve as a road-map for the discovery and development of EV markers and EV-RNAs specific to other tissues. Ultimately, the use of tissue-specific EV-RNAs to probe disease state would provide a dynamic window into disease progression or regression with higher sensitivity and fidelity compared to currently available technologies.

细胞外囊泡 (EV) 及其中包含的 RNA (EV-RNA) 被每个细胞分泌到生物体液中。细胞类型。 EV-RNA 已成为多种疾病的潜在预后或预测生物标志物，提供有针对性的、准确的疾病状态实时表示。然而，电动汽车的进步 RNA 作为疾病生物标志物在临床上的应用一直受到其分离和检测方面的挑战的阻碍。表征。最值得注意的是，缺乏工具和技术来 i) 隔离和精确表征组织特异性 EV-RNA 群体； ii) 定义组织中表面标记和 RNA 含量的异质性- 特定电动汽车人群； iii) 确定与疾病状态相关的 EV-RNA 的变化。在这个多PI 根据提案，我们将采用协作和创新的方法来推进技术，从而实现隔离以及造血细胞、大脑和心脏的 EV 群体的颗粒特征。我们在 UG3 阶段的目标是使用以下方法识别用于分离 EV 的细胞/组织特异性标记：遗传小鼠模型中的计算分析、转录组学和 EV 跟踪；这种方法将允许用于以细胞特异性方式进行基于荧光/抗体的 EV 识别。还将获得信息使用新型定量单分子定位显微镜（qSMLM）方法对单个电动汽车进行研究。 qSMLM, 一种灵敏的基于荧光的成像方法，将用于量化亲和分离的 EV 的数量、它们的数量使用分子信标确定大小和关键 RNA 含量。已鉴定的组织特异性 EV 标记物和 EV-RNA 将用于 UH3 阶段，在各种人体模型中进行验证。我们 UH3 阶段的目标是确定组织芯片流出物中细胞/组织对 EV-RNA 的贡献；并评估动态变化来自患有急性疾病（冠状动脉缺血或脑血管意外）的受试者的人血浆的 EV，或生理过程（运动）。我们将验证关键的 EV-RNA（使用纳米流式细胞术和分子生物学）信标）并使用 qSMLM 和分子信标来提供单个囊泡上 EV 的定量分析解决。值得注意的是，拟议的实验将有助于确定不同组织对血浆的贡献基线时的生物流体 RNA 景观，以及对生理或疾病应激源的反应。总之，在 UG3 阶段开发并在 UH3 阶段验证的工具和技术将服务于作为发现和开发针对其他组织的 EV 标记和 EV-RNA 的路线图。最终，使用组织特异性 EV-RNA 来探测疾病状态将为了解疾病状态提供一个动态窗口。与现有技术相比，疾病进展或消退具有更高的灵敏度和保真度技术。