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

• 批准号: 10018945
• 负责人: Saumya Das
• 金额: $ 45.49万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2021-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10018945
• 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 warehouse; 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

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）和其中包含的RNA（EV-RNA）被每个分泌到生物流体中 细胞类型。 EV-RNA已成为广泛疾病的潜在预后或预测性生物标志物， 提供疾病状态的有针对性，准确的实时表示。但是，ev-的进步 诊所中的RNA作为疾病的生物标志物受到孤立挑战和 表征。最值得注意的是，缺乏工具和技巧i）孤立和精确的特征 组织特异性EV-RNA种群； ii）定义表面标记中的异质性和组织中的RNA含量 特定的电动汽车种群； iii）确定与疾病状态相关的EV-RNA的变化。在这个多pi中 提案，我们将使用一种协作和创新的方法来推进技术，以允许隔离 来自造血细胞，大脑和心脏的EV群体的颗粒状表征。 我们在UG3阶段的目标是识别使用细胞/组织特定标记，用于使用 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 用于基于荧光/抗体以细胞特异性方式鉴定EV。也将获得信息 在具有新型定量单分子定位显微镜（QSMLM）方法的单个EV上。 QSMLM， 一种敏感的基于荧光的成像方法将用于量化亲和力隔离电动汽车的数量 使用分子信标的大小和关键RNA含量。确定的组织特异性EV标记和EV-RNA 将在UH3阶段使用用于各种人类模型的验证。我们在UH3阶段的目标是 确定来自组织芯片废水对EV-RNA的细胞/组织贡献；并评估动态变化 来自急性疾病受试者（冠状动脉缺血或脑血管事故）的人类血浆中的电动汽车或 生理过程（运动）。我们将验证键EV-RNA（使用纳米流动细胞术和分子 信标）并将QSMLM与分子信标一起使用单个囊泡的evs定量剖面 解决。值得注意的是，提出的实验将有助于确定不同组织对等离子体的贡献 基线时生物流体RNA景观，并响应生理或疾病压力源。 一起，在UG3阶段开发的工具和技术，并在UH3阶段进行了验证将服务 作为针对其他组织的EV标记和EV-RNA的发现和开发的路线图。 最终，将组织特异性EV-RNA用于探测疾病状态将提供一个动态的窗口 与当前可用 技术。