mRNA Expression Profiling from Extracellular Vesicles (EVs): Generating a Rapid Diagnostic for Stroke

细胞外囊泡 (EV) 的 mRNA 表达谱分析：快速诊断中风

• 批准号: 10445743
• 负责人: Alison E Baird
• 金额: $ 61.54万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445743
• 关键词: Acute; Affect; Affinity; Alteplase; Appearance; Biological Assay; Biological Markers; Blood Tests; Blood flow; Brain; Brain Diseases; Brain hemorrhage; CD8B1 gene; Cause of Death; Cells; Cessation of life; Clinical; Clinical Sensitivity; Collaborations; Complementary DNA; Coulter counter; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Emergency Situation; Expression Profiling; Fluorescence; Genetic Transcription; Harvest; Hospitals; Image; In Vitro; Injections; Ischemic Stroke; Label; Lasers; Length; Leukocytes; Ligase; Light; Liquid substance; Magnetic Resonance Imaging; Measurement; Membrane; Messenger RNA; Molds; Molecular; Oligonucleotide Primers; Output; Patients; Peripheral; Phase; Plasma; Plastics; Process; RNA; RNA analysis; RNA marker; Rapid diagnostics; Reaction; Recovery; Research; Reverse Transcription; Sampling; Secure; Sensitivity and Specificity; Solid; Source; Stroke; Structure; Surface; Syndrome; System; Testing; Therapeutic; Time; Transcript; Validation; Vascular blood supply; Work; X-Ray Computed Tomography; base; blood-based biomarker; circulating biomarkers; clinically relevant; design; diagnostic platform; extracellular vesicles; in-vitro diagnostics; innovation; innovative technologies; mRNA Expression; multidisciplinary; nano; nanocolumn; nanopore; nanosensors; novel; operation; peripheral blood; point of care testing; single molecule; stroke event; stroke model; stroke patient; stroke therapy; success; time use; tool; transcriptome sequencing; vesicular release

Project Summary/Abstract Stroke is a disorder of the brain by which a part loses its blood supply and the affected region rapidly progresses to death if blood flow is not restored in time. Treatments to restore blood flow after acute ischemic stroke (AIS) are effective if administered <4.5 h from the onset of the stroke event. But, due in part to the lack of an in vitro diagnostic test for AIS, imaging (CT – clinical sensitivity ~26%) at the attending hospital is required for diagnosis. As a result, <7% of AIS patients receive treatment. Therefore, a critical need exists to develop strategies for diagnosing stroke in near real time that potentially can allow for point-of-care testing (POCT). One approach is a peripheral blood test using markers that quickly respond to changes in the brain induced by stroke. The proposed project will develop innovative technologies that uses peripheral blood markers for diagnosing stroke syndromes (AIS and hemorrhagic stroke) in near real time (~31 min for sample-to-answer) with an LOD of ~0.03 ng (total RNA). The research team has found that alterations in mRNA expression secured from white blood cell subsets can be used for stroke diagnosis and appear rapidly in peripheral blood following a stroke event. Resulting from a prior R01, CD15+ and CD8+ leukocytes were discovered as a predominant source of stroke- related mRNA biomarkers. This project seeks to realize the development of an innovative fluidic cartridge and the associated assay for the measurement of stroke-related RNA markers sourced from CD15 and CD8 expressing extracellular vesicles (EVs). The utility of EVs as a source of stroke-related RNA biomarkers is based on their rapid appearance and high abundance in plasma, potentially providing even faster stroke diagnosis compared to the cells from which they originate. This project will discover EV-RNA markers with high clinical sensitivity and specificity (>80%) for diagnosing ischemic and hemorrhagic stroke in <3 h from stroke onset. The cartridge, which consists of task-specific modules made from plastics via replication (i.e., injection molding) connected to a fluidic motherboard, will use the EV-RNA markers emanating from this project. Plasma will serve as the input from which surface-affinity selection of CD8 and CD15 EVs will occur using a specifically designed module. Following EV release from the capture surface via a photocleavable linker (cleaved using a blue-light LED), the cartridge will quantify the number of EVs selected using a label-free readout strategy. The fluidic cartridge also consists of a mixed-scale (nm → µm) module to read electrically copy numbers of stroke EV-RNA markers in a highly multiplexed fashion (>24 targets). This module will consist of in-plane nanopores made in a plastic via nano-injection molding and can identify RNAs using a distinct oligonucleotide primer pair querying a specific RNA using a solid-phase ligase detection reaction (spLDR). Single-molecule readout will allow for high analytical sensitivity to observe subtle changes in EV-RNA marker copy numbers. The utility of this cartridge will be evaluated in a clinical setting. Success of the project is leveraged by a strong multidisciplinary team, whom have a productive record of collaboration in developing stroke markers and diagnostic platforms for stroke.

项目概要/摘要 中风是一种大脑疾病，由于该疾病的一部分失去了血液供应，受影响的区域迅速发展 急性缺血性中风（AIS）后如果不及时恢复血流，可能会导致死亡。 中风事件发生后 4.5 小时内给药是有效的，但部分原因是缺乏体外试验。 AIS 的诊断测试、就诊医院的影像学检查（CT – 临床敏感性 ~26%）需要进行诊断。 因此，<7% 的 AIS 患者接受了治疗，因此迫切需要制定治疗策略。 近乎实时地诊断中风，可能可以进行即时检测 (POCT)。 使用可快速响应中风引起的大脑变化的标记物的外周血测试。 拟议项目将开发利用外周血标记物诊断中风的创新技术 近乎实时（样本到答案约 31 分钟）的综合征（AIS 和出血性中风），LOD 约为 0.03 研究小组发现，白细胞中的 mRNA 表达发生变化。 子集可用于中风诊断，并在中风事件后迅速出现在外周血中。 根据先前的 R01，CD15+ 和 CD8+ 白细胞被发现是中风的主要来源 - 该项目旨在实现创新流体盒的开发和 用于测量源自 CD15 和 CD8 的中风相关 RNA 标记物的相关测定 表达细胞外囊泡 (EV) 作为中风相关 RNA 生物标志物来源的用途是基于的。 它们的快速出现和血浆中的高丰度，有可能提供更快的中风诊断 与它们来源的细胞相比，该项目将发现具有高临床价值的 EV-RNA 标记。 中风发作后 3 小时内诊断缺血性和出血性中风的敏感性和特异性 (>80%)。 墨盒，由通过复制（即注塑成型）塑料制成的任务特定模块组成 连接到流体主板，将使用来自该项目的 EV-RNA 标记。 作为输入，使用专门设计的 CD8 和 CD15 EV 的表面亲和力选择 通过光可裂解连接器（使用蓝光裂解）从捕获表面释放 EV 后。 LED），该盒将使用无标签读出策略量化所选电动车的数量。 盒还包含一个混合尺度 (nm → µm) 模块，用于读取中风 EV-RNA 的电拷贝数 以高度多重方式标记（> 24 个目标） 该模块将由在平面内制成的纳米孔组成。 通过纳米注射成型塑料，并可以使用不同的寡核苷酸引物对查询RNA来识别RNA 使用固相连接酶检测反应 (spLDR) 检测特定 RNA 将实现高读数。 观察 EV-RNA 标记拷贝数细微变化的分析灵敏度将提高该试剂盒的实用性。 在临床环境中进行评估 该项目的成功取决于强大的多学科团队，他们 在开发中风标记物和中风诊断平台方面拥有富有成效的合作记录。