Acoustofluidic Separation (AFS), Purification and Raman Spectral Fingerprinting of Single EVs: From Cell of Origin to Target Cell and Biofluids

单个 EV 的声流分离 (AFS)、纯化和拉曼光谱指纹识别：从起源细胞到目标细胞和生物流体

• 批准号: 10357985
• 负责人: Ji Yeong An
• 金额: $ 89.39万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10357985
• 关键词: Apoptotic; Area Under Curve; Biological Markers; Blood; Cancer Detection; Cells; Clinical; Communication; Communication Programs; Communication Research; Communities; Coupling; Credentialing; Detection; Development; Diagnostic; Disease; ERCC2 gene; Early Diagnosis; Fingerprint; Foundations; Funding; Goals; Heterogeneity; Homing; Human; Immunoblotting; Label; Malignant Neoplasms; Manuscripts; Metabolic Diseases; Molecular Analysis; Non-Invasive Cancer Detection; Organ; Outcome; Performance; Phase; Preparation; Procedures; Protocols documentation; Public Health; Publishing; RNA; Raman Spectrum Analysis; Reproducibility; Saliva; Salivary; Salivary Glands; Seminal; Sorting - Cell Movement; Source; Specificity; Surface; System; Systemic disease; TSG101 gene; Technology; Testing; Therapeutic; Tissues; United States National Institutes of Health; base; biomarker development; exosome; extracellular; extracellular vesicles; human tissue; innovative technologies; liquid biopsy; malignant stomach neoplasm; microvesicles; nervous system disorder; new technology; novel; scale up; tool

Project Summary/Abstract: This UG3/UH3 application is responsive to the NIH Common Fund initiative RFA-RM-18-028 “Advancing Extracellular RNA (exRNA) Communication Research: Towards Single Extracellular Vesicle (EV) Sorting, Isolation, and Analysis of Cargo”. This proposal is built on a foundation of a 5-year NIH Common Fund “Extracellular RNA Communication Consortium Stage 1 (ERCC1)” project to develop salivary exRNA biomarkers for gastric cancer detection where a panel of highly discriminatory salivary extracellular RNA (exRNAs) have been developed (discovered and definitively validated) for gastric cancer, scientifically and translationally credentialed salivary exRNA for systemic disease detection. This ERCC Stage 2 project is to develop an innovative technology, AcoustoFluidic Separation (AFS) coupling with Surface Enhanced Raman Spectroscopy (SERS), towards single EV isolation and to characterize exRNA cargos associated with specific EV subpopulations based on the cells of origin, intended target cells and biolfuids. Eight Specific Aims with twelve quantitative milestones in two phases (UG3, UH3) are in place to test the central hypothesis that exosomes are the EV from gastric cancer tissue/cells of origin harboring the nine validated discriminatory salivary exRNA biomarkers, transported through vasculature, homing into salivary glands (target organ) and into saliva. Four Specific Aims in the UG3 Phase are to develop the AFS technology as a standard operating procedure (SOP) for rigor and reproducibility (Aim 1); SERS development for EV fingerprinting and co-localization of exRNA targets to singe EV (Aim 2); two independent “Rigor and Reproducibility Labs (R&R Labs)” to evaluate the AFS SOP (Aim 3) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 4). In the UH3 Phase four Specific Aims are in place to optimize, refine and scale up the AFS SOP (Aim 5); perform sorting, tracking of validated salivary exRNA biomarkers for gastric cancer detection from cells of origin, to blood, to salivary glands and to saliva (Aim 6); “R&R Labs” to optimize AFS to other human biofluids (Aim 7) and approaches to share strategies, protocols, tools with broader scientific community with DMRR (Aim 8). Completing these Aims and goals based on the outcome of the ERCC1 project is logical and highly impactful as the outcomes of the ERCC2 project will deliver a set of novel technologies, AFS in tandem with SERC, for rapid, high yield (6X over current technologies) and single EV level isolation for salivary biomarker development for systemic disease detection. These will constitute the foundation of “exRNA Saliva Liquid Biopsy (exRNA- SLB)” where the diagnostic and therapeutic functionality of exRNAs can be fully realized when the range of EV subpopulations from a given cell source can be characterized and analyzed for molecular cargos.

项目摘要/摘要： 此 UG3/UH3 申请响应 NIH 共同基金倡议 RFA-RM-18-028“推进 细胞外 RNA (exRNA) 通讯研究：针对单个细胞外囊泡 (EV) 分选， 货物的隔离和分析”。该提案建立在 5 年期 NIH 共同基金的基础上 “细胞外RNA通讯联盟第一阶段（ERCC1）”项目开发唾液exRNA生物标志物 用于胃癌检测，其中一组具有高度辨别力的唾液细胞外 RNA (exRNA) 已被开发（发现并最终验证）用于胃癌，科学和转化 经认证的唾液 exRNA 用于全身性疾病检测。 该 ERCC 第 2 阶段项目旨在开发一项创新技术：声流分离 (AFS) 耦合 使用表面增强拉曼光谱 (SERS)，实现单个 EV 分离并表征 exRNA 与基于起源细胞、预期目标细胞和生物油的特定 EV 亚群相关的货物。 八个具体目标和两个阶段（UG3、UH3）中的十二个定量里程碑已经到位，以测试 核心假设是，外泌体是来自胃癌组织/细胞的 EV，其中含有 9 个基因 经验证的歧视性唾液 exRNA 生物标志物，通过脉管系统运输，归巢到唾液中 腺体（目标器官）并进入唾液。 UG3阶段的四个具体目标是开发AFS技术 作为严格性和可重复性的标准操作程序 (SOP)（目标 1）； EV 的 SERS 开发 exRNA 目标的指纹识别和共定位以单一 EV（目标 2）；两个独立的“严谨和 再现性实验室（R&R 实验室）”来评估 AFS SOP（目标 3）和共享策略的方法， 具有更广泛科学界的 DMRR 协议、工具（目标 4）。 UH3 阶段的四个具体目标是 优化、完善和扩大 AFS SOP（目标 5）；对经过验证的唾液进行分类和跟踪 用于胃癌检测的 exRNA 生物标志物，从起源细胞到血液、唾液腺和唾液（Aim 6); “R&R 实验室”优化 AFS 以适应其他人类生物体液（目标 7）以及共享策略、方案、 具有更广泛科学界的 DMRR 工具（目标 8）。 根据 ERCC1 项目的成果完成这些目的和目标是合乎逻辑且具有高度影响力的 因为 ERCC2 项目的成果将提供一系列新技术，AFS 与 SERC 协同， 快速、高产量（是现有技术的 6 倍）和单 EV 水平分离，用于唾液生物标志物开发 用于全身性疾病检测。这些将构成“exRNA唾液液体活检（exRNA- SLB）”，当 EV 范围达到一定范围时，可以充分实现 exRNA 的诊断和治疗功能 可以对给定细胞来源的亚群进行表征并分析分子货物。