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： 该UG3/UH3申请对NIH共同基金计划RFA-RM-18-028响应 细胞外RNA（EXRNA）交流研究：朝向单个细胞外囊泡（EV）分类， 该提案建立在5年NIH普通基金的基础上 “细胞外RNA通信联盟1（ERCC1）”项目，以发展唾液ExRNA生物标志物 对于胃癌检测 在科学和翻译上开发了（发现和确定验证）胃癌 用于全身性疾病检测的唾液exrna。 这个ERCC 2阶段项目是开发创新技术，Acoustofluidic分离（AFS）耦合 具有表面增强的拉曼光谱法（SER），朝单个EV隔离并表征Exrna 与特定EV亚群相关的Cargos基于原始细胞，预期的靶细胞和生物流体。 在两个阶段（UG3，UH3）中具有十二个定量里程碑的八个具体目标，可以测试 中央假设是外泌体是胃癌组织/含有九个的胃癌的EV 通过脉管系统运输的歧视性唾液exrna生物标志物 腺体（靶器官）进入唾液。 UG3阶段的四个具体目标是开发AFS技术 作为严格和可重复性的标准操作程序（SOP）（AIM 1）； EV的SERS开发 ExRNA靶标与Singe EV的指纹和共定位（AIM 2）；两个独立的“严格和 可重复性实验室（R＆R Labs）”来评估AFS SOP（AIM 3）和共享策略的方法， 协议，具有更广泛科学界的工具，具有DMRR（AIM 4）。在UH3阶段中的第四个具体目标是 以优化，完善和扩展AFS SOP（目标5）；执行分类，跟踪经过验证的唾液 从原始细胞，血液到唾液腺和唾液的胃癌检测的ExRNA生物标志物（AIM） 6）; “ R＆R实验室”以优化其他人类生物流体（AIM 7），并分享策略，协议， 具有更广泛的科学界的工具（AIM 8）。 基于ERCC1项目的结果完成这些目标和目标是合乎逻辑且有影响力的 随着ERCC2项目的结果将提供一系列新型技术，AFS与SERC协同 快速，高收率（比当前技术高6倍）和唾液生物标志物发育的单EV水平隔离 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范围时，可以完全实现EXRNA的诊断和治疗功能 可以将来自给定细胞源的亚群来表征和分析分子量。