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: 10009490
• 负责人: Ji Yeong An
• 金额: $ 41.62万
• 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 2021-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009490
• 关键词: Abbreviations; 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; Transducers; 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. 1

项目摘要/摘要： 此UG3/UH3申请是对NIH共同基金倡议RFA-RM-18-028“Advance”的响应 细胞外RNA(ExRNA)通讯研究：走向单胞外囊泡(EV)分选， 货物的分离和分析“。这项提议建立在为期5年的美国国立卫生研究院共同基金的基础上 “细胞外RNA通讯联盟第一阶段(ERCC1)”计划开发唾液外源RNA生物标志物 用于检测胃癌，其中一组高度区分的唾液细胞外RNA(ExRNAs)具有 已被开发(发现并最终证实)用于胃癌的科学和翻译 获得认证的唾液exRNA用于系统性疾病检测。 该ERCC第二阶段项目旨在开发一种创新技术--声流分离(AFS)耦合 用表面增强拉曼光谱(SERS)分离单个EV并鉴定exRNA 根据来源细胞、预定目标细胞和生物液体与特定EV亚群相关的货物。 在两个阶段(UG3、UH3)制定了8个具体目标和12个量化里程碑，以测试 中心假说，外切体是来自胃癌组织/细胞的EV，其中含有九种 经过验证的唾液exRNA生物标志物，通过血管系统运输，归巢到唾液中 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)(目标1)；电动汽车的SERS开发 针对单个EV的exRNA靶标的指纹和共定位(AIM 2)；两个独立的“严格性和 可再生性实验室(R&R实验室)“评估AFS SOP(目标3)和分享战略的方法， 与DMRR的更广泛科学界合作的协议、工具(目标4)。在UH3阶段，四个具体目标是 到位以优化、细化和扩大AFS SOP(目标5)；对验证的唾液进行分类、跟踪 从来源细胞、血液、唾液腺和唾液检测胃癌的外源RNA生物标志物(目的 6)；“R&R实验室”，以优化用于其他人体生物体液的AFS(目标7)和分享战略、方案、 与DMRR的更广泛科学界合作的工具(目标8)。 在ERCC1项目成果的基础上完成这些目的和目标是合乎逻辑和极具影响力的 由于ERCC2项目的成果将提供一套新技术，AFS与SERC合作，用于 快速、高产量(是当前技术的6倍)和单一EV级分离，用于唾液生物标记物的开发 用于系统性疾病检测。这些将构成“exRNA唾液活组织检查(exRNA- SLB)“其中exRNA的诊断和治疗功能可以完全实现当EV的范围 来自给定细胞来源的亚群可以针对分子货物进行表征和分析。 1