Exhaled small RNA biomarkers to detect and monitor airway disease

摘要 “呼出的小RNA生物标志物用于检测和监测气道疾病” 哮喘和慢性阻塞性肺疾病（COPD）共同影响全世界超过6亿人， 强调需要开发新的早期诊断和干预工具。因为航空公司 直接与外部环境沟通，基于气道的采样提供了一个独特的机会， 捕获肺部疾病特异性生物标志物。因此，我们选择询问呼出气体冷凝物 (EBC)我们的多实验室合作团队已经证明，来自>500名供体的未分级EBC含有 完整的microRNA，可通过qPCR和下一代测序（NGS）测量。最近，使用 优化的基于抗体（抗CD 63）的纯化试验，我们确定了呼出的小外泌体的存在 在EBC中，通过电子显微镜和纳米颗粒跟踪、蛋白质印迹和小RNA NGS证实。 我们对呼出外泌体标本的初步临床分析表明，我们独特的方法可能允许 使用定性和定量方法检测显著的哮喘和COPD病例对照判别信号 RT-PCR法我们工作的下一个阶段是从技术上完善我们优化的分子的组合适用性， 方法，并评估在诊断时和治疗期间识别哮喘/COPD特异性呼出生物标志物， 急性加重，测试气道组织特异性抗体以选择呼出的外泌体用于分析。在这款R33 我们假设：优化EBC衍生的小RNA的回收和定量 生物标志物将促进非侵入性气道（哮喘、COPD）疾病病例对照鉴别， 疾病监测，超越现有技术。对于这些NGS和qPCR小RNA分析， 为了验证，我们将比较呼出的完整和外泌体分区的EBC样品，选择内部参考， 交叉验证检测/定量方法，并确认EBC生物标志物来源的气道水平。我们将 通过NGS和qPCR开发精细的小/microRNA面板，并引入自动化平台， 在两个病例对照试点的临床标本的吞吐量评价。该R33项目的主要影响将是 将完善用于哮喘和COPD检测的呼出小/miRNA生物标志物的开发，目的是 识别早期恶化鉴别器。这项研究将为多机构的前景奠定基础 这两种常见肺部疾病的呼出小核酸生物标志物的队列验证。

ABSTRACT “Exhaled small RNA biomarkers to detect and monitor airway disease” Asthma and chronic obstructive pulmonary disease (COPD) together affect over 600 million people worldwide, highlighting the need for development of novel tools for early diagnosis and intervention. Because airways communicate directly with the external environment, airway-based sampling provides a unique opportunity to capture lung disease-specific biomarkers. Therefore, we have opted to interrogate exhaled breath condensate (EBC), and our multi-lab collaborative team has demonstrated that unfractionated EBC from >500 donors contain intact microRNAs, measurable by both qPCR and next generation sequencing (NGS). More recently, using an optimized antibody-based (anti-CD63) purification assay, we identified the presence of small exhaled exosomes in EBC, confirmed by electron microscopy and nanoparticle tracking, western-blotting, and by small-RNA NGS. Our pilot clinical analyses of exhaled-exosomes specimens suggest that our unique approach may allow detection of significant asthma and COPD case-control discriminant signals, using qualitative and quantitative RT-PCR. The next phase of our work is to technically refine the combined applicability of our optimized molecular approaches, and to evaluate the identification of asthma/COPD-specific exhaled biomarkers at diagnosis and at exacerbation, testing airway tissue-specific antibodies to select exhaled exosomes for analyses. In this R33 proposal, we hypothesize that the: Optimized recovery and quantification of EBC-derived small-RNA biomarkers will advance non-invasive airway (asthma, COPD) disease case-control discrimination and disease monitoring, beyond existing technology. For these NGS and qPCR small-RNA analyses and validations, we will compare exhaled whole and exosome-partitioned EBC samples, select internal references, cross-validate detection/quantification methods, and confirm airway level of origin of EBC biomarkers. We will develop refined small/microRNA panels via NGS and qPCR, and introduce automation platforms for higher throughput evaluation of clinical specimens in two case-control pilots. The primary impact of this R33 project will be to refine the development of exhaled small/miRNA biomarkers for asthma and COPD detection, with intent to identify early exacerbation discriminators. This study will establish the basis for a multi-institutional prospective cohort validation for exhaled small nucleic acid biomarkers for these two common lung disorders.