Exhaled small RNA biomarkers to detect and monitor airway disease

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.