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To Develop a Technological Solution for Automated Detection of Circulating and Exosomal miRNAs


OUSD (R&E) CRITICAL TECHNOLOGY AREA(S): Military Operational Medicine OBJECTIVE: To develop a reliable, rapid, sensitive, multiplex method to quantify the levels of small RNA molecules such as exosome and circulating microRNAs (miRNA) in biological samples to explore their potential as diagnostic and prognostic tools. DESCRIPTION: The volume of in vitro diagnostics continues to grow steadily due to increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of small non-coding RNA molecule microRNAs have recently gained attention in healthcare management for its potential as biomarkers for human diseases. MicroRNAs (miRNAs) are evolutionary conserved, ~18–24 nucleotides long non-coding RNA, playing a significant role in controlling human gene expression by post-transcriptional gene regulation or silencing. Each miRNA can regulate up to 200 predicted target genes, and one mRNA may be influenced by multiple miRNAs. miRNAs are abundant in many cell types, exosomes, and even occur as extracellular circulating molecules in blood and other biological fluid. A growing number of reports have shown that subsets of miRNAs may have clinical relevance as biomarkers. These biomarkers can be used to indicate presence of a pathology and even the stage, progression, or genetic link of pathogenesis (1). In certain situations, one miRNA biomarker may be sufficient to identify a health outcome such as acute injuries to Warfighters in the operational environment; however, in other cases, a well-defined panel of miRNAs is necessary for increased diagnostic sensitivity and/or specificity such as traumatic brain injury (TBI), post-traumatic stress disorder (PTSD) etc. These investigations have been undertaken in preclinical animal models and in human cohorts. For example, multi-omics investigation of PTSD patients’ blood samples identified a diversified panel including miRNAs (miR-133a-3p, miR-192-5p, mir-424-3p and miR-9-5p) (2). Data from our lab have also shown exosome derived miRNA are involved in chronic neuropathic pain (3) as well as early impacts of irradiation was underscored by the large number of miRNAs in total body radiation pre-clinical model (4). However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing involves cascade of operations including sample processing, miRNA quantification can be cumbersome and crippled by serious flaws at all stages of the process. In addition, these methods require that the low abundance miRNA be several folds greater than background to give a significant result. Therefore, the current topic is about the possibility and feasibility to develop a reliable, rapid, sensitive, multiplex method to quantify the levels of exosome and circulating miRNA in biological samples. The ultimate goal is to translate technological developments into diagnostic and prognostic tools. 1) The development of a robust and portable device. 2) To conduct an integrated sample collection-to-assay-to-detection architecture including exosomal and cell-free miRNA. 3) The amount and character of sample requirements. Consider minimally invasive clinical samples, such as blood, urine, saliva. 4) Device should have multiplexing capability and should be flexible to adapt new miRNA panels. 5) The sensitivity and specificity of the assay should be addressed. 6) The robustness and simplicity of the method. 7) The simplicity of software for analysis and interpretation of the data. 8) Minimal use of specialized equipment and reagents. 9) Low turn-around time to result 10) Assay cost 11) Capable to differentiate between exosome-derived vs cell-free miRNA PHASE I: To establish feasibility for a quantitative molecular diagnostics technology based on the detection of exosomal and cell free circulating miRNA using readily available clinical or pre-clinical samples. Current in vitro approaches require extensive preparation involving extraction, reverse transcription of miRNA into cDNA, amplification followed by data analytics. To devise specific technological bricks to release these low molecular weight RNA molecules before proceeding to detection and analysis. Here, we are seeking experimental evidence of the proof-of-concept explaining methodologies to detect multiplexed miRNA panel (exosomal and/or circulating) from a single input of biomatrix of choice with minimal human handling. The proposed device should be able to conduct the entire process starting from the biomatrix collection to analysis in a rapid fashion. Molecular diagnostics assay will have a potential for more sensitive, more accurate, and more objective clinical judgments. Use of human or animal subjects is not intended, nor expected, in order to establish/achieve the necessary proof-of-concept in Phase I. Further noting, animal or human use research shall not occur during Phase I as the period of performance does not allow enough time for required approvals to be received. In addition, descriptions of data analysis and interpretations concept and concerns should be outlined. Phase I should also include the detailed development of Phase II testing plan. PHASE II: The Phase I proposed protype shall be validated in Phase II. During this Phase, technology should undergo testing using a panel of miRNA (exosomal and circulating) for evaluation of the operation and effectiveness of utilizing an integrated system. A complete demonstration from biomatrix to detection of miRNA quantification is expected. Accuracy, reliability, and usability should be assessed. The device should be easy to use and interpret. The testing should be controlled and rigorous. Statistical power should be adequate to document initial efficacy and feasibility of the assay. This phase should also demonstrate evidence of commercial viability of the tool. Any information about risk and its mitigation should be discussed. We encourage to have a data driven analysis of the proposed capability tested using biomatrix that can inform us about the feasibility of next steps. Lastly, shall develop a clear regulatory strategy on how FDA clearance will be obtained. PHASE III DUAL USE APPLICATIONS: The product developed is intended to be suitable for use and potential procurement by all Military Services and for civilian. The dual-use technology would be applicable via securing funds from other sources. The successful transition path of the technology is expected to include close engagement with military medical acquisition program managers during product commercialization to ensure appropriate product applicability for military field deployment. This assay format should also be seamlessly integrated into the device for its potential be used as monitoring tool for short- or long-term health assessment. Once developed and demonstrated, the technology can be used for identification of risk, diagnostic, prognostic, monitoring and/ or predictive biomarkers for diseased state. The broader/commercial impact of this project will be to enhance current diagnostic and prognostic tools for early detection of disease. REFERENCES: 1. Hanna Johora, Hossain Gazi S., Kocerha Jannet. The Potential for microRNA Therapeutics and Clinical Research. Frontiers in Genetics 2019 2. Dean, K.R., Hammamieh, R., Mellon, S.H., Abu-Amara, D., Flory, J.D., Guffanti, G., Wang, K., Daigle, B.J., Gautam, A., Lee, I. and Yang, R., 2020. Multi-omic biomarker identification and validation for diagnosing warzone-related post-traumatic stress disorder. Molecular psychiatry, 25(12), pp.3337-3349. 3. Sosanya NM, Kumar R, Clifford JL, Chavez R, Dimitrov G, Srinivasan S, Gautam A, Trevino AV, Williams M, Hammamieh R, Cheppudira BP, Christy RJ, Crimmins SL. Identifying Plasma Derived Extracellular Vesicle (EV) Contained Biomarkers in the Development of Chronic Neuropathic Pain. J Pain. 2020 Jan-Feb;21(1-2):82-96. doi: 10.1016/j.jpain.2019.05.015. Epub 2019 Jun 19. PMID: 31228575. 4. Chakraborty N, Gautam A, Holmes-Hampton GP, Kumar VP, Biswas S, Kumar R, Hamad D, Dimitrov G, Olabisi AO, Hammamieh R, Ghosh SP. microRNA and Metabolite Signatures Linked to Early Consequences of Lethal Radiation. Sci Rep. 2020 Mar 25;10(1):5424. doi: KEYWORDS: miRNA, Biosensors, exosomes, Biomarkers, non-coding RNA, small RNA, microfluidics,
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