OUSD (R&E) CRITICAL TECHNOLOGY AREA(S): Military Operational Medicine OBJECTIVE: To meet an innovation gap in rapidly detecting multiplexed multi-omics library of gene-epigene-protein-metabolite from single input of minimally invasive biomatrix in austere condition. DESCRIPTION: High throughput multi-omics readout and Systems integration galvanize our understanding about the molecular interplay and their roles in manifesting phenotypes. This interactive molecular landscape encompasses different layers of omics, namely epigenomics, transcriptomics, proteomics and metabolomics1, which operate in synchronized fashions to carry out biological functions. For instance, an epigenetic information flows through transcriptomics and proteomics layers to modulate metabolite landscape. Evidently, disease pathophysiology leaves footprints in any or all these layers of omics; hence a robust panel of disease biomarkers should include candidates from every layer of omics. Indeed, the current trend in biomarker discovery is progressively shifting from finding a single biomarker to a group of multi-omics biomarkers that can collectively define a clinical event2. A growing number of studies have identified multi-omics markers for psychological diseases like PTSD3,4 and somatic illnesses like rectal and prostate cancer1. Rapid probing of multi-omics molecular landscape is expected to enhance the diagnostic performance2, however such capability is yet to be fully materialized. This is the core innovation gap that we are poised to address here. To develop this capability, the pilot prototype of IVD platform will detect multiplexed multi-omics PTSD biomarkers3,4 as a proof of its capability. We will ensure maximum flexibility in this prototype development process, so that the prototype could be easily repurposed in future to diagnose additional diseases including, but not limited to sepsis, TBI, infection, exposure to CBRN and cancer. Diseases like PTSD is a good target for developing the pilot prototype due to two primary reasons. First, PTSD adversely impacts entire system; hence holistic screening of multi-omics landscape is imperative for PTSD subtyping, biomarker discovery and predicting comorbidities. For instance, DNA methylation markers were reported to biotype PTSD patients3. Moreover, multi-omics PTSD blood diagnostic markers included differentially methylated contigs (cg01208318, cg20578780, and cg15687973), miRNAs (miR-133a-1-3p, miR-192-5p, and miR-9-1-5p) and metabolites (gammaglutamyltyrosine)4. Although, we are yet to identify most robust panel of biomarkers for PTSD diagnosis and bio typing, a trend is rather apparent- the final product is likely to have representations from different omics layers, and this trend essentially justifies the proposed STTR program. The second reason to select PTSD is because many of its biomarkers are available in public domain3,4, ensuring an easy access to the Phase I awardees. A web search of SBIR.gov (dated January 25, 2023) found existing solicitations to develop multiplexed multi-omics tools to primarily reconstruct the cellular motifs with high resolution; all these prototypes are expected to be used in sophisticated laboratory settings and preclude any pursuit to make these assays rapid, automated and operatable in austere condition. There are several ongoing STTR efforts to rapidly screen individual omics layer in a field-rugged platforms. Clearly, there is a vast innovation and capability gap in developing a platform enabled to support rapid detection of pan-omics panel in austere condition. Present proposal is poised to meet this innovation gap. PHASE I: Provide experimental evidence of the proof-of-concept explaining methodologies to detect multiplexed multi-omics panel from a single input of biomatrix of choice. The expectation is that the biomatrix should be minimally invasive, such as blood, saliva, urine etc. We further expect that the proposed IVD platform should be able to conduct the entire process starting from the biomatrix collection to analysis in a rapid fashion. It is also important to note that different biomatrix is enriched by different omics components. For instance, whole blood is the preferred biomatrix for extracting maximum amount of mRNA and DNA, while the cell free serum or plasma is the preferred biomatrix for extracting maximum amounts of proteins and metabolites. Therefore, if an IVD prototype targets blood for molecular extraction, it should be able to handle whole blood and serum/plasma concurrently from single input volume. Target PTSD biomarkers could be curated from the public domain3,4. Use of human or animal subjects is not intended, or expected, in order to establish/achieve the necessary proof-of-concept in Phase I. At the end of this phase, a working prototype of the device should be demonstrated with reasonable sensitivity and feasibility. In addition, descriptions of data analysis and interpretations concept should be outlined. Phase I should also include the detailed development of Phase II testing plan. In summary, our expectations from Phase I is the following 1. A plan to develop an IVD device that can detect multiplexed multi-omics biomarker panel to map phenome of interest. For the pilot prototype, we plan to detect multi-omics PTSD biomarkers that are available in public domain. However, the final product should be flexible to diagnose other diseases, such as sepsis, traumatic brain injury, pathogenic infection, exposure to CBRN and cancer. 2. The expected device should be an automated and portable IVD platform enable to be used in far forward lab or at bedside in an energy inexpensive manner. 3. The device is expected to support an end-to-end methodology e.g., an integrated sample collection-to-assay-to-detection protocol. 4. Multiplexing capability of multi-omics panel from single input volume is essential. Should the device select blood as the input biomatrix, the platform should be able to simultaneously handle whole blood and serum/plasma from single input volume. PHASE II: The knowledge/ prototype generated in Phase I should be ready to be improved during Phase II. Phase II should start with a plan to assay the biomatrix of choice to detect a panel of multi-omics biomarkers. A comprehensive testing is expected to determine the feasibility of the platform to be operated with minimum hands-on time and least supervision. Suitable biomatrix should be finalized. The mode of endpoint reading should be finalized, and this process should be easily interpretable. Finally, we expect to have clear indications of the prototype's operational capability in real-world situations; some knowledge about the risks, source of confounders and concerns should be outlined, and pertinent mitigation plan should be furnished. 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. This phase should also deliver a plan for commercialization. In summary, our expectation from Phase II is the following: 1. The input and output modus operandi should be finalized. 2. Assay sensitivity and specificity should be characterized. Screening of limit of detection (LOD) profile in presence of potential confounders and contaminates is expected. 3. A turn-around time should be finalized. Herein the assay time includes the sample collection, assay and detection. 4. Potential risk factors and mitigation plan should be discussed. 5. Probable assay cost should be estimated. 6. Plan for commercial production and a plan 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 for primary use in the field/prehospital environment, including austere, prolonged care scenarios. At this phase, target diseases and pertinent biomarkers should be determined. As mentioned previously, the target disease might not be relevant to the health issues exclusive to active duty members. Realization of a dual-use technology applicable to both the military and civilian use could be achieved via securing funds from third party. Therefore, the successful transition path of the technology is encouraged to include close engagement with military medical acquisition program managers during product commercialization to ensure appropriate product applicability for military field deployment. Accuracy, reliability, and usability should be assessed. This testing should be controlled and rigorous. Statistical power should be adequate to document final efficacy and feasibility of the assay. FDA submission and approval is a goal for this phase. REFERENCES: 5. Subramanian I, Verma S, Kumar S, Jere A, Anamika K. Multi-omics data integration, interpretation, and its application. Bioinformatics and biology insights. 2020 Jan;14:1177932219899051. 6. Vincent, J.-L., Bogossian, E. & Menozzi, M. J. C. c. c. The Future of Biomarkers. 36, 177-187 (2020). 7. Yang, R., Gautam, A., Getnet, D., Daigle, B.J., Miller, S., Misganaw, B., Dean, K.R., Kumar, R., Muhie, S., Wang, K. and Lee, I., 2021. Epigenetic biotypes of post-traumatic stress disorder in war-zone exposed veteran and active duty males. Molecular psychiatry, 26(8), pp.4300-4314. 8. 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. KEYWORDS: In vitro diagnostic device, multi-omics biomarker detection, multiplexing capability, targeted molecular identification, minimally invasive biomatrix, rapid diagnosis, austere environment-friendly, minimum hands-on time
