# data[].attributes.title
Combined pathogen and host-based diagnostic to identify etiology of lower respiratory tract infection

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Lower respiratory tract infection (LRTI) is the leading infectious cause of death globally. Despite its prevalence, the exact etiology of LRTI is unknown in the vast majority of cases. Even when identified, bacteria or viruses in nasopharyngeal (NP) or sputum samples may be colonizers in the upper tract rather than the cause of infection in the lower tract. Unclear LRTI etiology results in the overprescription of antibacterials, which in turn drives the global crisis in antibacterial resistance. Antibacterial overprescription and resistance are greater in low- or middle-income countries (LMICs), where basic diagnostic capacity is limited. Host-based diagnostics, which assess the host immune response to infection, have recently emerged as a complementary method to pathogen-based diagnostics for identifying the class of respiratory infection. Our team has developed host- based gene expression classifiers using peripheral blood samples to differentiate viral versus bacterial respiratory infection. The goal of the current application is to develop an integrated diagnostic that uses a single, non-invasive NP sample to detect both pathogen and host response to identify LRTI etiology. The following specific aims will be conducted at a collaborative research site in Sri Lanka: 1) Develop a novel NP- based gene expression classifier to identify viral versus non-viral LRTI, and 2) Design and validate an integrated pathogen and host gene expression test to identify viral versus non-viral LRTI using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. For aim 1, we will use previously collected NP samples from clinically adjudicated viral and non-viral LRTI patients in Sri Lanka and conduct low-input RNA sequencing. Machine-learning approaches will identify host gene expression classifiers that discriminate viral versus non-viral LRTI. For aim 2, the genes identified in the NP-based classifier, as well as nucleic acid targets for two respiratory viruses that are frequently implicated in true infection as well as asymptomatic colonization (SARS-COV-2 and human rhinovirus [HRV]), will be migrated onto TaqMan Low-Density Array (TLDA) cards. A prospective cohort of patients will be enrolled in Sri Lanka, and etiological testing and clinical adjudications will be performed as the reference standard to identify viral (including SARS-CoV-2 and HRV) and non-viral LRTI. Using an optimally retrained and parsimonious viral versus non-viral classifier, we will perform a feasibility analysis of incorporating pathogen detection and host-response classifier. Among samples with TLDA-based pathogen detection for SARS-CoV-2 or HRV, performance of the host- response classifier to distinguish viral versus non-viral LRTI will be assessed. Successful completion of these aims will result in the development of a novel diagnostic that integrates host and pathogen detection using a single, non-invasive NP sample to identify the etiology of LRTI. Translation of this assay to a rapid platform will help shift the current diagnostic paradigm for LRTI.

# data[].attributes.title
UTS-1401: A Novel Mitigator of Radiation Injury

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.awardee_organization.name
HENRY FORD HEALTH + MICHIGAN STATE UNIVERSITY HEALTH SCIENCES

# data[].attributes.abstract
Abstract: Our long term objective is to develop a new class of radiation mitigating agents with attractive chemical, physical and biological characteristics required to be an effective drug that can be distributed widely. We have identified a small molecule, UTS-1401 [5-(methylthiomethyl) isoxazole-3-carboxylic acid] which demonstrates mitigation of hematopoietic stem cell death when administered at either 24h or 48h following whole body irradiation (WBI). Using the endogenous spleen colony assay we demonstrated a significant mitigating effect (ratio of colony number with and without UTS-1401) when drug was given 24h or 48h after radiation. We have also recently demonstrated a significant radioprotection for both mouse survival and hematopoietic stem cells for this compound for up to 72h before irradiation (Valeriote et al, Radiation Research, 202:16-25, 2024). In this application, we propose to examine solely the mitigating effect to both the hematopoietic acute radiation syndrome (H-ARS) in specific aim 1 and the gastrointestinal acute radiation syndrome (GI-ARS) in specific aim 2 following WBI (with 5% bone marrow protection for specific aim 2). Swiss mice will receive a series of graded doses of WBI around the LD50 for both syndromes with and without the administration of 150 mg/kg UTS- 1401. The single dose of UTS-1401 being used in all studies is the highest dose administrable due to its aqueous solubility (in tartrate buffered saline). The radiation mitigation factors will be calculated as the ratio of the LD50 for radiation plus UTS-1401 versus radiation alone. The degree of mitigation will be examined at 24, 48 and 72 h following WBI to determine the timeframe of mitigation after radiation exposure. Three routes of drug delivery, intravenous (iv), oral, and subcutaneous (sc), will be examined and compared. For all specific aims, both male and female mice will be separately studied. Radiation will be delivered by electrons from a Linac. In specific aim 3, we will examine the pharmacokinetics (PK) for 150 mg/kg UTS-1401 comparing the iv, oral, and sc routes to obtain a determination of both the drug kinetics and bioavailability. The AUC values will be correlated with the extent of mitigation. Finally, in specific aim 4, we will address the mechanism of action with studies focused on the role of specific cytokines induced by radiation in the so-called “cytokine storm”. We will assess the time course changes of TNF-α, IL-1β, IL-6, CSF and TGF-β in blood as well as bone marrow and intestinal mucosa over 20 days following: UTS-1401 alone, 10 Gy irradiation, and the combination of UTS-1401 and radiation at a 24h interval. The results from these studies are expected to demonstrate an effective first-in-class compound, UTS-1401, which has a small molecular weight, is chemically stable, nontoxic, aqueous soluble and inexpensive with radiation mitigating properties which extend for a number of days following irradiation. The mechanism studies are expected to demonstrate UTS-1401 as a new class of agents for mitigating the cytokine storm consequent to the irradiation.

# data[].attributes.keywords[]
United States National Institutes of Health

# data[].attributes.title
UTS-1401 as a Medical Countermeasure to H-ARS Consequent to a Radiation Mass Casualty

# data[].attributes.awardee_organization.name
HENRY FORD HEALTH + MICHIGAN STATE UNIVERSITY HEALTH SCIENCES

# data[].attributes.abstract
Abstract:  Our long term objective is to develop a new class of radiation mitigating agents with attractive chemical, physical and biological characteristics required to be an effective drug that can be distributed widely. We have identified a small molecule, UTS-1401 [5-(methylthiomethyl) isoxazole-3-carboxylic acid] which demonstrates mitigation of hematopoietic stem cell death when administered at either 24h or 48h following whole body irradiation (WBI). Using the endogenous spleen colony assay, we demonstrated a mitigating effect in that the colony number with and without UTS-1401 was 3.5 ± 0.4 for a 24h interval and 2.3 ± 0.5 for a 48h interval. We have recently demonstrated a significant radioprotection for both mouse survival and hematopoietic stem cells for this compound when administered up to 72h before irradiation (Valeriote et al, Radiation Research, 202:16- 25, 2024). In this application, we propose to further examine solely the mitigating effect on the hematopoietic acute radiation syndrome (H-ARS) using survival as the endpoint in specific aim 1. Groups of Swiss mice will receive a series of graded doses of WBI (in 0.5 Gy increments) around the LD50 for this syndrome (approximately 7.5 Gy in females and 8.5 Gy in males) with and without the administration of 150 mg/kg UTS-1401. The single dose of UTS-1401 being used in all studies is the highest dose administrable due to its aqueous solubility (in tartrate buffered saline). The radiation mitigation factors will be calculated as the ratio of the LD50 for radiation plus UTS-1401 versus that for radiation alone. The degree of mitigation will be examined at 24, 48 and 72 h following WBI to determine the radiation mitigation fraction as a function of time after radiation exposure. Three routes of drug delivery, intravenous (iv), oral, and subcutaneous (sc), will be examined and compared. Radiation will be delivered by 16 MeV electrons from a Linac. In specific aim 2, we will examine the pharmacokinetics (PK) for 150 mg/kg UTS-1401 comparing the iv, oral, and sc routes to obtain a determination of both the drug kinetics and bioavailability. The AUC values will be correlated with the extent of mitigation. For both specific aims, both male and female mice will be separately studied. The results from these studies are expected to demonstrate an effective first-in-class compound, UTS-1401, which has a small molecular weight, is chemically stable, nontoxic, aqueous soluble and inexpensive with H-ARS radiation mitigating properties which extend for a number of days following WBI. The mechanism studies (not proposed here) are expected to demonstrate UTS- 1401 as a new class of agents for mitigating the cytokine storm consequent to the irradiation.

# data[].attributes.keywords[]
United States National Aeronautics and Space Administration

# data[].attributes.title
Establishing Human 2D and 3D Testicular Models to Elucidate Monkeypox Virus Tropism and Pathogenic Mechanisms in the Testes

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
One of the most important lessons learned from the recent global outbreak of the monkeypox virus, now called Mpox virus (MPXV, clade II), is the recognition that males are at a much higher risk for infection and higher occurrence of genital rash. Transmission via sexual contact is one of the main routes of virus spread. However, whether MPXV constitutes a sexually transmitted infection and can infect the male reproductive tract is still being debated, thus affecting the strategies to minimize transmission risk. Confirmed reports of MPXV shedding in seminal fluid for long after the clearance of viremia suggest the ability of MPXV to establish a productive infection in genitourinary organs. Poxviruses can also cause testicular complications, including azoospermia and seminiferous tubule atrophy. More recently, MPXV antigens have been detected in the testis of nonhuman primates both during the acute and convalescent stages, and the presence of testicular inflammation and necrosis in these macaques collectively suggests testis-tropism of MPXV, like Zika and Ebola viruses. However, direct evidence of MPXV infection in human testis is currently lacking, including cell targets of the virus and downstream consequences. Relevant human in vitro models are needed to characterize MPXV testicular infection. Human testis immune homeostasis is tightly governed by an elaborate communication network between different cells including testosterone-producing Leydig cells (LC) and Sertoli cells (SC) that form the blood-testis barrier (BTB). We recently established a 3D human testicular organoid (HTO) system comprised of undifferentiated spermatogonia cells, SC, LC, and peritubular myoid cells that closely recapitulates the cell diversity and function of the human testis to study Zika virus and SARS-CoV-2 infection. We have also established 2D cultures of primary SC, LC, and mixed seminiferous tubule cells and an in vitro BTB model to delineate cell-specific responses to viruses. Therefore, the goal of this study is to utilize our 2D and 3D testicular culture systems as an effective in vitro surrogate to model testicular infection of MPXV and understand downstream consequences. In Aim 1, we will assess MPXV infection in the 2D and 3D HTOs, identify cell targets, and characterize key infection pathologies, including cytopathic effects, antiviral response, and effect on BTB integrity. Aim 2 will utilize single-cell RNA sequencing to determine relative infectivity in each cell type and key pathways, including antiviral and inflammatory response, cell death, and spermatogenesis. Collective data will provide much-needed evidence of the testis as one of the target organs of MPXV replication after it is cleared from blood and skin lesions and lay the foundation for future in vivo studies of transmission via the sexual route. The knowledge of whether MPXV is a sexually transmitted infection is critical in providing clinical management and transmission guidelines, especially in men who have sex with men (MSM), an underrepresented group in biomedical research.

# data[].attributes.keywords[]
United States National Institutes of Health

# data[].attributes.title
Programmable RNA-Based Sensors for In Situ Cell Type Detection and Response

# data[].attributes.funder_divisions[]
National Institute of Biomedical Imaging and Bioengineering (NIBIB)

# data[].attributes.abstract
There is a technology gap in currently developed tools that simultaneously monitor, compute, and respond to both coding and non-coding RNA in real-time within living cells or patients. The continued existence of this gap represents an urgent unmet need because, until it is filled, the accuracy of RNA-based therapeutics remains limited in complex and evolving biological systems like differentiation or cancer. The long-term goal of this proposal is to develop safe, universal, and programmable synthetic biology tools that using both coding and non- coding RNAs as disease marker inputs and program outputs to trigger therapeutic responses in patients. The objective of this particular application is to develop an RNA-based sensor (using mRNA as the delivery modality) that detects integrated changes in both mRNA and miRNA for in situ therapeutic responses within living cells and mouse models, given the crucial role of ncRNAs, especially microRNAs (miRNAs), as key regulators of post- transcriptional gene regulation, which allow only the correct set of genes to be active in each cell type. The central hypothesis is that an RNA-based sensor integrating both mRNA and miRNA inputs, using Boolean logic gate computation, can improve the specificity of cell type identification in complex biological systems. This proposed work builds on our and other’s recent works on sensing individual RNA species like mRNA in live cells. The rationale for the proposed research is that a deeper understanding of disease progression, derived from the vast RNA sequencing resources now available in user-friendly databases, creates a timely and unique opportunity for synthetic biologists to develop tools that can precisely identify diseased cells based on their RNA species and levels in living cells or even in patients. This allows for the development of treatments that specifically target diseased cells while minimizing off-target effects on healthy cells. Additionally, the success of COVID-19 mRNA vaccines using lipid nanoparticle delivery systems highlights the potential to translate RNA-based genetic circuits into practical medical applications. Given these advances, we plan to develop two independent and complementary aims for in situ cell state sensing using endogenous mRNA and miRNA as inputs: AND logic gates (requiring both inputs for an output) in Aim 1 and NOR logic gates (requiring neither input for an output) in Aim 2. This platform has broad biomedical potentials. As a proof of concept, we will demonstrate its ability to distinguish breast cancer cells from normal breast epithelial cells, evaluating its translational potential using a syngeneic mouse model of triple-negative breast cancer, which lacks key cell surface targets in current therapies. The proposed platform is innovative because it develops new platform by integration of existing miRNA sensing and RNA detecting approaches in a previously unproven combinatorial logic computation format to address a significant unmet need for accurate cell type identification for basic and translational applications. The proposed research is significant, because in situ monitoring and intervening based on endogenous RNAs will be key to addressing this unmet need, transforming disease detection and treatment.

# data[].attributes.keywords[]
National Institute of Biomedical Imaging and Bioengineering

# data[].attributes.keywords[]
United States National Institutes of Health

# data[].attributes.title
Design principles for engineering therapeutic macrophages

# data[].attributes.funder_divisions[]
National Institute of Biomedical Imaging and Bioengineering (NIBIB)

# data[].attributes.abstract
Genetically engineered immune cells are an exciting and promising frontier for treating a wide range of complex diseases. However, hyperinflammatory toxicities such as cytokine release syndrome plague clinical trials, stymieing their widespread clinical adoption. Macrophages are innate immune cells that fulfill many roles in tissue repair, regeneration, and homeostasis and are important regulators of inflammation. However, they are significantly under-utilized as engineered immune cell therapies because significant knowledge gaps exist in understanding how to engineer synthetic gene circuits that work robustly in human macrophages. Tools do not yet exist for determining how macrophages should be biologically manipulated to activate desired effector functions (biological design principles). Tools also do not exist for determining what gene circuit architectures are needed to robustly induce desired gene circuit behaviors (gene circuit design principles). The overall goal for this proposal is to create a human macrophage design toolkit for engineering therapeutic macrophages. Our published and preliminary data demonstrate that we have developed tools that enable us to discover cell signaling interventions that can control macrophage effector functions (biological design principles) and gene circuit architectures that can exert robust behaviors in human macrophages (gene circuit design principles). Here we will apply both these approaches to elucidate biological and gene circuit design principles that can be used to engineer therapeutic macrophages that can suppress inflammatory cytokine secretion or induce anti- inflammatory cytokine secretion in inflamed tissues. We will elucidate biological design principles using an interpretable machine learning approach that we previous developed. This approach combines biochemical screening with predictive network modeling and machine learning to discover network mechanisms causally regulating cell phenotypes. We will elucidate gene circuit design principles using a recently developed ultra-high- throughput genetic screening approach (CLASSIC). This approach synthesizes and screens large, barcoded gene circuit libraries to associate gene circuit architectures with gene circuit behaviors. With these design principles we will engineer gene circuits for controlling IL-1β or IL-10 secretion in inflamed tissue contexts and validate these synthetic gene circuits in human monocyte-derived macrophages and THP-1 cells. In its entirety, this Trailblazer R21 project is a first step towards addressing the unmet need for design principles for engineering therapeutic macrophages. We envision that insights gained by this project will help establish engineered macrophages as a platform technology for treating a wide range of complex human diseases.

# data[].attributes.title
Development of Tissue Engineered Tregs as a Treatment for Acute Ischemic Stroke

# data[].attributes.funder_divisions[]
National Institute of Neurological Disorders and Stroke (NINDS)

# data[].attributes.abstract
In the U.S., nearly 800,000 individuals experience a stroke each year, predominantly ischemic strokes. The economic burden of stroke is staggering, with projected stroke-related medical costs in the U.S. expected to surpass $94 billion by 2035. This underscores the urgent need for effective therapies to address this significant public health challenge. While the potential of regulatory T cells (Tregs) in promoting stroke recovery has been recognized, translating this promise into clinical success has been hampered by several obstacles. Traditional Treg therapies face challenges in manufacturing, phenotypic instability, and a lack of tissue specificity. This proposal focuses on the development of allogeneic engineered tissue Tregs (EngTregs) as a novel off-the-shelf therapeutic approach for stroke. Overexpression of ST2, the receptor for the alarmin IL-33, enhances the ability of EngTregs to: (i) sense and respond to tissue damage (ST2-expressing EngTregs efficiently migrate to sites of inflammation and injury); (ii) suppress excessive inflammation (EngTregs exert potent anti-inflammatory effects through multiple mechanisms, including direct suppression of immune cells and modulation of the inflammatory environment); and (iii) actively participate in tissue repair (EngTregs produce growth factors and interact with other cells to promote tissue regeneration). Overexpression of FOXP3 ensures a stable and suppressive Treg phenotype, crucial for long-term therapeutic efficacy. A chemically induced signaling complex (CISC) enables tunable IL-2 signaling, promoting Treg survival and function while facilitating scalable manufacturing. These innovations culminate in a first-in-class allogeneic tissue EngTreg product with advantages in manufacturing scalability, cost-effectiveness, and therapeutic potential compared to conventional Treg therapies. Preliminary studies demonstrate the ability of allogeneic EngTregs to accumulate in the injured brain and improve motor skills, sensory function, learning, and memory following ischemic injury induced in the transient middle cerebral artery occlusion (tMCAO) mouse model of stroke. This proposal outlines three aims to further advance the preclinical development of EngTregs for stroke: Aim 1: Evaluate the therapeutic efficacy of EngTregs in two preclinical stroke models (permanent middle cerebral artery occlusion and photothrombosis) in both adult and aged mice, assessing a comprehensive range of functional and histological outcomes. Aim 2: Characterize the mechanism of action and define a comprehensive in vitro profile of the human EngTreg drug product, including assessment of cytokine sequestration, T cell suppression, macrophage polarization, and transcriptomic analysis. Aim 3: Assess the immunotoxicity and immunogenicity of human EngTregs to ensure clinical safety, including evaluation of cytokine release syndrome and allo-immunogenicity. Successful completion of these aims will provide critical preclinical data supporting the clinical translation of EngTregs as a novel and promising therapeutic strategy for stroke, addressing a significant unmet medical need.

# data[].attributes.keywords[]
Receptors  Tumor Necrosis Factor  Type II

# data[].attributes.title
Pathogenic mechanisms of chronic lung sequelae following respiratory viral infection

# data[].attributes.funder_divisions[]
National Heart Lung and Blood Institute (NHLBI)

# data[].attributes.abstract
The COVID-19 pandemic has brought the world’s attention on respiratory viral infections. It is not a question of if another respiratory viral pandemic will arise, but rather when. The diversity of viral pathogens presents a tremendous challenge for designing virus targeted preventions and therapeutics. However, respiratory viruses cause similar lung injury that drives morbidity and mortality. A focus on host mediated lung injury pathways in this context is warranted, as there is potential for therapeutic interventions that would be effective across a range of respiratory viral infections. The COVID-19 pandemic has drawn attention to a poorly characterized phenotype of persistent lung inflammation that lasts well beyond clearance of live virus. Long-COVID and long-Flu are clinical diagnoses that lack an understanding of the molecular underpinnings of disease. In this application, we focus on understanding the mechanism by which respiratory viral infection causes lasting tissue damage covering antigen persistence, presentation, and a positive feedback inflammatory cell network. We propose that the persistent presence of viral antigen in pulmonary endothelial cells is a major driver for the prolonged activation of CD8+ T cells, consequently leading to chronic pulmonary sequelae, through reinforced interaction of CD8+ TRM cells and pro-inflammatory macrophages. This hypothesis will be tested in two Aims: 1) we will investigate the contribution of persistent activation of CD8+ T cells by pulmonary endothelial cells to the development of chronic pulmonary sequelae, and 2) we will interrogate the T cell and macrophage inflammatory axis in persistent inflammation during recovery from respiratory viral infection. To examine these pathways, we will employ the SARS-CoV-2 and influenza virus mouse models, and we will confirm findings using tissue from human patient transbronchial biopsies. Discovery of causative host inflammatory pathways that are critical to post-viral lung sequelae would provide pre-clinical evidence to support clinical trials. If pathogenic mechanisms are indeed similar between multiple respiratory viruses, then host-directed therapeutics would potentially have a tremendous impact. Currently, there are no specific therapies indicated for the treatment of post-viral lung inflammation.

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Post-Acute Sequelae of SARS-CoV-2 Infection

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Multidrug-resistant bacteria (MDRB) contribute increasingly to morbidity, mortality, and healthcare costs. Extended-spectrum beta-lactam- and carbapenem-resistant Enterobacterales (ESBL-E and CRE) are MDRB of particular concern due to their demonstrated ability to evolve into highly transmissible clones and acquire and spread antibiotic resistance determinants. Traditional epidemiological surveillance typically focuses on outbreaks of MDRB causing clinical infections, underestimating the burden of these pathogens within hospital systems. Broader efforts that account for asymptomatic carriage and environmental and community reservoirs would be ideal to track and mitigate the spread of MDRB. Wastewater surveillance has proven an effective tool for public health pathogen monitoring, as shown with SARS-CoV-2, but has not been established in clinical settings. This proposal will develop new systems to leverage wastewater for clinically applicable, proactive, and readily deployable MDRB monitoring. In Aim 1, we will establish standardized longitudinal surveillance strategies to inform infection control responses. We will use long-read metagenomics and novel bioinformatic approaches to rapidly identify significant changes in relative or absolute abundance of ESBL-E or CRE compared to site-specific baselines. We will also establish methods to translate wastewater testing data into interpretable “action thresholds” for use by hospital and clinical teams. In Aim 2, we will identify factors enabling the emergence of novel ESBL-E and CRE genotypes in the wastewater environment. Wastewater sampling can identify novel resistant genotypes before detection of clinical infections. We will further develop our novel Metapore-C technique to link bacterial hosts with resistance gene-harboring mobile elements and will use this approach to identify environmental factors such as wastewater antibiotic levels and plumbing design associated with acquisition of resistance. Lastly, in Aim 3 we will devise wastewater testing methodologies suited to resource-limited clinical settings. Given the costs and infrastructure needed for comprehensive clinical surveillance, wastewater testing is better poised to aid in mitigation of MDRB under resource constraints. Yet, current wastewater surveillance approaches are often impractical in such settings. Our strategies for reducing per-sample costs and the analytical burden of wastewater data interpretation, as piloted at a pediatric hospital in Gaborone, Botswana, will serve as a proof-of-concept for wastewater MDRB testing in diverse contexts. Overall, this project will significantly broaden the ability of wastewater surveillance to inform hospital and clinical care efforts, while establishing best practices for global surveillance of antimicrobial resistance in hospital wastewater.

# data[].attributes.title
Ultra-precision diagnostics for ALK+ non-small cell lung cancer

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Background & Significance: Identifying ALK-positive lesions, which are highly responsive to treatment with Crizotinib or Alectinib, is a high-priority for managing patients with ALK+ Non-Small Cell Lung Cancer (NSCLC). A blood-based companion diagnostic (liquid biopsy) could monitor NSCLC patients post-treatment for tumor recurrence without tissue re-biopsy, and also identify non-symptomatic ALK+ individuals for earlier, more effective treatment with ALK kinase inhibitors. Expansion of this approach to other major oncogenic mutations has potential as a non-invasive cancer screening tool that would be transformative for healthcare. Objective & Innovation: AleloPharma Inc. is achieving solutions for personalized precision medicine using AleloMAX, an innnovative high-dimensional molecular detection system that allows ultra-specific detection of target nucleic acids with an impressive 1-absolute copy per reacting assay limit-of-detection (LOD) that effectively eliminates both false positives and false negatives. We aim to offer clinicians a cutting-edge diagnostic capable of accurately detecting ALK translocations among cell-free RNA in the blood plasma of affected patients. Approach: A proof-of-principle study, supported by preliminary LOD data obtained with synthetic ALK constructs, is proposed to develop a liquid biopsy test to detect ALK translocation variants in a kit for use in clinical labs.  • Specific Aim 1: Develop an ultra-specific and ultra-sensitive molecular detection platform for  EML4-ALK gene fusion translocations in ALK+ NSCLC. We will probe EML4-ALK fusion RNAs using  synthetic, in vitro-transcribed mRNAs encompassing the distinct EML4-ALK oncogenic variants.  • Specific Aim 2: Demonstrate AleloMAX-ALK’s superior resolution power in ultra-specific  molecular probing and limit-of-detection (LOD) analysis in a clinical proof-of-principle study. The  EML4-ALK diagnostic platform will be tested using plasma- and/or blood-derived nucleic acid samples  obtained from ALK+ NSCLC patients, ALK- NSCLC patients, and healthy control volunteers. Team & Commercialization: Led by a distinguished team with a track record of groundbreaking research in molecular pharmacology, oncology and cellular biology, we are uniquely positioned to tackle this challenge. Our clear roadmap includes patenting all IP and aspires to launch a diagnostic that will have significant clinical utility. Feasibility & Impact: The assay is expected to detect low levels of EML4-ALK mutations in blood of individuals with ALK+ NSCLC. AleloMAX demonstrated diagnostic superiority in prior studies of the NSCLC biomarker POGLUT-1, SARS-CoV-2 and RSV with impressive results. Assay parameters established in this project will be developed as a kit for use in a Phase II study to establish its clinical utility as a companion diagnostic for NSCLC. Successful development of an ALK+ cancer diagnostic will be expanded to include assays for RET, ROS1 and other onco-mutations with additional AleloMAX liquid biopsy assays to monitor a broader panel of cancers. Conclusion: Combining innovation and tangible clinical benefit, our initiative represents a transformative shift in early detection and management of cancer that will positively impact patient survival.