Research Inst Nationwide Children'S Hosp

NIH Research Projects · FY 2026 · 2026-06

Congenital malformations are the leading cause of death for children under the age of one and 1/33 babies is born with such a birth difference. A significant proportion of these affect the developing face and cleft lip/palate occur in ~1/700 births making it one of the most common structural birth defects. Most craniofacial deficits have a genetic component, but our understanding of the genes and mechanisms involved is woefully incomplete. The overall objective of this application is to use forward genetics (i.e., moving from phenotype to causal gene) in the mouse to identify novel alleles important for multiple aspects of craniofacial development. We will also use sophisticated genetics to both predispose some of the embryos to a perinatal phenotype and to immediately assess if the crucially important hedgehog pathway is disrupted during development. Our rationale is that by taking an unbiased, forward genetic approach we will uncover new and fundamental discoveries in the genetics of craniofacial and brain development. We will utilize N-ethyl-N-nitrosourea (ENU), a chemical mutagen causing random, single base-pair substitutions in the genome. Treatment with ENU is an effective tool for generating heritable changes in the genome with low morbidity and/or mortality. We have previously used this technique to identify many new genes to be important for craniofacial development. One of these discoveries we have made from a previous screen is that P4hb is crucial for proper palate and skeletal development. We have not studied this allele in depth and here we further explore the underlying pathogenic mechanisms of this genetic variant. The major outcome of this proposed research is that new genetic determinants which control craniofacial development will be revealed through the use of ENU mutagenesis in mice. By completing these studies, we will be positioned to use this information together with the newly developed animal models to develop novel hypotheses guiding mammalian craniofacial development. We will accomplish the goals of this application by pursuing the following two specific aims. In Aim 1, we will perform ENU mutagenesis in the mouse and conduct a forward screen for novel genes important for craniofacial development. To further enrich a portion of our screen and quickly assess a potential molecular mechanism, we will incorporate the Patched1-lacZ null/reporter allele into our breeding scheme. This will allow us to both predispose some of the mutants to craniofacial anomalies and quickly assess if the malformation(s) are due to altered hedgehog signaling. We aim to clone and experimentally validate 15 novel mutations over the course of this grant period. Aim 2 will follow up on the P4hb variant previously discovered through ENU mutagenesis and determine the molecular basis for the malformations seen in these mutants. In addition to learning more about the role of this particular gene, the details of the experimental approaches in this aim serves as a benchmark for how we might pursue studies in other alleles identified in Aim1. Together, these studies will identify several genes essential for mammalian craniofacial structure.

NIH Research Projects · FY 2026 · 2026-05

Summary Hepatitis E virus (HEV) infection is a major cause of hepatitis worldwide and is associated with a mortality rate in pregnant women. The virus can often persist when the immunity is compromised leading to serious liver disease if untreated. Currently no HEV-specific treatments are available, necessitating a better understanding of the HEV infectious cycle. During infection, HEV produces two forms: naked virions (nHEV) that are shed into feces and transmitted to new hosts, and quasi-enveloped virions (eHEV) that circulate in the bloodstream responsible for cell-cell spread. The entry mechanism of both types of HEV virions remains poorly understood. Our previous work shows that both forms of HEV particles enter cells via clathrin-mediated endocytosis. However, entry of the nHEV is independent of low pH, Rab5 or Rab7. In stark contrast, eHEV requires low pH, Rab5 and Rab7. In addition, entry of eHEV uniquely requires NPC1 and LAL. NPC1 is an endosomal protein involved in cholesterol extraction from lipids, and LAL (lysosomal acid lipase) is the only lipase in the lysosomes that hydrolyses cholesteryl esters and triglycerides. Based on these findings, we proposed that the removal of the quasi-envelope is a prerequisite for the HEV capsid to interact with a putative endosomal receptor for productive entry. In addition, new studies show that cathepsins are required for eHEV entry, raising the possibility that degradation of eHEV-associated proteins and/or proteolytic processing of the capsid may be critical for this process. The goal of this project is to further elucidate the novel entry mechanism used by eHEV. Aim 1 will determine the fate of the quasi-envelope during eHEV entry. We will test the hypothesis that lysosomal membrane degradation is required for eHEV entry to reveal the HEV capsid for receptor interaction. Aim 2 will define the role of host proteases in HEV infection. We will test the hypothesis that proteolytic processing of HEV capsid in the endosome upon eHEV entry exposes a hydrophobic peptide that is essential for endosomal membrane penetration. Successful completion of the proposed study will shed new light on the life cycle of HEV, an understudied virus with an expanding list of closely related viruses that infect a variety of host species. Deciphering the progression of the envelope removal may identify viral and/or host targets for therapeutic intervention. The knowledge acquired through this research may have implications for other quasi-enveloped viruses including hepatitis A virus, poliovirus, and other enteroviruses.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Many tumors are driven by chromosomal translocation-derived fusion transcription factors (TFs), which combine an intrinsically disordered low complexity domain (LCD) with a DNA-binding domain (DBD) from a TF. A major class of oncogenic fusion proteins involves a member of the FET family (FUS, EWSR1, or TAF15) fused to a TF DBD. These fusion oncoproteins exhibit neomorphic functions, including altered sequence-specific DNA binding and biomolecular condensate formation, allowing them to act as master regulators that reshape the genomic and epigenomic landscape essential for tumorigenesis. However, the molecular mechanisms governing their activity remain poorly understood. Our research seeks to define the biochemical and biophysical principles that drive fusion oncoprotein function in cancer. A major breakthrough in our work is the successful purification of full-length EWSR1::FLI1, the oncogenic driver of an aggressive pediatric tumor called Ewing sarcoma. This fusion protein, which contains the LCD from EWSR1 and the DBD from the ETS transcription factor FLI1, exemplifies how FET fusion oncoproteins acquire new regulatory functions through the interplay of their LCD and DBD. Our data indicate that fusion to the EWSR1 LCD fundamentally alters the DNA and nucleosome interactions of the FLI1 DBD, while features of the FLI1 domain in turn influence the ability of the EWSR1 LCD to nucleate transcriptional hubs at key regulatory sites. These findings support our hypothesis that fusion oncoproteins require an as-yet-undiscovered intramolecular crosstalk between their LCD and DBD. Our ability to produce high-quality EWSR1::FLI1 protein enables us to integrate biochemical, biophysical, single-molecule, and functional genomics approaches to dissect this mechanism. We will systematically investigate how interactions between FET LCDs and ETS DBDs confer neomorphic properties across multiple levels of chromatin regulation: DNA binding (Aim 1), nucleosome invasion (Aim 2), and 3D chromatin organization in transcriptional hubs (Aim 3). These mechanistic insights will not only advance our understanding of EWSR1::FLI1 but will also illuminate broader principles governing FET fusion oncoproteins as a class, laying the groundwork for novel therapeutic strategies targeting fusion-driven cancers.

NIH Research Projects · FY 2026 · 2026-05

Project Description/Summary Reticulocyte maturation, the final stage of erythropoiesis, is critical for blood formation. Despite its importance in disease pathology, including inherited and acquired disorders of the erythrocyte, it is an understudied area. A major aspect of reticulocyte maturation is gradual loss of salt and water over time. Maintenance of water and solute homeostasis is critical to survival of both the reticulocyte and the mature erythrocyte. We have identified a novel regulator of erythrocyte hydration in erythropoiesis. In conjunction with the kinase WNK1, this TGF beta- stimulated clone (TSC) 22 family member controls potassium-chloride cotransport in reticulocytes and mature erythrocytes, shrinking reticulocytes to their normal volume in mature red blood cells. The goal of specific aim one is the identification of interacting proteins and characterization of regulator-WNK1 interactions and their influence on cellular hydration. The goal of specific aim two is elucidation of the influence of genetic variants on erythrocyte phenotype in humans and murine in vivo models of perturbed erythrocyte hydration. The overall goal of this proposal, which combines state of the art cellular, molecular, and genetic technologies in an innovative, multidisciplinary manner, is to characterize the structure and function of regulatory complexes in erythroid cells to better understand the mechanisms regulating erythrocyte volume homeostasis. These studies will extend our knowledge of normal erythropoiesis, particularly reticulocyte maturation. They will also provide a better understanding of the contribution of regulatory proteins to erythroid cell traits and its contribution to erythroid cell phenotype in inherited and acquired disorders of the erythrocyte.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Pediatric allogeneic hematopoietic stem cell transplant (PAHCT) is an intensive and lengthy inpatient treatment followed by an exceedingly complex and frequently changing outpatient medication regimen. Caregivers assume responsibility for this complex medication regimen during the transition from inpatient to outpatient care making adherence extraordinarily challenging. Indeed, non-adherence to critical medications is common in the first months following discharge and places patients at risk for life-threatening graft-versus-host-disease (GVHD). Recognizing the need for better adherence-focused care, our multidisciplinary team of PAHCT experts developed MedMemos, a novel 4-session tailored adherence promotion intervention comprised of 2 inpatient and 2 outpatient sessions. MedMemos will provide caregivers with a combination of tailored and standardized medication and adherence education videos and a “Medication Management Kit” that provides tailored strategies to target identified barriers to successful medication management. Informed by the ORBIT model, the goals of this study are to 1) iteratively refine the content, timing, dosing, and delivery of MedMemos with patients, caregivers, and providers (Phase Ib) and 2) establish behavioral (adherence) proof-of-concept (Phase IIa). To achieve the first goal, MedMemos will be delivered to cohorts of 5 caregiver/patient dyads and refined based on participant and provider feedback using a mixed-methods, rapid cycle testing design (Aim 1). After completing all 4 MedMemos sessions, caregivers, and patients ≥8 years-old will provide feedback via acceptability, feasibility, and usability measures and brief structured interview to inform the refinement of MedMemos content and procedures. Following each revision, 10 providers will provide their perspective of the acceptability, feasibility, and usability of the revised MedMemos. MedMemos will then be delivered to the next cohort and evaluated until no feasible changes are indicated by participants or providers. To achieve the second study goal, the final MedMemos version will be administered to 10 caregiver/patient dyads in a quasi- experimental, within-subjects design (Aim 2). Acceptability will be demonstrated by caregivers rating MedMemos a mean of ≥ 4 as measured by the intervention acceptability questionnaires (H1). Feasibility of the refined MedMemos program will be assessed by calculating enrollment (≥80% enrollment rate), data completion (≥80% completion of all follow-up assessments), and intervention fidelity (≥80% of fidelity) and completion rates (>80% will attend all MedMemos Sessions) (H2). Finally, proof-of-concept will be demonstrated by ≥75% children taking ≥75% of their immunosuppressant doses at all follow-up time points (i.e., 1-, 2-, & 3-months post PAHCT discharge) as measured by electronic monitor (H3). The proposed study will lay important groundwork for a fully powered randomized clinical trial to test the efficacy of MedMemos. MedMemos will be developed to be a clinically integrated adherence promotion intervention that optimizes medication adherence and minimizes preventable adverse outcomes in children who receive a PAHCT.

NIH Research Projects · FY 2026 · 2026-05

Project Summary/Abstract Large genomic cohorts are a key tool for identifying genetic factors contributing to rare diseases, but the scattered and heterogeneous nature of genomic cohort datasets limits their interoperability for synthetic cohort construction. The NHGRI’s AnVIL platform provides access to diverse genomic resources, including data from the GREGoR and CSER consortia, which focus on rare and undiagnosed diseases. However, integrating and analyzing data across multiple cohorts remains a challenge due to differences in variant representation and data structure. This project aims to develop computational methods and workflows to overcome these challenges and support standardized multi-cohort analyses, enabling efficient reuse of AnVIL data for rare disease research. Aim 1 focuses on enhancing workflows for multi-cohort data integration by extending our existing tools for standardized cohort allele frequency (CAF) generation from GREGoR cohort data. We will implement a plugin-based architecture to accommodate diverse dataset structures, develop indexing strategies to efficiently integrate variant data, and enable phenotype-informed synthetic cohort creation. This work will provide tools that are interoperable across studies, supporting scalable and reproducible genomic analyses. Aim 2 will evaluate the utility of synthetic multi-dataset cohorts for studying diseases suspected to have a rare genetic cause. We will address potential issues associated with multi-dataset cohort construction, such as assessing such cohorts for the presence of duplicate patient samples. We will assess the combined CSER and GREGoR consortia data using one-sided matching strategies, and compute ACMG variant pathogenicity evidence codes for reuse of these AnVIL data in downstream variant interpretation workflows. This work will assess and refine best practices for rare disease research from synthetic multi-dataset cohorts. Aim 3 focuses on disseminating tools and methods to the broader research community. We will develop training materials, Jupyter notebooks, and protocol papers, provide hands-on demonstrations at AnVIL and GA4GH meetings, and contribute to genomic standards development. These efforts will promote widespread adoption of standardized workflows for rare disease genomics research. By improving interoperability and scalability of multi-cohort analyses on AnVIL, this project will enhance the discovery and interpretation of rare disease variants, advancing genomic medicine and precision health.

NIH Research Projects · FY 2026 · 2026-04

Summary: Neuroblastoma is the most common type of cancer in infancy and causes as much as 15% of childhood cancer mortality. The 5-year survival rates for patients with high-risk neuroblastoma remain at about 50% even with intensive multimodal therapies. Developing novel precision- and immuno-therapies is imperative to augment current therapy with minimal toxicity, prevent disease recurrence, and achieve durable cures in high-risk neuro- blastoma patients. Thisproposal addresses major obstacles to understanding oncogenesis and developingnovel neuroblastoma therapies, including establishing and characterizing clinically relevant immunocompetent mouse models of high-risk neuroblastoma, targeting metabolic vulnerabilities, and reprogramming the immune-suppres- sive tumor microenvironment. We have recently developed neuroblastoma genetically engineered mouse mod- els (GEMMs) that resemble genomic, histological, and immunogenic characteristics of human high-risk neuro- blastoma. We analyzed metabolic gene signatures in high-risk human neuroblastoma and metabolic flux in neu- roblastoma-bearing animal models. We revealed that γ-aminobutyric acid (GABA) is a characteristic metabolite produced, metabolized, and excreted by neuroblastoma. GABA enhances mitochondria metabolism in neuro- blastoma and is excreted as a paracrine molecule, acting on T cells through GABA receptors to suppress CD8 T-cell effector functions. Accordingly, modulating the GABA metabolism reduces neuroblastomacell proliferation and immune-suppressive capacity. Hence, we hypothesize that GABA metabolism cooperates with onco- genic signaling to promote tumorigenesis and foster the immune-suppressive microenvironment in high-risk neuroblastoma; therapeutically targeting GABA metabolism may maximize immunotherapies. To test our hypothesis, we propose the following specific aims: 1) Determine how GABA metabolism controls and impacts neuroblastoma development; 2) Develop clinically relevant immune-competent neuroblastoma mouse models and test GABA-targeting strategies to improve immunotherapy. Our proposed innovative study is among the first major attempts to modulate the tumor’s immune-suppressive metabolic environment, which will help better understand how the altered metabolic landscape of the pediatric tumor’s microenvironment im- pacts anti-tumor immunity.

NIH Research Projects · FY 2026 · 2026-03

Project Summary/Abstract Leukodystrophy with vanishing white matter (VWM) is a severe, progressive neurodegenerative disease that most commonly afflicts infants and children. There are no disease modifying treatments. VWM is caused by autosomal recessive mutations in the five subunit genes of the Eukaryotic Initiation Factor 2B (eIF2B) complex, with most mutations occurring in EIF2B5. eIF2B is required for the first steps of protein translation, but also regulates the integrated stress response (ISR). The ISR can be triggered by minor stressors such as viral infection or trauma that decrease eIF2B activity; but in the context of VWM, results in acute and devastating neurological deterioration. Recent work, including ours, has shown abnormal, persistent activation of the ISR in VWM. This is caused by increased expression of stress response genes, such as ATF4, selectively in astrocytes. Concurrent work has shown that VWM astrocytes inhibit oligodendrocyte precursor cells (OPCs) from maturing, leading to decreased production of myelin, the core “vanishing white matter” pathology. Our preliminary data suggests that these two principal features of VWM pathogenesis, deregulated ISR and astrocyte-mediated oligodendrocyte impairment, are associated. However, studies of a therapeutic compound ISR inhibitor (ISRIB) in an EIF2B5-mutant mouse model failed to normalize disease pathologies and showed only partial efficacy. Our goal is to combine scientific insights from an interdisciplinary team of three VWM investigators to develop a targeted and highly translatable therapy for VWM. Due to VWM’s loss of function and monogenic nature, we are investigating adeno-associated virus (AAV) EIF2B5 gene replacement therapy. Advances in AAV vectors have led to safer and more efficient viral vehicles to deliver transgenes, and AAV serotype 9 has become the most widely used for neurological indications. In 2019, AAV9 gene therapy for spinal muscular atrophy achieved FDA approval based on preclinical and clinical work conducted at Nationwide Children’s Hospital, demonstrating the resources and expertise at our disposal to successfully translate a therapy from proof-of-concept to regulatory approval. Our project is to develop and test AAV-mediated transgene rescue of EIF2B5, with the ultimate goal to prevent or mitigate VWM disease. In Aim 1 (R61 phase) we will determine a lead AAV construct by comparing cell-specific and ubiquitous promoters, including a novel astrocyte-specific promoter. We will then ensure translatability of our therapeutic by validating expression and attenuation of disease markers in VWM human patient-derived organoids. Upon selection of a lead candidate, in Aim 2 (R33 phase) we will determine a safe and efficacious dose in two VWM mouse models using clinically relevant outcome measures, including magnetic resonance imaging and electroencephalography. In summary, we detail a rigorous approach to develop and optimize a lead candidate, targeting the underlying astrocytic VWM pathogenesis, and validate it in three models to enhance translation.

NIH Research Projects · FY 2025 · 2025-12

PROJECT SUMMARY / ABSTRACT This NIH K08 proposal describes a four-year career development training program in autism spectrum disorder (ASD) genomics research. With this research program, Dr. Mo will develop expertise in human genetics, analysis of next-generation sequencing data, and interpretation of somatic variants in non-neoplastic tissue. These skills complement Dr. Mo’s prior research and clinical training and ideally position her to transition to an independent investigator position studying somatic mutations in ASD. Dr. Mo’s mentor for this proposal is Dr. Christopher A. Walsh, a Professor of Neurology at Harvard Medical School, an HHMI Investigator at Boston Children’s Hospital, and a leader in the genetics of human neurological diseases. With over 25 years of mentorship experience, Dr. Walsh has an established track record of mentoring trainees to successful academic careers in biomedical research. Dr. Mo will be supported by a scientific advisory team and collaborators with complementary expertise in autism genetics, computational genomics, genotype-phenotype correlations of somatic mutations, and career mentorship. The institutional resources available at Boston Children’s Hospital, which is affiliated with Harvard Medical School, are world- class and provide an ideal environment to foster the development of a physician-scientist career. The primary scientific objective of the proposed research plan is to study the role of somatic (post- zygotic) variants in ASD. Dr. Mo’s central hypothesis is that somatic variants contribute to ASD risk. Dr. Mo provides pilot data indicating that somatic single nucleotide variants (sSNVs) are increased in gene exons in ASD probands compared to controls, particularly in highly constrained genes with loss-of-function intolerance. Furthermore, Dr. Mo shows that sSNVs in non-coding gene regulatory regions can be efficiently detected using ATAC-seq, which allows the detection of non-coding sSNVs in larger sample sizes than previously possible using whole genome sequencing. To achieve the research objective, a combination of whole exome sequencing, ATAC-seq, whole genome sequencing, amplicon sequencing, and computational analysis will be used. These strategies will systemically examine two independent, but related, aims: (1) the burden of sSNVs in functionally-relevant genes in ASD compared to neurotypical individuals; and (2) the distribution of sSNVs in non-coding gene regulatory regions in postmortem human brain neurons from ASD and neurotypical individuals. Findings from this study may improve our understanding of the genetic architecture and mechanisms of ASD as well as the genetic diagnosis of ASD in clinical practice.

NIH Research Projects · FY 2025 · 2025-09

Project Summary High-grade gliomas (HGGs), including diffuse intrinsic pontine glioma (DIPG), are a leading cause of cancer- related death in children, adolescents, and young adults. Despite intensive multimodal therapy, prognosis for pediatric, adolescent, and young adult patients with these aggressive brain and/or spine tumors remains dismal, with 5-year overall survival (OS) <10%. Genome-wide sequencing analyses have identified recurrent somatic alterations of receptor tyrosine kinases, cell cycle regulation, DNA repair, and/or PI3K/AKT/mTOR signaling pathways within molecularly distinct subgroups of pediatric HGG/DIPG, with therapeutic and prognostic implications. However, lack of consistent, prospective and comprehensive tumor molecular profiling in pediatric early phase trials of targeted agents and insufficient assessment of correlative biomarkers of response and resistance have hampered the interpretation of trials’ results and impeded the rational development of successive studies. Motivated by this critical need to develop novel, effective, and well-tolerated therapies for pediatric HGG/DIPG and guided by recent discoveries which have improved understanding of their genomic landscape, we have developed and recently opened TarGeT, an innovative molecularly-guided, multi-arm, multi-institutional phase II umbrella trial, with central, comprehensive molecular characterization using a multi-omic approach (whole exome sequencing, RNA-based fusion panel, DNA methylation array) with rapid return of results (within 3 weeks) to guide assignment to one of eight biologically-targeted treatment arms, all with upfront radiotherapy (RT). Efficacy of these targeted therapy arms will be assessed through evaluation of survival outcomes, compared to molecularly-stratified, historical controls. Furthermore, we will collect serial, well-annotated biospecimens (peripheral blood, cerebrospinal fluid, tumor tissue), as well as neuro-imaging to determine multimodal biomarkers predictive of response and resistance. Longitudinal genomic and immunologic profiling will be performed, and integrated with imaging data for correlative analyses, focused on (a) validating “liquid biopsy” molecular assays, (b) identifying genetic alterations which confer the greatest sensitivity to investigated targeted therapies within each arm, and (c) determining genomic and immunologic signatures that predict radiographic response to RT, including distinguishing between inflammatory pseudoprogression and true tumor growth. These samples provide an unprecedented opportunity to systematically characterize and evaluate biological determinants of therapy response. Finally, submission of serial MRIs at standardized disease evaluation timepoints will allow prospective validation of volumetric radiographic response assessments using artificial intelligence methodology, comparison to conventional bidimensional measures, and correlation with outcomes. If feasible and effective, this precision medicine approach, utilizing genomic, immunologic, and imaging biomarkers validated here may be incorporated into the treatment of pediatric HGG/DIPG to expand molecularly-guided therapy options and improve outcomes.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Social media (SM) use is ubiquitous among adolescents, and typical developmental factors during these formative years put adolescents at exceptionally high risk for harmful effects from SM. Social media challenges (SMC) (e.g., Tide Pod Challenge) are dangerous events where people engage in behaviors to share on SM platforms while “challenging” others to participate. While media and peer-reviewed journals have described many SMC and associated injuries, data on U.S. adolescents participating in SMC is scarce, including identified risk factors or proposed elements for an intervention. Dr. Middelberg is a board-certified pediatric emergency medicine pediatrician whose research focuses on pediatric injuries from consumer products to inform prevention. This Mentored Patient-Oriented Research Career Development Award (K23) focused on understanding the determinants and mitigators of SM risk-taking will allow Dr. Middelberg to gain additional skills to be a productive clinician-scientist focusing on pediatric injury and prevention. She has built a strong mentorship team, including national experts in pediatric injury, health behavior, decision psychology, and biostatistics. This team will provide methodological and content expertise while guiding her Training and Career Goals to acquire needed skills to be a successful independent investigator, which include gaining knowledge and proficiency in 1) study design and statistical analysis, 2) using a health behavior framework to guide intervention development, and 3) elements of injury prevention interventions specific to the adolescent age group. The rationale is that a better understanding of adolescents participating in SMC, and how their behavior is similar or different from other risky behavior, will add to evidence-based interventions and prevention measures for those irresponsibly using SM. The specific aims are as follows: Aim 1: Determine predictive sociodemographic, SM use, and digital status seeking characteristics, of adolescents who have participated in a SMC. Aim 2: Evaluate risk-taking behaviors of an enrolled cohort of adolescents. Aim 3: Define key elements for a future intervention. The approach is a prospective, cross-sectional survey study of adolescents (ages 13-21), followed by qualitative interviews, and utilization of the health intervention planning framework, Intervention Mapping, to identify evidence-based components of a proposed intervention. This award will provide Dr. Middelberg with the opportunity and training to become an independent investigator with the goal of future applications for independent National Institutes of Health funding.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY The long-term goal of this work is to develop a translatable imaging paradigm that enables comprehensive assessment of multiple anatomical and physiological parameters in patients with peripheral artery disease (PAD). Patients with lower extremity PAD are at increased risk of lifestyle limiting claudication, non-healing wounds, amputation, and death, which is often attributed to deficits in limb perfusion and high levels of vascular calcification above and below the knee. PAD patients with diabetes mellitus (DM) have accelerated progression of peripheral vascular calcification, which reduces lower extremity muscle perfusion, limits options for revascularization, reduces technical success rates of revascularization procedures, and increases risk for amputation. Despite this knowledge, an ongoing challenge for the vascular medicine community is the lack of standardized non-invasive imaging tests that quantify regional perfusion abnormalities as well as vessel- specific calcific disease progression in PAD patients. Our team recently developed a novel dual-phase (i.e., combined dynamic and static) PET/CT imaging approach using 18F-NaF that quantifies both regional muscle perfusion and vessel-specific active arterial microcalcification in the lower extremities during a single imaging session using a single radioisotope dose injection. In the proposed projects, we will characterize the stages of peripheral atherosclerosis targeted by 18F-NaF using an existing biobank of human arterial specimens possessing varying degrees of calcium density and stiffness. We will then prospectively apply dual-phase 18F- NaF PET/CT imaging to PAD patients with and without DM to quantify muscle-specific perfusion abnormalities and vessel-specific calcium burden, and to predict vessel-specific calcium progression across a 1.5-year study period. Following clinical evaluation of our approach, we will test the performance of deep learning image segmentation methods for enabling efficient analysis of vessel-specific measures of active and established arterial calcification as well as muscle-specific deficits in lower extremity perfusion from PET/CT images. Completion of the proposed studies would lead to a comprehensive and scalable imaging approach that quantifies multiple anatomical and functional parameters associated with PAD severity, calcium progression, and clinical outcomes in PAD patients, which would improve the evaluation, monitoring, and risk assessment of PAD patients who are at elevated risk of adverse limb and cardiovascular events.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract During bacterial or fungal infections, neutrophils engulf the pathogens by phagocytosis and initiate a signaling process leading to the activation of NADPH oxidase. NADPH oxidase utilizes NADPH as an electron donor to produce superoxide (O2●-), which is subsequently converted to H2O2 and highly microbicidal hypochlorous acid. NADPH is produced from glucose metabolism primarily through the pentose phosphate pathway. Defects in the subunits of the NADPH oxidase and the pentose phosphate pathway are associated with chronic granulomatous disease. Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione (GSH) utilizing NAPDH as an electron donor. GSH protects cytoplasmic components from oxidative damage by reactive oxygen species (ROS). Rare patients deficient in GSR activity have been reported to have slower recovery after infections, hemolysis after consuming fava beans, and cataracts at a young age. Gsr-deficient (Gsr-/-) mice with a deletion from intron 1 to intron 5 in the Gsr gene failed to contain bacterial and fungal infections, despite the recruitment of larger number of leukocytes to the sites of infections and production of greater amounts of cytokine and chemokines than wildtype mice. Gsr-/- neutrophils phagocytosed fewer pathogen particles upon encountering either E. coli or C. albicans than did wildtype neutrophils. Gsr-/- neutrophils also produced substantially less ROS and had attenuated pentose phosphate pathway, and exhibited lower microbicidal activity and markedly greater protein tyrosine phosphorylation than did wildtype neutrophils. We hypothesize that during neutrophil-pathogen interaction, Gsr protects the enzymes in the glucose metabolic pathways from oxidative damage thus facilitating the production of ATP for phagocytosis and NADPH for respiratory burst. We postulate that Gsr also facilitates the microbicidal program by regulating protein phosphorylation. We will test these hypotheses through two Specific Aims. Aim 1 will assess contribution of hematopoietic, neutrophil, vs parenchymal/stromal cell Gsr to host defense against bacterial and fungal pathogens using bone marrow swaps between wildtype and Gsr-/- mice, adaptive transfer of wildtype neutrophils into Gsr-/- mice, and Gsr restauration in hematopoietic stem cells of Gsr-/- mice. Aim 2 will assess the effects of Gsr deficiency on: 1) glucose metabolism (glycolysis, Krebs cycle, and pentose phosphatase pathway) through metabolomics and Seahorse assays; 2) protein oxidation via OxiCAT and protein phosphorylation through proteomics. These studies will identify the proteins that are dependent on Gsr for protection against oxidative damage during neutrophil- pathogen interactions and reveal the critical function of Gsr in facilitating ROS production, cellular metabolism, and cell signaling during the execution of the microbicidal program. Successful completion of these specific aims will uncover the underlining mechanisms by which Gsr regulates phagocytic functions.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) typically results from mutations in the DMD gene that disrupt the open reading frame, resulting in no dystrophin protein production. In contrast, the milder Becker Muscular Dystrophy (BMD) typically results from mutations that allow expression of a partially functional dystrophin protein. Current therapeutic approaches in trials are directed toward expressing shorter, “micro-dystrophin” proteins, likely ameliorating DMD into a BMD-like phenotype. An alternative approach is to use exon skipping. This strategy uses antisense oligonucleotide sequences (AONs) to modulate pre-mRNA splicing of DMD transcripts to restore the reading frame and to express a truncated but functional dystrophin. An alternative approach is to use a small virus, such as Adeno-Associated Virus, containing a small promoter expressing the AON (also referred as vectorized exon skipping [VES]), alleviating the need to reinject the AONs as they get degraded over time. Based on our published studies in mice and our publicly presented data in infant Dup2 patients, VES for DMD exon 2 demonstrate a more robust exon skipping and protein expression than the alternative exon skipping approach using AONs. Exon 44 is among the mutational hotspots of the DMD gene, and its skipping would benefit around 6-12% of DMD patients. Our long-term goal is to develop a vector that maximizes exon 44 skipping to provide the best potential outcome for this patient subpopulation. Our central hypothesis is that VES will provide a robust exon-skipping response, leading to therapies with significant efficacy. We have already generated promising preclinical data for this program, but a new vector design is required for the next phase, especially if this vector becomes commercially available. In Aim 1 (R61 phase, year 1), we will compare our current VES with a newly designed one that will have manufacturing advantages compared to our current vector both in vitro and in vivo. In Aim 2 (R61 phase, year 1-2), we will check if the designed vectors have splicing off-targets by RNA seq analyses. In Aim 3 (R33 phase, years 2-3), we will test a minimal efficacious dose of our lead candidate to generate data for a pre-Investigational New Drug (pre-IND). Our rationale for this project is that evaluation of potential therapeutic vectors, assessing their efficiency and confirming absence of off-targets, measuring minimal efficacious dosage, and early engagement with the FDA will lead to a streamlined path toward vector development. The immediate impact of our work will be data to support our ongoing interaction with the FDA in developing rapid VES personalized gene therapies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Sports-related concussions (SRCs) affect nearly two million US children annually, posing significant acute and long-term risk to the developing brain. The actual number of SRC incidents is likely higher, as up to one-third go unreported. Most existing SRC education and prevention programs transmit knowledge effectively but fail to change reporting behaviors, leaving youth athletes failing to recognize or report sports-related concussions (SRC). Innovative approaches to improve SRC reporting behaviors among youth athletes are urgently needed. The proposed intervention, Make Play Safe (MPS), is a two-component program grounded in behavioral theory aimed at enhancing SRC recognition and reporting among youth soccer athletes. MPS leverages virtual reality (VR) technology to allow youth athletes to virtually “experience” SRC events and symptoms. It also provides effective communication strategies for parents and coaches to facilitate and enhance discussions about SRC reporting and safety. The long-term goal of this project is to reduce negative and potentially long-lasting SRC- related health consequences by increasing SRC reporting among youth athletes. Specifically, this study will determine if the MPS intervention increases soccer athletes’ SRC recognition and reporting intentions (Aim 1) and SRC reporting behaviors (Aim 2). Additionally, it will evaluate whether the efficacy of the MPS intervention is mediated by the frequency of parent-child or coach-team communication about SRC (Aim 3). Boys’ and girls’ soccer athletes aged 9-12, along with their parents and coaches, will be recruited from eight youth recreational soccer leagues in central Ohio (n = 960 athletes, 960 parents, and 64 coaches) and enrolled in four waves. In each wave, two of the eight leagues will be randomly selected and assigned to either the Intervention or Control group. The intervention includes a VR-based SRC education session for youth soccer athletes at the beginning of the season and a weekly message notification program for parents and coaches throughout the season. Both groups will also receive the standard SRC education currently provided by their soccer league. Study out- come assessments will be conducted with athletes, parents, and coaches pre-season, during the season, post- season, and six months after the season ends. This project is significant because it addresses the underreporting of SRC in youth soccer, a popular sport for both boys and girls with a high rate of SRCs. The project is innovative in its use of VR technology to interactively teach youth athletes about SRCs in a safe and distraction-free, yet realistic environment. The findings of this study will have a significant impact, as the intervention is theory-driven, developmentally appropriate, and readily scalable for wide dissemination and immediate use at minimal cost by youth sports organizations, schools, and community groups. When adopted, this intervention has the poten- tial to vastly improve youth athletes’ SRC recognition and reporting behaviors, thereby reducing the negative and long-lasting health consequences.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT: The purpose of this Mentored Patient-Oriented Research Career Development Award (K23) is to provide Dr. Matthew J. Kielt, MD, Assistant Professor of Pediatrics at The Ohio State University College of Medicine and Nationwide Children’s Hospital with the requisite mentorship, training, and research experience to become an independent clinician-scientist with niche expertise in precision medicine clinical trials. His long-term goal is to identify precision therapeutics that restore normal pulmonary function in infants with bronchopulmonary dysplasia (BPD). His immediate goal is to develop the skills needed to lead multi-center biomarker-driven clinical trials of anti-inflammatory therapeutics for infants with BPD. To achieve these goals and transition to independence, Dr. Kielt and his mentorship team have developed a comprehensive career development plan that includes: (1) intensive mentorship from an R01 funded team with a proven record of K-award mentorship; (2) didactic and hands-on training in precision medicine research methods, advanced longitudinal statistical analyses, and the conduct and design of clinical trials; and (3) an innovative research proposal to develop biomarker-informed prognostic models for infants with BPD. Preterm infants with the most severe form of BPD, grade 3, are ventilator-dependent near term corrected age and suffer from chronic pulmonary insufficiency throughout life. Infants with grade 3 BPD exhibit striking heterogeneity in the time required to wean from mechanical ventilation (MV). Infants with grade 3 BPD who are difficult-to-wean from MV, defined as unsuccessful liberation from MV by 2 months’ corrected age, are at 3-fold increased risk of death as compared to infants who are simple-to-wean. Biologic predictors that discriminate grade 3 BPD infants who are and are not difficult-to-wean remain unknown. In adult critical care patients, pathogenesis-informed biomarkers of lung inflammation discriminate patients who are and are not difficult-to- wean. Additionally, biomarker-driven clinical trials in adults demonstrate that budesonide, an inhaled cortico- steroid that reduces pulmonary inflammation, decreases the expression of pro-inflammatory biomarkers, and facilitates weaning from MV. Whether these findings are generalizable to infants with grade 3 BPD constitutes an important knowledge gap. Dr. Kielt's research proposal will address this knowledge gap by (1) determining the discriminatory performance of pro -inflammatory biomarkers for grade 3 BPD infants who are and are not difficult-to-wean from MV and (2) demonstrating the feasibility of a biomarker-driven clinical trial of budesonide in infants with grade 3 BPD. The expected outcomes of Dr. Kielt's K23 studies will yield novel insights into grade 3 BPD disease progression that will inform the design of pioneering R-series biomarker-driven randomized controlled trials of anti-inflammatory therapeutics. His career development plan outlines a clear path to gain the knowledge, skills, and experience needed to become an independent and innovative clinician- scientist who leads multi-center BPD precision medicine clinical trials.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Salmonellae are Enterobacteriaceae that cause a spectrum of disease in humans and animals, including enteric (typhoid) fever and gastroenteritis. Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S. Typhi), results in a life-threatening systemic disease that is responsible for significant morbidity and mortality worldwide. Approximately 5% of individuals infected with S. Typhi become chronic carriers with the gallbladder (GB) as the site of persistence, and these asymptomatic carriers represent a critical reservoir for further spread of disease. We have demonstrated that gallstones (GSs) aid in the development and maintenance of GB carriage in a mouse model (utilizing S. Typhimurium, which causes a typhoid-fever like disease in mice) and in humans, serving as a substrate to which salmonellae attach and form a protective biofilm. Thus, biofilm formation is a key step in the establishment of carriers. Until recently, typhoid fever has lacked a direct in vivo model, as S. Typhi is host-restricted to humans. Recently, the Collaborative Cross project has produced a mouse strain (CC003) that is permissive to S. Typhi infection, which we have utilized to create a model of chronic S. Typhi infection. This model will be more physiologically relevant to human disease, as it will utilize the causative infectious agent and not a closely related serovar. We hypothesize that the CC003 mice are permissive to S. Typhi chronic infection through biofilm formation on gallstones and immune suppression. Further, we hypothesize that treating chronically infected mice with our lead anti-biofilm agents will clear the carrier state. In Aim 1, we will determine whether S. Typhi chronic infection is mediated by biofilm formation on cholesterol GSs and characterize the inflammatory response within the GB of infected mice. Studies in Aim 2 will determine whether anti-biofilm compounds in combination with antibiotics can clear the chronic carrier state of S. Typhi in the CC003 mouse model. These experiments will develop a more physiologically relevant model of chronic typhoid fever and characterize potential therapeutics.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY / ABSTRACT Traffic crashes are the leading cause of death among teens aged 15-17 years, with the national death rate for teen drivers increased 26% from 2019 to 2022. Research on supervised driver training for novice teen drivers is limited, particularly interventions using smartphone technology and involving parents. Therefore, we propose a hybrid randomized controlled trial to simultaneously test the effectiveness and implementation of a theoretically informed smartphone app, Drive Ready Venture (DRV). The aims of this project are to determine the effective- ness of the DRV app intervention in improving driving safety scores among novice teen drivers (Aim 1); and to identify contextual factors associated with the adoption and implementation of the DRV app intervention (Aim 2). We propose a prospective, randomized, controlled, parallel-group, three-arm trial. A total of 465 novice teens drivers (15.5-17 years) beginning their learner period of licensure and one of their parents will be randomized to receive: (1) DRV only. The app tracks driving performance while providing individualized feedback and educa- tional materials on driving errors and a driving safety score with gamification-centered activities and incentives (e.g. badges). (2) DRV + parent engagement. In addition to the above features, parents will participate in situa- tional supervised driving practice with their teens, utilizing their teen’s driving data and motivational interviewing (MI) based communication strategies (e.g., MI-based conversation starters). or (3) active control. The app will only track driving performance and be paired with passive education materials on car maintenance (sham DRV app). We hypothesize that teens in the DRV-only and DRV + parent engagement groups will demonstrate higher driving safety scores compared to controls. Participants will be studied for six months. Outcomes will be driving safety scores and high-risk driving events as measured by the DRV app. In addition, we will employ quantitative and qualitative approaches to identify contextual factors associated with the adoption and implementation of the DRV app intervention. Guided by strong preliminary data, this study is innovative because, although app have been used to track driving behavior, they have yet to be tested comprehensively for behavior change. This study is significant, because it will establish the effectiveness of the DRV app with teen-parent dyad intervention in improving driving safety scores and reducing high-risk driving events, speeding incidents, and phone use, while also enhancing driving skills. The findings will provide critical insights into the facilitators and barriers to imple- menting this intervention among the general population of teen drivers. If successful, the DRV app will be dis- seminated at no cost to families across all socioeconomic levels and to driver’s licensing and education systems as part of learning-to-drive practices.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Prolonged diagnostic delays, or “diagnostic odysseys,” in neonatal intensive care units (NICUs) represent a significant burden for patients and families while posing challenges for clinicians, particularly when genome sequencing (GS) is delayed or omitted. Up to 20% of critically ill neonates may have a genetic disease, yet many diagnoses are made only after extended uncertainty, leading to worse outcomes, longer hospital stays, and higher healthcare costs. These issues are especially pronounced in underserved populations, such as racial and ethnic minorities, who face barriers to GS due to healthcare disparities, further compounding diagnostic delays and worsening outcomes. Our long-term goal is to eliminate health disparities in genetic testing, ensuring that no child with a genetic disease—regardless of racial, ethnic, or socioeconomic background—experiences a prolonged diagnostic odyssey. The overall objective of this application is to develop a machine learning (ML)-based approach that reduces health disparities by objectively identifying neonates from underserved populations who require genomic testing, using documented clinical data to mitigate provider- and system-driven biases that often contribute to unequal access to genetic services. Our central hypothesis is that the combined analysis of maternal and infant health records will enable efficient identification of neonates in Level III NICUs likely to benefit from early GS, facilitating faster and targeted diagnosis of genetic diseases. To test this hypothesis, our specific aim is to develop and evaluate an interpretable ML model that leverages both structured and unstructured data from neonatal and maternal electronic health records (EHRs) to systematically identify neonates most likely to benefit from early-life GS. The ML model will integrate data from clinical notes—encoded as Human Phenotype Ontology terms—and structured data elements such as ICD codes (mapped to PheCodes), laboratory results, clinical characteristics (e.g., gestational age, birth weight), neonatal critical care management (e.g., intubation, medications), and relevant maternal factors (e.g., maternal age, parity, prenatal care). Developed within a privacy-preserving environment, the model will be designed to integrate seamlessly into existing clinical workflows and EHR systems to provide clinicians with real-time decision support. By developing ML that integrates maternal and infant health data, this project introduces an innovative, data-driven approach to identifying at-risk neonates while minimizing human bias. The rationale is that early detection of genetic diseases triggered by predictive analytics will enable timely interventions, reduce health disparities, and improve outcomes in all populations, not just those with ready access to Level IV NICUs. This aligns with funding opportunity PAR-21-255 and helps the NHGRI advance its mission by addressing critical gaps in neonatal genomic medicine and reducing diagnostic disparities. Our team’s unique expertise in neonatal genomics, ML, and clinical decision support positions us to implement this transformative approach successfully, ultimately improving health outcomes and reducing healthcare costs for vulnerable neonates.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT Variants in tubulin genes cause many severe malformations of human forebrain development and these are called “tubulinopathies.” Multiple tubulin genes are highly expressed in the developing brain and the human genetics studies now show there is a spectrum of disease arising from tubulin mutations. Completed sequencing of multiple mammalian genomes reveals there are multiple highly similar tubulin genes. These include the genes of the TUBA1* group (TUBA1A, TUBA1B, TUBA1C) and the TUBB2* group (TUBB2A and TUBB2B). All human tubulin mutations identified to date with malformations of cortical development are de novo, heterozygous missense mutations. This suggests that a tubulin monomer protein is being produced, but acting in an inappropriate manner within the cell. We have performed the only complementary loss of function studies to date on any of the mouse Tuba1* or Tubb2* genes. A deeper understanding of the etiology of the human malformations and requirements for individual tubulin genes is essential to any therapeutics. The overall objective of this application is to understand why and how tubulin mutations cause malformations of cortical development. Our central hypothesis is that the human tubulin missense, de novo variants cause cortical malformations through dominant-negative effects on microtubule function and/or tubulin monomer protein-protein interactions. We will test this central hypothesis and accomplish the goals of this application by pursuing the following three specific aims: (1) use novel epitope-tagged alleles to determine the unique sites of expression for Tuba1 and Tubb2 genes, (2) determine cellular and embryonic molecular mechanism(s) of phenotypes caused by a series of mutations in Tuba1a/b/c, and (3) determine the unique requirements for Tubb2a and Tubb2b for mouse neural development. We expect these studies to have the following outcomes: First, we will determine for the first time the cellular expression patterns of a series of tubulin genes crucial for mammalian nervous system development. Second, we will determine the levels of genetic redundancy within the Tuba1* and Tubb2* gene families. Third, we will understand how human pathogenic mutations in tubulin genes lead to complex malformations of cortical development. By answering some of these basic questions about tubulin biology, we are poised to contemplate the best therapeutic intervention(s). Recent studies have demonstrated that treatments of cortical malformation syndromes in an already malformed brain can ameliorate some symptoms. Thus, a greater understanding of the tubulinopathy developmental disorders could open some promising doors to fruitful treatments.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in the pediatric population. A lethal form of this tumor is driven from MYOD1 mutations occurring in conserved residues in the DNA binding domain. These soft tissue sarcomas are almost universally lethal, frequently metastasize, rapidly progress, do not respond well to standard therapy, and are an unmet medical need. Identifying effective treatments for these tumors is critical, as are correlative or “reverse translational” studies to identify tumor-driving mechanisms. The primary goal of this application is to establish a robust preclinical/clinical pipeline (bench-to-bedside and back) to rapidly develop and test new therapies for this deadly malignancy. Our effort will harness the specialized expertise of both clinical investigators at the NCI and extramural experts in RMS biology and translational epigenetics, and will leverage the unique resources of the NIH Clinical Center. Our project builds upon strong preliminary and published genomics and functional data from our team, suggesting that PI3K is a unique vulnerability in MYOD1-mutant RMS. These insights will be used to develop rational single agent and combination therapies and will be tested in robust preclinical RMS models. These insights will then be used to perform clinical trials in RMS patients with an emphasis on correlative studies to understand the precise genetic contexts where the clinical agents are effective therapy within the same trial within the Intramural NIH Clinical Center. This will allow for more timely identification of active agents and will allow patients to have more treatment options available to them. The preclinical to clinical translation will be complemented by comprehensive genomic analyses of tumor samples obtained prior to treatment and on treatment with novel agents to identify mechanisms of response and resistance to therapy. Insight and samples from clinical trial participants will serve as the foundation for correlative studies, including single cell multi-omics and cell-free DNA analysis, to understand therapy-resistance and develop improved therapies. Our project also addresses fundamental questions in cancer etiology. We have exciting preliminary data revealing that MYOD1-mutant RMS tumors are dependent upon PI3K activity and have unique epigenetic reprogramming. Our extramural basic science team will integrate highly innovative technologies to understand how and why chromatin structural alterations and PI3K can together drive oncogenesis. Thus, the project’s broad long-term objectives are to address an unmet medical need and provide mechanistic insight that will reveal new vulnerabilities and clinical development. We have assembled a multi- disciplinary team of basic and clinical scientists to develop and translate promising therapies for individuals with MYOD1-mutant RMS. This project will allow more effective and rapid translation of promising new therapies for RMS and will expand the type of therapies that are developed. These studies have the potential to develop a new standard of care for patients with MYOD1-mutant RMS.

NIH Research Projects · FY 2026 · 2025-06

The mammalian neocortex is a vast cell network with thousands of connections and is susceptible to numerous genetic congenital brain anomalies impacting cortical structure and connectivity. This application leverages forward genetics in mice to identify new genes influencing forebrain neurodevelopment with a specific focus on cortical structure, lamination and axonal pathways. The cerebral cortex and the basal ganglia comprise the two major telencephalic brain regions controlling voluntary movement and intellectual capacities. Our rationale is that by taking an unbiased, forward genetic approach we will uncover new and fundamental discoveries in the genetics of development and neural circuit formation in these two telencephalic brain regions. The major outcome of this proposed research is gene discovery to study both typical neurodevelopment and neuropathology. We will find novel genes which control the formation of forebrain structure and circuits through the use of ENU mutagenesis in mice carrying genetically encoded reporters expressed at early stages of neural development, a novel application of these existing genetic tools to a basic neuroscience question. By completing these studies, we will be positioned to use this information together with the newly developed tools to develop novel hypotheses concerning the etiological mechanisms of mammalian forebrain development and neural circuit formation. We will accomplish the goals of this application by pursuing the following two specific aims: 1) Identify novel genes responsible for proper gross cortical development, lamination, and layer-specific axonal trajectories and 2) Identify novel genes responsible for proper development of the internal capsule and the corticospinal tract. The aims are accomplished through an ENU mutagenesis approach in the mouse. The mutations are then cloned and validated through a number of functional studies. These studies will identify several genes essential for mammalian forebrain structure and function and thus linking genetics to neural phenotypes. The significance of this work is found in the specific application to cortical structure and circuitry, and that an unbiased approach such as this has the capability to implicate entirely new pathways in neurological disease. Such knowledge is not only critical to further understand the basic mechanisms of neurodevelopment, but also has immediate clinical relevance through identification of a number of potential therapeutic targets. Furthermore, these mouse models are novel genetic tools which provide a reusable resource to directly characterize the role of the mutated gene in neurodevelopment, and potentially serve as a tool to test future therapeutic interventions. Taken together, these findings are therefore applicable to basic developmental neurobiology, pediatric and adult neurology, human genetics and genetic counseling.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY Congenital heart disease (CHD) is the most common birth defect and a leading cause of mortality in children. Despite a well-established heritable componentto this disorder, genetic testingis often unsuccessful. Some 60% of patients fail to receive a molecular diagnosis; for patients with isolated CHD, the failure rate is closer to 90%. Without a precise diagnosis, it is difficult to provide any patient with an accurate prognosis or personalized interventions. The main reason that diagnostic testing fails is that our knowledge of the genetic underpinnings of CHD remains woefully incomplete. CHD is not only genetically heterogeneous, but also notorious for incomplete penetrance (variants that do not always cause a condition). In this proposal we aim to quantify the role of incomplete penetrance in CHD risk genes. We will source a large cohort of CHD families fromthe Gabriella Miller Kids First resource, the Undiagnosed Disease Network, Genomics England, and an internal research study and performtwo complementary analyses to rank CHD genes. First, we will apply a genetic association study of CHD probands to prioritize genes with an increased burden of rare genetic variation among CHD patients irrespective of the inheritance pattern. This will be achieved through an innovative new resource: a remote control database of 40,000 healthy, diverse controls and a method to securely select matched controls for any given case cohort. Second, we will performpedigreeanalysis of all CHD families to identify genes in which rare large-effect variants segregate with disease. Finally, we will combine the results of both strategies into a single master table of CHD genes and use the segregation patterns observed in all families to quantify the extent of incomplete penetrance. We expect our results will serve as a reference for the diagnostic initiatives in CHD and will inform clinical geneticists about the likelihood of disease causality for genes challenged by incomplete penetrance. Our results should provide a framework for integrating association and segregation analyses that could be adapted to other genetically heterogeneous conditions.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY/ABSTRACT Urinary tract infections (UTI) are a morbid condition affecting a significant proportion of individuals; yet there has been minimal progress made in their treatment and even prevention beyond antibiotic use. The host-pathogen interaction is an important relationship in UTI pathogenesis and interventions promoting the host innate immune response to reduce UTI susceptibility would be ideal therapeutic measures. The urothelium serves essential roles in the detection and elimination of invading bacteria through expression of bacterial receptors, production of cytokines and chemokines, and secretion of antimicrobial peptides (AMPs). We have previously demonstrated in a murine model of UTI that the interleukin (IL)-6/Signal Transducer and Activator of Transcription (STAT) 3 signaling pathway in the urothelium is important in producing AMPs and eliminating uropathogenic Escherichia coli (UPEC) from the urinary tract. While the transcription factor STAT3 is a central mediator in this process, its contributions to urothelial antimicrobial properties and transcriptional targets are incompletely understood. The objective of this proposal is to define STAT3 mediated host defense mechanisms in human urothelium. This proposal tests the hypothesis that STAT3 and its downstream targets contribute to UTI defense by limiting intracellular and extracellular bacteria urothelial reservoirs necessary for UTI pathogenesis, with the goal that these host mediators could be leveraged in treating and preventing UTI. The novelty of this proposal is the use of CRISPR/Cas9 gene editing in human urothelial cells to study STAT3 biology and the use of RNA-seq to reveal transcriptional targets of STAT3 signaling. The anticipated outcome of this research is to identify the mechanisms by which STAT3 confers host immunity against UTI and reveal its therapeutic targets. Aim 1 will define mechanisms by which STAT3 mediates urothelial defense against UTI in human urothelial cell culture. Aim 2 will identify transcriptional targets of STAT3 that serve instrumental roles in limiting UTI. Together, these Aims seek to implicate STAT3 and its downstream targets as integral components of the host innate immune response to UTI. The completion of this award will broaden our understanding of the mechanisms by which STAT3 signaling impacts UTI and provide candidate transcriptional targets which would be further interrogated in an R01 application.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT Congenital cytomegalovirus (cCMV) infection is the most common congenital viral infection in the U.S, affecting 1 in 200 liveborn infants annually, and can cause permanent neurodevelopmental and hearing impairment. In contrast, postnatal CMV infection in full-term infants causes no clinical illness or neurologic sequelae. The immunologic differences underlying the clinical manifestations induced by cCMV but not postnatal infection are undefined. Our group recently found that cCMV infected infants expressed a spectrum of CD8+ T cell responses, ranging from naïve-like T cells (TN) resembling those of CMV uninfected newborns, to highly differentiated cells with features of T cell exhaustion (TEX). By comparison, the CD8+ T cells of postnatally infected infants were differentiated and lacked TEX characteristics. Importantly, cCMV-induced developmental delay was associated with the TN-like phenotype, whereas progressive SNHL was associated with persistent TEX-like cells, underscoring the clinical relevance of understanding these T cell responses to cCM infection. This project will test the central hypothesis that cCMV infection impairs the infant’s T cell response to CMV, impeding effective viral control and increasing risk for neurologic injury. We will compare CD4+ and CD8+ T cell responses to CMV between a large enrolled cohort of cCMV infected infants and a group with early postnatal CMV infection acquired through breastfeeding. Aim 1 will identify pathways inhibiting Th1 cell responses after cCMV infection by assessing several possible causes for the lack of neonatal Th1 responses during cCMV infection: (1) Th1 cell differentiation occurs, but cytokine-secreting function is limited by inhibitory signals; (2) Th2/Treg cells develop preferentially, inhibiting Th1 cell differentiation; (3) TLR signaling by dendritic cells is inhibited by cCMV infection, reducing cytokine and proliferative signals to CD4+ cells. Comparisons between cCMV and postnatally infected infants will identify key differences in the CD4+ T cell responses and their upstream regulators. Aim 2 will identify differences in CD8+ T cell responses induced by congenital and postnatal infection and the T cell characteristics associated with cCMV-induced neurodevelopmental impairment. In this Aim, the surface receptor and transcriptional profiles of antigen-specific CD8+ T cells will be compared between cCMV and postnatally infected infants to discern distinctive features generated during cCMV infection in the absence of CD4+ T cell help. Anti-PD-1 antibodies will be used in-vitro to test if function can be restored to CD8+ TEX cells induced by cCMV infection. The CD4+ and CD8+ T cell parameters associated with cCMV-induced neurodevelopmental impairment will be identified. Together, these studies will advance our fundamental understanding of human T cell responses to a congenital viral infection and their potential impact upon neurologic outcomes. This knowledge may inform development of new therapeutic approaches to reinvigorate T cells and improve neurodevelopmental outcomes among cCMV infected infants.