Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2025 · 2025-06

ABSTRACT Familial platelet disorder with associated myeloid malignancy (FPDMM) due to haploinsufficiency of the transcription factor RUNX1 is a rare orphan disorder affecting hundreds of people in the United States. FPDMM is associated with both quantitative and qualitative defects in platelets (Plts), resulting in a moderate bleeding diathesis, and an increased risk of developing myelodysplastic and myeloleukemic transformation. At present there is no treatment that reduces the risk of bleeding or developing leukemia in FPDMM. We have identified a small compound RepSox, known to block transforming-growth factor-β (TGFb) type I receptor kinase (ALK5), that corrects both the quantitative and qualitative defects in FPDMM megakaryocytes (Mks) and Plts. These findings are based on studies of both patient-derived induced-pluripotent stems cells (iPSC)- and short-hairpin (sh) RNA suppression of RUNX1 levels in healthy CD34+-hematopoietic stem and progenitor cells (shRx)-derived human (h) Mks, the terminal hematopoietic cells that gives rise to Plts. Testing other inhibitors that suppress the TGFb pathway did not replicate these findings, suggesting that RepSox affects FPDMM hMks by a separate pathway. We propose that RepSox increases RUNX1 level/activity or modifies distinct RUNX1 isoform usage, correcting both the Plt bleeding diathesis and leukemic risk. We propose the following studies to bring RepSox or a RepSox analog towards clinical application in patients with FPDMM: Specific Aim (SA) #1: Studies of the effects of RepSox on RUNX1 in FPDMM. We will examine whether RepSox affects RUNX1 expression level, specific activity and/or RUNX1 isoform usage in FPDMM hematopoietic progenitor and definitive cells. SA#2: Pharmacologic studies of RepSox and RepSox-related drugs. A series of modified RepSox compounds have already been generated and will be examined to optimize RepSox effects in FPDMM for potential therapeutic application and to gain additional insights into the role of ALK5 inhibition in this process. This will be accompanied by murine studies to define the best mode/dosage of administration, levels achieveable, excretion and toxicity. SA#3: Efficacy of RepSox and RepSox-related drugs in murine models of FPDMM. A newly- described murine model of RUNX1 haploinsufficiency develops mild thrombocytopenia and Plt defects with an increased risk of leukemic transformation, especially after a “second hit”. This Runx1R188Q/+ murine model will be the focus of in vivo therapeutic studies. RepSox and/or related compounds will be tested in this system to see if they can correct the two major phenotypes. We believe that at the end of our studies that we will have better defined how RepSox corrects Mk/Plt biology and reduce leukemic risk in patients with FPDMM and will have identified candidate drugs to move forward to clinical application utilizing a PARTNERSHIP PLAN STEERING COMMITTEE. We envision an initial clinical study of the drug in the setting of acute bleeds that will be then extended to intermediate-length studies to limit bleeding manifestations and finally to long-term studies to examine efficacy to prevent accelerated leukemic evolution in FPDMM.

NIH Research Projects · FY 2025 · 2025-06

Abstract: Metachromatic leukodystrophy (MLD) is a rare, fatal, progressive neurologic disease. Therapeutic and diagnostic innovations are changing the landscape of this rare disease. However, identifying which patients should receive preventative treatments is currently unknown and is a critical area of need. The foundation of understanding of the natural history of disease is derived from case series and single institutional reports. This siloed research approach in a rare disease and lack of data harmonization and sharing has slowed scientific progress. With MLD newborn screening pilot studies underway at multiple international sites, there is a clinical urgency for early disease stratification and characterization, when children are still minimally symptomatic and eligible for life- saving interventions. We hypothesize that risk prediction models and sensitive tools capable of measuring function across the lifespan can be used to accurately determine who is at high risk for early onset MLD and assist in determining appropriate interventions. To address the needs, in the R61 phase, we propose to harmonize and curate the MLD-CORE Project to create a rigorous clinical-trial ready natural history database using pooled data from 10 major leukodystrophy centers. This harmonization project represents the Rare Diseases Clinical Research Network (RDCRN) Global Leukodystrophy Initiative Clinical Trials Network (GLIA- CTN; U54TR002823), an NIH-funded research consortium for leukodystrophy network of 8 large US-based academic institutions, University of Minnesota, and University of Pittsburgh Medical Center Children’s Center for NeuroGenomics [formerly known as the Neurodevelopment in Rare Disorders (NDRD)]. From each subject, longitudinal medical records from birth, diagnostic information, and research clinical outcome assessments will be collected, and entered into a rigorous regulatory-ready database with source documentation. We anticipate that this rigorous Natural History platform (MLD-CORE) will inform future clinical (CCNG) trials and be capable for use as non-concurrent control arm. Next, we will work with stakeholders to create a publicly accessible web-based platform for research transparency (MLD-LINK). We anticipate that this aim will facilitate novel collaborations and grow the network of MLD researchers. In the R33 phase, we will use MLD- CORE to create and validate a risk stratification tools and develop a novel clinical outcome assessment (COA) fit for presymptomatic monitoring. In pursuit of these aims, we address the NINDS’ mission and respond to the need for a harmonized rare disease database. The MLD-CORE platform will support collaboration and will be used to validate clinical outcome assessments in novel populations and to develop data-driven statistical methodology for validated clinical outcome assessments and constructing prediction models. Furthermore, because the fundamental methodology is not disease-specific, our approach could be used to enable similar exploration for other rare diseases.

NIH Research Projects · FY 2025 · 2025-06

Next Generation T cell therapies for childhood cancers [NexTGen] Current treatments fail to cure many children with solid cancers. Recent advances in adult cancers such as checkpoint blockade and targeted small molecules have made little impact in childhood disease. Engineered T-cell therapies can achieve durable responses in refractory lymphoid cancers without long-term toxicity. These are precisely the characteristics required for new treatments for pediatric solid cancers. In contrast to hematologic malignancies, solid cancers are challenging due to a lack of targets, tumor heterogeneity, and hostile tumor microenvironment (TME). We posit that through advanced cellular engineering we can overcome these challenges. Our vision is that engineered T-cell therapy for childhood solid cancers will become routine within a decade. Our central hypothesis is that coupling of advanced cellular engineering along with progressive clinical development is the fastest route to developing effective T-cell therapies for pediatric solid tumors. In NexTGen, we combine detailed studies of primary tumors to discover new targets and understand how the TME subverts T- cell function. This, along with a closely coupled clinical development program will guide the progressive engineering of T-cells to result in transformative therapies. NexTGen is composed of 6 inter-connected work-packages (WPs) with work initially focused on pediatric sarcomas and brain tumors. AIMS: WP1: To identify suitable targets for engineered T-cells. WP2: To understand the TME in pediatric solid cancers. WP3: To develop receptors and other engineering components which target tumor cells and resist or modulate the TME. WP4: To evaluate the function of engineered T-cells developed in WP3. WP5: To translate approaches from WP4 and test them in clinical studies designed for maximal impact. Cancer Grand Challenges - Full Application - 2021 WP6: To promote data sharing across all WPs. METHODS: Target discovery (WP1) and TME studies (WP2) will utilize mass spectroscopy and chip cytometry respectively. Component engineering (WP3) will use protein engineering methods. To model engineered cell function, WP4 will mostly use intact tumor models such as immune PDXs. In WP5, clinical product generation will involve autologous closed system semi-automated manufacturing. WP6 uses standard and custom databases and data sharing platforms. USE OF RESULTS: Tumor target and TME data from WP1 and 2 will be uploaded to databases developed by WP6 for widespread distribution. Engineering components from WP3 and functional data from WP4 will be available for incorporation into therapeutic T-cell strategies by the entire community. Clinical study data from WP5 should lead to registration studies, improving cure rates and mitigation of long-term toxicity to realize our Vision.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY / ABSTRACT Pediatric sepsis is the leading cause of death of hospitalized children worldwide. While sepsis is defined as life-threatening organ dysfunction that develops in the setting of immune dysregulation, optimal management of immune dysregulation in pediatric sepsis is a fundamental knowledge gap. A successful precision medicine approach to pediatric sepsis requires that we understand the molecular events that cause organ failure, which of these events are potentially reversible, and how to identify this reversible patient pathobiology in real-time. We recently identified features of cellular immune dysregulation in children with sepsis and used proteomics to define three molecular subphenotypes which predict the severity of organ failure and mortality. Reproducible, validated molecular sepsis subphenotypes are viable candidates for translation to clinical trials, as models based on clinical data and plasma proteins could be used to identify “treatable traits” in real-time for both prognostic and predictive enrichment. Our preliminary data also identifies a pathologic and potentially reversible role for STAT3 hyperactivation in our most severe sepsis subphenotype. The IL-6/JAK/STAT3 signaling pathway is a canonical inflammatory pathway associated with capillary leak, endothelial dysfunction, emergency granulopoiesis, and lymphocyte dysregulation. Selective and non-selective JAK inhibitors have been used clinically to treat COVID-19 ARDS and pediatric hemophagocytic lymphohistiocytosis (HLH). It is not known if JAK inhibition can reverse immune dysregulation in sepsis, despite its related pathobiology. We propose to address these current knowledge gaps by defining and validating molecular subphenotype that predicts sepsis severity and testing the reversibility of T cell STAT3 hyperactivation. The overall objectives of this application are to define and prospectively evaluate a model to identify prognostic molecular sepsis subphenotypes (Aim 1) and to determine the impact of ex vivo JAK/STAT inhibition to reduce immune dysregulation in pediatric patients with sepsis (Aim 2). My central hypothesis is that immune dysregulation in pediatric sepsis can be identified in the plasma proteome and reversed with targeted immunomodulation. By validating a model to prospectively identify prognostic molecular sepsis subphenotypes and determining the impact of JAK/STAT inhibition on immune dysregulation, this study will support a precision approach to immunomodulation in pediatric patients with sepsis. My long-term goal is to design precision approaches to immunomodulation in critically ill children as a translational critical care physician-scientist trained in computational human immunology. I am optimally situated to perform these studies given my background as a clinician, my experience in prospective cohort development and proteomic subphenotyping, and the strong mentorship and supportive environment at CHOP/UPenn. The K23 award will provide new skills in translational research and computational analysis, facilitating my development as an independent investigator with expertise in translational human immunology.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY The central objective of this MIRA project is to harness the power of long-read transcriptomics to elucidate transcriptome complexity and advance genomic medicine. Mammalian cells generate remarkable regulatory diversity and complex phenotypes from a finite set of genes. Pre-mRNA alternative splicing (AS) is an essential mechanism for generating this regulatory diversity. Widespread changes in AS occur in both normal and pathological processes, resulting in transcript and protein isoforms that vary in their sequences and functions. However, determining the full-length transcripts that arise from AS on a transcriptome-wide scale has been a long-standing challenge in transcriptomics. Today, transcriptomics is on the cusp of a major technological transformation. While short-read RNA-seq has been the standard approach for transcriptome analysis over the last 15 years, the advent of long-read RNA- seq platforms holds the potential to revolutionize transcriptome research. Long-read RNA-seq enables end-to- end sequencing of full-length transcripts, offering unprecedented insights into transcriptome complexity and its impact on gene products. Furthermore, long-read RNA-seq greatly enhances our ability to identify and interpret splice-altering variants, by providing a complete view of splicing in full-length transcripts and linking mis-spliced transcripts to disease-associated alleles. Despite its potential, long-read RNA-seq remains substantially under- utilized compared to short-read RNA-seq, primarily due to its higher base error rate, lower throughput, and the associated experimental and computational challenges. My lab has a long-standing interest in developing and applying genomic technologies to study RNA processing and regulation. In recent years, we have spearheaded multiple projects to overcome the primary technological hurdles and demonstrate the innovative biomedical applications of long-read RNA-seq. Specifically, we developed experimental and computational approaches to enable robust transcript analysis using long-read RNA-seq. We also developed TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq, achieving ultra-high sequencing coverage for any gene panel of interest. Building on these technological advances, over the next 5 years we plan to: 1) develop technologies to delineate haplotype- resolved, full-length transcriptomes in both bulk tissues and single cells; 2) characterize transcript isoforms of haploinsufficient genes to discover novel therapeutic targets; and 3) establish a program for RNA-guided genetic diagnosis of rare diseases. Overall, my MIRA program combines technology development with innovative applications in genomic medicine. The novel technologies and resources developed from this project will empower researchers to study AS and transcript isoform variation across a wide range of biomedical contexts. The proposed research will also propel us towards our long-term goal of using RNA-based tools to improve the diagnosis and treatment of patients with rare and complex diseases.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Worldwide, more than 500,000 children become blind each year, and the majority of blind children live in low- and middle-income countries (LMICs), where rates of preventable vision loss and blindness are significantly higher. Amblyopia is the leading cause of vision loss in children worldwide. Vision loss from amblyopia can be prevented through early, effective amblyopia screening, though current screening devices have high sensitivity and low specificity which leads to a high number of false positive referrals. This ultimately limits the success of amblyopia screening programs due to increased cost and resource utilization, which is of particular concern in LMICs and low resource settings. This proposal will evaluate the efficacy and cost-effectiveness of two amblyopia screening devices and screening strategies: a current standard device, autorefractor (AR), which only detects amblyopia risk factors, and a novel device, retinal birefringence scanner (RBS), which detects amblyopia directly. Aim 1 of this proposal will compare the performance of the AR and RBS devices for detecting amblyopia in a clinic-based setting in Nepal. Aim 2 will compare the devices in a community-based setting in Nepal. Aim 3 will compare the cost-effectiveness of these two amblyopia screening strategies and devices. The feasibility of these Aims is anchored by collaboration with the Village Integrated Eye Worker II (VIEW II) trial, a cluster-randomized trial led by the candidate’s primary mentor, Dr. Jeremy Keenan. The VIEW II trial randomizes adults in peri-urban communities in Nepal to undergo a vision screening intervention by trained community outreach workers. This proposal incorporates several innovations. First, it uniquely leverages a well-established adult vision screening infrastructure to create and evaluate a screening program for children. Second, it will be the first study to directly compare the performance and cost of two amblyopia screening strategies. The candidate, Dr. Julius Oatts is a pediatric ophthalmologist at the University of California, San Francisco (UCSF), whose long-term goal is to become an independent investigator with expertise in diagnostic accuracy studies for novel technologies to diagnose and monitor preventable vision loss in children nationally and internationally. To successfully complete this research, Dr. Oatts will focus on four relevant domains of training: diagnostic accuracy studies, clinical trial design, cost-effectiveness analysis, and epidemiology and biostatistics. His exceptional mentorship team includes his primary mentor, Dr. Jeremy Keenan, Director of International Programs at the Proctor Foundation and PI of the VIEW II trial, and co- mentors Dr. David Hunter, Chief of Ophthalmology at Boston Children’s Hospital, and Dr. Ying Han, Director of the UCSF Glaucoma service. This team, combined with the environment of the Proctor Foundation and UCSF Department of Ophthalmology, will support his development into an NIH-funded independent investigator.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY/ABSTRACT Macroglossia (severe tongue overgrowth) leads to catastrophic morbidity and mortality in infants with Beckwith-Wiedemann Syndrome (BWS). Respiratory distress and airway obstruction are often present at birth, and the only current treatment is surgical resection of the tongue. Surgery can have significant side effects, including risk for lingual nerve injury and infection and is not always curative. Tongue muscle is considered to be similar to skeletal muscle, but in-depth analysis is currently limited in both human and mouse models. Several other skeletal muscle diseases have been linked to defective signaling in satellite cells and the interaction between specific muscle cell types. The specific signaling pathways causing macroglossia have not been defined. There are two critical genes altered in BWS that may play a role in tongue overgrowth (Cyclin dependent kinase 1C (CDKN1C) and/or Insulin-like growth factor 2 (IGF2)). Clinical observation of hundreds of patients with BWS suggests that the macroglossia phenotype can be divided based on the increased state of IGF2 versus decreased state of CDKN1C in each patient. This work will focus on how the genetic changes seen in BWS lead to distinct BWS tongue phenotypes based on the cell composition, expression, and function of these BWS genetic subtypes. We have the largest collection of BWS tongue samples in the United States, including tongue samples with alterations in each of these genes, which will allow us to study the contribution of each gene to macroglossia. Through our collaborative research team, we have assembled the expertise to define the cell types, cellular drivers and the pathways causing macroglossia. Our approach includes single-nuclei transcriptomic analysis of cell composition and interaction and in situ analysis of the interface between the key cell types through immunohistochemistry. We will separate these cell types in the tongue through sorting and characterize satellite cell proliferation and differentiation potentials based on their genetic alterations (IGF2HIGH, CDKN1CLOW, IGF2HIGH+CDKN1CLOW). Finally, we will define the in vivo role of the satellite cells from the different BWS subtypes (IGF2HIGH, CDKN1CLOW, IGF2HIGH+CDKN1CLOW) in a xenograft tongue model. This proposal will define the cell composition and cell-specific contributions to tongue overgrowth at the individual and the organ level. This work represents a critical step towards developing targeted non-surgical therapeutics for infants with BWS and macroglossia along with related muscle diseases.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT Neonatal and infant mortality remains a significant public health issue in the United States (US). One key driver of this disparity in infant mortality rates is the increased risk of very-low-birth weight (VLBW) in women of minority racial/ethnic status and racial/ethnic disparities in preterm-related infant mortality rates. The outcomes of VLBW infants are improved if they deliver at a hospital with a “high- level” neonatal intensive care unit (NICU), which is a unit with the clinical expertise to manage the sickest of infants. Unfortunately, there are studies that suggest that high-risk black infants are more likely to deliver at poor quality obstetric hospitals, especially in urban areas, and bypass hospitals with high-level NICUs for delivery because of the proliferation of delivery hospitals without such services who deliver high-risk infants. As a result, 60% of states have enacted legislation to encourage the transfer of mothers and babies to such centers, with wide variation in the specific types of policies. There is no research to identify the association between such policy elements and patient outcomes, nor how state, hospital, and patient characteristics modify these associations. The overall goal of this project is to determine how specific maternal and infant transfer policies affect racial/ethnic disparities in (1) where VLBW infants deliver, and (2) neonatal mortality and morbidity. We will apply a modification of the Anderson/Aday behavioral model of health care use that we have published to an innovative 17-year population-based cohort of VLBW infants whose data are prospectively collected as part of Vermont Oxford Network which is composed of 870 US NICUs that cover nearly 90% of VLBW births in the US. Using marginal structural models and formal mediation analyses, this study will characterize (1) how the presence of any perinatal policies surrounding neonatal and maternal transfer and specific policy elements differentially affect access to high-level neonatal care for VLBW infants of different racial/ethnic status; (2) determine how transfer policies and characteristics, through changes in access to high-level NICU care, differentially affect rates of neonatal mortality and morbidity in VLBW infants of different race/ethnicity; and (3) define how the combination of specific characteristics of maternal and infant policies and policy load moderate these associations. At the end of this study, we will provide information to stakeholders about how transfer policies related to a unique population, the mother-baby dyad, reduce disparities in infant mortality and improve infant health.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY/ABSTRACT Training: This K23 proposal will equip Dr. Elizabeth (Betsy) Salazar for a career as an independent physician-scientist. Her long-term goal is to inform policies and interventions to reduce drivers of maternal and infant outcome variation and associated disparities in NIH-prioritized subpopulations at the hospital level. She will perform rigorous retrospective studies with advanced econometric methods to elucidate how two policy-relevant hospital characteristics, hospital financial health and neonatal intensive care unit (NICU) strain, influence outcome variation and associated subpopulation disparities. Dr. Salazar and her mentorship team have created a career development plan incorporating: 1) comprehensive, collaborative mentorship from successful investigators with diverse experience in perinatal care, health economics and finances, biostatistics, and health disparities, in an exceptional research environment for this topic; 2) advanced training in hospital finances and operations, econometric analyses, and disparities analyses; and 3) innovative research examining the role of 2 hospital factors in neonatal outcome variation and subpopulation disparity. Research: One tenth of infants in the United States require care in the neonatal intensive care unit (NICU) with extensive associated costs. Hospitals demonstrated over 100-fold variation in neonatal morbidity and mortality, as well as significant outcome disparities in NIH-prioritized subpopulations of infants born extremely prematurely at high risk of disability, as well as infants born to birthing parents residing in rural environments, facing lower socioeconomic status as indicated by public insurance. Hospital factors contribute significantly to this observed variation and disparity. Two hospital factors, hospital financial health, and NICU strain, or the availability to meet the daily demand for high-quality care to optimize outcomes, are associated with adult outcomes and disparities, but have not been studied in neonates despite their relevance for hospital-staffing and disproportionate share payment legislation. Dr. Salazar’s mentored research will address this gap. By creating an enhanced administrative cohort, she will: 1) define the relationship between hospital financial health and neonatal morbidity and mortality considering hospital differences in case-mix, 2) examine the association of NICU strain with neonatal morbidity and mortality considering effect of the hospitalization and whether strain serves as mediator for the effects of hospital financial health, and 3) assess whether hospital financial health and NICU strain have differential effects in NIH-prioritized subpopulations. This study will directly address top NICHD research priorities “understanding of the contribution of social, economic, and regional factors” in neonatal health. Summary: Findings will inform future R01 proposals to examine the role of hospital financial health and NICU strain on neonatal quality of care and maternal outcomes. Through this award, Dr. Salazar will become a national expert on the role of hospital finances and operations in neonatal care and their policy implications.

NIH Research Projects · FY 2026 · 2025-03

ABSTRACT In response to inflammatory stimuli, neutrophils (PMN) release neutrophil extracellular traps (NETs), webs of negatively-charged cell-free (cf) DNA complexed with positively-charged histones, which capture pathogens. However, the release of NETs (NETosis) can inflict tissue damage on the host and has been implicated in the severity of conditions like sepsis, linked with acute NETosis, and sickle cell disease (SCD), associated with chronic NETosis. While many advocate for treatments degrading NETs or inhibiting NETosis, these approaches may inadvertently release endothelial-damaging NET-degradation products (NDPs) or increase susceptibility to infection. An alternative strategy is NET stabilization—wherein NETs are preserved but modified to bolster microbial capture and diminish NDP release. This approach may be therapeutic in both sepsis and SCD. Platelet factor 4 (PF4) is a positively-charged chemokine released by activated platelets that binds to and cross- aggregates polyanions like heparin and DNA. PF4 similarly binds to NETs, causing them to become physically compact and resistant to nuclease digestion. PF4 also binds to negatively-charged molecules on the bacterial surface and markedly enhances their capture by NETs. An Fc-deglycosylated murine monoclonal antibody (moAb) targeting human PF4 complexed to polyanions enhances PF4's protective effects, improving survival in murine sepsis models. This proposal is designed to fill knowledge gaps in our understanding of the role of NETs in host defense to define when NETs should be preserved, providing justification for further exploration of NET- stabilizing therapeutics, such as PF4 and Fc-modified anti-PF4:polyanion antibodies. Specific Aim (SA) #1: Characterize the role of NETs in immune homeostasis. This aim is structured to clarify the role of NETs in host defense, exploring how impaired NETosis alters inflammatory cytokine levels, PMN gene expression, and gut microbiome composition in murine models. The impact of these changes on enteric bacterial pathogenicity and murine survival will be assessed using a polymicrobial sepsis model to clarify the role of NETs in host defense. SA#2:Define the mechanism by which PF4 and Fc-modified KKO limit bacterial dissemination. Studies in this aim will define the mechanisms by which PF4 and anti-PF4:polyanion antibodies enhance NET- mediated bacterial capture and determine whether these interventions promote bacterial killing. Comparative studies of KKO variants will be conducted in vitro and in murine infection models utilizing bacterial strains with variation in surface electrostatic charge. Murine studies will examine how different interventions affect the release of NDPs, bacterial dissemination, and microvascular damage. SA#3:Investigate the potential of NET-directed therapeutics in SCD. The final aim will investigate the use of NET-directed therapeutics in SCD, examining their potential to prevent endothelial cell injury and reduce bacterial infection risk. Patient plasma samples will be analyzed for NET levels, NDP concentrations, and endothelial cell damage markers, and therapeutic efficacy will be evaluated in an endothelial-lined microfluidic system.

NIH Research Projects · FY 2026 · 2025-03

ABSTRACT Although over 90% of human genes undergo RNA alternative splicing, most studies fail to delineate the various isoforms that are functionally important in the biological or pathological process under study. Past efforts to identify isoforms were limited by incomplete sequencing read length, depth, or both. Meanwhile, alterations of exon usage are critical regulatory mechanisms in biology with dramatic clinical consequences in embryonic anomalies and cancer transformation. This failure to delineate the phenotype-causing RNA isoform derangement is a particular barrier when investigating epithelial-mesenchymal transition (EMT), as we and others have shown that Epithelial-splicing regulatory proteins ESRP1/2 are key regulators of RNA alternative splicing and EMT. Loss of Esrp1/2 function abrogates alternative splicing in the embryonic epithelium termed periderm, resulting in global alteration of mRNA isoforms in key EMT genes causing dramatic phenotype of orofacial cleft malformation in humans, mouse and zebrafish. Since orofacial cleft is one of the most common human birth structural anomalies, there is an urgent scientific and clinical unmet need to understand the role of RNA alternative splicing and EMT with isoform level resolution and specificity. This proposal leverages next generation long-read single-cell technologies and computation approaches based on rigorous prior research to comprehensively identify RNA isoforms transcriptome-wide. This proposal tests the central hypothesis that Esrp1/2 and Esrp-regulated isoforms modulate cell-cell interactions necessary for EMT, translating to tissue morphogenesis changes in craniofacial morphogenesis. To address this hypothesis, we applied long-read bulk RNAseq in zebrafish wildtype and esrp1/2 mutants and identified tp63, gsk3bb and other candidate genes important in EMT. We will functionally investigate these genes and their isoforms in complementary in vitro and in vivo models to mechanistically elucidate their function in EMT. We will further identify lineage specific isoforms at a whole-transcriptome scale using long-read single-cell RNAseq. We also developed a CleftTeq isoform panel using TEQUILA-seq, a rapid and cost-efficient isoform identification and quantification technology we pioneered, to analyze 103 isoforms of 464 cleft genes in a proof-of-concept translation for clinical diagnosis of orofacial cleft. The expected outcome of this project is to gain a mechanistic understanding of EMT with cell-type specific isoforms and their dynamic function during EMT. This work will have a broad impact by identifying key molecular drivers of EMT shared across biological contexts in development and disease. We will also translate isoform identification and quantification to clinical diagnosis. Moreover, we will contribute public and searchable single-cell RNA isoform database during EMT and inexpensive isoform gene panel technology, to broadly share data and tools to catalyze investigations into the fundamental biological underpinnings of EMT.

NIH Research Projects · FY 2025 · 2025-03

Workshop Title: “Body MRI: Unsolved Problems & Unmet Needs” Workshop Overview and Aims Functional body MRI biomarkers are underused in research and clinical practice due to the need for dedicated in-house expertise and development. Transferring solutions to other centers is therefore a challenge, leading to a significant duplication of efforts, a lack of standardization in the methods, and difficulties in comparing results between centers. This also limits commercial buy-in, hinders the set-up of multi-center trials, and overall slows down their translation into clinical practice. The proposed workshop aims to bridge this gap. The purpose of this workshop is to bring together internationally recognized scientists and clinicians along with new investigators who are currently developing and applying advanced renal MRI techniques to investigate the causes and consequences of body diseases. The program of this workshop integrates clinical presentations outlining the needs for non- invasive testing and biomarkers and covers advances in contrast-based MRI, new applications of relaxometry, diffusion, and elastography, and application of Artificial Intelligence techniques. We intend to include interactive sessions. With an important focus on the next generation, the workshop will include mentoring sessions for trainees and new investigators, called “Meet the Experts”. These mentoring sessions will provide specific information on clinical trials design, funding and regulations, as well as a hands-on “How-to” session on available imaging post-processing software. A previous NIDDK supported ISMRM workshop on Renal imaging held in September 2021 was attended in-person by 80+ attendees. Thus, we expect robust interest in the community for the proposed workshop.

NIH Research Projects · FY 2025 · 2025-02

PROJECT SUMMARY Irritable bowel syndrome (IBS) is a disorder of gut-brain interaction affecting about 11% of the global population and ~ 10-20% of adults in Western countries, and highly prevalent in children, impairing social development and quality of life (QoL). Abdominal pain, bloating, constipation and/or diarrhea are characteristic symptoms of IBS, often associated with food intake. Disease mechanisms include increased pain sensation from visceral nerves (hypersensitivity), altered bowel motility, and alterations in the gut microbiota. Treatments are limited and often do not completely relieve pain. Identifying factors involved in IBS pathophysiology as potential therapeutic targets is essential. Bowel motility may play a critical role in IBS pathophysiology, but its role relative to dietary triggers is unknown. Current diagnostic methods have limited ability to address spontaneous symptoms during daily life. Diagnoses solely based on symptoms are non-specific and may lead to suboptimal therapies. We use a new non-invasive diagnostic Wireless Patch System (WPS) to simultaneously evaluate real-time gastric, small intestine, and colonic myoelectric signals as a measure of organ motor activity over a 7-day period alongside symptoms and dietary intake. Up to 90% of patients with IBS report symptoms related to food intake. Specifically, food rich in fermentable, oligosaccharide, disaccharide, monosaccharide, and polyols (FODMAP) can exacerbate IBS symptoms. Low-FODMAP diets can improve symptoms and quality of life, however, poor adherence is common resulting in failure to fully resolve symptoms, and when imbalanced, can adversely impact overall nutrition. Identifying individual symptom triggering foods (FODMAPs and others) and their effect on bowel motility could improve adherence and care. This prospective, longitudinal, observational study of children with IBS and unaffected controls will simultaneously collect bowel activity, diet, GI symptoms, and psychosocial health data and evaluate contributions to IBS symptoms in real-time. In Aim 1 we simultaneously assess gastric, small bowel, and colonic motility in children with IBS and controls using the non-invasive WPS. We hypothesize bowel activity will differ between IBS and controls, and between IBS-C, IBS-D, and controls; and between symptomatic and asymptomatic periods. In Aim 2 we evaluate the effect of diet on bowel motility and symptoms in children with IBS and controls. We hypothesize that high intake of FODMAP foods and other known dietary triggers will associate with distinct patters of bowel activity, and symptom severity in children with IBS but not in controls. We further hypothesize that stress and anxiety exacerbate the association between diet, motility, and symptom severity. Our innovative approach employs a non-invasive measure of bowel activity, and deep phenotyping of dietary intake and symptoms under free-living conditions to elucidate the relationship between diet, bowel motility, and IBS symptoms, addressing several knowledge gaps in our understanding of mechanisms driving IBS symptoms. This study will provide the foundation for larger trials testing how motility and diet assessment are essential in developing personalized therapies for children with IBS.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY Addiction is a chronic, relapsing disorder, characterized by persistent drug-taking behavior despite negative consequences. New therapies are needed that prevent relapse.One trigger for relapse is the context where drug use previously occurred. When a rewarding drug is taken in a particular context, a “reward-context memory” can be formed. Reward-context memories are powerful; placing a human or rodent back into a reward-linked context can provoke drug craving. Disruption of reward-context memories is thought to prevent addiction. However, to disrupt reward-context memories, we have to first understand their neural basis.The neural basis of reward-context memories can be informed by literature on fear-context memories. Fear-context memories are reliant on the hippocampal dentate gyrus (DG) and a discrete population of DG context memory cells—termed an “engram”. Inhibition of DG fear-context memory engrams disrupts fear memory recall. It is unknown whether the DG is a site of reward-context engrams or if inhibition of the DG disrupts reward-context memory recall. Stimulation of the DG prior to context-fear memory encoding enhances fear-context memory. It is unknown if stimulation of the DG prior to reward-context memory enhances reward-context memory recall. My objective is to assess if DG reward-context memories can be manipulated, with the long-term goal of diminishing the salience of context cues and preventing relapse. The Aims of this proposal are to 1) test if DG neurons active during reward-context memory encoding can be inhibited during re-exposure to the reward-context, reducing or inhibiting drug-linked context memory recall and 2) determine if stimulation of DG neurons during reward-context memory encoding improves drug-linked context memory recall. When completed in two years, these experiments will determine whether or not the DG can serve a novel therapeutic target for diminishing the salience of drug-context memories. Further, regardless of experimental outcome, we will be able to elucidate novel properties of reward linked memories. Starting my postdoc during Covid-19 and moving into a new field (from context fear memory to addiction), it took time for me to establish a baseline knowledge and generate pilot data in order to develop these aims. Through this fellowship, I will accomplish defined Training Goals: learn hippocampal circuit manipulation, develop expertise in the field of substance abuse in the context of disordered learning/memory, use machine learning for the characterization for reward behaviors, and gain further professional development skills including project and personnel management. I have a supportive and ideal mentoring team (Drs. Amelia Eisch and Julie Blendy) to guide my training of both conceptual and technical skills. At the Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, I have exceptional resources and a stimulating environment in which to accomplish these goals.The completion of these Aims, and the training provided within, will provide the foundation for my path to becoming an independent investigator at an academic institution.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY/ABSTRACTS Severe subglottic stenosis, the narrowing of the airway just below the vocal folds, develops as a response to intubation in close to 10% of the > 20,000 premature births per year in the United States. Severe cases require laryngotracheal reconstruction (LTR), in which surgeons split the cricoid and add a piece of autologous patient- derived cartilage to expand the airway and restore proper airflow. However, in children, the success rate is as low as 50% with a high incidence of restenosis requiring revision surgery. Graft failure is tied directly to the lack of sufficiently sized autologous cartilage in the child, and tissue engineering has been proposed to develop alterative grafting options for pediatric LTR. Some approaches, including some of our previous work, have been effective in producing functional cartilage, but the overall timeframe required for the construct to match the mechanical properties of native cartilage (>24 weeks) is not compatible with clinical translation (<8 weeks). Furthermore, current cell sources such as expanded autologous chondrocytes and mesenchymal stem cells frequently result in hypertrophic and calcified tissue. Our objective is to engineer a new type of cartilage implant that is populated with patients’ cells, mechanically viable and suitable for LTR within a clinically relevant timeframe. Our approach is to exploit the blood vessels and elastin fibers that are uniquely present in the fibro-elastic cartilage of the meniscus to form microchannels for effective recellularization after enzymatic decellularization. Our patent-pending Meniscal Decellularized scaffold (MEND) technology can indeed be easily recellularized and has mechanical properties of the same order as native tracheal cartilage. Furthermore, cartilage progenitor cells have been proposed as a rapidly proliferating, highly chondrogenic cell source. To harness these cells, we have developed a minimally invasive biopsy procedure to harvest ear Cartilage Progenitor Cells (eCPCs). Our overarching hypothesis is that MEND and eCPCs can be combined to create cartilage implants with suitable mechanical strength, dimensions, and phenotypic stability for personalized, minimally invasive LTR. We propose to use MEND recellularized with eCPCs to engineer cartilage with tissue properties matching those of native cartilage. We will then validate the MEND-engineered cartilage in a miniature pig LTR model. We expect that our findings will provide strong pre-clinical evidence of functional laryngotracheal cartilage repair by our innovative eCPC-MEND technology and will thereby prompt follow up long term studies to eventually apply this technology to restore children’s airway.

NIH Research Projects · FY 2026 · 2025-02

PROJECT ABSTRACT Human B cells are exquisitely sensitive to changes in SPI1, a master transcriptional regulator. Loss- and gain-of-function SPI1 gene mutations were recently described as causes of agammaglobulinemia and Waldenström macroglobulinemia. Preliminary data indicates other changes in SPI1 activity/dose may cause additional clinical phenotypes including common variable immunodeficiency (CVID) and autoimmune diseases. The long-range goal of the proposed work is to determine SPI1’s full role in human health and disease(s). Our objective is to establish mechanisms of SPI1 gene regulation in human germinal center B cells and the consequences of dysregulation. The working hypothesis is that SPI1 has a critical role throughout B cell development, including germinal center (GC) B cell maturation, and that the culmination of rare SPI1 coding mutations and common non-coding variants determine disease state. To explore this hypothesis, we propose the following aims: 1) Identify non-coding elements regulating SPI1 gene expression in human GC B cells 2) Delineate effects of coding variants in SPI1 on autoregulation and expression 3) Determine maturational and transcriptional consequences SPI1 LOF and SPI1 GOF mutations to differentiated B cells. The contribution is significant because SPI1’s role in human GC responses is unexplored but is clearly affected in several patient groups carrying SPI1 gene mutations. The proposed work is innovative because we are using only human and human derived cells, including bona fide tonsillar GC B cells for proposed experiments. We are comprehensively investigating the role of coding and non-coding DNA on SPI1 regulation using crystallography, NMR, long-read sequencing, prime editors, base editors, ATAC-, RNA- and ChIP-seq. Finally we are utilizing tonsillar organoids to model the effects of altered SPI1 dose on GC B cell maturation in human lymphoid follicles.

NIH Research Projects · FY 2026 · 2025-01

Project Summary I am passionate about identifying novel approaches to treating antimicrobial resistant (AMR) infections in children and adults. During my Pediatric Infectious Diseases fellowship, I completed a Masters in Clinical Investigations that enabled me to lead pivotal studies to move the field of AMR treatment forward (e.g., piperacillin-tazobactam is associated with greater mortality than meropenem for ESBL-E infections). These studies have informed clinical guidelines and eventually led to me being the Chair of the Infectious Diseases Society of America AMR Treatment Guidance. It quickly became clear to me; however, that to effectively treat AMR infections, I needed to improve my knowledge of molecular mechanisms of resistance to develop translational treatment concepts that can be applied at the bedside. I was fortunate to receive a Career Development Award with a focus on rapid WGS to predict susceptibility testing results for clinical application. My research lab focuses on sequencing isolates and analyzing bacterial genomes to identify AMR genes and mutations in critical regions of the genome. I have been fortunate to be the PI on over $40 million in federal funds to conduct AMR research. These funds enabled me to develop a large AMR biorepository with >7,000 isolates that includes comprehensive WGS and clinical data that serves as secondary data sources for current and future mentees. Given my passion for mentoring, I have mentored dozens of trainees from graduate students to Assistant Professors in the aforementioned work. Four of my mentees have Career Development Awards and 2 are currently submitting K23/K08 awards. However, my administrative roles have grown significantly (i.e., Director of Antibiotic Stewardship and Fellowship Program Director). This makes it challenging for me to take on new mentees and is limiting my growth in research skills (particularly lab skills) which are needed to ensure I am training mentees in the most cutting edge and clinically relevant science. In my K24 proposal, I discuss plans to advance my knowledge of constructing site-directed mutants of key β- lactamase enzymes to identify amino acid residues responsible for resistance to specific β-lactams followed by molecular modelling to visualize critical amino acid substitution sites. Additionally, I would like to incorporate pharmacodynamic hollow-fiber models with drug exposures simulating humanized plasma concentrations for clinically prescribed antibiotic dosing regimens into my work to better understand the role of changes in antibiotic concentrations (e.g., dosages, infusion times, combinations) in the emergence of AMR. Furthermore, in my proposal, I outline plans to apply for additional federal funding to support mentees in two tracts of AMR research: clinical-based AMR research (e.g., K23 candidates) and laboratory-based AMR research (e.g., K08 candidates). I also discuss my specific plans to enhance my mentoring skills and my mentoring plans for trainees with support from a team of mentors, collaborators, and an external advisory committee - all of whom are internationally recognized leaders in their respective fields.

NIH Research Projects · FY 2026 · 2025-01

Project Abstract Pediatric cardiac arrest affects >15,000 children each year. Less than half of these children survive. High- quality cardiopulmonary resuscitation (CPR) can save lives, but to date, there is an imbalance in this research area with much more known about optimal chest compression techniques as compared to ventilation strategies. Given that the majority of pediatric patients have respiratory disease at the onset of cardiac arrest, the lack of rigorous investigation into optimal CPR ventilation represents a significant roadblock to progress. In 2020, the American Heart Association (AHA) increased the recommended CPR ventilation rate from 10 breaths per minute (bpm) to a range of 20 – 30 bpm, a target associated with better outcomes. Unfortunately, providers do not achieve this recommended target in actual practice. As such, innovative training strategies to this CPR parameter are potential therapeutic interventions to rescue more children from cardiac arrest. To that end, we developed an OPTImizing VENTilation (OPTI-VENT) bundle consisting of provider education and point-of-care CPR ventilation rate guidance (CPR cue cards and a ventilation rate metronome). Our bundle demonstrated efficacy to train providers to a specific ventilation rate with high compliance. In this application, we propose an adaptive, parallel-stepped-wedge hybrid cluster-randomized trial to determine if the OPTI- VENT bundle can improve cardiac arrest outcomes by training providers to the 2020 CPR ventilation rate. To achieve our objectives, we will leverage the existing infrastructure of the Pediatric Resuscitation Quality Collaborative (pediRES-Q), a network specifically designed to study in-hospital pediatric CPR. The following aims are proposed: 1) Evaluate the effectiveness of the OPTI-VENT bundle to improve survival to discharge with favorable neurological outcome among children receiving at least 1 minute of CPR; 2) Determine the association between ventilation rate and a) survival outcomes and b) intra-arrest physiologic surrogates of vital organ perfusion among children receiving at least 1 minute of CPR; and 3) Determine the association between Airway Opening Index (AOI) – a newly discovered ventilation metric – and positive end-expiratory pressure, effectiveness of ventilation during CPR, intra-arrest hemodynamics, and patient outcomes using novel multidimensional machine learning algorithms. In Aim 1, we will execute a randomized trial to determine if training to the 2020 CPR ventilation rates with the OPTI-VENT bundle can improve patient outcomes. In Aim 2, an embedded secondary observational study will determine if an alternative rate target exists. In Aim 3, we will continue our investigation into AOI – a transformative line of work that may lead to a paradigm shift in cardiac arrest algorithms. In short, even if the OPTI-VENT rate target tested in this trial is not ideal, we will have conducted the multicenter observational study to substantially advance our evidentiary support for pediatric CPR guidelines, and potentially, discover a new target that will lead to fundamental changes in pediatric CPR ventilation recommendations.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY This proposal addresses mechanisms used by adenovirus (AdV) to manipulate host RNA binding proteins (RBPs) in order to exploit cellular RNA processing pathways while overcoming host defenses. DNA viruses are reliant on cellular RNA processing machinery to generate viral transcripts and synthesize viral proteins. Productive viral infection is achieved by redirecting cellular RBPs through changes in post-translational modifications and subcellular localization. Although RNA viruses are known to co-opt or counteract RNA-protein complexes, there is much less knowledge about the fundamental mechanisms employed by DNA viruses to harness cellular pathways for efficient mRNA transcription, transport, and translation. Adenovirus presents a highly tractable model DNA virus which replicates in the cell nucleus and harnesses host RNA processing machinery. The transcriptionally complex pattern of gene expression from the AdV DNA viral genome provides a powerful system to study temporal regulation of RNA splicing and RNA fate. Our preliminary studies employed mass spectrometry proteomics and RNA Binding Region Identification (RBR-ID) to monitor post-translational modifications and RNA-protein interactions during infection. Integrating these unbiased proteome-wide approaches revealed a unique set of nuclear RBPs which are decreased for both arginine methylation and RNA- binding during infection with AdV serotype 5 (Ad5). We discovered that the multifunctional Ad5 late protein L4- 100K is methylated, binds cellular methylase Protein Arginine Methyltransferase 1 (PRMT1), and causes PRMT1 cytoplasmic relocalization. We propose that L4-100K redirects arginine methylation to alter RNA-binding capacity and function of diverse cellular RBPs. In Aim 1 we will define mechanisms by which modulation of RBP methylation promotes infection through changes to splicing and RNA processing. Insights into mechanisms for redirecting RBPs during AdV infection have also come from preliminary data using viral mutants that are defective for splicing (DE4 lacking early region E4) and protein translation (DVA lacking the viral associated RNA). We found that infection with these mutants induces signaling pathways that lead to assembly of two distinct ribonucleoprotein (RNP) complexes in cytoplasmic granules known to have antiviral activity against other viruses. The field has lacked a suitable viral system for selectively inducing granules, and here we demonstrate that AdV can be used to identify the RBP composition of distinct cytoplasmic granules and define their antiviral properties. In Aim 2 we will examine viral evasion of cytoplasmic granules and the impact of manipulating host responses. Together these studies will employ innovative technologies to define RNA binding and localization of host RBPs during infection, with systematic approaches of global proteomics and high-throughput imaging. Understanding changes in RNA-protein complexes mediated by RBP modification during infection will provide fundamental knowledge of virus-host interactions and will point to new antiviral therapeutic targets.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract This proposal presents a five-year research and career development training program focused on the potential of mRNA-lipid nanoparticles (mRNA-LNP) and lipid nanoparticle (LNP) adjuvants to overcome maternal antibody interference. The candidate is currently an Attending Physician and Postdoctoral Research Fellow in Adult and Pediatric Infectious Diseases at the University of Pennsylvania (Penn) and Children’s Hospital of Philadelphia (CHOP). The current proposal builds on the candidate’s previous research and clinical experience in innate and humoral immunology and adjuvant research with aims and training activities that emphasize the analysis of humoral and cellular immune responses to mRNA-LNP platforms and LNP adjuvants in the setting of maternal antibodies. The proposed aims and training plan will prepare the candidate with an array of skills that will accelerate and strengthen his transition to independence as a physician-scientist studying maternal-fetal factors impacting infant vaccine responses. Despite over a century of advances in vaccine technology, we still lack vaccines that effectively induce protective antibody (Ab) responses in young infants. This leaves a vulnerable group reliant on maternal antibodies (matAbs). MatAbs provide infants with early-life protection against pathogens. However, matAbs also interfere with Ab responses to vaccines. MatAb interference (MAI) has been a major barrier to early immunization against influenza (flu), measles, and others. The foundation of this proposal is our preliminary data showing that mRNA- lipid nanoparticle (mRNA-LNP) vaccines and protein-subunit vaccines administered with empty lipid nanoparticles (eLNP) overcome MAI in mice. However, the mechanism(s) that allow mRNA-LNP and eLNP- containing vaccines to overcome MAI, and the degree to which they could do so for other vaccine types, are not understood. The specific objective of this proposal is to use mouse models of MAI to determine the mechanism by which mRNA-LNP and eLNP-containing vaccines overcome MAI and to determine whether those properties could be more broadly applied to other vaccine types. My central hypothesis is that LNPs overcome MAI through interferon-alpha-dependent adjuvant activity. This hypothesis will be investigated in two aims. 1: Determine if mRNA-LNP and eLNP-adjuvanted flu vaccines alter intracellular B cell signaling pathways associated with MAI, 2: Determine if mRNA-LNP and eLNP-adjuvanted live attenuated vaccines can overcome MAI in a mouse model of measles vaccination. Successful completion of these aims will determine if adjuvant properties of LNP are important for overcoming MAI with the vision that the knowledge gained can inform new approaches to vaccine and adjuvant development that may impact rational vaccine design for newborns and young infants.

NIH Research Projects · FY 2026 · 2025-01

Growth, structure and regeneration of the skeleton remain areas of intense research activity, due to their importance and relevance to body function and quality of life. The major engines of skeletal growth are the growth plates that are composed of a top reserve zone rich in stem cells followed by proliferating, pre- hypertrophic and hypertrophic zones of chondrocyte maturation and ossification, with cells organized in columns. Growth plate-like structures also form during skeletal repair and regeneration as observed in bone fracture healing. Growth plate activity is regulated by multiple mechanisms including signaling proteins such as hedgehog and fibroblast growth factor family members; transcription factors including Runx2 and Mef2c; and local and systemic hormones. The growth plates normally remain active until the end of puberty and then undergo involution and close, establishing a fully grown skeleton. However, trauma, genetic disorders or drug treatment of certain pediatric pathologies can have side effects on growth plates, derange proliferation and maturation rates and cause slowdown and involution, often with severe skeletal consequences. For example, about 10% of adolescents with a growth plate injury in long bones develop asymmetric bone growth and require challenging surgical intervention. An example of genetic conditions is children affected by Crouzon in which the cranial base growth plates malfunction and cause skull defects. Drug-induced growth plate malfunction is observed in pediatric patients treated with pharmaceuticals that can have unwanted side effects on their growth plates, causing growth retardation. In sum, the growth plates are vulnerable to malfunction in several acquired and congenital pathologies, representing an unmet clinical need in search of solutions. In this new project, we have used a standard model of growth plate defect in juvenile mice that mimics a pathology seen in pediatric patients. Preliminary data suggest that growth plate dysfunction was caused by concurrent changes in mechanisms normally fine-tuning growth plate activity and functioning. Additional data suggest that systemic treatment with a drug targeting one such mechanism was able to maintain growth plate function and restore skeletal lengthening. Our hypothesis is that growth plate dysfunction is amenable to drug-based rescue. Aim 1 will assess growth plate dysfunction and genes involved in it. Aim 2 will delineate the cellular mechanisms underlying dysfunction and rescue. Aim 3 will test the overall effectiveness and safety of the pharmacological treatment and its range of action. We will use transgenic mouse lines, in situ hybridization, protein analysis and treatment, biochemical approaches, and cultures of primary cells to delineate the mechanisms of growth plate defects and means to counter them. We will utilize strict and unequivocal rules to ensure scientific rigor and robust and unbiased design, methodology, analysis, interpretation, and reporting of data. The project is geared to further test mechanisms of growth plate activity and will exploit the resulting information to test new tools of therapeutic intervention on growth plate defects.

NIH Research Projects · FY 2026 · 2025-01

Project Summary: Our goal is to find new ways to prevent and treat Hirschsprung disease-associated enterocolitis (HAEC), a form of bowel inflammation. HAEC is a life-threatening manifestation of Hirschsprung disease (HSCR) characterized by explosive (often bloody) diarrhea, abdominal distension, fever, and risk of bacterial translocation from the gut lumen to the blood, causing sepsis. Sepsis is the most common cause of death in children with HSCR. HSCR is a first trimester developmental anomaly defined by absence of enteric nervous system (ENS) at the end of the bowel. Bowel lacking ENS is called “aganglionic”. Aganglionic bowel tonically contracts and lacks propagating contractions causing functional obstruction. Because ENS also controls blood flow, epithelial, and immune cell activity in the bowel, HAEC mechanisms are complicated, and incompletely understood. HSCR treatment (devised 75 years ago) is surgical removal of aganglionic bowel and re-attachment of “good” ENS-containing bowel near the anal verge (called “pull-through surgery”). In theory, pull-through surgery should result in a perfect cure, but ~35% of children have HAEC after surgery and ~10% undergo “re-do” surgery because of persistent problems. If we understood why some children with HSCR appear healthy after pull-through surgery and others have recurrent HAEC, we might then be able to devise new ways to treat or prevent HAEC. Interestingly, HSCR symptoms vary dramatically before any treatment. Some children are critically ill within days of birth. Others appear well for months or years before HSCR symptoms become clear. For example, recently a 53-year-old man was newly diagnosed with HSCR in Japan. Because HSCR is always present at birth, variable symptom onset and severity suggests HSCR symptoms (including HAEC) are influenced by factors beyond ENS biology. Serendipitously, we discovered in an inbred HSCR mouse model (Piebald lethal, sl/sl) that diet alters survival 3.4-fold within a single mouse colony. Exploiting this observation, we discovered the “Protective” diet reduces Enterobacteriaceae (bowel inflammation-associated bacteria), increases stool butyrate (energy source for colonocytes and a histone deacetylate inhibitor), increases epithelial production of anti-microbial peptides and fucosyltransferases (which protect from inflammation), and reduces gut epithelial oxygen levels (facilitating growth of beneficial obligate anaerobes near colon epithelium). These mechanistic observations suggest new therapies (e.g., butyrate supplementation or elimination of Enterobacteriaceae), but we need to know which mechanisms in mice are relevant for humans with HSCR. To do this we will analyze stool metabolites, stool microbes, and gut epithelial biology in bowel removed from children during pull-through surgery (Aims 1 and 2). We will also examine ENS anatomy near the proximal resection margin using a new tissue clearing and 3D imaging method we invented that provides exceptional data about ENS anatomy (Aim 3). Our studies test specific mechanistic hypotheses and may suggest potentially effective HAEC prevention or treatment strategies.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY The etiology of bronchiolitis obliterans syndrome (BOS), a serious complication that affects the terminal airways of the transplanted lung and is the main cause of long-term graft dysfunction and failure after lung Tx, is unknown. We hypothesize that loss of lymphatic function after lung Tx is critical to the development and progression of BOS and, post-reconstitution is affected by disruption of interactions between lymphatic endothelial cells (LECs) and Foxp3+ T-regulatory (Treg) cells. This hypothesis is based on our preliminary data. First, using orthotopic murine models of lung Tx, we have shown that deletion of lymphatics (loss of function, LOF) within the transplanted lung leads to acute inflammation and promotes BOS, as does conditional deletion of lymphotoxin-beta receptor (LTbR) within LECs. Second, we found stimulation of lymphangiogenesis in a gain of function (GOF) model involving conditional deletion of the lipid phosphatase, Pten, can protect against BOS. Third, adoptive transfer of LTb+ but not LTb- Treg cells can protect against lung injury and the development of BOS post-Tx. Specific Aim 1. Determine the mechanisms of lymphatic vascular function after lung Tx. Hypothesis: Lymphatic function post-Tx is mediated by immunomodulatory adaption of donor LECs and signaling via the LTbR/LTa1b2 axis. Using lymphatic reporter mice, i) we will determine the contribution of donor versus host-derived LECs to the lymphatic vasculature post-Tx. ii) We will also map LEC heterogeneity and subtypes that define the lymphatic vascular adaptation in lung Tx. Finally, iii) we will use mice deficient in LTbR, LTa or LTb to determine the mechanistic role of these pathways in lymphatic function post-lung Tx. Specific Aim 2. Strategies to promote lymphangiogenesis in the lung Tx. Hypothesis: GOF strategies to promote lymphangiogenesis within the transplanted lung will protect against the development of BOS. This will involve 3 independent lines of investigation, i) administration of an agonistic mAb directed against LTbR expressed by LEC; ii) delivery of recombinant VEGF-C by AAV; and iii) blockade of epsins to reduce VEGFR3 endocytosis and degradation and thereby increase VEGFR3 protein expression and promote lymphangiogenesis. Specific Aim 3. Strategies to promote CLAD-free Treg-dependent lung allograft survival. Hypothesis: Therapeutic strategies that enhance endogenous Treg numbers and/or function will promote CLAD-free Tx survival. Accordingly, we will test the ability of therapy with i) IL-2 muteins alone or ii) in conjunction with Cdk8/19 inhibitors (Cdk8/19i) to expand the recipient Foxp3+ Treg cell population and promote beneficial outcomes in lung Tx models; and iii) assess whether Foxp3 mRNA delivery can likewise expand the host Treg population and promote lung allograft survival. Our proposed research will unravel the complex interactions between lymphatics, host immune responses, and the development of key complications of lung Tx, with the ultimate goal of improving patient outcomes and long-term lung Tx survival.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY/ABSTRACT Most patients with high-risk neuroblastoma (NB), the most common extracranial solid tumor of childhood, die despite intensive cytotoxic therapies and immunotherapy with dinutuximab, a chimeric monoclonal antibody targeting the disialoganglioside GD2. This underscores the need for new immunotherapeutics, with GPC2 emerging as one promising target for chimeric antigen receptor (CAR) T cells and antibody-drug conjugates (ADC). Nevertheless, alternative targets will undoubtedly be needed to safeguard against intra- and intertumoral heterogeneity and epitope loss. Given the limitations of proteomic approaches, such targets are likely to emerge from sophisticated transcriptomic analyses. The Gabriella Miller Kids First Pediatric Research Program (GMKF) has already generated high-quality bulk, short-read RNA-seq datasets corresponding to high-risk NB. The value of this unique resource could be further increased by accounting for widespread variations at the level of mRNA processing, including alternative splicing. Focusing on surface proteins as potential CAR/ADC targets, we compared and contrasted splicing patterns in 150+ NB samples vs adrenal gland (where NB often originates) and filtered for highly expressed genes and in-frame events affecting extracellular (or ecto-) domains. We identified dozens of local splicing variations (ectoLSVs) giving rise to potentially targetable neo-epitopes. Notably, many of these ectoLSVs mapped to microexons, very short (<51 nt), conserved, dynamically regulated cassettes, which account for much of the surfaceome diversity in the nervous system. As rigorous and reproducible as they are, our preliminary RNA-seq have two major limitations. The first limitation reflects the inherent shortcomings of short-read technologies, which are not very good at resolving complex mRNA splicing events. This limitation can be overcome by long-read RNA-seq, as implemented in the PacBio and Oxford Nanopore Technologies platforms. However, as commonly utilized, these platforms do not offer single-cell resolution capabilities. The second major limitation of bulk RNA-seq is the paucity of insights into tumor heterogeneity and evolutionary trajectories of individual clones. The goal of this proposal is to overcome these limitations by first performing bulk long-read RNA-seq on 20 high-risk NB patient samples and patient-derived xenografts (PDXs). Then single-nuclei isoform RNA sequencing will be performed on a subset of these specimens. Prioritizing microexon-related events will help filter out aberrantly spliced transcripts unlikely to yield functional proteins and elucidate alternative splicing events with possible roles in NB pathogenesis. Therefore, upon completion of this 2-year project we will be in the position to a) integrate all the long-read sequencing data (bulk and single-cell) with the GMKF ecosystem; and b) nominate additional non-canonical proteoforms, for immunotherapy with CAR T cells and ADCs.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Characterizing neurocognitive risk parameters in children is critical to the study of developmental psychopathology and paves the way for novel treatment and interventions. Heightened threat sensitivity – the recognition, interpretation, and response to real or potential threat cues in the environment – is a strong neurocognitive predictor of anxiety risk. However, little is known about how heightened threat sensitivity develops and is maintained across time. The limited extant work suggests parental characteristics and behaviors influence children's anxiety risk and may shape the development of children's threat sensitivity. However, we lack knowledge about intergenerational influences on the transmission of heightened threat sensitivity. The current study uses a longitudinal intergenerational framework to identify factors that influence the neural, biological, and behavioral correlates of threat sensitivity across time and generations. The current project will be the first intergenerational study of the Philadelphia Neurodevelopmental Cohort (PNC), a racially-diverse cohort of ~10,000 participants assessed at age 8–21 between 2009-2012, with many participants becoming parents in the last 5 years. In 300 mother-child dyads (children aged 4-8 years), we use a multimethod assessment framework with psychiatric (clinical interview, self-report), behavioral (computer tasks), observational (parent-child interactions), neural (electroencephalogram: EEG), and physiological (electrocardiogram: respiratory sinus arrhythmia) measures to examine the pathways underlying longitudinal and intergenerational influences on threat sensitivity and anxiety. First, we leverage data from the original PNC to examine factors that influence the presence of a multimethod threat sensitivity construct in mothers, 10- years later, and examine longitudinal links between threat sensitivity and anxiety symptomatology. Second, we focus on the child's threat sensitivity and anxiety, investigating the intergenerational influence of maternal threat sensitivity and, in an available subsample, the influence of paternal/secondary caregiver (n=200) threat sensitivity, on the child's threat sensitivity and anxiety. The study also examines the role of environmental threat exposures (e.g., trauma, discrimination, and poverty) on individual differences in threat sensitivity, with the hypothesis that increased threat exposures will increase susceptibility to abnormal threat processes. Lastly, we investigate a possible mechanism underlying the intergenerational transmission of threat sensitivity: negative parental communication. We will examine real-time transmission of threat through parental communication (e.g., verbal threat promotion, expressed anxiety) across a series of parent-discussion tasks. This project aligns with NIMH programmatic goals by charting mental health symptoms and mechanisms of risk across the lifespan. Results from the multimethod approach will inform multisystem mechanisms of intervention and prevention at the level of the parent and child. Our proposed expansion of the PNC to a longitudinal intergenerational cohort is an unprecedented opportunity to transform knowledge on anxiety risk.