Research Inst Nationwide Children'S Hosp

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Left ventricular noncompaction cardiomyopathy (LVNC) is a rare morphological abnormality of the myocardium occurred during early heart development characterized by excessive trabeculae with deep inter- trabecular recesses, but thin free wall. LVNC is classified as the third most common cardiomyopathy, with a prognosis ranging from asymptotic to preterm death, cardiac arrythmia, and heart failure. Recent advances in imaging technology have led to increased detection of LVNC, although the underlying cellular and molecular causes remain elusive. Previous studies have mainly focused on intrinsic defects in myocardium, yet mounting evidence also suggests that LVNC is associated with impaired function of non-muscle cell types in the developing heart including cardiac endothelial cells (CECs), which are highly heterogenous, composed of both endocardial and coronary endothelial cells. Previous studies have shown that both endocardial endothelial cells (EECs) and coronary endothelial cells (CoECs) are required for proper trabecular and compact myocardial morphogenesis, although the underlying cellular and molecular mechanisms remain elusive. We discovered that global knockout (KO) of Zip8, a zinc ion importer, results in LVNC and preterm lethality in mice, suggesting that Zip8 plays indispensable roles during myocardial trabeculation and compaction. However, it is unclear through what cellular and molecular mechanisms Zip8 impacts myocardial formation. Using various Cre driver mouse lines, our preliminary data demonstrated that only endothelial deletion of Zip8 recapitulated the LNVC phenotype seen in global Zip8 KO hearts. Surprisingly, the proportions of EECs and CoECs in Zip8 endothelial KO (Zip8eko) hearts were skewed; compared to control littermates, EECs were significantly more abundant, whereas CoECs were less abundant in Zip8eko hearts. We also found that certain paracrine growth factors such as Insulin-like growth factor 2 (IGF2) were significantly upregulated in Zip8eko hearts, which is consistent with an increased EEC population, and may contribute to the hypertrabecuation phenotype. Conversely, we also found that coronary vessels were significant fewer in Zip8eko hearts, accompanied by decreased VEGF signaling, a major angiogenic pathway during early heart formation. These results are consistent with a reduced CoEC population, which together account for the noncompaction phenotype. Accordingly, we hypothesize that Zip8/Zn in CECs orchestrates myocardial morphogenesis by determining endothelial cell identity and modulating trabecular growth and coronary angiogenic signaling. We will test this hypothesis using multiple genetic and biochemical approaches. Upon completion of this study, we will gain novel insights on the pathogenesis of LVNC.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Adolescent onset of major depressive disorder (MDD) is a major public health concern causing greater risk for a chronic and recurrent depression course, and an increased risk for suicide. Offspring of parents with a history of MDD are at particularly high risk for developing depression. As depression rates among adolescents have been steadily rising in the past decade, research on the discovery of biological markers of youth depression onset, particularly among highly vulnerable populations, is crucially needed to develop early interventions targeting malleable factors. Sleep changes are a hallmark of depression emergence, with sleep disturbances and changes in sleep architecture serving as a precursor to depression onset in youth. Adolescents show high rates of poor sleep and strong vulnerability to emotional dysregulation driven by poor sleep. This is paired to youth cortical neurodevelopment being crucial to emotional regulation, and signatures of sleep brain dynamics changing in parallel with neurodevelopment. While current research shows associations between depression and subjective sleep complaints, these studies rely on subjective measures and are mostly cross-sectional investigations, which limits information on causal mechanisms of MDD emergence in youth. One promising objective marker of sleep disturbances linked to adolescent depression is altered sleep brain oscillations. Specifically, youth with MDD show increased frontal slow wave activity (SWA) and reduced frontal sleep spindles. Furthermore, high risk (HR) youth of depressed mothers show changes similar to those found in MDD, albeit less pronounced, suggesting that these sleep profiles may represent the substrate of a biological vulnerability that precedes the development of depression. This imbalance in sleep oscillations is functionally relevant, as cortical Slow Oscillations (SOs, dynamic contributors to SWA), spindles, and their coordination are associated with health, restorative properties of sleep, cognitive processing, average intelligence, and brain homeostasis. However, there is currently no data on the predictive potential of quantifiers of sleep oscillations in depression emergence in youth. Furthermore, recent work from PI Malerba introduced novel space-time analyses of SOs and spindles enabling enhanced understanding of their biophysics and coordination. Since these properties are crucial to SO and spindle functional roles, structural changes in SOs and spindles related to MDD risk in youth could inform on mechanisms of depression emergence and novel targets for intervention. The proposed study will: (Aim 1) identify the space-time profiles of SO and spindles, and contrast-compare the resulting properties across 92 youth at high and low risk for depression; then (Aim 2) establish the predictive role of these space-time profiles in relation to increases in depression symptoms at 3 and 6 months, and finally (Aim 3) explore the potential mediating role of emotional regulation in the relation between sleep profiles and depression trajectories in youth. These findings will inform future research on early detection of MDD in youth and the development of interventions that can manipulate these oscillations in youth with MDD.

NIH Research Projects · FY 2026 · 2025-03

Approximately 180 million people globally, including 2-3 million in the United States, live with chronic hepatitis C virus infection. Persistent HCV replication causes progressive liver injury including cirrhosis and cancer. Direct acting antivirals (DAA) that cure chronic HCV infection have been available for a decade but new infections outpace treatment. New cases of HCV infection doubled between 2013 and 2022 and it is unlikely that treatment alone will interrupt virus transmission. A vaccine to prevent chronic HCV infection could provide a cost-effective solution to this problem and is supported by substantial evidence. Spontaneous resolution of infection in 25-30% of humans and chimpanzees provides life-long immunity that sharply reduces the risk of persistent infection with diverse HCV genotypes. Vaccine-mediated protection will likely require broadly neutralizing antibodies and a T cell response that is sustained until after apparent clearance of the virus. The objective of this proposal is to define the protective CD4+ T cell signature that supports effective CD8+ T cell immunity. Human CD4+ T cells are challenging to study. CD4+ T cells target multiple class II epitopes and circulate at low frequency. They are therefore difficult to visualize and enrich with class II tetramers. Blood sampling is typically infrequent in prospective studies of those at risk of infection and liver is rarely sampled during acute hepatitis C. Here, protective T cell immunity will be defined with historical blood and liver samples collected from chimpanzees between 2005 and 2013 with NIH approval and funding. Preliminary studies support this approach. Co-expression of the PD-1 and ICOS activation antigens was established as surrogate marker for direct ex vivo visualization and enrichment of HCV-specific CD4+ T cells, a significant advance for feasibility of the proposed studies. The blood response coincided with peak ALT and infiltration of multifunctional HCV-specific CD4+ T cells into liver. HCV-specific CD4+ T cell responses will be defined by single cell RNA sequencing (scRNAseq) and spatial genomic analysis of liver, technologies that did not exist when the samples were collected. An innovative Central Hypothesis was developed from preliminary scRNAseq studies: CD4+ T cells infiltrate liver to form lymphoid aggregates, where they attract and regulate intrahepatic CD8+ T cell responses to HCV. Lymphoid aggregates were first defined in liver during the non-A,non-B hepatitis era. Their function and significance in acute hepatitis C remains unexplained. Here, we propose that they are a site for close range CD4+:CD8+ T cell communication that determines infection outcome. This hypothesis is supported by identification of a unique HCV-specific CD4+ T cell population that differentially expressed CXCL13 and LGALS9, which encodes the galectin-9 inhibitory ligand implicated in CD8+ T cell exhaustion. These CD4+ T cells had a transcriptional profile consistent with formation of tertiary lymphoid structures. Transcriptional and spatial characterization of HCV- specific CD4+ T cells in blood and liver, facilitated by identification of a key surrogate marker of the response, is expected to fill a substantial gap in knowledge critical for understanding HCV control and vaccination of humans.

NIH Research Projects · FY 2026 · 2025-03

Project Summary Human early-onset dilated cardiomyopathy (DCM) is associated with significant morbidity and mortality in utero and after birth. The majority of DCM in fetus is considered to be idiopathic, with a strong association with genetic mutations in sarcomeric genes. Pathogenic sarcomere protein variants often arise from post-transcriptional regulation. However, whether post-transcriptional dysregulation contributes to the pathogenesis of DCM in fetus has not been determined, presenting a significant knowledge gap to understand the etiology of early-onset DCM. In eukaryotic mRNA, N6-methyl-adenosine (m6A) is the most abundant post-transcriptional modification and affects mRNA processing (i.e., stability, splicing) and translation. m6A is deposited by the m6A methyltransferase complex composed of several catalytic subunits including methyltransferase 3 (METTL3) and methyltransferase 14 (METTL14). METTL3 and METTL14 play important roles in adult heart injury and repair, although their potential function during early myocardial development and whether their dysregulation contributes to the pathogenesis of fetal DCM is undetermined. In this proposal, we will address this by leveraging mouse genetics, biochemistry, molecular biology, RNA biology, and histology. First, we will determine the functions of METTL3 and METTL14 in embryonic mouse myocardium, and whether their loss in cardiomyocytes induces fetal DCM in mice. Secondly, we will identify the underlying molecular mechanisms by which METTL3 and METTL14 regulate mRNA fates and early sarcomerogenesis and myofibrillogenesis. Successful completion of the proposed study will reveal key roles for the methyltransferases and post-transcriptomic profile of cardiac mRNAs during early cardiac myogenesis, and uncover the novel pathogenesis of fetal DCM, and thus shed new lights on therapeutic strategies.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY: There are almost half a million childhood cancer survivors in the United States. Over two-thirds of survivors will develop at least one late effect. Young childhood cancer survivors (YCCS; age at diagnosis <7 years) are at the highest risk for developing neurocognitive late effects, given their developmental vulnerability and absence from early education. YCCS miss critical opportunities to develop foundational school readiness skills, including preacademic, social-emotional, and self-regulatory skills. Neurocognitive risk is exacerbated for YCCS living in rural and Appalachian areas, who are at elevated risk due to poverty and distance from specialty care. Digital health interventions (DHIs) targeting positive parenting skills show promise in promoting neurodevelopmental outcomes in other neurological conditions. The proposed research is the first to co-design a positive parenting DHI for YCCS (3-6 years), called Preparing for Life and Academics for Young survivors (PLAY) program, to improve their neurodevelopmental trajectory. Following the ORBIT model phases, PLAY has been adapted in partnership with caregivers and oncology healthcare providers from parent-coaching DHIs developed by the mentoring team. PLAY involves 6 self-directed web-modules and 8 virtual coaching sessions for caregivers of YCCS (Phase Ia). The rationale and central hypothesis for this project is that PLAY will improve parent-child interactions and reduce parenting stress, which will lead to improvements in child school readiness, or prevent future declines.68,71 In Aim 1, the research team will iteratively refine the PLAY program through rapid cycle testing (Phase 1b) until three families sequentially endorse above-average usability, feasibility, and acceptability, or until 15 families have completed the intervention. In Aim 2, a single arm trial (Phase IIa) will be conducted with 15 families to evaluate the feasibility, acceptability, and proof-of-concept of the refined PLAY program in improving (or maintaining at normative levels) observed positive parenting skills, parenting stress, and school readiness at 3-and-6 month follow ups. In Aim 3, a feasibility trial (Phase IIb) will be conducted by randomizing families to either PLAY or an internet resource comparison group (n = 15 per group) to determine acceptability of randomization and retention. With the guidance of an exceptional mentorship team, the K08 candidate, Dr. Moscato, will acquire training in: 1) mixed-methods assessment approaches and intervention strategies for young children, 2) community-engaged research with rural and Appalachian populations, 3) iterative digital health co-design and refinement, and 4) the design, conduct, and analysis of behavioral trials based on the ORBIT model. This training will facilitate Dr. Moscato’s long-term goal to become an independent clinician-scientist focused on developing widely available DHI for childhood cancer survivors. This K08 proposal will provide preliminary data and the expertise needed to conduct a large scale RCT. The ultimate goal is to inform contextually responsive, available psychosocial care that enhances quality of life across the lifespan for childhood cancer survivors.

NIH Research Projects · FY 2026 · 2025-02

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and worldwide, and it remains disease incurable in part due to an incomplete understanding of its cellular mechanisms. Small airway (SAD) and emphysema are important pathological phenotypes of COPD. SAD is characterized by excessive mucus production and reduced alveolar attachment in the small airways (<2mm in diameter). SAD may also worsen emphysema, the progressive destruction of alveolar tissue. However, the mechanisms underlying the pathogenesis of small airway mucin overproduction and alveolar destruction are not well understood. Importantly, mucin is produced by an epithelial progenitor, the club cell, which can differentiate into alveolar type II cells for tissue repair. Here, we have discovered that overproduction of a polymeric gel-forming mucin MUC5B may be linked to impaired alveolar regeneration by club cells in a murine COPD model. Therefore, this proposal aims to test the central hypothesis that alveolar maintenance and repair by club cells in health and COPD is dependent on MUC5B expression. Specific Aim 1 will test the hypothesis that MUC5B expression inhibits differentiation of club cells into ATII cells during homeostasis. I will employ organoid assays to test steady state progenitor functions of MUC5B-expressing and non-expressing club cells from healthy mice with tamoxifen-induced lineage tracing of club cells and fluorescent labeling of endogenous MUC5B. Further studies will employ mice with genetic deficiency or transgenic overexpression of Muc5b. Specific Aim 2 will test the hypothesis that overproduction of MUC5B inhibits alveolar repair in a mouse model of COPD. Mice from Aim 1 will be treated with porcine pancreatic elastase (PPE) to test whether excessive MUC5B in club cells impairs alveolar differentiation in vitro and in vivo, and whether conditional depletion of Muc5b ameliorates SAD and emphysema. Finally, Specific Aim 3 will test the hypothesis that disrupting stimulated mucin overproduction prevents SAD and emphysema by restoring progenitor function of club cells. I will investigate the mechanism of how extra- versus intra-cellular excessive MUC5B impairs progenitor functions of club cells, and I will identify novel genes that regulate club cell mucous and progenitor functions and expand on translational studies in human COPD. These studies will fill gaps in our knowledge of heterogeneous functions of club cells in small airways in homeostasis and disease. Aim 1, 2 and part of Aim 3 will be completed during the K99 training phase. Key elements of my training will be learning new fields of mucin biology and bioinformatics that will help me develop my career as a translational COPD researcher. Results of my studies will supply a foundation for strong lines of independent research on pathways regulating epithelial phenotypes in COPD and development of novel therapeutics for SAD and emphysema in the R00 phase. With my outstanding mentoring team and an exceptional training environment, I am ideally positioned to achieve the proposed scientific training and career development goals and mature into an independent investigator.

NIH Research Projects · FY 2023 · 2025-01

Abstract The congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic diseases of red blood cell production marked by hyporegenerative anemia with the presence of multinucleated red blood cell precursors in the bone marrow. Numerous gene loci have been associated with CDA, the most common being codanin-1 (CDAI) and SEC23B (CDA II). Although a common cause of CDA worldwide, the function of codanin-1 is largely unknown. We have demonstrated that codanin-1 is a chromatin binding protein whose expression is maintained during erythroid differentiation. The objective of this application is to identify the function of CDA-associated proteins in erythropoiesis, with a particular focus on codanin-1 and a newly identified CDA protein, MACF1. This will be explored by examining three Specific Aims: (1) study of mechanisms of codanin-1 regulation of gene expression during erythropoiesis; (2) characterization of a murine CRISPR/Cas9 knockin model of a codanin-1 CDA- associated point mutations; and (3) characterization of the role of MACF1 in erythropoiesis and in the pathophysiology of CDA, including development of a knockin and knockout mutant MACF1-linked murine model of CDA. The central hypothesis is that codanin-1 and MACF1 are involved in regulation of erythroid development and differentiation. The experimental plan focuses on understanding this regulation using cell lines, human and murine primary erythroid cell systems, and mouse models. Preliminary data support the hypotheses and proposed studies. Diminished expression of codanin-1 in CDA I by siRNA and overexpression of a patient-derived point mutant codanin-1 both result in erythroid precursor multinuclearity and decreased hemoglobinization. Genetic analyses strongly support the role of MACF1 in CDA. The investigators have created numerous important tools for the proposed studies. The rationale for this proposal is that by understanding the pathophysiology of a rare disease like CDA, we will gain broad knowledge of mechanisms controlling erythropoiesis, resulting in insights applicable to development of therapeutic strategies for inherited and acquired disorders of red blood cell production. This project takes advantage of a wealth of expertise using the multiple PI format, joining 2 experienced hematologic researchers in Pediatrics at Yale. Dr. Gallagher has experience studying mechanisms of erythropoiesis and its perturbation in genetic disease as well as genomics of erythroid development and differentiation. Dr. Kupfer has studied fundamental aspects of hematopoiesis and its perturbation using Fanconi anemia as a model. Together these investigators are uniquely qualified to study diseases associated with dysfunctional erythropoiesis. The co-PIs are actively involved in molecular hematology research at Yale; both are co-PIs of the Yale Cooperative Center of Excellence in Hematology (YCCEH), providing support for some of the studies in this proposal. Together, these studies will shed important light on erythropoiesis and its perturbation in inherited and acquired disorders of erythrocyte production.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Wilms’ tumor 1, or WT1, is a transcription factor that plays an essential role in both nephrogenesis and maintenance of the adult podocyte. Variants in WT1 can cause Wilms tumors, progressive glomerulopathy, and disorders of sex differentiation in humans. WT1 also plays a role as either a tumor suppressor or oncogene in a context-dependent manner in both hematological malignancies and solid tumors. There are at least 36 different WT1 isoforms generated by alternate start sites and splicing. Two common isoforms, designated WT1-KTS and +KTS, differ in the presence or absence of three amino acids, lysine-threonine-serine, between the protein’s third and fourth zinc fingers and result from alternate splice sites in intron 9. Interestingly these two isoforms exhibit different nuclear localization patterns and DNA-binding affinity, and have been shown to have distinct yet overlapping cellular roles. Although the role of WT1 in the developing and mature kidney has been the subject of many research endeavors, there remain several critical gaps in our understanding of WT1 biology: 1) the factors that regulate WT1 transcriptional activity are unknown; 2) the function of the WT1+KTS isoform has not been fully characterized; and 3) the mechanisms by which human WT1 mutations disrupt WT1+KTS function have not been studied. We aim to apply novel proteomic approaches including proximity labeling and quantitative mass spectrometry to address these knowledge gaps. In Specific Aim 1, we propose to apply proteomic techniques to identify overlapping and unique interaction partners of the WT1-KTS and WT1+KTS isoforms. In Specific Aim 2, we will apply similar techniques in addition to other relevant cell biological assays to determine how WT1 patient variants disrupt protein-protein interactions and cause disease. Completion of these aims would provide the first successful proteomic approach to identify WT1 interaction partners, thereby providing a better understanding of the factors that modulate WT1 transcriptional activity, insight into the differential functions of the WT1-KTS and WT1+KTS isoforms, and, ultimately, to the development of novel therapeutic strategies for patients with WT1-related kidney disease.

NIH Research Projects · FY 2024 · 2024-12

Elucidating the cellular and molecular basis of LYST-mediated TEVG stenosis ABSTRACT A tissue-engineered vascular graft (TEVG) that resists stenosis and grows with the patient has the potential to transform the surgical management of infants and children requiring congenital heart surgery. However, initial clinical evaluation of TEVG efficacy in children revealed an unexpectedly high incidence of graft stenosis or vessel narrowing. In our pursuit to understand the mechanism underlying stenosis, our lab observed reduced rates of TEVG stenosis in mice carrying a mutation in the lysosomal trafficking regulator gene (LYST), which codes for an immunomodulatory protein important for innate immune cell function. As high macrophage infiltration correlated with high rates of stenosis, we initially expected that macrophages were responsible for causing LYST-mediated stenosis. However, macrophage specific LYST dysfunction did not prevent stenosis in mice. In the foreign body reaction to the TEVG, platelet signaling occurs upstream of macrophages, and a growing body of evidence suggests platelets play a critical role in stenosis. Therefore, we hypothesized that the inflammatory response to the TEVG requires LYST-mediated platelet specific signaling that recruits other immune cells to assist in remodeling the developing thrombus into collagen-rich neotissue that persistently occludes or narrows the vessel. Understanding the initial signals driving LYST-mediated stenosis would allow us to harness the therapeutic potential of LYST modulation to prevent graft complications in human patients. In this proposal, we will i.) assess whether LYST dysfunction in megakaryocytes, the precursor cell for platelet production, will prevent TEVG stenosis in murine models, ii.) determine the contribution of purinergic signaling to TEVG inflammation and remodeling, and iii) characterize the time course of LYST involvement in shaping the TEVG immune microenvironment. Successful completion of these studies will deepen our understanding of the LYST-mediated foreign body response to the TEVG and support the development of next generation TEVGs that resist stenosis.

NIH Research Projects · FY 2026 · 2024-09

SUMMARY: Worldwide, up to 25% of premature infants born prior to 31 weeks gestation, and up to 50% of those born prior to 28 weeks gestation, have neurodevelopmental impairment (NI). The causes of NI are multifactorial, but neuroinflammation is now recognized as a key component. Early life stresses commonly encountered by premature infants, including hypoxia, forced formula feeding, and hypothermia, have the potential to trigger inflammation throughout the body. This inflammation can originate in the intestine, e.g. during neonatal necrotizing enterocolitis (NEC), where the microbiota incite a dysregulated inflammatory response. Excessive inflammation in the periphery propagates neuroinflammation and subsequent long-term NI. We have developed a novel, tunable probiotic delivery system in which the probiotic Limosilactobacillus reuteri (Lr) is administered in a protective biofilm on biocompatible porous microspheres that provide Lr with cargos that induce beneficial properties. Administration of Lr in this biofilm state reduces the incidence of NEC and subsequent neuroinflammation in a rat model after just a single dose. The central hypothesis of this proposal is that identifying the extent to which Lr antimicrobial and/or anti-inflammatory properties attenuates NI can be leveraged to tune these Lr properties to optimally prevent, or even treat, NEC-induced NI. Studying the neurodevelopmental impacts of optimized dosing regimens on both NEC and healthy control pups will demonstrate the contributions of these properties towards not only preventing and treating NEC-induced NI, but also healthy neurodevelopment. These studies are in line with the recent NOFO (PAR-23-130) for Translational Research in Maternal and Pediatric Pharmacology and Therapeutics, where we intend to develop an FDA approved probiotic formulation which can be administered to all premature infants at risk of developing NEC. AIM 1: Determine the extent to which Lr antimicrobial pathways can prevent NEC-induced neurodevelopmental impairment (NI). Bacterial dysbiosis can incite inflammatory responses in the periphery and brain. We hypothesize that tuning Lr antimicrobial activity can prevent NEC-induced NI. AIM 2: Determine the extent to which Lr anti-inflammatory pathways can prevent NEC-induced NI. We will determine the extent to which Lr anti-inflammatory pathways prevent neuroinflammation and subsequent NI. We hypothesize that tuning Lr anti-inflammatory properties can prevent NEC-induced NI. AIM 3: Determine the dominant Lr-dependent pathways for the prevention and treatment of NI using all proven cargos. We will determine whether Lr antimicrobial or anti-inflammatory pathways are dominant, antagonistic, additive or synergistic in the prevention and treatment of NI. We hypothesize that tuning the best combinations of Lr pathways can be used to both prevent and treat NEC-induced NI. The significance of the proposed research is that it will lead to a better understanding of the ability of Lr to prevent and treat NEC-induced NI, leading to improved novel therapeutic approaches.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect in which the left side of the heart is underdeveloped, with structural malformations to the ascending aorta, aortic/mitral valves, and a characteristically small left ventricle. These anatomical abnormalities restrict proper blood circulation in infants and can be lethal if left untreated. While palliative procedures have improved the prognosis of HLHS patients, many still suffer downstream morbidities and a diminished quality of life. Unfortunately, the etiology of HLHS remains elusive to scientists and clinicians. Genetic and environmental factors are thought to contribute to the development of the disease; however, the exact cellular processes that modulate HLHS pathogenesis still need to be elucidated. An inadequate understanding of the syndrome is exacerbated by limited experimental models that can recapitulate and manipulate key features of HLHS. Restricted ethical access to human embryos, and fundamental differences between animals and humans have failed to produce models that mimic clinical phenotypes. Furthermore, standard 2D in-vitro cell culture spatially restricts tissue growth, which does not fully recap three-dimensional development in nature. When compared to 2D culture, cardiomyocytes grown in 3D have shown enhanced sarcomeric structure, contractility, mitochondrial respiration, cellular alignment, and electrophysiological characteristics. Therefore, there exists a need to develop alternative strategies to study abnormal cardiac development in-vitro. Tissue engineering techniques such as 3D bioprinting offer a unique platform to deposit cells and biomaterials that mimic the extracellular matrix of tissues in an architecturally controlled fashion. In addition to geometric control, the properties of these cell-laden hydrogels, such as stiffness, can be controlled. Moreover, advances in iPSC technology offer an in-vitro biological system that retains genetic- and disease-specific information from donors which provides a tool to probe genetically inheritable and developmental diseases. Leveraging these technological advances, our proposed study aims to utilize a 3D- bioprinted heart tube patterned into an endocardial layer (with iPSC-derived endothelial cells) and myocardial layer (with iPSC-derived cardiomyocytes) to interrogate how dysregulated fluid-induced biomechanics and microenvironmental stiffness impedes cardiac proliferation in-vitro. In this regard, we hypothesize aberrant biomechanical forces induce stress-related endocardial-myocardial signaling that ultimately impedes cardiomyocyte proliferation. We will test our hypothesis by (1) selectively varying endocardial stiffness within our disease-specific 3D model and assessing intercellular signaling that dysregulates cardiomyocyte proliferation, and (2) by applying varying degrees of flow-induced shear stress to the endocardial layer and studying transcriptional shifts that occur in response to these perturbations at the single-cell level. Successful completion of these aims will give novel etiological insights into how dysregulated biomechanical cues in HLHS impedes cardiac growth and could shed light into alternative approaches to treat ventricular hypoplasia.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Severe lower respiratory tract infection (LRTI) due to viral pathogens remains one of the most common causes of death for young children worldwide. Current management of patients with viral-induced LRTI focuses on supportive care, but outcomes may be improved by augmenting the host immune response to fight the virus and regain immune homeostasis. A growing body of literature suggests that children with severe viral LRTI have an impaired adaptive immune response, specifically decreased T cell proliferation and cytokine production. Myeloid- derived suppressor cells (MDSC) are a heterogeneous cell population that expand during inflammatory conditions and potently inhibit T cell proliferation and function. MDSC have been extensively studied in adult oncologic populations where clinical trials show promising results of improved outcomes by inhibiting MDSC as part of chemotherapy and anti-tumor vaccine treatment regimens. Our group has identified significant increases in the frequency of MDSC populations in children with septic shock and COVID-19 and demonstrated associations between increased percentages of MDSC and worse clinical outcomes. However, it is unknown what role MDSC play in children with viral-induced severe LRTI. The overall goal of this proposal is to identify MDSC as contributors to the immune response in children with viral LRTI and identify mechanistic pathways as potential therapeutic targets for future investigations. Our central hypothesis is that increased frequency of MDSC will be associated with worse clinical outcomes and decreased numbers of CD4+ and CD8+ T cells in children with viral LRTI, and that pediatric MDSC-induced T cell suppression is reversible through inhibition of the PD-1 pathway. The proposed experiments will involve blood sampling of children with PCR-confirmed viral LRTI to characterize the dynamics of MDSC and lymphocyte populations during hospitalization. We will also use our in vitro model of MDSC induced from pediatric peripheral blood mononuclear cells and select patient samples to establish the PD-1 pathway as an important mechanism of MDSC-mediated inhibition of T cell proliferation and cytokine production in children. This career development award will generate the necessary data to inform the design of future studies of the pediatric immune response in severe lower respiratory tract infections and will equip me with the necessary tools to achieve independence as a patient- oriented clinician-scientist.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Background: Antibiotic overuse results in antimicrobial resistant (AMR) bacteria and unnecessary adverse events including Clostridioides difficile infections (CDI) killing up to 150,000 and specifically 29,000 Americans. Without intervention these numbers are likely to increase. Surgical antibiotic prophylaxis is a common area for unnecessary antibiotic use among hospitalized children following life-saving surgery. The CDC surgical site infection (SSI) guidelines and the AAP Choosing Wisely Campaign recommend no postoperative antibiotic prophylaxis for procedures considered low risk for an SSI. Our current stepped-wedge cluster randomized trial shows antimicrobial stewardship program (ASP) teams participating in a facilitation workshop significantly reduced inappropriate post-operative antibiotic use at the hospital level. Goal: To disseminate our virtual facilitation workshop to ASP teams and surgeons at 20 US children’s hospitals to aid to de-implement unnecessary postoperative antibiotic use in children while assessing important implementation and clinical outcomes. Methods: For Aim 1, we will adapt our current theoretically and evidence-based facilitation strategy, a problem-solving approach to integrating evidence-based care, with feedback we received from current study participants and updated scientific literature for synchronous and asynchronous online workshop. For Aims 2 and 3, we will recruit 20 children’s hospitals that are members of both the SHaring Antimicrobial Reports for Pediatric Stewardship (SHARPS) Collaborative and the National Surgical Quality Improvement Program- Pediatric (NSQIP-P). ASP teams and their surgeon champions will participate in the updated facilitation workshop. Individuals participating in the workshop will complete pre/post surveys and interviews to assess the immediate and long-term implementation outcomes including feasibility, appropriateness, guideline integration through order set changes, and reach across surgical subspecialities (Aim 2). Each children’s hospital will provide NSQIP-P clinical data three years pre- (2022-2024) and post-intervention (2025-2027). Utilizing these data, a time-series analysis will evaluate the impact of the intervention on appropriate post-operative antibiotic use and the rate of SSI and CDIs (Aim 3). Innovation and Impact: This study is innovative and impactful because it will: 1) combine expertise from surgeons, antimicrobial stewards and implementation scientists to disseminate an effective de-implementation strategy to eliminate unnecessary antibiotic use; 2) assess the impact of this strategy on surgeries in children, an underrepresented group; 3) utilizing a time-series analysis, clinical outcomes (e.g. antibiotic use, SSI, CDI) important to ASPs and surgeons will be evaluated; 5) collect implementation outcomes that will inform the dissemination of the strategy to more hospitals; and 6) synergy between two large collaboratives (SHARPS & NSQIP) that will enhance pediatric ASPs and improve the care of children requiring surgical procedures.

NIH Research Projects · FY 2025 · 2024-08

Abstract The Center for Gene Therapy at the Abigail Wexner Research Institute of Nationwide Children’s Hospital (NCH) hosts a robust and active research community in the muscular dystrophies, with extensive expertise in unraveling disease pathogenesis and developing new treatment paradigms that can be translated from the bench to the bedside. We propose an MDSRC with Cores designed to share this expertise with the Wellstone and broader muscular dystrophy research communities, with Projects intended to address questions raised or left unanswered by current therapies, such as microdystrophin gene therapy. Project 1 (PI, Paul Martin, PhD) seeks to utilize a novel bicistronic AAV vector to both prevent muscle disease and simultaneously build new muscle function in muscular dystrophy patients with Limb Girdle Muscular Dystrophy 2I (LGMD2I). Project 2 (PI, Kevin Flanigan, MD) will develop vectors containing U7 small nuclear RNAs (U7snRNAs) reprogrammed with antisense sequences directed toward splice site or splice enhance elements to restores highly functional dystrophin expression. Project 3 (PI, Nizar Saad, PhD) seeks to identify novel circulating biomarkers through the investigation of circulating extracellular vesicles (EVs) and to develop a new generations of gene therapy that increase transduction efficiency, dystrophin expression, and reduce the development of humoral immunity against AAV gene therapies. All three projects make use of the critical resource core, the Muscular Dystrophy Cell and Serum Banking Core (Director, Nicolas Wein, PhD) by establishing primary fibroblasts from patients with muscular dystrophies, a critical resource for early therapy development, while collaborating with Penny Gilbert, PhD, at the University of Toronto, to develop improved protocols leading to superior reagents for Center Projects. We have a longstanding dedication to serving as a national training resource, as demonstrated by our Myology Course, held annually since 2012, and our Training Core (Director, Scott Harper) will expand the number of attendees and the range of topics covered in the Course, offer individualized career development guidance to Wellstone Fellowship trainees, and develop new curricula of benefit to the community. Our Administrative Core will implement a meaningful community outreach program and develop a highly functional MDSRC website to provide access to the muscular dystrophy community. Altogether, our longstanding efforts and the highly collaborative multidisciplinary environment of our two institutions will be synergized by a MDSRC that leverages existing structures and collaborations, and develops outward-facing resources of benefit to the broader Wellstone and muscular dystrophy research communities.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT While hematopoietic cell transplant (HCT) is an effective curative treatment for children with various malignant and non-malignant diseases, it is also associated with several complications, such as oral mucositis (OM) and bloodstream infections (BSI). The loss of mucosal integrity in OM not only results in ulcerative lesions that are a significant source of pain, but also, the compromised mucosal barrier becomes susceptible to bacterial translocation and predisposes patients to BSI. Several studies have reported disruptions of the oral microbiome in patients undergoing HCT, and this was linked to OM. In addition, bacterial species present in the oral cavity (i.e., viridans group streptococci) have been implicated in BSI. While this establishes the role of oral microbial species in OM and BSI pathogenesis, nearly all of this research is limited to adults, despite the high prevalence of OM (70-80%) and BSI (15-65%) in children. Furthermore, the current standard for diagnosing BSI is culture- based method that is not comprehensive or high enough resolution to distinguish all species, including the oral streptococci. It is crucial to accurately define the blood microbial signature of this high-risk cohort to determine if the oral cavity is a reservoir of organisms causing BSI, and if so, to guide preventive approaches. For Aim 1 we will conduct a longitudinal study in children undergoing HCT at Nationwide Children’s Hospital to understand the impact of oral microbial species during the development and progression of OM and BSI. Our pilot study revealed overall microbial community composition changed as children underwent HCT. We will now expand our sample size to define the oral microbial community changes at the species level and determine the relationship of these changes to oral mucositis and bloodstream infections. Site-specific oral samples (plaque, tongue, mucosa) will be collected at a minimum of four time points. Both species level bacterial and fungal composition by 16S/ITS2 amplicon sequencing, and absolute abundance by qPCR will be analyzed. OM incidence and severity will be scored, and all BSI occurrences will be recorded. The relationship of microbial community composition with time course, oral hygiene, OM score, and BSI occurrence will be examined. Our preliminary 16S amplicon data revealed polymicrobial profiles containing microbial species common to the oral cavity in the bloodstream of subjects with BSI. For Aim 2 we will comprehensively define the bloodstream microbial profile in children receiving HCT by longitudinally collecting blood samples from all subjects and comparing their bloodstream microbial profile in the presence and absence of BSI. To determine if the oral cavity is the likely source of bloodstream infection-associated microbes, we will use high-resolution bioinformatic tools (DADA2) on our amplicon data and analyze the concordance of species detected in blood samples with those detected in the oral cavity. The most likely oral source (plaque, tongue or mucosa) will also be examined. The results of these studies will inform future preventive and therapeutic strategies to reduce OM and BSI burden. They will also lay a foundation for future investigations of therapies and mechanistic studies of bloodstream invasive oral microbes.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT. Neonatal opioid withdrawal syndrome (NOWS) is a major public health problem. While existing findings suggest an increased biological risk of neurodevelopmental impairment, the direct developmental effects are nearly impossible to disentangle fromsocioeconomic, environmental, and family factors. Children with NOWS are often exposed to multiple adverse childhood experiences (ACEs) with known detrimental effects on long-term health, however, positive childhood experiences (PCEs) can buffer the effects of ACEs, promoting positive adaptation. In this proposal our objective is to identify PCEs associated with optimal developmental outcomes as meaningful and feasible future intervention targets. To achieve this goal, we propose incorporating lived experience through an active partnership with a local community advisory board and an innovative, strengths-based focus on positive experiences. We will capitalize on our established, prospective longitudinal cohort of children with a history of NOWS, adding a school-age assessment to establish a more complete developmental trajectory with a comprehensive evaluation of socioeconomic, parental, and environmental variables. We will accomplish this study through the following specific aims: (1) Engage key community members as active partners in the research process to participate in identification of key patient- centered outcomes and explore acceptability, appropriateness, and feasibility of potential interventions, (2) Assess the independent and moderating effects of total PCEs/ACEs and longitudinal measures of parenting on school-age neurodevelopmental outcomes in a prospective cohort of infants with NOWS, (3) Examine the association between the individual PCEs and components with neurodevelopmental outcomes in children with a history of NOWS. We use several novel concepts that differentiate this proposal and line of research from others including an analysis plan directed by positive psychology principles and incorporation of key community members in all phases of the project to improve study processes, reduce barriers, and facilitate meaningful results. I will work with a team of experienced mentors (Drs. Freisthler, Taylor, Sege, Breitenstein & Rausch) to increase my competency in three critical areas: (1) community-engaged research, (2) design and analysis of longitudinal studies, (3) methods for assessment of and intervention to promote PCEs and prevent ACEs. These three training objectives will be accomplished through formal coursework, interactive workshops, presentations and conference attendance, completion of my research aims, and hands-on mentorship activities with my diverse, multi-disciplinary group of mentors and collaborators. This early career development award will provide the necessary candidate training and foundation for an R01 testing the efficacy of an intervention to increase relevant PCEs identified during the K23, propelling an independent, federally funded program of research designed to promote optimal developmental outcomes in children with in-utero substance exposure.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY The long-term goal of this work is to develop a non-invasive imaging strategy for quantifying chemotherapy- induced vascular toxicity to better understand the potential role of image-derived measures of venous inflammation for predicting risk for venous thromboembolism (VTE) in pediatric, adolescent, and young adult (AYA) patients undergoing cancer treatment. Chemotherapy is known to have off-target vascular effects that increase the risk for VTE. In spite of this recognized risk, risk models are not regularly used to guide preventative anticoagulation in young cancer patients and preventative thromboprophylaxis is traditionally avoided due to a high risk of bleeding associated with cancer and concurrent chemotherapy. Therefore, there is a need for biomarkers that can assess chemotherapy-induced toxicity, identify patients at highest risk for VTE, and detect the potential anti-inflammatory vascular effects of thromboprophylaxis. In this proposal, we will validate and test a molecular imaging approach for characterizing chemotherapy-induced venous toxicity from PET/CT images acquired as part of routine care in pediatric and AYA patients undergoing treatment for lymphoma. To accomplish this overarching goal, we will first pre-clinically validate 18F-FDG PET/CT imaging as a non-invasive biomarker of chemotherapy-induced venous toxicity. We will then clinically evaluate the prognostic value of our imaging approach for predicting risk for VTE by collecting whole-body PET/CT images from a large multi-site cohort of pediatric and AYA patients with lymphoma and prospectively monitoring occurrence of VTE events. Following pre-clinical validation and clinical testing of our approach, we will assess the utility of 18F-FDG PET/CT imaging for detecting the anti-inflammatory actions of standard thromboprophylaxis methods. Validation of PET/CT imaging for detecting venous toxicity, risk for VTE, and responses to thromboprophylaxis could provide a non-invasive approach for enabling real-time monitoring of adverse vascular effects associated with cancer treatment in pediatric and AYA patients and identifying those at high risk for VTE.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Syphilis infection remains endemic among adults worldwide. Rates are rising among women of childbearing age in the U.S., resulting in a concomitant increase in congenital syphilis cases. Untreated congenital syphilis can result in permanent neurologic impairments and osteoarticular deformities. Although some infants have clinical symptoms at birth, many are asymptomatic at delivery. For these asymptomatic, syphilis-exposed infants, diagnostic testing does not reliably identify those with true congenital infection. Because of this, a large number of syphilis-exposed infants undergo extensive diagnostic testing, are treated with antibiotics, and require long- term outpatient follow-up. Tests to discriminate infected infants from those who are exposed but uninfected are necessary to improve clinical management. Our group has a longstanding interest in congenital cytomegalovirus (CMV) infection, and we have shown that infants with congenital CMV infection have evidence of global immunologic activation in peripheral blood that differs markedly from the naïve immune signatures of uninfected neonates. These differences encompass the whole blood transcriptional profiles, differentiation and activation of innate and adaptive immune cell subsets, and serum cytokine profiles. Importantly, the immune profile of congenital CMV is observed among both symptomatic and asymptomatically infected infants and is significantly different from uninfected infants. Human immune responses to congenital syphilis infection are largely unstudied, but responses in adults and animal models show monocyte activation and sustained B and T cell responses. In this project we will test the hypothesis that infants with congenital syphilis infection express markers of global immune activation that distinguish them from healthy, uninfected infants and syphilis-exposed, uninfected infants. Aim 1 will identify characteristics of innate and adaptive immune responses to congenital syphilis among an established cohort of infants with symptomatic congenital syphilis infection using advanced CITE-seq and flow cytometry immunophenotyping methods, which will greatly expand our limited understanding of congenital syphilis immunopathogenesis. Leukocyte markers expressed during congenital syphilis infection will then be applied to an enrolled cohort of asymptomatic, syphilis-exposed infants to determine if these immunologic signatures can distinguish their likelihood of having congenital infection. Aim 2 will establish serum cytokine and chemokine profiles induced by congenital syphilis and apply this profile to asymptomatic, syphilis- exposed infants to discern their likelihood of congenital syphilis infection. As an exploratory objective, biomarkers from Aims 1 and 2 will be synthesized to generate an immune profile for likelihood of congenital infection among asymptomatic, syphilis-exposed infants that can be tested in future studies. Together, these data will provide new insights into the immunopathogenesis of congenital syphilis infection and seek to identify potential biomarkers to discern likelihood of congenital infection among asymptomatic, syphilis-exposed infants.

NIH Research Projects · FY 2025 · 2024-07

PROJECT ABSTRACT/SUMMARY Leukodystrophies are genetic neurodegenerative diseases that affect the white matter within the central nervous system (CNS) and commonly manifest during childhood. They have a range of cellular etiologies, with some resulting from astrocytic dysfunction; two of these include Vanishing White Matter Disease (VWM) and Alexander’s Disease (AxD). While both diseases lead to demyelination, motor abnormalities, intellectual disability, seizures, and pre- mature death, AxD can be inherited or sporadic, and is caused by autosomal dominant, toxic gain-of-function mutations in glial fibrillary acidic protein (GFAP). VWM, however, is caused by loss-of-function mutations in the subunits [1-5] of eukaryotic initiation factor 2B (eIF2B), most commonly in EIF2B5. Both have no treatments. Due to VWM’s monogenic and recessive nature, it is a good candidate for adeno-associated virus (AAV)- mediated gene supplementation therapy. Therefore, my dissertation has focused on characterizing and treating the severe Eif2b5I98M murine model with an astrocyte-targeted AAV9-EIF2B5 gene therapy. We designed constructs to drive astrocyte-specific or ubiquitous therapeutic EIF2B5 transgene expression. Ongoing efficacy studies indicate that these vectors partially rescue body weight and motor function, and significantly extend survival of Eif2b5I98M mice; however, these animals are now experiencing severe and life-limiting seizures. Preliminary data in treated VWM mice shows we were able to restore EIF2B5 expression, however analysis of the integrated stress response (ISR)—a pathway that is mediated by eIF2B and rescues homeostasis after cell stress—remained dysregulated. Therefore, we hypothesize that uncorrected molecular pathways (ISR) in primary and secondary cell types are leading to incomplete rescue. We propose that synergistic combination utilizing the clinically relevant ISR modulator, Integrated Stress Response Inhibitor (ISRIB), and our gene therapy will provide a targeted and more comprehensive correction of disease. ISRIB has been shown to lessen VWM pathologies by stabilizing the mutated eIF2B complex and is now in a clinical trial for VWM [NCT05757141]. Herein, in the F99 phase (Aim 1), I propose to learn and utilize cutting edge approaches including state-of-the-art sequencing and multiomic analyses to elucidate the molecular profile of incomplete treatment, to then provide a more durable, comprehensive, and targeted therapeutic approach for VWM. To continue my academic career and to strengthen my training in neuroscience-focused gene therapy, in the K00 phase (Aim 2) I propose to diversify my scientific repertoire with novel therapeutic approaches that are applicable to more complex white matter disorders—such as toxic gain-of-function, AxD—as well as advance my training in in vitro and in vivo model characterization and pre-clinical efficacy testing through the implementation and testing of AAV-mediated astrocyte-targeted gene knockdown of toxic GFAP. The goal is to provide a durable, safe, and efficacious treatment for AxD.

NIH Research Projects · FY 2026 · 2024-07

Abstract Diseases affecting the heart valves are among the most common type of cardiovascular medical condition affecting 2.5% of the population in the United States. The aortic valve is the most often affected and encompasses both congenital and acquired forms. Congenital aortic valve disease is the result of a malformed valve and represents the most common type of congenital heart defect when including bicuspid aortic valve (BAV), which has a prevalence of 1-2% in the population. At birth, diseased aortic valves are often myxomatous with bicuspid valve morphology and display congenital aortic valve stenosis (AVS). If untreated, congenital AVS results in progressive left ventricular hypertrophy and ultimately heart failure. Severe congenital AVS is treated with surgical or catheter-based valve intervention or replacement. Accordingly, there is a critical need for pharmacologic therapies for AVS that will obviate the need for surgical intervention. We were the first to discover that pathogenic variants in NOTCH1 were associated with BAV and AVS in humans, and we subsequently found that Notch1 signaling is critical for heart valve development in murine models. Furthermore, our prior publications have demonstrated a genetic and molecular link between Notch1 and nitric oxide (NO) signaling. We and others also found pathogenic variation in GATA5, a zinc finger transcription factor, was associated with BAV in humans. Interestingly, Gata5-null mice display BAV and decreased expression of Nos3 and Notch signaling in the embryonic heart. These findings support the prevailing view about the critical roles of NOTCH1, GATA5, and NOS3 in the pathogenesis of human BAV and congenital AVS. We recently generated Notch1 and Gata5 compound mutant mice (Notch1+/-;Gata5-/-); these mice display highly penetrant congenital AVS and survive to adulthood with progressive aortic valve stenosis. This exciting, new murine model recapitulates the human disease condition and will allow us to decipher the mechanisms of disease development and progression. The long-term goal of this research is to understand how molecular pathways regulated by NOTCH1, GATA5, and NOS3 in the aortic valve can be manipulated to treat congenital AVS and progressive valve stenosis. The objectives are to utilize this new highly penetrant and clinically relevant congenital AVS mouse model for the discovery and testing of mechanism-based pharmacologic therapies. We will achieve this in the following aims: Specific Aim 1. To define the cell-type specific molecular pathways underlying development of congenital AVS using the Notch1;Gata5 compound mutant mouse model. Specific Aim 2. To determine the cellular and molecular mechanisms for disease progression in congenital AVS using the Notch1;Gata5 mutant mouse model. Specific Aim 3. To test novel therapeutics for congenital AVS and stenotic disease progression.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY Treatment with the chemotherapy drug, Cisplatin, frequently results in developing and/or progressing chronic kidney disease (CKD). Cisplatin accumulates in renal proximal tubular cells, which reabsorb nutrients and metabolize Cisplatin to reactive thiols that cause oxidative damage. Injured tubular cells that fail to repair are known drivers of progressively declining kidney function and show impaired mitochondrial oxidative metabolism. We have supportive evidence demonstrating that GC4419, a mitochondrially targeted superoxide (O2•–) dismutase (SOD) mimetic, protects the kidney against oxidative stress and cisplatin-induced CKD in mice and humans. GC4419 also reverses electron transport chain (ETC) disruptions and improves mitochondrial oxidative metabolism. Building on recent breakthroughs from our group, we will test the hypothesis that increasing mitochondrial O2•– dismutation in renal tubular cells will reduce cisplatin-induced renal injury and promote renal repair by reversing mitochondrial ETC disruptions and increasing NADPH regeneration by the pentose phosphate pathway. In Aim 1, we will determine how tubular mitochondrial O2•– mediated interactions with ETC complex II disrupt mitochondrial ETC function in cisplatin-induced CKD. In Aim 2, we will establish how O2•– dismutation impacts renal NADPH metabolism in cisplatin-induced repair. In Aim 3, we will determine renal biomarkers that predict the beneficial effects of GC4419 in vivo. The overall objective of this proposal is to increase our understanding of how tubular O2•– dismutation protects from cisplatin-induced CKD and to develop translational tools that will facilitate risk stratification strategies for future SOD-mimetic clinical trial design in patients fighting cancer with Cisplatin. To that end, we will incorporate the following innovative methods using 1) newly developed mouse model to delete mitochondrial SOD in tubular cells for pre-clinical studies, 2) novel experimental design to interrogate the link between renal metabolomic adaptations, oxidant formation, and antioxidant responses in cisplatin-induced CKD mouse models; 3) the mitochondrially targeted SOD mimetic (GC4419) currently pending approval by the US FDA in patients undergoing Cisplatin treatment, and 4) evaluating tubulointerstitial injury biomarkers to facilitate risk stratification strategies for future clinical trial design. If successful, this proposal will translate into novel diagnostic and therapeutic approaches to promote renal repair in cisplatin-induced kidney toxicity.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract: Idiopathic Nephrotic Syndrome (INS) is the most common type of nephrotic syndrome in childhood, yet its cause remains unknown. Most authorities consider the disease to be mediated by an unknown circulating factor that directly binds the podocyte, causing podocyte activation and proteinuria. However, our group has shown that the disease may not be that simple and that there is also injury to the glomerular endothelial cells (GEnC) that line the inside of the capillary. We recently showed evidence of circulating endothelial biomarkers in ~90% of INS patients. In preliminary studies, we found that one specific endothelial protein, CD93, was especially important, which may relate to its ability to activate focal adhesion kinase (FAK), a protein that is activated in INS and causes podocyte injury. We found high CD93 expression in GEnC from kidney biopsies of INS patients and that levels of its soluble form were high in serum and urine in ~90% of ~300 patients. We also found that sera in relapse stimulate cultured GEnC to release CD93; and importantly, that activation of podocytes by relapsing sera could be blocked with an antibody to CD93. Our preliminary work also identifies urinary CD93 as predictor of kidney disease progression. Our data suggest that the pathogenesis of INS involves an intermediary step of GEnC activation that facilitates podocyte injury. Our central hypothesis is that circulating factors activate GEnC to release the soluble form of CD93, which, in turn, acts as (1) a predictive biomarker for kidney disease progression, and (2) mediator of podocyte activation via FAK signaling. In aim 1, we will study whether CD93, in glomeruli, urine or serum, predicts clinical outcomes and associates with structural and/or biological changes affecting glomeruli. This will be complemented by pilot studies to delineate the patterns of soluble CD93 across disease states, and its specificity across glomerular diseases. In aim 2, we will combine multiple in vitro approaches to identify the source of soluble CD93 and its effects on human podocytes. In aim 3, we will study the CD93 involvement in 2 classic animal models of INS (puromycin and adriamycin), and whether CD93 blockade prevents and/or mitigates proteinuria. Our hypothesis is innovative because GEnC are not considered to have an important role in INS. The combination of several approaches to assess endothelial injury and the inclusion of a large population provide rigor to the clinical studies. Integrating multiple cell systems and classic models of INS also provide a rigorous approach to test causality. Our proposal is highly translational and could lead to the identification of a novel prognostic and therapeutic target in INS. A four-year mentored career development plan has also been devised, which incorporates training in endothelial biology, precision medicine, and leadership by an experienced mentoring and advisory teams. The candidate’s long-term career goal is to become an independent investigator discovering biomarkers, mechanistic pathways, and targeted therapies in INS.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY The somatostatin subtype 2 receptor (SSTR2) is expressed in 80-100% of NETs. SSTR2 targeted alpha-emitter radioligand therapy (α-RLT) is emerging as an exciting alternative to beta-emitter radiopharmaceuticals due to the higher linear energy transfer and dense ionization tracks that result in enhanced formation of double stranded DNA (dsDNA) damage with up to 80% tumor response rates in early clinical studies. However, preclinical studies show that α-RLTs can cause a dose dependent late nephrotoxicity characterized by late tubular injury, fibrosis, and inflammation. Building on recent breakthrough from our group we will test the overall hypothesis that [212Pb]Pb-VMT-α-NET (212Pb-VMT) tumor responses can be maximized through a treatment scheme that incorporates biomarkers of tubular injury, dosimetry, dose fractionation, and a nephroprotective superoxide dismutase mimetic to mitigate nephrotoxicity. In Aim 1, we will develop a detailed understanding of the role of superoxide dismutation in tubular dsDNA repair following [212Pb]Pb-VMT-α-NET treatment. In Aim 2, we will identify [212Pb]Pb-VMT-α-NET dosing regimens to achieve maximal tumor responses and reduce nephrotoxicity using a superoxide dismutase mimetic. We will incorporate the following innovative methods: (1) a novel α-RLT [203Pb/212Pb]Pb-VMT-α-NET theranostic pair targeting SSTR2; (2) innovative SSTR2 expressing tumor models; (3) urine biomarkers for non-invasive detection of tubular injury; (4) dsDNA injury/repair tools to study mechanism of early subclinical tubular dsDNA damage following α -RLT; and (5) a novel nephrotoxicity mitigation strategy based on superoxide scavenging that shows promising tumor response improvement to external beam radiation. If successful, we expect these findings and advances be transferable to a broad range of α-RLTs, not only for pancreatic NETs and neuroblastoma, but also for prostate, breast, melanomas, and other difficult to treat cancers amenable to α-RLT.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY: End-stage lung disease is the third leading cause of death worldwide. Lung transplantation is often the only option for patients with advanced lung disease, yet 50% of recipients die within five years due to the development of chronic lung allograft dysfunction (CLAD). This project seeks to examine the basis for the loss of region-specific stem cells and impaired airway regeneration, with a long-term goal of improving cell-based regenerative medicine approaches. We will make use of innovative transgenic ferret models in our well-established orthotopic lung transplantation model. We will investigate the role of glandular myoepithelial cells (MECs) in renewing submucosal gland (SMG) cells and generating abnormal surface basal stem cells (BSCs) in CLAD. We hypothesize that sustained regenerative pressures drive MECs to exit their SMG stem cell niche to reconstitute surface BSCs with abnormal lineage properties that promote an immune response. Additionally, this project will determine the regenerative function of KRT7+ glandular duct cells (DCs) during the progression of CLAD. We hypothesize that the gland duct is a maturation point for MEC stem cells to adopt a surface BSCs phenotype that is bypassed under sustained regenerative stresses in CLAD. Finally, we will determine the relationship of antigen-experienced B-cells in promoting alloimmune and autoimmune reactions against airway stem cells to drive CLAD. We hypothesize that prolonged regeneration of surface BSCs by reserve MEC stem cells leads to the retention of MEC proteins on the airway surface. This ectopic expression of self-antigens promotes an autoimmune response against the SMG stem cells niche. Currently, end-stage lung disease is a significant cause of morbidity. However, we expect that by clarifying the processes that deplete stem cells in transplanted lungs, we will improve the likelihood of developing long-term objectives of developing effective stem cell therapies to sustain lung function and resilience to prevent CLAD.

NIH Research Projects · FY 2026 · 2024-06

Abstract Approximately 30% of hepatitis C virus-infected persons spontaneously clear the virus within six months. The remaining 70% develop chronic infection. Of those with chronic HCV infection, the risk of cirrhosis ranges from 15% to 30% within 20 years. The viral and immune correlates of these different outcomes of infection and pathogenesis remain poorly studied, mainly due to the lack of an informative animal model. We developed surrogate animal models for HCV using a rodent HCV-like virus isolated from feral brown rats (Rattus norvegicus), RHV-rn1 (Rn-1). So far, studies of Rn-1 infection and immunity in lab mice have revealed that normal immunocompetent mice do not develop prolonged or chronic HCV-like infections. Notably, chronic HCV and RHV infections can be studied in their natural host, humans and rats, respectively. However, how HCV evades immunity during the acute phase, a prerequisite for developing chronic infection, requires animal models that allow comparative analyses of HCV-like clearance and persistence and mechanistic analysis of innate and adaptive immune responses. To create such models, we isolated several new RHV variants from feral rats and used these as a pool for serial passaging and adaptation in mice. After several passages, we identified a mouse- adapted variant (Rn-2m) that produced delayed clearance or chronic infection in normal lab mice. Subsequently, we identified the parent rat virus Rn-2, and its infection cleared within 3-4 weeks in mice. The project seeks to identify the viral and immune determinants of heterogenous outcomes of HCV infection and pathogenesis. Considering the strengths and weaknesses of the rat and mouse models, we propose a comparative biology approach where the new Rn-2m infection in the mouse model, due to the availability of vast resources, will be used to develop and test hypotheses, and the rat model will be used for validation of key findings. Specific Aim 1 is to identify the viral correlates of spontaneous clearance and chronic infection. Our new data indicate that Rn-2m constantly evolves and modulates innate immune cells in the liver of infected mice to establish a prolonged or chronic infection. We will compare the nature of infection, evolution, and immunogenicity of clone- rescued Rn-2 and Rn-2m to identify viral correlates of acute clearance, delayed clearance, and chronic infection in mice and subsequently in rats. Specific Aim 2 is to identify the immune correlates of chronic infection and liver diseases. We will use RHV-specific mouse and rat MHC tetramers and E2 protein tetramers, ex vivo antigen stimulation, passive antibody, and in vivo T and B cell transfer, and cell depletion experiments to define the role of T and B cells in determining different outcomes of RHV infection and pathogenesis. We expect that the solid rationale behind our approach will yield a biologically relevant and widely accessible lab mouse model for HCV and gain novel and mechanistic insights into HCV immune evasion and pathogenesis. This knowledge is crucial for developing new strategies to prevent chronic HCV infections and associated liver diseases.