Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY/ABSTRACT Uncertainty in our understanding of the functional impact of variation in the human genome has grossly impeded our ability to realize the full potential of now mature sequencing technologies. There is a universal need in genomics for the development of tools to support high-throughput functional characterization of genomic variants in disease genes. This proposal will develop Multiplexed Assays of Variant Effects (MAVEs) to functionally assess genomic variation of over 10,000 variants in a ligand and receptor pair, JAGGED1 (JAG1) and NOTCH2, that result in the autosomal dominant disorder, Alagille syndrome, a pediatric cause of liver, cardiac, vertebral, renal, ocular, and facial anomalies. Functional haploinsufficiency is the mechanism for JAG1-related disease and the likely mechanism for NOTCH2-related disease, which is supported by the high- frequency of protein-truncating and large gene deletions seen for JAG1-related disease and a by a high intolerance for loss-of-function variants observed in population databases for NOTCH2. Missense variants, which are identified in 15% of patients with a JAG1 variant and >50% of patients with a NOTCH2 variant, are notoriously difficult to classify, but those that are pathogenic show defects related to protein misfolding, intracellular retention, and an inability to heterodimerize. Moreover, a high incidence of missense variants of uncertain consequence in both genes identified during routine panel testing for general cholestatic disease confuses both patients and providers. Resolution of the functional consequence of all missense variation in JAG1 and NOTCH2 through the development of high-throughput tools would greatly improve diagnostics and reduce reporting of non-disease-associated variants. We propose the development of MAVEs to simultaneously test the functional consequence of all disease- relevant missense variants in JAG1 and a subset of those in NOTCH2. In Aim 1 we will design two separate MAVEs to analyze membrane expression and NOTCH2-binding ability of all JAG1 nucleotide permutations as readouts of protein function. In Aim 2 we will modify these MAVEs for use in studying all nucleotide permutations across a region of interest in NOTCH2 that contains the JAG1-binding interface. In Aim 3 we will test the fidelity of our assay system by studying variant effects in liver cell lines, including both a high- throughput MAVEs approach and a low-throughput, but high-fidelity induced pluripotent stem cell (iPSC) model approach. Aims 1 and 2 will include complementary structural modeling and binding assays while Aim 3 will interrogate the role of cellular environment using disease-relevant cell lines, all of which we expect will validate and extend our findings, providing insight into the biochemical and cellular consequences of pathogenic variation. Ultimately, these data will resolve uncertainty in JAG1 missense variant function that will benefit diagnostics, improve our understanding of the functional consequence for NOTCH2 variants, and provide guidance for how to incorporate this functional data as useable evidence during clinical variant interpretation.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY - OVERALL Kidney stone disease (nephrolithiasis) is characterized by painful, recurrent symptomatic events and is rising disproportionately in youth. Structural and scientific barriers have impeded strengthening the evidence base for management of pediatric kidney stone disease. The overarching objective of the Personalizing Outcomes of Nephrolithiasis in Youth (PONY) P20 Center is to generate knowledge that improves outcomes for youth with nephrolithiasis through an intentional and focused initiative to accelerate the research careers of early-stage investigators and thereby enhance the benign urology research community. To achieve this mission, this proposal strengthens partnerships among Dr. Gregory Tasian (PI at Children’s Hospital of Philadelphia, Dr Jonathan Ellison (early-stage investigator at Children’s Wisconsin), Dr. David Chu (early-stage investigator at Lurie Children’s Hospital of Chicago), and Dr. Jing Huang (biostatistician at Children’s Hospital of Philadelphia). In the proposed PONY P20 Center, we address critical structural and scientific barriers that have impeded strengthening the evidence base for management of pediatric nephrolithiasis. We will accomplish our objectives by leveraging the resources generated by Dr. Tasian’s research program, in particular the robust infrastructure of the Pediatric KIDney Stone (PKIDS) Care Improvement Network and data generated by the NIDDK-supported Urinary Stone Disease Research Network. Central to the PONY P20 Center is the PKIDS Care Improvement Network, which will support the Research Project and provide the means to disseminate the knowledge generated by the research directly into real-world care. In Aim 1, we will generate new knowledge responsive to patient-identified gaps in kidney stone research by conducting two distinct but complementary research studies that use data from PKIDS trial, the Prevention of Urinary Stones with Hydration (PUSH) trial, and the STudy to Enhance uNderstanding of sTent-associated Symptoms (STENTS). In Aim 2, we will accelerate the research careers of early-stage investigators in benign urology. The Administrative Core will create opportunities for Drs. Ellison and Chu to lead the execution of the Research Project through operational and logistical support as well as data access. PKIDS will also provide the scientific environment to support the R01 applications led by Drs. Ellison and Chu that will arise from the Research Project. In Aim 3, the PONY P20 Center will enhance the benign urology research community. We will share our expertise in stakeholder (particularly patient and caregiver) engagement with members of CAIRIBU other benign urologic researchers. We will participate in existing CAIRIBU forums by contributing our lessons learned on stakeholder engagement in clinical investigation, patient-centered trial design, and conceptual innovations in learning health systems. Overall, the PONY P20 Center will generate knowledge that addresses critical priorities identified by youth with nephrolithiasis and accelerates the career of early-stage investigators who will be leaders in benign urological research.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT In the United States, over 60,000 infants are born very preterm (VPT, ≤32 weeks’ gestation) each year; more than 15% of these infants die in infancy, while ~20% survive with severe morbidity. Despite the sobering statistics, not all preterm infants face these consequences. A crucial factor in determining outcomes is the individual infant's response to any given therapy. However, our understanding of factors causing variation in response, beyond immaturity itself, is limited and impedes our ability to customize therapies. The perinatal period, is a critical time window when multiple changes and adaptations in the fetus/newborn set the foundation for the future. Perinatal events could therefore be important determinants of variation in later life health and could be used to identify therapeutically relevant phenotypes. Previous phenotyping efforts in preterm birth have either based the categorization on the presentation leading to preterm delivery (aimed at prevention of preterm birth); or used neonatal severity of illness markers (aimed at mortality prediction); or made prediction models for specific complications (aimed at improving management of said morbidity). These approaches intentionally exclude perinatal interventions such as delivery mode and perinatal antibiotics to minimize impact of practice variation. However, maternal care practices are critical exposures that affect the infant’s response to interventions. Our central hypothesis is that perinatal variables combining reason for birth with intrapartum care variables and neonatal presentation can be used to categorize infants into finite ‘phenotypes’ early in life and can explain variation in multiple later health outcomes. In aim 1, we will use a novel conceptual model to create perinatal phenotypes by integrating the variables using clustering techniques. In aim 2 and 3, we will determine the contribution of perinatal variables in explaining the variation of 2 key outcomes: timing and type of late-onset infection and abnormal growth trajectory. In experimental models, both late-onset infection and weight gain are causally linked with early-life colonization, which, in turn, is influenced by multiple perinatal factors. Thus, perinatal factors, and any derived phenotypes from these factors, may explain the risk variation for these outcomes. To perform this study, we will leverage a unique registry of over 3600 very preterm infants and their mothers with manually adjudicated reason for birth and in-patient neonatal course, that we can link with longitudinal pediatric records from birth to 5 years. The study team and PI have extensive experience in modeling perinatal variables, creating linked datasets, clustering techniques and expertise the field of preterm birth and neonatal infections. The expected result of our study is to create a much-needed and novel patient selection framework for early life interventions, specifically those targeting early colonization, infection and inflammation. If successful, such a framework could change the paradigm of how trials are designed and ultimately how clinical care is customized.

NIH Research Projects · FY 2025 · 2024-08

Abstract The temporomandibular joint (TMJ) controls the movement of the jaw but is susceptible to acquired diseases, including osteoarthritis (OA). Osteophytes, referred to as fibrocartilage-capped bony spurs, are one of the radiographic hallmarks of TMJ degenerative disease and cause skeletal deformities, chronic pain, and limited condyle movement. Current treatments are limited, and patients may undergo reparative surgical procedures, which are not always remedial. The etiology and pathogenesis of osteophyte formation in TMJ OA remain unresolved and interfere with basic, translational, and clinical progress toward new effective treatments. Recently, arthropathy, including in the TMJ, was reported for the first time in a patient with compound heterozygous mutations in the PRG4 gene. Our preliminary studies show that Prg4-mutant mice reproduce some key phenotypes in TMJ OA patients, including synovial hyperplasia and osteophytes. Whereas heterozygous Prg4-deficient (Prg4+/-) mice did not exhibit a strong TMJ phenotype, osteophytes did form in homozygous Prg4-null (Prg4-/-) mice, initiating ectopic chondrogenesis. The latter data indicate that local normal anti-chondrogenic mechanisms had been deranged, possibly causing greater availability of chondrogenic factors and leading to osteophyte onset. We tested this novel thesis and found that before osteophyte formation, the expression of the heparan sulfate (HS) synthesizing gene EXT1 was downregulated in the TMJ fibrocartilage. In addition, we found that the local levels of heparanase (HSPE), the primary enzyme responsible for extracellular HS degradation, were markedly increased in osteophyte-forming regions of TMJs. These preliminary data suggest that local decreases in HS and increases in HSPE cause a significant drop in HS levels and create a robust prochondrogenic-inducing environment. In support of this thesis, BMP signaling was more greatly distributed within the mutant condyles and developing osteophytes. These and other data lead to our central hypothesis that a steep local loss of HS triggers osteophyte formation. Aim 1 will test whether conditional Ext1 ablation causes osteophyte formation or worsens the TMJ phenotype in Prg4+/- mice. We will conduct single-cell RNAseq studies to assess cellular phenotypic characteristics, developmental trajectories, signaling pathways, and networks during osteophyte formation. Aim 2 will test whether Bmpr1a ablation will inhibit osteophyte formation in Prg4-null and other mutant mice above. To expand treatment options and make them clinically relevant, we will test whether administering a BMP signaling antagonist LDN-193189 prevents or inhibits osteophyte development. This project is based on new data and insights into the mechanisms of osteophyte formation in the TMJ and will test the efficacy of drug therapy. Given its novelty, the project is in its early stages but will undoubtedly have major and broadly relevant implications for basic research and translational medicine for TMJ and other joint-related musculoskeletal diseases.

NIH Research Projects · FY 2026 · 2024-07

Schizophrenia (SZ) is a heterogenous neurodevelopmental disorder in which the complex and interacting influences of genetics and environment drive neuropathological processes leading to symptom development. While the precise neuropathological underpinnings of SZ remain undetermined, and multiple brain regions are likely to be involved, both genetic and neuropathological evidence suggest that disrupted glutamatergic synaptic function in the cerebral cortex can be an important component of symptom development in SZ. Studies, including from the contact PI Stewart Anderson’s, lab have also suggested that mitochondrial weakness may contribute to the development of schizophrenia. Since a major locus, perhaps the major locus, of mitochondrial ATP dependence for neural function occurs in the presynaptic terminal, it stands to reason that mitochondrial weakness could be one influencer of the glutamatergic synaptic disruption contributing to SZ. The most common genetic risk factor for SZ is the 22q11.2 deletion syndrome (22qDS), occurring in about 1:3000 births, roughly one quarter of which develop SZ. We previously used IPSC-derived glutamatergic neurons (iNeurons) and lymphoblastoid cell lines to demonstrate that while the condition of 22q+SZ is associated with weaker ATP production via oxidative phosphorylation (OXPHOS), 22qDS without SZ (22q(-)SZ) is associated with elevated levels of mitochondrial-biogenesis related transcripts that may denote a compensatory mechanism reducing SZ risk. Treatment of iNeurons from the 22q+SZ group with the medication bezafibrate resulted in enhanced expression of mitochondria biogenesis-related genes as well as normalization of their ATP production. Here we propose to extend these studies with the following aims: Aim 1: Study of mitochondrial biogenesis and mitophagy in IPSC-derived neurons from 22qDS with or without SZ and unaffected controls. We hypothesize that there will be enhanced mitochondrial biogenesis and turnover in the 22q(-)SZ group relative to both controls and 22q+SZ. Aim 2: Study of glutamatergic synaptic release in IPSC-derived neurons from 22qDS with or without SZ and unaffected controls. We hypothesize that OXPHOS deficits in IPSC-derived neurons from 22qDS+SZ will result in reduced glutamatergic synaptic release and reduced synaptic vesicle cycling relative to neurons from 22qDS without schizophrenia and to healthy controls, and that activation of mitochondrial biogenesis with bezafibrate will normalize synaptic vesicle cycling and glutamate release in the 22qDS+SZ group. Success in showing that 22q(-)SZ is associated with enhanced mitochondrial biogenesis/turnover would bolster the rationale for targeting this system to prevent or ameliorate SZ symptoms in 22qDS. Since synaptic energetic weakness, potentially interacting with genetic and environmental factors that also affect synaptic function, has been implicated in non-syndromic SZ and other neuropsychiatric disorders, the novel human-based experimental paradigms we apply in this proposal could be broadly applicable to other IPSC-based studies of brain disease.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Recombinant adeno-associated virus (rAAV) vectors have demonstrated efficient gene transduction in preclinical gene therapy studies and are approved for clinical use in the US. Although we know the relevant viral genes that facilitate rAAV replication and packaging, we have very little understanding of the cellular proteins required to facilitate AAV replication. We also know very few of the host cell interactions with the vector genome which impact the efficiency of rAAV transduction in target cells. Recruitment of cellular proteins to AAV genomes promotes replication in producer cells, whereas host antiviral factors limit rAAV transduction in target cells. Production of rAAV vectors and their use for transduction and gene therapy will benefit from a deeper understanding of the cellular factors that interact with AAV genomes to facilitate gene expression, DNA replication, genome encapsidation, and vector transduction. In this proposal we aim to identify cellular factors that associate with AAV genomes. Knowledge of proteins that interact with AAV genomes will be harnessed to improve both rAAV production and effective gene delivery. We will employ a novel proteomics technology that identifies proteins on replicating DNA by coupling Isolation of Proteins on Nascent DNA (iPOND) with Mass Spectrometry (MS). Our lab has extensive experience using iPOND-MS to identify cellular factors on replicating viral DNA genomes, with existing experimental and bioinformatic pipelines for acquiring these proteomics data and analyzing results. We are the first to employ iPOND-MS for AAV, and our preliminary data demonstrate feasibility with an innovative approach to label AAV genomes. In Aim 1 we will adapt the iPOND-MS technique to identify cellular factors associated with replicating wild-type AAV genomes. We will optimize labeling of AAV genomes and identification of associated proteins, and determine their impact on AAV replication. In Aim 2 we will identify cellular proteins associated specifically with DNA genomes of rAAV vectors. We will employ iPOND- MS to define proteins on replicating rAAV genomes in producer cells. We will also label rAAV vector genomes and define factors associated with labeled genomes during rAAV transduction of target cells. We will use cellular assays to determine the impact of these host proteins on rAAV transduction. In this way we anticipate that our results will identify the cellular factors associated with rAAV genomes that regulate both vector production and efficient transduction of target cells. This knowledge will suggest ways that production and transduction can be improved. This application is based on innovative proteomics methodology we have developed to identify the repertoire of proteins associated with DNA of viral genomes. Our combined expertise and reagents make us ideally suited for this project which has the potential to provide insights into cellular factors that can be harnessed to improve AAV replication during rAAV vector production and gene expression during rAAV vector transduction.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Obesity can pose a lifelong threat to health and quality of life for many autistic people. Across the lifespan in autism, obesity prevalence is increased relative to non-autistic peers. One third of autistic adults have obesity and there are concerning associations between obesity and in-hospital mortality, risk of type II diabetes, and cardiovascular disease. Prevention in childhood should be a priority; however, ~100,000 autistic youth will transition to adulthood each year in the United States, and there is no existing comprehensive treatment for obesity designed specifically for autistic individuals transitioning to adulthood and greater independence. The overall objective of this study is to identify stakeholder-generated and testable treatment components to inform obesity intervention specifically designed for transition-age autistic youth. Comprehensive obesity treatment programs are prime candidates for the engineering-inspired Multiphase Optimization Strategy (MOST) framework that optimizes multicomponent behavioral treatment packages to expedite implementation and maximize scalability. The proposed online study, grounded in activities for the MOST Preparation Phase, will use a simultaneous exploratory mixed methods study design to gather perspectives on obesity treatment directly from transition-age autistic youth with obesity. Thirty transition-age autistic youth with obesity (16-25 years) will complete qualitative interviews to identify stakeholder-generated factors that impact obesity treatment (Aim 1). Transition-age autistic youth with obesity (N=120), caregivers of transition-age autistic youth with obesity (N=60), and clinical providers (N=60) will rate and prioritize the perceived effectiveness of obesity treatment components (e.g., diet modifications, text message prompts, parent training, goal setting) that could be included in a comprehensive obesity intervention program tailored for transition-age autistic youth (Aim 2). Then, in partnership with a Community Advisory Board, we will integrate Aim 1 qualitative and Aim 2 quantitative findings to iteratively develop a set of treatment components specifically tailored for transition-age autistic youth with obesity that can be subsequently tested in a MOST framework optimization trial. The resulting Obesity Treatment Blueprint for Transition-Age Autistic Youth will also provide initial steps toward adaptation of existing interventions that can be leveraged for use within the proposed tailored treatment package. Aligned with NICHD Scientific Theme 4, ‘Improving Child and Adolescent Health and the Transition to Adulthood,’ this program of work will meet the pressing need for effective obesity treatment to improve health and quality of life for autistic individuals across the lifespan.

NIH Research Projects · FY 2026 · 2024-07

PROJECT ABSTRACT The millions of brain MRIs acquired each year in clinical settings are a vastly underutilized scientific resource. Meanwhile, brain MRIs collected in research studies often struggle to recruit cohorts fully representative of populations of interest, particularly in the case of rare neurodevelopmental disorders. One clear opportunity for discovery science are pediatric MRIs where gross pathology has been ruled out in children with headache, concern for head trauma, or neurodevelopmental symptoms such as delayed milestones, autism, or psychosis. One reason why brain morphology phenotypes derived from clinical brain MRIs have been underutilized is due to the expected technical differences across sites and scanners, which pose methodological and statistical challenges. However, our study team recently developed brain growth charts based on one of the largest aggregated neuroimaging datasets to date, which provide an unprecedented model for brain MRI developmental imaging phenotypes -- including quantitative measures of brain volumes of cortical and subcortical brain regions – to be accurately benchmarked against population norms while controlling for technical differences. In preliminary investigations, we have now further developed this approach to benchmark individual patients' brain anatomy against hospital norms – so-called “clinical controls” – to provide high-quality imaging metrics to combine with data on genetics, environmental exposures, and neuropsychiatric outcomes. Using advanced, fully-reproducible image processing, deep learning, and statistical modelling, we will develop a clinical radiomics reference that allows precise quantification of where an individual clinically-acquired brain MRI lies in reference to expected developmental variation, using 20,000 clinical MRIs without gross pathology obtained by Children's Hospital of Philadelphia care teams (Aim 1). By integrating records of clinical MRIs with large-scale CHOP biobanks, we will identify developmental imaging phenotypes associated with high genetic risk for neuropsychiatric and neurodevelopmental disorders (Aim 2). By integrating secondary analysis of clinically-acquired scans with prospective behavioral phenotyping and neuroimaging, we will identify developmental imaging phenotypes associated with psychiatric vulnerability in individual patients (Aim 3). Throughout, we will generate practically useful brain chart resources that will facilitate analysis of clinically- acquired brain scans by other researchers (Aim 4). By leveraging clinically-acquired brain MRIs, this proposal harnesses untapped neurogenomic information to investigate neurodevelopment trajectories in high-risk youth, capitalizing on the PI and assembled team's expertise in psychiatric and developmental brain imaging, imaging-genetics and neuroinformatics. The project will provide the necessary infrastructure to support future HIPAA-compliant multi-site collaborative projects across health systems with expected sample sizes on the order of millions of scans.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor T-cells (CART) have revolutionized cancer therapy. However, CART cause two common and severe toxicities: Cytokine Release Syndrome (CRS) and Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS). CRS and ICANS are overlapping phenomena that occur with a spectrum of severity from mild to life threatening. Effective therapies for CRS currently exist but severe refractory cases still occur. Effective therapies for ICANS are lacking. The pathophysiology of CRS and ICANS have not yet been fully defined, precluding development and translation of novel therapies. In particular, the role of the host innate immune system in these conditions is poorly understood. The specific objective of this project is to identify the cellular and cytokine initiators of CRS and the role of complement and the inflammasome in the biology of ICANS. In Specific Aim 1, the candidate will determine the number and transcriptional activation state of different monocyte and non-monocyte populations associated with CRS using single cell RNA sequencing (scRNAseq) and test the role of novel monocyte produced cytokines in the priming and initiation of CRS. In Specific Aim 2, the candidate will dissect the role of the inflammasome and complement dysregulation in the development of ICANS, and test individual and combined approaches for inflammasome and complement blockade in preventing and treating ICANS. The candidate is a pediatric oncologist committed to understanding immune dysregulation disorders that occur as a consequence of cancer immunotherapy. The proposed training plan and research project will be conducted at the Children's Hospital of Philadelphia (CHOP) and the University of Pennsylvania (UPENN). CHOP and UPENN are leaders in cellular immunotherapies and basic and clinical immunology and will provide an outstanding environment in which to conduct the proposed research. Throughout the period of the award the candidate will be mentored by Dr. David Teachey and a very strong advisory committee including Drs. Ed Behrens, Marcela Maus, Wenchao Song and Stephan Grupp. The long-term goal of the candidate is to develop a research program devoted to understanding the role of the innate immune system in immune dysregulation related to cancer immunotherapies, and improving the health of patients by facilitating translation of less toxic, more effective immunotherapies. Completion of the proposed research project and the complementary training plan will provide a robust foundation to achieve this goal.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT In the United States, the prevalence of youth-onset type 2 diabetes (T2D) diagnosed in adolescents has nearly doubled in one decade and is projected to increase four-fold by 2050. Young adults less than 40 years old now represent 15-20% of adults with T2D. Early-onset T2D in adolescents and young adults (AYAs) is associated with earlier and higher rates of failed glycemic control, cardiovascular disease, microvascular complications, and mortality compared to T2D that develops after age 40 years old. Accelerated decline in b-cell function is thought to underlie the aggressive T2D phenotype in AYAs. The T2D glucagon-like peptide 1-receptor agonist therapies (GLP-1 RAs) achieve glycemic control by stimulating glucose-dependent insulin secretion. However, the real- world effectiveness and utilization of GLP-1 RAs in young adults with T2D compared to other second-line non- insulin diabetes therapies are unknown. Furthermore, recent GLP-1 RA shortages, known racial/ethnic disparities in access to novel diabetes medications, and the disproportionally increased risk of T2D among minority and socioeconomically disadvantaged AYAs, all raise additional concerns regarding AYA access to GLP-1 RAs. The overarching goal of this training program is to evaluate the real-world effectiveness and utilization of GLP-1 RAs in AYAs with T2D using the TriNetX network database, a large-scale electronic health record database. In Aim 1, a retrospective incident user cohort design will be used to emulate a target trial to compare the real-world effectiveness of GLP-1 RAs, sulfonylureas, dipeptidyl peptidase-4 inhibitors, and sodium- glucose cotransporter-2 inhibitors in achieving and maintaining glycemic control in young adults aged 18-30 years. In Aim 2, an interrupted time series will be used to examine changes in utilization of GLP-1 RAs in AYAs 10 to 30 years old since FDA approval of the GLP-1 RA, liraglutide, for adolescents in June 2019 and use multivariable logistic regression to identify patient characteristics associated with lower utilization of GLP-1 RAs. The proposed study will provide insights in how to best optimize treatment of T2D in AYAs and lead to future studies evaluating interventions to increase utilization of these treatments. This grant will provide Dr. Chu with valuable training in epidemiologic research methods. She will obtain intensive training in complex observational study design, biostatistics, and database management by accomplishing the above aims and through formal coursework as part of her Master of Science in Clinical Epidemiology degree program. Her research training will also involve mentors with complementary expertise in pharmacoepidemiology and patient-oriented research in diabetes who are focused on her career development into a physician-scientist. The data obtained from this proposal will inform the design of future studies in optimizing treatment and access of medications in AYAs with T2D to prevent long-term diabetes-related complications, which will be the focus of her K23 application.

NIH Research Projects · FY 2025 · 2024-07

Project Abstract Pediatric in-hospital cardiac arrest (IHCA) occurs in >15,000 children annually in the United States, most of whom do not survive to hospital discharge. As less than 5% of pediatric cardiopulmonary resuscitation (CPR) guideline recommendations are supported by high-quality evidence, there are significant knowledge gaps regarding optimal resuscitation techniques. Since pediatric IHCA represents a heterogenous process that results from the progression of many disease states, CPR physiology and the response to therapies also vary among patients. Therefore, our group has aimed to advance CPR beyond “one-size-fits-all” care and toward more patient-specific methods with an overarching objective of developing personalized, physiology-directed CPR strategies that account for both known patient characteristics and real-time physiology. A central component of in-hospital CPR is the administration of epinephrine, which is universally recommended during cardiac arrest to augment systemic vascular resistance, thereby increasing diastolic blood pressure (DBP) and coronary perfusion pressure to enhance the likelihood of return of spontaneous circulation (ROSC). Recent laboratory data and our 2023 clinical study identified substantial variability in the DBP response to epinephrine and found that a more robust increase in DBP after administration of the first dose of epinephrine is associated with higher rates of ROSC. As data are conflicting regarding ideal dosing strategies during pediatric IHCA, we postulate that epinephrine is beneficial during CPR in some clinical scenarios and potentially deleterious in others. Furthering our understanding of this variability in physiologic response will facilitate the development of methods of personalized, physiology-directed CPR to improve IHCA outcomes. Due to limitations in currently available datasets, a prospectively designed study is necessary to address this topic. Thus, we will leverage the existing infrastructure in 20 sites of the Pediatric Resuscitation Quality Collaborative (pediRES-Q), a network specifically designed to study IHCA, to prospectively collect extensive granular data including epinephrine dose timing and physiologic waveforms from bedside monitoring systems. In Aim 1, we will examine the physiologic response to epinephrine in greater detail and will build on exciting preliminary data to expand the applicability of this work to patients without invasive monitoring, investigating the pulse oximetry waveform as a non-invasive marker of CPR physiology and of epinephrine response in particular. In Aim 2, we will determine how the physiologic response to epinephrine impacts the relationship between epinephrine dosing strategies and outcomes. Finally, in Aim 3, through a well-established collaboration with machine learning experts, we will develop models to predict the response to epinephrine and subsequent CPR outcomes. The successful completion of these aims will expand our understanding of how epinephrine dosing and resuscitation in general can be tailored to individual patients and will facilitate the design of future interventional trials of physiology-directed CPR.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Pediatric cardiac arrest occurs in over 20,000 children annually in the United States, with mortality rates as high as 50%. In children who survive their arrest, post-cardiac arrest syndrome (PCAS) manifests as secondary organ injury with the return of circulation, including brain injury, ischemia-reperfusion, and myocardial dysfunction. Pediatric PCAS is relatively understudied, specifically in the domains of post-cardiac arrest myocardial dysfunction and systemic and cardiac inflammation. Preliminary data, though in small cohorts and retrospective in nature, have demonstrated associations between myocardial dysfunction and elevated markers of inflammation and injury with post-cardiac arrest mortality. This K23 proposal leverages the robust research infrastructure within the Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (UPENN) to conduct a larger, single-center prospective observational study in a total of 200 critically ill children after cardiac arrest. Advanced echocardiographic imaging after arrest will include global longitudinal strain (GLS), a more sensitive measure of myocardial dysfunction compared to standard measures of ejection and shortening fraction. Systemic and myocardial inflammation will be assessed early after cardiac arrest by circulating biomarkers in a customized panel of proteins associated with outcomes in other disease processes. These measures will be individually examined for their association with hospital mortality, and survival to hospital discharge with unfavorable neurologic outcome. Together, with clinical data, these measures will be assessed with latent cluster analysis to create pediatric PCAS phenotypes which will be distinctly associated with outcomes. This phenotyping will allow for better understanding of pediatric PCAS to allow for trial enrichment in future interventional studies. The principal investigator of this study, Dr. Monique Gardner, is uniquely positioned to complete this study with her training in pediatrics, pediatric cardiology, and pediatric critical care, as well as her experience to date with research in critically ill children with cardiac disease. Supported by her mentorship team and research environment, she will leverage the opportunity to study advanced biostatistical techniques, echocardiography, and biomarker assessment to mature into an independent patient-oriented clinician-scientist to improve outcomes for critically ill children.

NIH Research Projects · FY 2023 · 2024-07

Abstract Craniofacial abnormalities are some of the most commonly occurring human birth defects worldwide, with up to 200,000 children born every year with some type of craniofacial defect. These defects can occur as part of complex syndromes that involve multiple tissues and organs. The syndromic forms of these disorders have been successfully linked to nearly 500 genes including TWIST1 for craniosynostosis and IRF6 for orofacial clefting. However more frequently no other part of the body is directly involved (50% of craniosynostoses, 70% of orofacial clefts). Genome wide association studies indicate heritability for such defects, however the vast majority of associations fall outside of genes suggesting defective gene regulation is a major contributor to incidence of such defects. Gene regulatory elements can be located throughout the genome and typically have tissue-specific activity, making them difficult to identify and predict what gene they control. The overall objective of this application is to identify the cell types present in the developing human face and functionally characterize important regulatory elements that specify them as single base pair resolution. In Aim 1 we propose to systematically identify populations of cell types using single-cell based methods for measuring the transcriptome and active regulatory sites across the genome in human craniofacial tissue from 4 to 5 weeks of gestation and mouse from embryonic days 10.5 and 11.5. In Aim 2 we propose to identify physical interactions between regulatory sequences and target genes in these same tissue types. Finally, in Aim 3 we will identify regulatory elements from these tissues that can be tested in a cell culture model of cranial neural crest cells. These enhancers will be assessed for effects on gene expression when repressed or removed from the genome. Those with significant effects on gene expression will be tested for every variant to identify important locations within them. Our proposed studies will generate the most comprehensive view of the cell types active in the developing human face and reveal the contributions individual noncoding variants make on gene expression.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY 300,000 Americans suffer paralysis from spinal cord axon injury. Glia permit axon regeneration by clearing debris, partly through autophagy. Inappropriate release of glial debris can inhibit axon regeneration. To treat axon injuries, it is essential to understand mechanisms of glial recovery after axon injury, permitting glia to facilitate axon regeneration. Larval fly sensory neurons are a genetically-tractable model for axon regeneration in the mammalian peripheral nervous system, with regrowth dependent on wrapping glia. In my postdoctoral experiments, I have discovered that fly sensory glia release large exopher-like vesicles (ELVs) (~3 µm) at the injury site, a phenomenon previously undescribed in glia. Glia also display membrane damage and subsequent repair at the injury site, and loss of wound healing pathway components results in diminished axon regeneration. These data suggest that glia may adapt to the stress of axon injury through ELV release and membrane repair. Exophers, large (~3 µm) vesicles so far documented in C. elegans neurons and mouse cardiomyocytes, sequester oxidatively-stressed mitochondria for release. In mice, the autophagic pathway is required to produce exophers. My preliminary experiments in fly larvae showed loss of an essential autophagy regulator results in smaller glial ELVs and reduced axon regeneration. Together, these data suggest that the wound healing and autophagic pathways are important for glial ELV production and axon regeneration. As previous work in exophers revealed modes of stress recovery, understanding the origins of fly glial ELVs could uncover novel mechanisms of glial recovery from the stress of axotomy, likely necessary for glia to facilitate axon regeneration. This proposal aims to define the origins of this novel glial ELV response and its effects on axon regeneration. Aim1 of this proposal will identify the cell responsible for autophagy-mediated glial ELV release and axon regeneration by utilizing glial- and neuronal-specific RNAi knock-downs. Furthermore, Aim1 will use electron microscopy and fluorescent reporters to determine if ELVs isolate stressed mitochondria, akin to exophers, in an autophagy-dependent manner. Aim 2 of this proposal will identify the cell-responsible for wound-healing mediated axon regeneration, interactions between wound-healing and autophagic pathways, and effects of the wound-healing pathway and extrinsic signals on glial ELV release. This will be achieved via glial- and neuronal- specific RNAi knock-down constructs for components of the wound healing pathway, antibody staining for wound healing components, and a genetic system to simulate axotomy without physical injury. This proposal will uncover novel mechanisms of glial recovery and glial facilitation of axon regeneration. These studies may help inform and improve methods to drive human axon regeneration in the CNS and PNS, aligning with the mission of NINDS. With the expertise of the Song lab and my postdoctoral advisory team, I will gain a comprehensive understanding of the relevant cell biology fields and necessary technical skills, uniquely positioning me to tackle my scientific proposal. I will expand these projects in my own research group as an independent investigator.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY CD4+ T cells (TCD4) orchestrate adaptive immune responses to infectious agents and cancers and instigate most autoimmune diseases. Thus, greater mechanistic insight into the drivers of TCD4 activation will be broadly bene- ficial. TCD4 are stimulated by antigen-derived peptides (“epitopes”) displayed at the surfaces of antigen-presenting cells (APCs) in combination with major histocompatibility complex class II (MHCII) molecules. By convention, peptide display entails internalization and proteolysis of extracellular antigen, loading of the resultant peptides onto MHCII in the late endosome, and transit of peptide:MHCII (p:MHCII) complexes to the cell surface. In truth, p:MHCII production is far more complex. The classical route is made up of many distinct pathways, and there are 3 multifaceted, nonclassical routes: 1) a recycling route: antigens that unfold in the early endosome are captured by MHCII in that compartment. 2) an endogenous network: antigens located to the APC cytoplasm, typically through biosynthesis, are converted to p:MHCII via an array of intracellular pathways, and 3) an “indi- rect” route: material from an infected non-APC is transferred to an uninfected APC. Despite their obscurity, these non-classical routes can play essential roles in TCD4 activation. Poor mechanistic understanding of all 4 routes, even the classical, has precluded their incorporation into many models of adaptive immunity or strategies to modulate TCD4+ responses. Here we propose to transform the MHCII processing and presentation landscape by testing the hypotheses that: (A) the degree to which cellular components contribute to epitope production and display is highly variable - from far reaching (impacting many epitopes) to highly focused (impacting only one epitope or a small set), and (B) processing and presentation pathways within one route or within functionally adjacent routes (e.g., classical and recycling), share more cellular components than disparate routes (e.g., clas- sical and endogenous). Drawing from our influenza (flu) antigen system, we have carried out an siRNA-based high throughput screen (HTS) for proteins involved in generation of one classical (“S1”) and one endogenous (“NA79”) epitope. Consistent with our hypothesis, hits are largely non-overlapping. Aim 1 of this project is to solidify this screen by validating key hits. Aim 2 is to expand the landscape by screening 6 flu epitopes that comprehensively encompass the 4 major routes. Instead of an siRNA screen, in Aim 2 we will use genome-wide CRISPR-Cas9 knockout and select hits via FACS using a panel of T cell receptor-like antibodies in concert with single-cell illumina sequencing. Outcomes from both aims will contribute to Gene Set Enrichment Analysis that will substantially revise the MHCII processing and presentation landscape with respect to both scope and reso- lution. Outcomes will launch many new avenues of investigation that explore: a) additional epitopes from flu and other pathogens, b) mechanisms underlying key hits and pathways, c) the impact on TCD4 activation of knocking out, in vivo, high priority hits, and d) development of therapeutics (e.g., small molecules) to enhance vaccine efficacy and anti-cancer immunity, and to diminish autoimmune diseases.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Long term survival of pediatric heart transplant (HT) patients remains unacceptably low and is driven in part by modifiable cardiovascular risk factors. Children with HT engage in low levels of moderate to vigorous physical activity (MVPA), which is a major health problem for a population already at increased cardiovascular risk and further contributes to their morbidity and mortality. Increasing MVPA may be a critical way to improve cardiovascular health, quality of life, and survival in this population. While environmental exposures are associated with physical activity in children without HT, their effects in those with HT are unknown, and likely different. To design effective, equitable, generalizable, and sustainable interventions to increase MVPA, we need to recognize the environmental determinants of MVPA and involve stakeholders in intervention development. Jonathan Edelson, MD, is a pediatric cardiologist and physician-scientist passionate about understanding how physical activity can improve outcomes of children after HT. With a mentoring team specializing in implementation science, Dr. Edelson will 1) develop expertise in mobile health technology and accelerometry; 2) hone the skills to design, launch, oversee, and complete a multisite, prospective interventional study; and 3) gain methodologic expertise in geospatial and qualitative methodology. This study proposes to leverage Dr. Edelson's existing mobile health studies of pediatric HT patients to evaluate the macro and micro environmental determinants of MVPA in pediatric HT and to pilot an exercise intervention using the Multiphase Optimization Strategy (MOST). Specific Aims are to 1) Determine the association of the macro-environment with MVPA in a socioeconomically diverse cohort of children after HT; 2) Use qualitative methodology to identify micro-environmental determinants of participation in physical activity in pediatric HT patients; 3) Determine the feasibility and preliminary efficacy of a digital exercise program in pediatric HT patients. Dr. Edelson will map participant addresses to neighborhood-level built and natural environmental exposures to estimate the association of MVPA patterns with macro environmental factors. He will then conduct semi-structured interviews informed by the COM-B (Capability, Opportunity, Motivation, Behavior) implementation science framework with patients and their families to identify determinants of physical activity and desirable components of a mobile health exercise intervention. Finally, he will apply the knowledge gained to the development and implementation of a prospective pilot longitudinal exercise intervention using MOST, a strategy for developing multi-component intervention which has successfully informed physical activity interventions. This K23 award will support Dr. Edelson's pathway to becoming an independent investigator in the interventional space with skills that will support future R level proposals using exercise in a large multisite cohort to improve long-term outcomes. This work will provide a novel framework for exercise interventions that will be applicable to a range of populations, further highlighting the public health significance of this proposal.

NIH Research Projects · FY 2025 · 2024-05

Project Summary/Abstract Leukodystrophies are a collection of rare genetic disorders that affect cerebral myelin development. Exciting recent therapeutic advances, including gene therapy development for adrenoleukodystrophy and metachromatic leukodystrophy, underscore the critical unmet need for therapeutic development more broadly across the leukodystrophies. The future of innovation, however, is limited by the pipeline of future translational researchers interested in rare diseases. The Rare Diseases Clinical Research Network (RDCRN) Global Leukodystrophy Initiative Clinical Trials Network (GLIA-CTN; U54TR002823) is an NIH-funded research consortium for leukodystrophy collaboration and innovation across a network of 8 large US-based academic institutions. This infrastructure provides a unique opportunity to recruit and support the next generation of rare disease research leaders. We hypothesize that a formal education program in rare disease research targeting junior research coordinators who are early in their career will facilitate long-term commitment and passion for rare disease research. This is a population that is enriched with young scholars interested in science who are often undecided about career paths or have had insufficient exposure to translational research. The proposed program represents a novel collaboration between the Leukodystrophy Center of Excellence (LCE) at the Children’s Hospital of Philadelphia (CHOP), a GLIA-CTN site, and the Institute for Translational Medicine and Therapeutics Education (ITMAT Ed) programs at the Perelman School of Medicine, University of Pennsylvania. In 2021-2022, the LCE and ITMAT Ed piloted the Predoctoral Preparatory Program (P3) with 8 junior research coordinators, known as P3 scholars, based exclusively within the Children’s Hospital of Philadelphia (CHOP). In 2022-2023, the program expanded to include 14 coordinators working at sites across the GLIA-CTN. Each year we aim to enroll 12-15 scholars who are junior research coordinators working within the GLIA-CTN. We recognize the importance of diversity among future translational research leaders and will continue to recruit from populations underrepresented in medicine (UIM). In this application, we propose an education program, the Predoctoral Preparatory Program (P3), which will (1) provide a curriculum related to responsible conduct of research and (2) prepare the next generation of physician scientists in translational research through professional development workshops and mentorship. This proposal is to further the development and implementation of a one-year program focused on research and professional skills development, community building, and mentorship which is fully integrated into a hands-on research experience through the GLIA-CTN research network.

NIH Research Projects · FY 2026 · 2024-05

Abstract Understanding how T cell receptors (TCRs) see tumor antigens presented by MHCs is necessary to fully understand how the immune system recognizes tumor antigens, and to reap the full potential of antigen-specific immunotherapy. To achieve this goal, a quantum leap forward is required in which the revolutionary advances in machine learning are combined with a large volume of structure, function, data on matched TCR-pMHC pairs. The development of accurate predictors of TCR-antigen recognition will be dependent on the creation and integration sequencing-based datasets with high-throughput structural and functional insights. Our proposal, submitted as a CRUK/NCI Grand Challenge team (MATCHMAKERS) will combine researchers with expertise in immunology, methods development, structural biology, and computation to enable generalized prediction and design of TCR recognition. This work will be spread across four Work Packages (WPs): WP1: Large-scale generation of TCR-pMHC pairs from naturally occurring sources. We will build datasets of naturally occurring TCR-pMHC pairs. Our team will use an array of approaches to collect these datasets, from humans and from mouse models, and in the context of both cancer and immunity more generally. WP2: Ultra-high throughput TCR-pMHC matching using molecular engineering. Efforts to create general models will require a broader array of data than feasible to collect from natural TCR systems. We will use an array of synthetic approaches developed by our team to comprehensively match TCRs with pMHCs to train computational models. WP3: Large-scale structural and biochemical analyses of TCR-pMHC interactions. A key to our team's vision is to match interaction datasets with high throughput structural and functional insights. A deep understanding of how the TCR contacts with MHC helices control function and orientation will be essential for training and testing computational models. WP4 AI-based prediction and design of TCR-pMHC interactions. We will integrate our data to train next- generation algorithms capable of generally predicting and designing TCR-pMHC interactions. These predictions will proceed through a reiterative testing and feedback circuits for further model optimization.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY/ABSTRACT Lower urinary tract symptoms (LUTS) and disorders of voiding are among the most common reasons for referral to the urologist and affect ~20% of school-aged children. LUTS can be caused by one of several different LUT conditions, including voiding postponement, and the prevalence of these conditions varies by sex. Neuropsychiatric comorbidities, especially depression and anxiety, occur in 20-40% of children with LUTS and school bullying can lead to bladder holding. Despite the known sex differences in terms of psychosocial factors and underlying LUTS etiology, almost all basic science research on LUTS and stress is limited by male only animal models. Chronic social defeat stress (SDS) in male mice leads to a voiding phenotype similar to that seen in children with voiding postponement, as well as to increased expression of the stress neuropeptide corticotropin releasing hormone (CRH) in Barrington's nucleus (BN), the brainstem region considered the voiding “command center.” Data from the candidate's NIDDK K08 aims identified a sexual dimorphism in voiding phenotype changes when the CRH neurons in BN are stimulated: female mice bladder sizes increase ~2x the amount seen in males. These clinical and basic science data fuel this R03's central hypotheses: that SDS will induce sex- specific voiding, bladder, and brain phenotypes and that gonadal hormones are required for these changes. The proposed research is an extension of the candidate's K08 grant conducted in primary mentor Dr. Stephen Zderic's laboratory using mice and will facilitate the candidate's research independence. In Aim 1, we will establish sex-specific effects of a novel chronic non-discriminatory social defeat stress (CNSDS) paradigm on bladder voiding, histology, and contractility, and on neurohormone and receptor expression in BN, a brain region that the candidate has studied extensively in his K08 grant. Through collaboration with Drs. Eisch and Yun (CHOP neuroscientists), the candidate will gain new skillsets in chronic social defeat stress models and behavioral assays in mice. CNSDS allows study of both male and female mice exposed to identical stress and will help overcome the knowledge gap that exists on sex-specific SDS effects on LUT dysfunction. In Aim 2, we will determine the effects of gonadectomy in female and male mice on CNSDS susceptibility to bladder and brain changes. Female and male mice will undergo gonadectomy prior to CNSDS followed by bladder and brain measures as in Aim 1. Successful completion of these Aims will result in the first characterization of an altered voiding phenotype in socially-stressed female mice, allow direct comparison with male mice, define neurohormone changes in BN, and reveal if gonadal hormones play a role in sex-specific stress-induced bladder voiding phenotype. As such, the data lay critical groundwork for future translational assessment of sex-specific LUTS and their personalized treatment in clinical populations. As a supplement to the candidate's K08 grant, these aims will provide preliminary data to help launch an R01 application and formalize collaborations with Drs. Eisch and Yun, who will support the candidate's research independence.

NIH Research Projects · FY 2026 · 2024-05

ABSTRACT Red blood cell transfusion remains a life-saving therapy for patients with sickle cell disease (SCD). A major problem is the high rate of alloimmunization (antibody formation against transfused red cells) that occurs in transfused patients with SCD. Alloimmunization leads to delays in care, increases costs, and makes transfusion therapy unsafe and impossible for some patients. The most common antibodies formed by patients with SCD are directed against the Rh blood group system. Studies performed by our group and others demonstrate RH genetic variants in patients with SCD and Black donors is a major risk factor leading to the high incidence of Rh alloimmunization, and the complexity of Rh antibody identification. Inheritance of variant RH alleles explains approximately one-third of Rh antibodies formed by patients with SCD. The remainder are stimulated by altered Rh proteins on Black donor RBCs, who share similar RH genetic heterogeneity. Recruitment of Black donors is necessary to support provision of C, E, K negative units for patients with SCD. In a pilot study, we demonstrated the feasibility of identifying and transfusing >300 RH genotype matched donor units to chronically transfused patients with SCD. Further studies are now required to demonstrate efficacy in preventing Rh alloimmunization. In addition, a higher-throughput RH genotyping method and interpretation are needed to facilitate testing donors, which are major barriers to widespread implementation. Lastly, the ability to identify the specific altered Rh protein that is the target of the patient's immune response and responsible for incompatibility is critical for selection of donor units. Precise identification of Rh epitopes responsible for antibody reactivity is hampered by the lack of appropriate reagent RBCs with uncommon Rh antigen expression. The major goal of this proposal is to provide a precision medicine approach to transfusion therapy for SCD by 1) providing RH genotype matched red cells to chronically transfused patients with SCD and determine the effect on Rh antibody formation, 2) to apply new next generation sequencing approaches and data analysis pipelines to identify a low-cost method to accurately identify the complex RH genotypes in African Blacks, and 3) genetically engineer induced pluripotent stem cells to generate red cells that express variant Rh proteins for precise antibody identification. In this application, we propose three integrated aims that can address the current challenges of transfusion therapy for SCD and will drive the field forward by providing innovative solutions to precisely match blood.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Human immunodeficiency virus (HIV) infections are dramatically climbing in adolescents, who are infected during a time of critical brain development characterized by significant myelination. Adolescents may therefore be particularly vulnerable to the white matter pathologies closely linked to HIV-associated neurocognitive disorders (HAND), which affect 30-50% of people living with HIV. Understanding the mechanisms resulting in HIV-related white matter deficits will be key in the development of therapies to promote myelin integrity and repair, which may greatly improve neurological function. One important factor predictive of both cognitive decline in HIV and a lack of myelin is altered brain lipid metabolism. Myelin is highly enriched in cholesterol and lipids, and both oligodendrocytes, the myelin-producing glial cells of the central nervous system, and astrocytes must synthesize large quantities of cholesterol and lipids for successful myelination to occur. Based on published and preliminary data, I hypothesize that glial lipid biosynthesis and transport essential for adolescent myelination are dysregulated by HIV or DTG/TDF/FTC antiretroviral therapy (ART), making lipid metabolism a potential therapeutic target for improving white matter integrity in adolescents living with HIV. To address this hypothesis, I will determine how HIV and ART affect 1) oligodendroglial lipid metabolism and resulting myelin lipid composition and adolescent myelination, and 2) astroglial lipid metabolism and release and its impact on myelin formation. Successful completion of the proposed aims will reveal whether HIV or ART affect lipid metabolism in either oligodendrocytes and/or astrocytes and whether this is detrimental to adolescent myelination. Results from the proposed studies will directly advance the mission of the NIMH by transforming our understanding of mechanisms of white matter pathology in adolescent HIV/HAND, revealing potential therapeutic targets for prevention of and recovery from HIV-related neuropathology.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Antibiotics are the most prescribed medication in neonatal intensive care units (NICU). Antibiotic stewardship efforts have reduced overall antibiotic use in the NICU, but preterm and low birth weight infants have not benefited proportionately. Preterm infants (≤35 week gestation) constitute ~45% of all NICU admissions, are admitted for extended periods, are frequently exposed to antibiotics, are at higher risk for antibiotic side effects and serve as reservoirs for multi-drug resistant pathogens. Thus, interventions to optimize antibiotic use in preterm infants can reduce unit-level selection pressure and improve patient safety. Despite recommendations from the American Academy of Pediatrics against its empirical use in the NICU, vancomycin is the most common antibiotic administered to preterm infants after 3 days of age, largely as empiric therapy for suspected late-onset sepsis (LOS). Studies by us and others demonstrate that use of vancomycin reducing protocols (VRP), designed to encourage the use of narrower spectrum antibiotics as first-line empiric and provide guidance for when vancomycin is an appropriate choice, can safely reduce vancomycin use in the NICU. Yet these regimens are not universally adopted, and when adopted are not universally applied, highlighting the need for more comprehensive strategies to implement change. Our long-term goal is to identify and promote practices that optimize antibiotic use in neonates. Our broad objective is to implement a VRP in NICUs for safe and sustained reduction of unnecessary vancomycin use in preterm infants (≤35 weeks). We hypothesize that VRP implemented by preparing local champions, educational outreach, introduction of a clinical decision support tool and providing unit-level audit and feedback will reduce vancomycin use (Aim 1); and that the use of additional external facilitation will improve fidelity and acceptability of VRP use (Aim 2). We used the updated Consolidated Framework for Implementation Research (CFIR) to design this study and simultaneously use CFIR constructs to understand barriers and facilitators of VRP use (Aim 3). We will comprehensively examine antibiotic use in the context of VRP implementation including evaluation of implementation outcomes, disparity drivers, and parent and clinician perspectives. We will conduct this study in 13 NICUs across two large healthcare networks. Our specific aims are 1) To reduce unnecessary vancomycin use in preterm infants by implementing a VRP in a quasi-experimental trial; 2) To determine the effect of external facilitation on the fidelity of using VRP and other implementation outcomes in a cluster randomized trial; and 3) To identify barriers and facilitators to implementation of the VRP across NICUs. By using an implementation science approach to reduce vancomycin in a multicenter study we will provide a road map for the broader implementation of VRP across NICUs in the United States. Additionally, our study will provide key insights into the factors influencing prescribing behavior in the NICU that can inform future antibiotic stewardship efforts in this high-risk setting.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Skeletal growth occurs during development in cartilage growth plates, bone template regions wherein chondrocytes actively proliferate, follow a stepwise maturation program, and produce an abundant extracellular matrix. Their activities are tightly regulated by numerous factors, including the fibroblast growth factor receptor- 3 (FGFR3), whose importance in humans is illustrated by the fact that FGFR3 gain-of-function variants (FGFR3Ach) cause achondroplasia (ACH), the most frequent form of short stature. The disorder markedly affects the appendicular skeleton (short limbs), axial skeleton (vertebral stenosis and lordosis) and craniofacial skeleton (frontal bossing and flat face) and also results in a large spectrum of neurologic issues. Various types of surgical and pharmacological treatments have been proposed and approved for this condition, but none addresses all clinical issues, and long-term outcomes are unknown. There is thus an urgent clinical need to develop new, safe and efficient treatments for ACH. We propose to help address this need by deepening our current understanding of the pathways acting upstream and downstream of FGFR3 in growth plate chondrocytes (GPCs) and by testing in preclinical models if targeting newly identified pathways may provide successful treatments. Aim 1 will test the hypothesis that the expression of FGFR3 is controlled by several cis-acting regulatory elements (CREs) and that the inactivation of key CREs could safely and effectively lessen the severity of ACH. This hypothesis and our research strategy rely on a solid scientific premise constituted by published and pilot data. We will create and analyze CRE reporter transgenic mice to determine to which extent individual CREs reproduce the expression pattern of Fgfr3. We will also determine which CREs most significantly contribute to Fgfr3 expression in mice by deleting the most likely ones in the Fgfr3 wild-type and ACH alleles and assessing phenotypic consequences. Aim 2 will test the hypothesis that FGFR3Ach perturbs a pathway critical to ensure the high-energy demands of GPC activities and whose link to FGFR3Ach was uncovered in pilot studies. We will analyze to which extent this pathway is perturbed and affects GPC activities in ACH. Further, we will use transgenic approaches to test if normalizing this pathway may safely and effectively lessen ACH features. This project is highly significant and innovative considering on the one hand the prevalence and severity of ACH and the current lack of fully satisfying treatments, and considering on the other hand that it will push forward the frontiers of scientific knowledge to propose novel, potentially life-changing strategies to treat people with ACH, and may also suggest novel mechanisms and treatments for other chondrodysplasias and still poorly treatable FGFR3-related cancers.

NIH Research Projects · FY 2025 · 2024-04

Project Summary/Abstract Children with medical complexity (CMC) are an extremely vulnerable population with significant morbidity and mortality risk. To facilitate the study of CMC, Complex Chronic Conditions (CCCs) were defined in 2000 as a set of ICD-9-CM codes specified as “CCCs” with categories (such as neurologic, cardiovascular, metabolic, and other congenital or genetic defect). Three important concerns regarding the widely used CCC system motivate this proposal. First, the CCC system is now used “off-label” for different tasks including adjustment of health status in studies not specifically focused on CMC, and for the identification of children who might benefit from comprehensive complex care services. Accordingly, the CCC system needs to be evaluated regarding performance accuracy for various tasks. Second, the CCC system needs to be assessed for potential embedded associations between ICD codes and patients’ socioeconomic status (which, for historical reasons, is associated with the structural bias markers of race and ethnicity), arising from differential access to healthcare and diagnostic testing, as well as genetic conditions related to genetic ancestry. Embedded associations might bias the aggregated CCC system, affecting the identification of or adjustment for medical complexity. Third, these two concerns must be considered jointly: the CCC system might be accurate but, due to any embedded associations, perpetuates disparities. To address these concerns, we propose the CCC EQUATE (Equitable Quantification And Task Evaluation) project. We will create scenarios using two data sources, Medicaid claims data and 52 children’s hospitals inpatient data. We will focus on the representative outcomes of hospitalizations (including readmissions) and mortality (specifically, inpatient mortality). Specifically, we aim to: Aim 1. Evaluate the performance of the CCC system across settings for these specific outcomes. Aim 2. Quantify the degree of embedded associations between ICD codes and patients’ race and ethnicity. Aim 3. Quantify potential perpetuation of bias in CCC system performance due to embedded associations in ICD codes. In sum, this project will safeguard against the CCC system having subpar task performance in certain settings for specific outcomes, and from inadvertently perpetuating bias. Furthermore, the methods and findings will be generalizable to all ICD-code based classification systems. Ultimately, the project will enhance equity, enable future research, and help improve care for CMC and their families.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY Children with high-risk neuroblastoma (NB) have a significant risk of treatment failure and death emphasizing the need to develop novel targeted therapies, which constitutes the long-term goal of this proposal. The proposed research focuses on the oncoprotein glypican-2 (GPC2), which is glycophosphatidylinositol (GPI)-linked to the cell surface of NBs and other cancers but is not found on vital normal tissues. To capitalize on GPC2’s differential expression, we developed GPC2 chimeric antigen receptor (CAR) T cells and performed Investigational New Drug (IND) application-enabling studies in NB showing that GPC2 CARs induce safe and robust tumor regression. These data led directly to the development of a first-in-human GPC2 CAR T cell Phase 1 clinical trial that opened in May 2023. However, NB cells can escape GPC2 CAR pressure leading to tumor relapse and limiting long-term efficacy. Thus, there is an urgent need to identify the mechanisms of GPC2 CAR T cell resistance in NB and develop next-generation CAR approaches to combat these processes. Towards this goal, our recent profiling of tumors that escaped GPC2 CAR pressure showed that NB relapse is associated with GPC2 downregulation and conversely upregulation of the GPI-cleaving angiotensin-converting enzyme (ACE). We have also found that GPC2 is a major component of NB-associated extracellular vesicles (EVs), modulation of which can significantly change GPC2 cell surface density. Together, our published and unpublished preliminary data suggest the following central hypothesis: A major mechanism of GPC2 CAR T cell resistance in NB is downregulation of cell surface GPC2 driven both by ACE-induced GPC2 cleavage and increased shedding of GPC2+ EVs and can be circumvented by a new bicistronic CAR dual antigen targeting approach. We propose to test this original concept in two integrated but independent Specific Aims. In Aim 1, we focus on validating GPC2 cell surface downregulation as a major mechanism of GPC2 CAR resistance in NB, including defining the role of ACE-induced GPC2 cleavage and the dynamic modulation of GPC2+ EVs under GPC2 CAR pressure. In Aim 2, we will define the efficacy and safety of a new CAR T cell dual antigen targeting approach we designed to overcome GPC2 CAR escape in NB; GPC2 CAR T cells simultaneously targeting the disialoganglioside GD2 via a secreted bispecific innate immune cell engager (BiCE) that also binds CD16a on NK cells and macrophages to activate antitumor innate immunity in a GPC2-independent manner. This innovative CAR.BiCE therapeutic approach synergizes dual antigen targeting and activating adaptive and innate immunity. Our results will yield critical insights into CAR T cell resistance and these new immunotherapeutic principles and technology can be applied to other potent CARs where antigen escape limits long-term efficacy. These studies will also produce IND application-enabling data for the clinical translational of a CAR.BiCE therapeutic approach to develop desperately needed new targeted therapies for children with NB.