Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2025 · 2021-02

Project Summary Drugs that inhibit Bcl2-family survival proteins promise to change the landscape of cancer care. Cancers activate stress signals in the form of BH3-domain proteins like Bim as they escape cellular growth constraints and invade hostile environments. To remain viable, tumors use survival proteins like Bcl2, Bclx, Bclw and Mcl1 to sequester these BH3 proteins through specific protein-protein interactions (PPIs). This blocks their apoptotic signal but also renders such tumors continually dependent on this function. Drugs that competitively displace BH3 proteins from the survival protein sequestering them unleash a potent apoptotic signal. Indeed, venetoclax is a Bcl2 inhibitor that has demonstrated striking clinical efficacy, garnering FDA-approval for the treatment of chronic lymphocytic and adult myelogenous leukemias.These tumor types do not have Bcl2-activatingmutations but are empirically defined to be dependent on Bcl2 for their survival. In contrast, many solid tumors have heterogeneity in which survival protein they use to block BH3 signals, and the absence of biomarkers that predict sensitivity to this emerging drug class remains a barrier. Our objectives are to identify predictive biomarkers and develop diagnostic tools to leverage Bcl2-family inhibitors for clinical use. We created a national infrastructure to generate patient-derivedxenograftand cell line models of the lethal childhood tumor, neuroblastoma, and utilized innovative functional assays to define the Bcl2-family protein they depend on for survival. We discovered that neuroblastomas have endogenously activated Bim neutralized through a PPI with a single dominant survival protein. This sequesters Bim's apoptotic activity, but also encodes a continual dependency, and defines a mechanistic biomarker defining the survival protein required for viability. An unanticipated finding is that this survival dependency is highly cancer cell intrinsic and stable: consistent in patient-matched tumors from primary and metastatic sites, and at diagnosis and relapse. In tumors with Bim bound by Bcl2 (Bim:Bcl2 PPI), Bcl2 inhibitors like venetoclax are highly active in vitro and in vivo. In tumors with Bim bound by Mcl1 (Bim:Mcl1 PPI), Bcl2 inhibitors have no activity. Further, we find that all neuroblastomas with MAPK pathway mutations are in the Bim:Mcl1 class. Surprisingly, Mcl1 inhibitors also have no activity for this subset, despite displacing Bim from Mcl1. In these tumors, Bim is re-sequestered by Bclx, and all are exquisitely sensitive to combined Mcl1/Bclx inhibition. This demonstrates the robustness of our predictive Bim PPI biomarker that we will exploit to identify all survival dependency classes in neuroblastoma. We will define the extent to which the biomarker remains a stable intrinsic tumor feature that predicts selective vulnerability to Bcl2-family inhibitors. We also leverage therapeutic MAPK inhibitors to antagonize Mcl1 dependency. Importantly, we seek to develop in vitro diagnostic tools to identify predictive BimPPIs using proximity-ligation assays, and to credential genomic MAPK biomarkers to define Mcl1 dependence. Collectively, our work applies precision medicine approaches to assign Bcl2-family inhibitors for clinical use, and inform clinical trial designs, including predicting rational combination therapies.

NIH Research Projects · FY 2025 · 2021-01

Project Abstract Overtreatment – health care in which the benefit does not outweigh risks – accounts for up to $200 billion annually in the United States, and is associated with worse outcomes and death. Unnecessary use of diagnostic testing is a primary driver of overtreatment. Diagnosing and treating suspected sepsis exemplifies this challenge. In hospitalized children, severe sepsis is common (8.2% prevalence) and deadly (25% mortality rate). Pediatric hospitals prioritize early sepsis recognition and rapid antibiotic administration as key performance metrics. Signs and symptoms of sepsis in children, however, are non-specific. The resulting diagnostic uncertainty may lead to harmful overtreatment. Blood cultures are the gold standard for diagnosing sepsis due to bacteremia. Frequently, clinicians obtain blood cultures and simultaneously start empiric broad-spectrum antibiotics. Only 5-15% of cultures will be positive, however, and up to 50% of those are actually falsely positive. False positive results lead to unnecessary antibiotics, increased lengths of stay, and increased costs. Reduction in unnecessary blood cultures in pediatric intensive care unit patients is feasible and safe, but current practice patterns for blood culture use vary widely, and culture use may be driven by reflexive behavior and fear of missing sepsis. With my Primary Mentor, I have been leading an AHRQ-funded 14-site PICU blood culture quality improvement collaborative called Bright STAR (R18 HS025642-01) since 2017. I will now leverage Bright STAR to 1) investigate what leads to blood culture overuse in the PICU, and 2) develop and test strategies to safely reduce blood culture overuse. I will use the Consolidated Framework for Implementation Research, the concept of cognitive bias, and the Proctor implementation framework to accomplish these objectives, in a series of qualitative, quantitative, and quasi-experimental research investigations. This proposal will give me experience in: applying frameworks of implementation/behavioral science, conducting mixed methods studies, designing implementation strategies, and conducting a clinical trial. These skills are critical to achieving my long term goal: to design and test strategies to safely reduce harmful overuse of other unnecessary diagnostic tests (and consequently, unnecessary treatments) in critically ill children. I will be mentored by a team of highly funded, successful researchers with expertise in infectious diseases, implementation science, behavioral science, trial design, and mixed methods research. I will have the full support of the Division of Critical Care at The Children's Hospital of Philadelphia and the University of Pennsylvania, with access to innumerable resources (such as the Penn Implementation Science Center, the Center for Pediatric Clinical Effectiveness research, and the Penn Mixed-Methods Laboratory) to help me accomplish my aims. I will use this award to become an independent implementation scientist improving patient outcomes by reducing harmful overuse of unnecessary tests in critically ill children on a large scale.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY Recently-described SCN3A-related neurodevelopmental disorder (SCN3A-NDD) is caused by pathogenic variants in the gene SCN3A, which encodes the sodium (Na+) channel subunit Nav1.3. SCN3A-NDD is a devastating condition defined by treatment-resistant epilepsy and severe/profound intellectual disability (ID); surprisingly, many patients also exhibit malformation of cortical development (MCD), a developmental disturbance in the structural formation of the cerebral cortex of the brain, suggesting functional roles for Nav1.3 during embryological development. How genetic variants in SCN3A leads to epilepsy and neurodevelopmental disability, and how SCN3A variants lead to MCD, is unknown. Research is required to clarify the functional role of Nav1.3 during early brain development and to progress towards novel therapies or preventative measures for SCN3A-NDD, which is currently and untreatable disorder. This 5-year collaborative application employs novel tools and innovative neuroscience approaches to test the hypothesis that pathogenic variants in SCN3A lead to a disorder that includes epilepsy and MCD via dysregulated Na+ currents in migrating neurons of the developing cerebral cortex. Electrophysiological recordings in heterologous cell systems indicate that pathogenic SCN3A variants found in patients with SCN3A-NDD largely produce Na+ channels that exhibit gain of function due to increased persistent current and alterations in the voltage dependence of channel activation, which increase channel activity. However, the mechanistic basis of observed variability in epilepsy severity and presence or absence of MCD, is unclear. And how altered channel activity impacts the function of neurons has not been investigated. Proposed experiments will determine the relationship between specific SCN3A variants and correlated clinical phenotype (epilepsy, MCD, severity of ID) in a large cohort of human patients with SCN3A-NDD. To link SCN3A variants to dysfunction of ion channels and neurons, we will compare the biophysical properties of normal Na+ channels to channels containing variant Nav1.3; test cell-intrinsic effects of SCN3A variants in neurons generated from induced pluripotent stem cells from human SCN3A-NDD patients; and test effects of variant overexpression via in utero electroporation of mouse embryo followed by electrical recording in brain slices (Aim 1). The impact of variant SCN3A on the morphology of immature neurons and cytoarchitecture of the developing cerebral cortex will inform the role of SCN3A in development (Aim 2). To translate these findings towards clinical applications, we will attempt to ameliorate features of SCN3A-NND in advanced model systems, including a newly generated conditional point mutant mouse, via targeted manipulation of pathogenic Nav1.3-mediated Na+ current (Aim 3). Results will provide novel information on the role of Nav1.3 during brain development, and will define the pathogenic mechanisms of SCN3A-NDD towards development of novel, targeted therapies in human patients.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY The overall goal of this five-year proposal for a Mentored Clinical Scientist Research Career Development Award is for me to develop into a productive, independent academic investigator in the field of reproductive immunology. I completed an MD and a PhD in the field of basic cellular immunology, and I now seek to apply my interest in dysregulated immunity to the public health threat of adverse fetal and maternal outcomes of pregnancy. I graduated from the American Board of Pediatrics Accelerated Research Pathway for Residency in General, and I completed my Fellowship in Neonatal-Perinatal Medicine at Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (Penn). I joined the faculty of CHOP and Penn as an Attending Physician and Instructor in the Division of Neonatology. My mentor for this award, Dr. Edward M. Behrens, is a physician- scientist with a longstanding track record of scientific innovation and providing exceptional training to mentees at all levels. As an internationally-recognized expert in innate immunity and inflammatory disorders, Dr. Behrens’s work complements my own, and we are thus poised for productivity. My scientific advisory committee includes scientists and physician-scientists with collective expertise in all aspects of the proposed work, from placental biology to next-generation sequencing. I am also extremely fortunate to have the unreserved support of CHOP and Penn, whose combined resources are unmatched. Scientifically, this proposal focuses on roles for novel macrophages that I discovered under the guidance of Dr. Behrens, called CD122+Macs, in normal and threatened pregnancy. Enriched in the uterus in mice and humans, CD122+Macs express high levels of CD122, the hallmark of responsiveness to interleukin-15 (IL-15). These novel Macs signal and function when exposed to IL-15, surprising because killer lymphocytes like natural killer (NK) cells, not Macs, are the classical targets of IL-15. Disrupted homeostasis of IL-15 is associated with numerous adverse outcomes of pregnancy, including preeclampsia and abnormal feto-placental growth but through unknown mechanisms. Based on prior literature and my preliminary data, my central hypothesis is: IL- 15 exerts its influence over outcomes of pregnancy not only by maintaining NK cells but also by modulating the inflammatory properties of novel CD122+Macs. The aims of this proposal will establish: 1) Mechanisms by which CD122+Macs respond biochemically and transcriptionally to IL-15 and 2) IL-15-dependent requirements for CD122+Macs in pregnancy in vivo. This proposal will close major gaps in knowledge regarding the mechanism by which IL-15 acts on a novel cellular target to ensure maternal and fetal health during pregnancy. In accordance with my career development objective to become a field leader in reproductive immunology, my scientific proposal complements my current proficiency in cellular immunologic methods with training in advanced reproductive biology and bioinformatic methods.

NIH Research Projects · FY 2025 · 2020-11

ABSTRACT Mechanical force is essential for T cell activation. It activates TCR signaling, and allows the T cell to sample the quality of TCR-pMHC interactions. This greatly expands the dynamic range of TCR responses and permits antigen discrimination during thymic selection, T cell priming, and effector responses. Our understanding of how force influences TCR-pMHC interactions has advanced significantly, thanks to biophysical studies at the single molecule level. However, there are large gaps in knowledge at the cell biological level. This project seeks to identify the biochemical and mechanical circuits within the TCR signal transduction network that permit the rapid translation of small differences in the physical characteristics of the TCR–pMHC interactions into distinct cellular responses. During the first project period, we showed that the T cell actin network exerts force on the integrin LFA-1 as well as the TCR, supporting mechanical crosstalk that influences the activation of both molecules. Interestingly, this process is sensitive to the biophysical features of the stimulatory surface, including ligand mobility and stiffness. These parameters are physiologically relevant, as they are regulated during DC maturation to optimize T cell priming. Further analysis reveals that this mechanobiology also impacts cytoplasmic signaling molecules that interact with the actin cytoskeleton. In particular, we find that T cell stiffness responses involve phosphorylation of the stretch-sensitive adapter protein CasL. On the basis of these findings, we hypothesize that TCR-induced actin polymerization allows the cell to sense biophysical cues provided by the interacting APC, initiating mechanical feedback loops that modulate force-dependent signaling of cell surface receptors and intracellular signaling molecules that interact with the actin cytoskeleton. To test this hypothesis, we will carry out 3 specific aims. First, we will determine how ligand mobility influences actin dynamics and TCR signaling. Using stimulatory glass coverslips, planar bilayers with different mobility properties, and mixed mobility patterned surfaces, we will ask how the agonist strength and mobility of pMHC complexes and integrin ligands influences actin dynamics and TCR signaling. As part of this analysis, we will use TCR tension probes to define how altering the mobility of TCR and integrin ligands influences the forces experienced by the TCR. Next, we will carry out similar studies to understand how substrate stiffness influences T cell activation. We will stimulate T cells on hydrogels of varying stiffness, and analyze the effects on actin dynamics, TCR tension, and TCR signaling events needed for full T cell activation. Finally, we will investigate the role of CasL, a prototypic force-sensitive signaling intermediate. Using T cells lacking CasL, we will study the function of CasL during T cell responses to changes in ligand mobility and substrate stiffness. In addition, we will probe the signaling pathways leading to CasL phosphorylation during stiffness responses, and use mass spectrometry to identify relevant binding partners.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY / ABSTRACT Rural areas have fewer, and less well trained, health care providers than non-rural areas. Schools have become more involved in the delivery of mental health services and hold great potential for increasing access to children and adolescents. Innovations in training and service delivery are needed to improve mental health care quality and availability in rural schools. Evidence-based practices (EBPs) can be incorporated into school- wide multi-tiered systems that are currently used to improve school climate and safety. School-wide Positive Behavioral Interventions and Supports (PBIS), a service-delivery strategy based on the public health model is one example. A growing number of schools in rural areas are employing PBIS. Our research team has used PBIS to incorporate EBPs at Tier 2 for children with, or at risk for, mental health disorders. Given the large service disparities for children in rural areas, offering EBPs through PBIS can improve access and lead to better long-term outcomes. School personnel, with or without prior mental health training, can implement Tier 2 interventions with fidelity and clinical effectiveness if given adequate consultation support. In urban and suburban schools, this support can be provided to school staff on site. However, providing ongoing on-site support is not feasible or sustainable in rural schools, due to their remote physical location. For this reason, telehealth technology has been recommended for the training of behavioral health staff (BHS) in under-served rural communities. The purpose of this study is to use an iterative process (Rapid Prototyping) to develop and evaluate the appropriateness, feasibility, acceptability, and preliminary efficacy of a remote training strategy that provides resources to support use of Tier 2 EBPs and effective support for care coordination practices in rural schools. Participants will be school personnel with and without prior mental health training and K-8 students in rural schools in Pennsylvania who are deemed at risk for externalizing and internalizing mental health disorders. The Aims of the study are to (1) obtain input from school stakeholders employing community-based participatory research, (2) use the iterative rapid prototyping approach to develop an asynchronous and synchronous remote training strategy based on preliminary studies and Aim 1 data, and (3) conduct a pilot trial of a remote training strategy vs. a control condition. The study will employ a pragmatic design comprised of a mixed-method approach for Aims 1-2, and a pilot randomized clinical trial for Aim 3. Participants will be 68 behavioral health staff and 156 children in grades K- 8. The interventions to be implemented by school personnel are EBPs for the most common externalizing and internalizing mental health disorders. Our guiding framework is the Interactive Systems Framework for dissemination and implementation research. We will use data from this study to submit an R01 grant proposal to test the effectiveness of a free-standing online training platform with a large number of rural schools.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Overuse of antibiotics in all settings accelerates the development of antibiotic resistance, a top threat to global health. Optimizing antibiotic use is a strategy for combatting resistance. Inter-hospital comparison of antibiotic use is recommended by national antibiotic stewardship implementation policies but requires accurate measurement of such use. Newborn infants admitted to neonatal intensive care units (NICUs) have high rates of antibiotic exposure. The purpose of this pharmacoepidemiology study is to provide a framework for safely optimizing neonatal antibiotic use. The project’s specific aims are (1) to develop a risk-adjusted method for benchmarking antibiotic use across NICUs; (2) to develop a neonatal antibiotic use metric that accounts for antibiotic spectrum of activity; and (3) to determine the associations between antibiotic use metrics and neonatal outcomes. Aims 1-3 will utilize neonatal data from the Premier Health Database and Aims 2 and 3 will also utilize University of Pennsylvania patient-level neonatal data for validation. This study will directly address a top AHRQ research priority, promoting appropriate antibiotic use in order to reduce the transmission of resistant bacteria. The proposal focuses exclusively on newborn infants, an AHRQ priority population. The results of this research will identify meaningful metrics of neonatal antibiotic use in order to allow hospitals and stakeholders to pinpoint antibiotic utilization practices that should be emulated or avoided, and ultimately improve the safety and quality of neonatal care. This career development award is designed to support the transition of Dr. Dustin Flannery, an Instructor of Pediatrics at the University of Pennsylvania and Children’s Hospital of Philadelphia, into an independent physician-scientist and to achieve his long-term career goal of becoming a national expert in neonatal antibiotic stewardship and leader in pediatric health services research. In addition, this award will allow him to obtain the training, mentorship, and research experience necessary to successfully compete for R01 grants supporting studies in this arena. Dr. Flannery will receive advanced training and experiential learning in pharmacoepidemiology research methods, advanced biostatistical techniques, and health services and policy. The University of Pennsylvania and Children’s Hospital of Philadelphia provide a stellar research environment to conduct such research. This project will be guided by a renowned mentorship team with a track record of successful collaboration and scholarship, including experts in neonatology, infectious diseases, pharmacoepidemiology, health services, biostatistics, and outcomes research.

NIH Research Projects · FY 2026 · 2020-09

Novel Ultrasound Indices of Intracranial Pressure and Brain Ischemia in Neonatal Hydrocephalus Project Summary Neonatal hydrocephalus affects 1-2 of every 1000 live births and results in neurologic deficits in up to 78% of treated patients. A wide spectrum of etiologies including intraventricular hemorrhage, congenital aqueduct stenosis, myelomeningocele, and brain tumors cause an abnormal accumulation of cerebral spinal fluid (CSF) in this disease, resulting in cerebral ventricular dilatation and elevated intracranial pressure (ICP). While ventricular shunting is used as a surgical treatment for CSF diversion, the decision to shunt is based on ventricular size and clinical judgement which are not sensitive markers of ICP and brain health. Thus, delayed diagnosis of elevated ICP and surgical shunting can result in permanent brain damage and long-term neurologic deficits. Invasive ICP monitors are not routinely inserted due to the associated risk of bleeding and regional brain ischemia in the vulnerable neonatal brain. To provide a solution to a significant gap in clinical care of neonatal hydrocephalus, we utilize the contrast-enhanced ultrasound (CEUS) technique for noninvasive detection of ICP and brain ischemia in a well-established porcine model of hydrocephalus. CEUS uses intravascular injection of microbubbles of 2-3 µm in average size for assessment of tissue perfusion. With the use of the novel particle tracking method (so-called particle image and/or tracking velocimetry), the underlying spatial and temporal changes in cerebral microcirculation can be quantified for assessment of elevated ICP and brain ischemia. To this end, the previous R01 cycle validated a promising cerebral microvascular flow marker of ICP and brain ischemia in the acute porcine hydrocephalus model. This renewal R01 will focus on expanding the imaging biomarker repertoire and applicability to hydrocephalus of varying duration and etiology. The central hypothesis of the proposal is that CEUS will be a robust biomarker of ICP, brain ischemia and oxygenation in neonatal hydrocephalus. The overall goal of the proposal is therefore to 1) expand the novel CEUS biomarker repertoire and applicability beyond the acute phase of elevated ICP and to 2) explore the complementary use of macro- vascular diagnostic tools. As an exploratory aim, we will conduct a pilot Investigational New Drug (IND) approved clinical study as a continuous laboratory-to-clinical and reverse feedback learning and improvement mechanism in biomarker development. Our work will set the stage for clinical translation of a new noninvasive tool for assessment of ICP and brain ischemia in neonatal hydrocephalus, which can ultimately improve the guidance of shunting and long-term neurologic outcomes.

NIH Research Projects · FY 2024 · 2020-09

Title: Using EHR Data to Evaluate the Burden of Diabetes Mellitus in a National Network of Children’s Hospital Health Systems Project Summary/Abstract Diabetes mellitus is a group of disorders characterized by hyperglycemia resulting from defects in insulin production, insulin action, or both. In children and adolescents 0-17 years-old, pediatric diabetes mellitus (PDM) is one of the more common chronic diseases. Mounting evidence suggests that rates of both Type 1 and 2 diabetes mellitus in children and adolescents have been increasing over the past 30 years. Nonetheless, there has been limited research on how these rates differ by sociodemographics (e.g., race/ethnicity, geography) and clinical characteristics (e.g., body mass index) across diverse regions of the US. To help fill this gap, the proposed project will use electronic health record (EHR) data to assess the prevalence and incidence of PDM, overall and by diabetes type and patient sociodemographic and clinical characteristics. Data will come from PEDSnet, a national pediatric clinical research network that has transformed EHR data to a common data model for over 6.5 million children. PEDSnet includes 8 pediatric medical centers that provide care to children in all 50 states; however, the 11 states with the greatest concentration of children are: CO, DE, FL, IL, IN, KY, MO, NJ, OH, PA, and WA. In addition to participating in the DiCAYA consortium, we propose: (Aim 1)—to evaluate and improve the quality of the EHR data that will be used for identifying patients with PDM; (Aim 2)—to implement an EHR-based computable phenotype methodology for each type of PDM to support accurate, efficient, and timely surveillance; and, (Aim 3) to compute prevalence and incidence rates of PDM, overall and by diabetes type and patient sociodemographic and clinical characteristics. Aim 1 (data quality) will take advantage of PEDSnet’s well-established data quality program that evaluates both structural and semantic data quality and works with institutional data contributors to remediate data quality problems. Aim 2 (Computable Phenotyping) will implement validated algorithms for identifying children and adolescents with PDM. And, the denominator population for Aim 3 (Rate Computations) will be patients who reside in one of the 62 counties for which PEDSnet has representative data and who have >1 contacts with a PEDSnet institution during the observation period. We plan to harmonize our methods with the rest of the DiCAYA consortium to enable standardized assessments of disease rates. Our team has extensive experience working in consortia, such as PCORnet and OHDSI, that share and execute each other’s data science methods. Our attention to evaluating and improving EHR data quality, constructing and testing pediatric EHR-based computable phenotypes, and use of a national network of major pediatric medical centers are key strengths of this proposal.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY – Overall In 2018, the NIH launched the INCLUDE (INvestigation of Co-occurring conditions across the Lifespan to Understand Down syndromE) Project, a transformative initiative focused on the study of Down syndrome (DS), the condition caused by trisomy 21, which is the most common chromosomal abnormality and a leading cause of intellectual and developmental disability. Importantly, individuals with DS display a different clinical risk profile, whereby they are protected from developing certain medical conditions, but are also highly predisposed to others, such as Alzheimer’s disease, autoimmune conditions, congenital heart defects, complications from lung infections, and autism. Therefore, the INCLUDE Project funds a large portfolio of investigations that could advance not only our understanding of DS, but also of many conditions that affect the general population. Within this framework, the mission of the INCLUDE Data Coordinating Center (DCC), is to accelerate discoveries that continuously improve and enrich the lives of people with DS through accessible data resources that enable interdisciplinary collaboration, connected communities, and the expansion of a global DS knowledge ecosystem. To fulfil this mission, the DCC is organized into three cores: the Administrative and Outreach Core (AOC), the Data Management Core (DMC), and the Data Hub Core (DHC), which will develop the following activities: 1. Provide administration and outreach functions to advance the mission of the INCLUDE Project. The AOC will provide management and administration to ensure mission-aligned DCC functions and engage a diverse community of stakeholders through outreach, training, and education. 2. Develop a platform for efficient data ingest, curation, harmonization, and sharing. The DMC will collaborate with the DS research community to jointly embrace data sharing best practices; modernize data infrastructure to improve speed and scalability; extend support to new data modalities and improve usability of data products; and build connections with key INCLUDE initiatives to maximize research and innovation. 3. Accelerate discoveries through a portfolio of online resources for data sharing and analysis. The DHC will expand the INCLUDE Data Hub’s capabilities to support FAIR-compliant, interoperable, and user-driven data sharing; advance cohort-building, analytics, and visualization tools for integrative, multimodal data exploration and hypothesis generation; support integration and collaboration across the INCLUDE program’s ecosystem of cohort-development initiatives; and engage and expand the INCLUDE research community through cross- platform interoperability, training, outreach, and partnerships. Altogether, these activities advance the mission of the INCLUDE Project by ensuring that all data generated by studies of DS are returned to the community in ways that are accessible to a broad range of secondary users, thus enabling for discoveries that elongate the lifespan and improve health outcomes for people with DS.

NIH Research Projects · FY 2024 · 2020-09

Abstract Tightly-regulated protein synthesis rates are critical for hematopoietic stem cell (HSC) maintenance and function. Mutations in ribosome proteins or genes that affect ribosome biogenesis cause “ribosomopathies”, a class of bone marrow failure (BMF) syndromes. As prominently illustrated by Shwachman-Diamond Syndrome (SDS), a BMF disease with progressive hematopoietic stem and progenitor cell (HSPC) failure and predisposition to myeloid malignancies, is driven by germline biallelic mutations in the assembly factors essential for the maturation of the 60S ribosome subunit. However, how ribosome assembly is regulated in HSCs remains poorly understood, as is its contribution to hematopoietic dysfunction. Importantly, other than allogeneic stem cell transplantation, therapeutic interventions that mitigate the HSPC defects in BMF do not exist. This application is based on our new studies that uncovered a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and self-renewal, concomitant with a reduction in global protein synthesis. The mechanism underlying HSC dysfunction upon Hectd1 deficiency is directly linked to aberrant ribosome assembly by ubiquitinating and regulating the stability of ZNF622, a critical biogenesis factor for the maturation of the 60S large ribosomal subunit in the cytoplasm. Depletion of HectD1 led to an accumulation of ZNF622 and the anti-association factor eIF6 on the 60S subunit, decreased 80S monosome to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, knockdown of ZNF622 in Hectd1-deficient cells restored protein synthesis and HSC reconstitution capacity. This finding represents a rare in vivo example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis. The implications of this novel pathway to the etiology of HSC failure and clinical treatment of “ribosomopathies”, mandates detailed mechanistic understanding. Here, we propose comprehensive and in-depth analyses on the role of HectD1 and ZNF622 in ribosome biogenesis and HSCs. In aim 1, we propose to investigate the roles of HectD1 and ZNF622 in HSCs and how they interact to regulate HSC function, using a combination of complementary genetics, genomics, and biochemical approaches. In aim 2, we will systematically analyze if HectD1/ZNF622 affects different aspects of protein translation controls. Moreover, we will perform quantitative proteomics to assess if ribosome levels or ribosome composition is affected by Hectd1/ZNF622 loss. In aim 3, we will interrogate potential dysregulation of HECTD1 and ZNF622 in human BMF syndromes and explore therapeutic potential of targeting ZNF622 for the treatment of BMF with dysfunctional ribosome biogenesis. Our study implicates a previously unappreciated role of ubiquitination in regulating HSC function via controlling ribosome biogenesis factors, which are dysregulated in ribosomopathies. Our findings will likely have significant impact on the therapeutic potential of modulating ubiquitination and/or ribosome biogenesis factors in restoring HSC functions in BMF syndromes.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Sepsis is a medical emergency defined as life-threatening organ dysfunction due to a dysregulated host response to infection. Septic shock is the most severe form, involving cardiovascular failure. More than 75,000 children in the US and four million children worldwide are hospitalized with sepsis or septic shock every year. Sepsis is currently the most expensive hospital condition in the US and, for critically ill patients— including children—is the most common cause of multiple organ dysfunction syndrome and hospital death.1,26 The World Health Organization and the US Centers for Disease Control and Prevention have called for optimizing therapies for sepsis.33 Because fluid resuscitation provides the best established benefit for septic shock (other than antibiotics), there has been an effort to identify the most effective fluid administration strategy. Despite progress, a remaining key gap in knowledge delineated by the Surviving Sepsis Campaign is which crystalloid fluid is the most effective and safest to use for initial resuscitation of septic shock. Two types of crystalloids are used for resuscitation in sepsis: 0.9% “normal” saline (NS) and balanced fluids (BF). BF have well-established biologic and physiologic advantages over NS and two recent adult trials found that BF reduced major adverse kidney events and mortality. However, in the absence of pediatric-specific data, NS resuscitation remains the overwhelming preference among pediatric emergency clinicians. We therefore propose the PRagMatic Pediatric Trial of Balanced versus NOrmaL Saline FlUid in Sepsis (PRoMPT BOLUS) study, a randomized, open-label, pragmatic comparative effectiveness trial, to test the relative effectiveness and safety of BF versus NS fluid resuscitation in children with suspected septic shock. Eighteen pediatric emergency departments in the Pediatric Emergency Care Applied Research Network (PECARN) will collaborate with sites in Canada, Australia, and New Zealand to enroll 8,800 children with suspected septic shock. Eligible patients will be enrolled either through prospective informed consent or, after appropriate ethical safeguards, “Exception From Informed Consent” for emergency research. Subjects will be randomized to fluid resuscitation and maintenance fluids with either NS or BF for 24-48 hours, with all aspects of care other than fluid type at the discretion of the care team. Our primary outcome is major adverse kidney events within 30 days from randomization (MAKE30), a patient-centered composite endpoint that includes persistent kidney dysfunction, initiation of dialysis, or death. This outcome addresses the biological advantages of BF to preserve renal blood flow and function compared to NS. Secondary outcomes include the individual components of MAKE30, hospital-free days, length of stay, 90-day mortality, and specified safety events. This pragmatic trial will provide the definitive evidence necessary for a “paradigm shift” to move clinical practice from 0.9% saline to balanced fluid-based resuscitation in children with septic shock.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT The purpose of this Mentored Patient-Oriented Research Career Development Award (K23) is to provide Nicolas A. Bamat, MD, MSCE, Instructor of Pediatrics at the Perelman School of Medicine at the University of Pennsylvania (Penn) and The Children’s Hospital of Philadelphia (CHOP) with the mentorship, training and research experience needed to become an independent clinical investigator. His long-term career goal is to improve outcomes for infants with neonatal lung disease by leading trials that identify evidence- based pharmacotherapeutic practices. His immediate career development goal is to acquire the skills needed to ensure a successful transition to research independence. To meet this goal, Dr. Bamat and his mentor team have devised a career development plan that integrates: (1) intensive mentorship from successful pediatric investigators; (2) focused training in clinical trials, clinical pharmacology and longitudinal data analysis; and (3) innovative research on furosemide treatment strategies for severe bronchopulmonary dysplasia (BPD). Complications of preterm birth are the leading pediatric contributor to disability-adjusted life years lost. BPD, or chronic lung disease of prematurity, is the most common major morbidity. BPD is particularly devastating for infants with severe BPD (sBPD), the worst severity class. Prolonged initial hospitalizations, co- morbid pulmonary hypertension, tracheostomy for prolonged ventilation and childhood death are common. Currently, no pharmacotherapies have proven clinical efficacy for improving respiratory disease course in sBPD. Despite this, medication exposures are common. The loop diuretic furosemide is most frequently used. Dr. Bamat’s mentored research will address key knowledge gaps surrounding furosemide use in sBPD. He will: (1) determine if and when tolerance to furosemide diuresis occurs in sBPD, (2) develop furosemide population pharmacokinetic models with covariate analysis for individualized dosage regimens, and (3) apply the knowledge gained in (1) and (2) to determine the comparative effectiveness of an informed furosemide treatment strategy versus standard of care for improving the respiratory severity score and decreasing the rate of furosemide-associated adverse effects. This research will be conducted by leveraging the existence of CHOP’s Chronic Lung Disease Program, a multidisciplinary referral program dedicated to infants with sBPD. Dr. Bamat’s findings will guide furosemide treatment strategies in sBPD, for testing in placebo- controlled multicenter trials, funded through R-series awards in his transition to research independence. The described career development and research activities will occur at Penn and CHOP, an ideal environment in which to train the next generation of scientists conducting NIH-supported patient-oriented research.

NIH Research Projects · FY 2024 · 2020-09

Summary/Abstract Preterm births occur in 10% of US births, cause 36% of infant mortality, and cost $26 billion each year. Repeat preterm births represent approximately 20% of total prematurity. Preventive care that effectively addresses modifiable risks (e.g. tobacco use, weight status, interpregnancy interval) among women with a prior preterm birth could plausibly reduce overall preterm birth rates by 10% or more. However, our preliminary work found that 39% of Medicaid-insured women with a prior preterm birth received no preventive care in the year after pregnancy. Leveraging existing contact between pediatric health systems and new mothers, this research proposes a pediatric-based nurse intervention as a novel strategy to reduce prematurity. We hypothesize that nurse care coordination will increase receipt of preventive care. In addition, we propose embedding motivational interviewing within the care coordination model to address modifiable health risks. We hypothesize that this care coordination plus motivational interviewing intervention will increase receipt of preventive care and reduce modifiable behavioral risks, thus improving subsequent birth outcomes. The proposed intervention builds on existing care coordination models and on prior work demonstrating feasibility of maternal risk screening in pediatrics. This intervention innovates over existing interconception (IC) care models by: (1) locating our intervention in the pediatric health care system to capitalize on existing interactions, (2) focusing on strategies to address needs and risks identified through screening, and (3) implementing motivational interviewing within care coordination to better address modifiable risks. The candidate, Dr. Gregory, has conducted prior maternal-child health research focused on the IC period. Her long-term goal is to become an independent maternal-child health researcher developing clinical programs to improve birth outcomes. This five-year mentored research proposal will support her goal through course work and completion of mentored research. Candidate training will focus on three objectives: (1) motivational interviewing, an evidence-based behavior change strategy, (2) implementation science, and (3) pragmatic clinical trials. Research will address two specific aims. (Aim 1) will adapt care coordination and motivational interviewing for women with a prior preterm birth. Using mixed methods, and starting with existing models of pediatric care coordination, adaptation will focus on the needs of women with a history of preterm birth and on integration of motivational interviewing. (Aim 1a: qualitative interviews; Aim 1b: iterative testing.) (Aim 2) will conduct a pilot pragmatic randomized trial of usual care vs care coordination plus motivational interviewing for women with a prior preterm birth. This trial will demonstrate intervention feasibility and estimate the effect size of the intervention on health care utilization. Findings will inform an R01 level trial testing this intervention as a strategy to reduce repeat preterm birth. This proposal aligns with NICHD research priorities of improving the health of women before, during, and after pregnancy and improving pregnancy outcomes.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Glycosylation is an essential, post-translational modification with complex and poorly understood roles in protein function. My long-term objective is to elucidate the neurobiological functions of glycosylation, including identifying the roles of critical glycosylation sites in neuronal protein function. The importance of glycosylation is emphasized by the congenital disorders of glycosylation (CDG), a group of genetic disorders that disrupt cellular glycosylation machinery. Affected patients exhibit severe neurological deficits. The genetic basis of CDG provides an opportunity to identify the neurobiological functions of glycosylation using mouse models and glycoproteomics. Understanding glycosylation in the nervous system will elucidate the pathophysiology of CDGs and other neurological diseases, enable therapeutic advances targeting glycosylation pathways, and inform normal function of glycosylation. GALNT2-CDG is a new CDG type caused by biallelic mutations in GALNT2, which encodes a critical glycosyltransferase initiating the first step in mucin-type O-glycosylation. GALNT2-CDG patients suffer from epilepsy and global developmental delay. Galnt2 constitutional knock-out mice recapitulate many of the patient neurological deficits. My central hypothesis is that site-specific loss of O-glycosylation on neural proteins contributes to neurological dysfunction. The specific objective of this project is to identify the cause of neurological dysfunction in GALNT2-CDG. This will be achieved by determining cellular origins of Galnt2 deficiency-mediated neurological deficits using Cre-mediated deletion of Galnt2 in neural cells and by identifying disrupted O-glycosylation in these cells using glycoproteomics. This proposed five-year career development plan focuses on achieving four objectives: develop research skills in mouse models and glycoproteomics, increase my knowledgebase in neuroscience and glycobiology, establish a body of work focusing on the role of glycosylation in the context of neurobiology, and obtain the necessary skills to transition to independence. Mentoring will be provided by Dr. Zhaolan Zhou, a recognized leader in the development and investigation of mouse models of genetic disorders that affect brain development and function, and Dr. Benjamin Garcia, a recognized expert in developing quantitative mass spectrometry techniques to interrogate post-translational modifications. The skill set developed through these investigations and career development plan will make me uniquely poised to uncover glycosylation-mediated mechanisms of CDG and other neurological diseases, as well as to elucidate the critical roles of glycosylation in human neurological function. These studies will generate new tools and a foundation to establish a long-term research program to investigate the pathophysiology of various glycosylation-related disorders in the nervous system and prepare me to become an independent R01-funded physician scientist.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY Multicellular systems tightly regulate cell division and differentiation. Cells require both signals that license them to take up the requisite nutrients for these processes as well as detect there is a sufficient environmental nutrient supply to meet this demand. The cooperation of these two circuits is critical, as metabolic environments fluctuate temporally, with diet, and during disease, while migratory cells transit between tissue contexts. While there is a large body of work explaining how these pathways support normal development in replete environments and how malignant cells coopt them to support transformation, the extent to which growth and differentiation are supported and modified when normal development faces the stress of nutrient depletion remains a fundamental knowledge gap. Presenting themselves as an ideal model system to study this in, T-cells are naturally migratory cells that as part of their biology must transit between nutrient replete environments, like blood, into dysregulated diseased tissues and do so while maintaining proliferative and differentiation capacity. Work during our previous funding period led us to discovered that these cells can sustain multiple rounds of division even when faced with the absence of essential amino acids and that this type of sustained stress modifies their future fate potential. Mechanistically, we have found that this occurs from the cooption of pro-proliferative (mitogenic) regulatory networks at two levels: 1) selective mRNA translation of a nutrient restricted pro-growth program; 2) the interaction of stress and mitogenic signaling to rewire transcriptional programming. Notably, these mechanisms are specific to stress and not observed in steady-state conditions. The goal of this proposal is to render the regulatory logic of these stress-modified networks – both the proteins driving these phenomena and the features conferring environmental-sensitivity to genes. Leveraging our knowledge of stress-sensitive regulatory proteins we have identified, we will investigate how cell growth and division is supported through distinct translational mechanisms during nutrient stress. We will both target the RNA binding proteins that drive this stress selectivity to define their translationally controlled targets as well as illuminate the landscape of untranslated regions that confer environmental sensitivity to transcripts. In parallel, we will dissect the how stress signaling modifies canonical pro-growth transcriptional programming through the formation of a new transcriptional factor regulatory architecture that alters cell fate trajectories. Completion of these studies will illuminate the molecular mechanism explaining how environmental stress is incorporated into normal lineage developmental programs, opening novel therapeutic strategies for cancer, inflammation, and disease.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY The microbiota is essential for human health and early life colonization by microbes is key for development and long-term fitness. Perturbations to assembly of this microbial community can have long-term consequences for the host. Moreover, disrupted community assembly can lead to outgrowth and long-term persistence of pathogens in the gut. The ecological rules that underlie establishment of a beneficial microbial community are not well understood and we still know very little about the molecular processes that govern early colonization and niche establishment by important early life microbes. Defining the fundamental principles of microbial colonization, assembly, and succession are critical to understanding what makes a healthy microbiota and will provide a roadmap for preventing the negative consequences of perturbation to this ecosystem. Here, we will i.) define the nutritional and environmental factors that shape early life microbiota colonization and assembly and ii.) determine strategies that early life colonizing pathogens use to establish a successful niche in the infant gut. This work will shed light on key axes of competition that shape the ultimate success of early colonizers and define the molecular strategies that pathogens exploit to establish niches early in life. Completion of this proposal will lead to the discovery of novel factors and molecular mechanisms shaping early life establishment of a healthy microbiome and provide new insights into previously unappreciated aspects of early life microbial ecology. Our ultimate goal is to generate a comprehensive understanding of early life ecology that will guide the creation of tools to prevent the effects of perturbation to host-microbiota homeostasis. Moreover, defining the niche space that pathogenic organisms inhabit in the early life gut will lead to innovative therapeutic strategies to prevent establishment and promote decolonization of pathogens in early life.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT Our objective is to identify genes that regulate development of bone density, quality and strength in childhood. Childhood is a critical window for lifelong musculoskeletal health. Failure to achieve optimal bone accrual during childhood results in suboptimal peak bone mass and bone fragility later in life. In excess of 50 million older US adults have osteoporosis or low bone mass. Osteoporosis has a strong heritable component, yet only 20% of adult bone mineral density (BMD) variability is explained by genetic variants discovered to date. Pediatric studies should be highly effective in distilling the genetics of this complex phenotype, given (a) the duration of environmental influences is shorter, and (b) the genetic determinants of growth, body composition and maturation also influence bone accrual. Uncovering the genetic architecture of childhood bone accrual is critical for understanding lifelong skeletal health and identifying targets for preventing and treating bone fragility. Dual energy x-ray absorptiometry (DXA) measures of areal BMD are widely used in genetic studies. With DXA software advances, elements of bone quality and structural strength can be extracted, along with body composition parameters known to influence bone accrual. These deeper DXA-derived phenotypes have great potential to shed further important, novel insights into genetic determinants of the developing skeleton. We have genome-wide genotyped the NICHD Bone Mineral Density in Childhood Study (BMDCS) cohort which is unique for its large size, broad age range, high data quality, diversity and longitudinal design. We will derive new phenotypes from existing DXA and radiograph images, and apply advanced multidimensional phenotyping and multivariate GWAS methods to identify new loci. GWAS only reports genomic signals associated with a given trait and not necessarily the precise location of culprit genes. Therefore, we will use high-resolution `variant to gene mapping' techniques established in our `Center for Spatial and Functional Genomics' to investigate both previously reported pediatric novel loci and our anticipated new loci. Our approach first prioritizes putative causal SNPs using open chromatin and enhancer epigenetic signatures, and then identifies 3D genomic contacts between these prioritized SNPs and their target gene promoters, using a high-resolution promoter-based chromatin conformation capture technique. To validate these target genes, we will use CRISPR/Cas9 to edit the putative regulatory SNPs and use siRNA to target genes and show an effect on bone-relevant phenotypes. We will apply our techniques in primary pediatric human mesenchymal progenitor cell (MSC)-derived osteoblasts, a very relevant bone cellular model for understanding pediatric bone mass accrual. Thus, our proposal is an unparalleled opportunity to interrogate novel phenotypes and functionally characterize the actual effector genes using high resolution chromatin conformation capture approaches at these new, as well as previously known bone-related loci.

NIH Research Projects · FY 2024 · 2020-08

Project Summary / Abstract Pediatric antibiotic stewardship programs (ASPs) in hospital and outpatient settings optimize the use of antibiotics to improve clinical outcomes, decrease adverse drug events, and reduce the emergence of antibiotic resistant bacteria. However, stewardship for patients at the transition from hospital discharge to home, or “discharge stewardship,” is an unmet need for several reasons. First, few pediatric stewardship programs perform discharge stewardship. Second, approximately 30% of pediatric patients receive antibiotics at hospital discharge. Third, the majority of antibiotic days prescribed for hospitalized patients occur after discharge. Fourth, up to half of discharge antibiotic prescriptions are suboptimal, which includes choosing the wrong drug, dose, route, or duration of therapy. This project will use an implementation science framework to develop, implement, and test the effectiveness of a multifaceted discharge stewardship intervention for hospitalized children with the three most common indications for antibiotic prescribing in hospitalized children - community-acquired pneumonia (CAP), urinary tract infections (UTI), and skin/soft tissue infections (SSTI) - at four children's hospitals to establish a foundation for future expansion to additional target populations. Antibiotic choice, dose, route, and duration of therapy will be addressed. Aim 1 is to develop, locally adapt, and implement a discharge stewardship intervention across the four participating sites. The integrated Promoting Action on Research Implementation in Health Services (i-PARIHS) framework will guide a rapid formative evaluation to identify contextual factors likely to facilitate or hinder the implementation of a discharge stewardship intervention at each site. Based on these results, local facilitators will work to develop and implement a discharge stewardship intervention comprised of consensus driven clinical prescribing guidelines for CAP, UTI, and SSTI plus quarterly feedback of prescribing data based on these guidelines. Aim 2 is to measure the impact of the discharge stewardship intervention on antibiotic prescribing (the primary outcome) and patient-centered balancing measures. For the primary outcome, suboptimal antibiotic prescribing, we will use retrospective data collection leveraging validated diagnostic code-based algorithms to maximize consistency and feasibility for future dissemination. For the balancing metrics, treatment failure and post- discharge adverse drug events, we will use prospective data collection from parents of patients with CAP, UTI, and SSTI to maximize their capture. Both sub-aims will utilize a time series analysis based on 18 months of pre-intervention data followed by 30 months of post-intervention data. This project will form the foundation for future dissemination of discharge stewardship to a broader array of patient populations. Investigators on this proposal form the leadership of the Sharing Antimicrobial Reports for Pediatric Stewardship (SHARPS) Collaborative, a network comprised of more than 60 children's hospitals across North America that is uniquely positioned to adopt antimicrobial stewardship interventions designed to target prescribing at hospital discharge.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT Hypoplastic left heart syndrome (HLHS) is characterized by incomplete development of the left heart and affects over 1,000 live born infants in the United States per year. Staged surgical treatment allows children with HLHS to survive and flourish, but the right ventricle (RV) remains the sole functioning ventricle and the tricuspid valve (TV) is their only functional atrioventricular valve. Tricuspid regurgitation is thus highly associated with heart failure and death. Thirty percent of HLHS patients require surgical intervention to treat tricuspid regurgitation. Despite incremental progress, understanding of the relationship between TV structure and tricuspid regurgitation in HLHS is limited, and results of surgical repair are suboptimal. 3D echocardiography and quantitative 3D echocardiography based analysis have dramatically improved adult mitral valve surgical planning and repair. However, despite preliminary work with basic tools suggesting relationships between the 3D structure of the TV and patient survival, and the unmet need for precise structural information to guide TV repair, there is no commercial or readily available method sufficiently adaptable to model and quantify the unique and highly variable TV anatomy in HLHS. As a result, the information in 3D echocardiography images remains latent, limiting understanding of the relationship between TV structure and tricuspid regurgitation, as well as the design of patient-specific repairs informed by 3D structural analysis. This proposal builds upon Dr. Jolley’s experience with modeling of atrioventricular valves in congenital heart disease and the development of flexible open-source modeling tools. The specific goals are to 1) investigate methods for quantifying TV structure from 3D echocardiography images using metric based tools as well as novel shape parameterization and statistical shape analysis; and 2) use these tools to identify TV structural correlates of TV dysfunction in 100 HLHS patients who have completed staged surgical repair. The methods developed will be incorporated into the open-source 3D Slicer imaging processing platform to catalyze future studies of congenital and adult structural heart disease, particularly those not possibly using current modalities. These studies are the first step toward individualized surgical repair of the TV informed by 3D structural analysis, and a detailed understanding of structural features of TV dysfunction in HLHS. The proposal leverages the existing infrastructure and extensive expertise at the Children’s Hospital of Philadelphia, Queen’s University, and Kitware Inc. Overall, this study represents a significant advance in the understanding of tricuspid valve disease in HLHS, as well as a flexible, and extensible armamentarium of tools for the future image-based investigation of congenital and adult structural heart disease.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT The overall goals of this proposal are to investigate the mechanisms of bladder injury (hemorrhagic cystitis) associated with BK polyomavirus (BKPyV) infection in children and young adults undergoing bone marrow transplantation (allogeneic hematopoietic cell transplantation, HSCT). BKPyV-associated hemorrhagic cystitis is a complication for up to 25% of allogeneic HSCT recipients. Hemorrhagic cystitis leads to patient morbidity from painful urination, prolonged hospitalizations, increased blood transfusion requirements, and invasive procedures when urinary obstruction is present. Each episode of hemorrhagic cystitis accounts for 10 additional hospital days and costs $70,000. In its most severe presentation, hemorrhagic cystitis may be associated with a higher risk of death. BKPyV replication in the urine can be detected in 80% of HSCT recipients, but not all of these patients will develop hemorrhagic cystitis. There are no antiviral therapies with proven efficacy against BKPyV and current treatment strategies for hemorrhagic cystitis are limited to supportive care. A major barrier to developing and testing novel therapies is our lack of understanding the mechanisms of bladder injury in patients with BKPyV replication. This also prevents the identification of the patients at highest risk for bladder injury. The central hypothesis of this proposal is that the risk of hemorrhagic cystitis after HSCT is a function of both BKPyV subtype and the host response to the virus. To test our hypothesis, we will study two separate prospective observational cohorts: an existing group of 193 children and young adults who received an allogeneic HSCT at Cincinnati Children’s Hospital Medical Center or the Children’s Hospital of Philadelphia with already banked urine samples and an independent validation cohort of 200 children and young adults undergoing allogeneic HSCT to be enrolled during the award period at these same two centers. The project’s specific aims are: 1) to test whether the population or within-person BKPyV diversity is different between patients with and without hemorrhagic cystitis after HSCT and to enroll the validation cohort; and 2) to test whether host cellular responses or specific gene polymorphisms are different between subjects with and without hemorrhagic cystitis after HSCT using proteomics and transcriptomics approaches. This study’s results are expected to increase our understanding of the viral and host mechanisms of bladder injury after HSCT. The data from this application are critical for identifying the patients at highest risk of hemorrhagic cystitis who would benefit from cellular therapies and other targeted, future prevention or treatment strategies.

NIH Research Projects · FY 2024 · 2020-07

Abstract This project focuses on the design and evaluation of prodrugs with dual pharmacological selectivity, combining molecularly targeted mode of action with a tissue-specific, active uptake process (uptake-1) to enhance drug delivery to aggressive neuroendrocrine neoplasms, including high-risk neuroblastoma (NB) – the deadliest extracranial pediatric solid tumor currently lacking effective treatment options. Norepinephrine transporter (NET) driving accumulation of norepinephrine and its functional analogs is expressed by most solid tumors developing from sympathoadrenal precursor cells. However, tumor radiotherapy targeted to NET has shown limited efficiency, while causing serious adverse effects due to significant off-target distribution and extensive damage to healthy tissues. Centered on a dual-selective experimental drug delivery strategy integrating the uptake-1 process with a replication-dependent mode of drug action to confine the pharmacological effect to proliferative tumor cells expressing NET, this project aims to evaluate and optimize a pharmacotherapeutic approach designed to effectively combat refractory disease not responding to existing treatments, while minimizing toxicity to healthy organs and tissues. In our proof-of-concept experiments, a tripartite prodrug design integrating NET affinity with unique molecular targeting of a potent and selective topoisomerase I inhibitor was shown to be essential for achieving sustained intratumoral drug presence and markedly extended survival in clinically relevant models of aggressive neuroblastoma. Guided by these results, we hypothesize that dual-selective pharmacotherapy using NET-targeted prodrugs can provide a selective, safe and efficient way of treating different forms of high-risk disease. We also hypothesize that potency and selectivity of this approach will be enhanced by combining it with clinically proven small-molecule agents modulating tissue-specific expression of NET. These hypotheses will be tested by pursuing the following specific aims: Aim 1 studies will focus on comparative evaluation of polymeric carrier-linked prodrug constructs with regard to their cell uptake and growth inhibitory effects on primary NB cells and cell lines with different phenotypes, as a function of their molecular design and the potentiating action of the NET expression enhancing agents; Aim 2 and Aim 3 studies will comparatively evaluate the biodistribution profiles and therapeutic effectiveness of a series of tripartite prodrugs, with the goal to identify and optimize key construction variables, to establish feasibility of pharmacologically modulating NET expression for improving drug delivery and treatment outcomes, and to examine the roles of tumor phenotype and disease status in clinically relevant models of aggressive NB. The proposed research focusing on NET-targeted prodrugs equipped with dual pharmacological selectivity is significant by informing the development of a new strategy for treating aggressive NB and other refractory cancers.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Autosomal recessive mutations in the TBCK gene cause intellectual disability of variable severity. I have further characterized the neurologic phenotype of Puerto Rican children with a homozygous null mutation (p.R126X) in TBCK, which we designated the Boricua mutation. The biological mechanism underlying the genotype-phenotype correlations remain unclear. On one extreme, patients with the severe Boricua mutation develop progressive brain, cerebellar and motor neuron atrophy, coarse facial features and epilepsy. We named this severe syndrome TBCK-encephaloneuronopathy (TBCKE). On the other hand, other patients with biallelic TBCK mutations have clinical diagnosis of autism and/or intellectual disability, without evidence of neurodegeneration. The function of TBCK protein is unknown, but previous studies have shown absence of TBCK leads to downregulation of mTORC1 signaling. The mTORC1 pathway regulates autophagy, including the targeted degradation of mitochondria (mitophagy). I recently reported increased autophagic flux and impaired glycoprotein degradation in TBCKE patients’ fibroblasts, which was rescued by activating mTORC1 signaling with L-leucine. Our fibroblasts studies suggests that TBCKE patients have mitochondrial dysfunction and mitochondrial DNA (mtDNA) depletion. Furthermore, the degree of mtDNA depletion predicts the neurologic severity of TBCK disease. Therefore, I hypothesize that loss of function of TBCK in human neurons leads to mtDNA depletion and mitochondrial dysfunction due to excessive autophagic clearance of mitochondria. To test this hypothesis in more disease relevant models, I propose to generate TBCK-null induced pluripotent stem cell (iPSC) derived neurons (iNeu) and tbck-/- zebrafish. The goal of this proposal is to address whether loss of function of TBCK affects mitochondrial function in human neurons, and to determine whether mtDNA depletion modulates the severity of neurodegeneration in TBCK disease. Using novel disease models and unique tools to assay mitochondrial function, I propose to address the following questions: (Aim 1) Do human neurons lacking TBCK protein have excessive mitophagy? (Aim2) What is the function of TBCK? Can we define its protein-protein interactions in neurons? (Aim 3) Can TBCK- null zebrafish model the variable severity of TBCK disease? Can mtDNA depletion modulate the severity of the phenotype in vivo? The experiments outlined in this proposal will determine the role of mitochondria in TBCKE and whether mtDNA depletion is sufficient to drive the neurodegenerative phenotype or merely an epiphenomenon. This work will also provide training in novel techniques for assaying neuronal mitochondrial function in disease models in situ. I will also learn to develop and characterize zebrafish models of neurodevelopmental disease. Support from this K02 award will be instrumental in growing my independent research program as a physician scientist in a superb institutional environment towards my long-term goal of studying the role of mitochondrial dysfunction in pediatric neurodegenerative disorders.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract I am a physician/scientist focusing on the development of optical neuroimaging techniques to improve bedside detection of neurologic injury in critically-ill children. The goal of my mentored career development award is to acquire training in mouse models of hypoxia as well as in advanced statistical methods for the analysis of resting-state brain activity. This training will launch an independent research career with the aim to bring imaging biomarkers from bench to bedside. Many pediatric diseases that were once universally fatal (e.g., complex congenital heart disease and extreme prematurity) now have relatively good survival rates. However, neurodevelopmental outcomes have improved only marginally. With timely intervention, it is possible to minimize hypoxic injury, but current bedside tools are insensitive and inadequate for this purpose. Furthermore, the heterogeneity of clinical populations limits clinical studies. Each patient has a unique injury and treatment, and neuroimaging is performed at varying times after injury; thus, it can be difficult to rigorously analyze such data to determine the best, most generalizable biomarkers. The present work aims to solve these problems by using mouse models of hypoxemic neurologic injury, robust statistical methods, optical functional neuroimaging techniques, and resting-state hemodynamic assessment (e.g., functional connectivity). The optical methods are similar to functional magnetic resonance imaging but have much lower cost, higher portability, and higher through-put. My proposal will test the hypothesis that resting-state hemodynamic metrics can serve as neuroimaging biomarkers of injury after acute and chronic hypoxia. Aim 1 will develop statistical methods adapted to optical neuroimaging to permit more robust noise filtering, brain segmentation, atlasing, and image analysis. Aim 2 will use resting-state hemodynamics in the hyperacute phase of ischemic stroke to identify the penumbra. Aim 3 will study the longitudinal development of functional connectivity networks across mouse development and the disruptive effects of chronic hypoxemia. This research will be conducted under the mentorship of Arjun Yodh, PhD, with co-mentorship by Daniel Licht, MD; both faculty are recognized leaders in the development of optical neuromonitoring techniques. In addition, I have assembled an interdisciplinary group of collaborators with expertise in mouse models of hypoxemia, neuroimaging statistics, and advanced network analysis methods. I will benefit from this excellent mentorship and research environment, and my unique optical neuroimaging methods offer a path to independence. I am a board-certified pediatric cardiologist, and my long-term career goals are to combine neuroimaging with a tenure-track position at a pediatric research hospital. The exceptional research environment at CHOP/Penn will enable future translational studies in the intensive care unit, as well as further animal models of disease. Page 1

NIH Research Projects · FY 2024 · 2020-06

ABSTRACT Low-grade glioma is the most common brain tumor in children and often involves one or more structures of the anterior visual pathway (i.e., optic nerves, chiasm and tracts). Nearly 20% of children with neurofibromatosis type 1 (NF1) will develop a low-grade glioma of the anterior visual pathway, which are called optic pathway gliomas (OPGs). NF1-OPGs are not amenable to surgical resection and can cause permanent vision loss ranging from a mild decline in visual acuity to complete blindness. Children with NF1-OPGs typically experience vision loss between 1 and 8 years of age and are monitored with brain magnetic resonance imaging (MRI) to assess disease progression. However, traditional two-dimensional (2D) measures of tumor size are not appropriate to assess change over time and how NF1-OPGs are responding to treatment. Our proposal addresses the lack of robust and standardized quantitative imaging (QI) tools and methods needed for NF1-OPG clinical trials. We will develop and validate a novel three-dimensional (3D) MRI-based QI application for automated and comprehensive quantification of these unique pediatric tumors. We will use machine learning algorithms to accommodate MRI sequences from different manufacturers and protocols. We hypothesize that the novel QI application will accurately assess treatment response in clinical trials. In this project, we will validate our QI software and machine learning methods to make accurate and automated measures of tumor volume and shape using data from a phase 3 clinical trial of NF1-OPGs. From these measures, we will create methods to assess response to therapy that will enable physicians to make informed and objective treatment decisions. Our specific aims are: 1) Develop a comprehensive QI application to perform accurate automated quantification of NF1-OPGs; 2) Determine and predict treatment response using our 3D QI measures of tumor volume; and 3) Validate our 3D QI measures using visual acuity outcomes. Upon study completion, our QI application could transform clinical care for NF1-OPG by identifying the earliest time to determine a favorable versus unfavorable treatment response. The QI application's ability to accurately measure treatment response, along with harmonizing data across MRI manufacturers and protocols, will standardize imaging assessments essential to NF1-OPG clinical trials.