Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2026 · 2013-08

Abstract One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease and thalassemia, and knowledge about regulatory processes is being translated into gene therapies and other therapeutic approaches. BCL11A is a critical player in the globin switch, but how it is regulated developmentally is surprisingly still largely unclear. Our preliminary data show that BCL11A is controlled predominantly at the transcriptional level. Via a CRISPR-Cas9 genetic screen we identified the transcriptional repressor HIC2 as a novel regulator of hemoglobin switching. HIC2 is expressed highly in fetal erythroid cells and extinguished in adult erythroid cells. Our preliminary data further suggest that HIC2 represses BCL11A transcription specifically in fetal type cells by directly decommissioning a fetal stage-specific BCL11A enhancer element. Together, these observations define the foundational hypothesis of this application: HIC2 expression is extinguished in adult red cells, allowing for the activation of a BCL11A enhancer to boost BCL11A expression in adult cells and trigger the silencing of fetal type globin genes. This places HIC2 upstream of BCL11A in the regulatory circuitry that controls hemoglobin switching. Additional preliminary data suggest that HIC2 promotes a broader fetal transcriptional program. In Specific Aim 1 we will examine the biology of HIC2 in gain- and loss-of-function experiments in vivo using a combination of cell culture models and whole animal studies. Specific Aim 2 is focused on mechanistic experiments, defining the way by which HIC2 regulates chromatin features and transcription factor binding at target genes, including BCL11A, and what HIC2 co-factors are involved. Specific Aim 3 is dedicated to defining the developmental control of HIC2 expression. This will be accomplished by characterizing the regulatory landscape of the HIC2 locus in fetal and adult erythroblasts in combination with developmental stage specific perturbative experiments. In sum, the proposed studies aim to understand the role of HIC2 in hemoglobin switching and in the establishment of a fetal erythroid state at an organismal and molecular level. This proposal is expected to produce results with ramifications for a broader understanding of developmental hematopoiesis as well as the treatment of sickle cell disease and thalassemia.

NIH Research Projects · FY 2026 · 2012-12

PROJECT SUMMARY Severe malaria due to infection by Plasmodium falciparum is a serious threat to global health with over a million deaths per year. New antimalarial agents are needed due to widespread resistance to existing therapies. A promising antimalarial drug target is the MEP pathway of isoprenoid biosynthesis, which is not found in humans. We have used forward genetic screening to identify malaria parasites resistant to MEP pathway inhibition. We have thus identified a new family of metabolic regulators in malaria, the HAD proteins. We now propose to determine the mechanism by which loss of HAD phosphatases confers drug resistance (Aim 1); establish the biological functions of HADs in parasite development (Aim 2); and use a new MEP pathway inhibitor to identify and characterize additional mechanisms of resistance (Aim 3). We will identify P. falciparum genes and pathways that genetically interact with the essential MEP pathway and our strong preliminary results support this approach. In addition, our results will inform our understanding of the basic biology of the HAD family of metabolic regulators and will determine whether HAD-mediated drug resistance can be transmitted.

NIH Research Projects · FY 2024 · 2012-09

PROJECT SUMMARY Cystic fibrosis-related diabetes (CFRD) not only burdens affected patients with a second, attention-demanding disease but threatens nutritional status, pulmonary function, and survival. Developing strategies to preserve β- cell function are crucial for interrupting CFRD development and its hazard to CF-relevant outcomes. The overall aims of this application are to better understand the emergence and progression of abnormal glucose tolerance in pancreatic insufficient CF (PI-CF) and to test a potential strategy for restoring β-cell function. This application extends our recent studies demonstrating that 1) insulin secretion defects are present at glucose thresholds traditionally considered normal (one-hour oral glucose tolerance test [OGTT] glucose >155 but <200 mg/dL; referred to as early glucose intolerance [EGI]), 2) such subtle glucose abnormalities associate with increased CFRD risk and may portend greater declines in pulmonary function, and 3) infusion of the incretin hormone, glucagon-like peptide-1 (GLP-1), but not glucose-dependent insulinotropic polypeptide (GIP), augments glucose- dependent insulin secretion in PI-CF. Our cross-sectional studies in CF demonstrate marked reductions in meal- related early-phase insulin secretion and β-cell secretory capacity in EGI. With worsening glucose tolerance, PI- CF subjects with impaired glucose tolerance (IGT, two-hour OGTT glucose >140 but <200 mg/dL) and CFRD exhibit further compromised meal-related early-phase insulin secretion and β-cell secretory capacity. The extent to which emergence and progression of glucose intolerance is a manifestation of worsening β-cell secretory capacity is not known and will be investigated in longitudinal studies of youth and adults with PI-CF in whom mixed-meal tolerance tests (MMTT) will be performed to characterize early-phase insulin secretion and glucose- potentiated arginine (GPA) tests will be completed to quantify β-cell secretory capacity. In Aim 1, we will leverage the genotyping and clinical phenotyping of our pediatric and adult CF cohort (n=350) to test the impact of T2D genetic variants, diet, CFTR modulator therapy, and pulmonary exacerbations on the emergence and progression of glucose intolerance and the relationship of glucose intolerance with nutritional status, pulmonary function, and body composition longitudinally over 4-5 years. In Aim 2 we will test whether our findings of β-cell responsiveness to acute GLP-1 infusion has the potential to be translated into the use of chronic GLP-1 therapy as a mechanism to preserve β-cell function. Specifically, we will pursue a proof-of-concept 6-week randomized, placebo-controlled cross-over study of the GLP-1 agonist, dulaglutide; the primary outcome will be the impact of dulaglutide upon meal-related early-phase insulin secretion, one of the earliest defects detected clinically. If successful, this work will provide the foundation for a multi-center study aimed at identifying and treating early insulin secretion defects in PI-CF and interrupting progression to CFRD.

NIH Research Projects · FY 2026 · 2012-09

The neuronal circuitry within the dentate gyrus is massively disrupted in temporal lobe epilepsy patients and in experimental models of this disorder. This proposal builds upon our laboratory’s previous findings, which demonstrate that the dentate gyrus circuitry within the epileptic hippocampus retains an embedded coding network of dentate granule cells which can reemerge and restore appropriate cognitive function following treatments to suppress degraded pathologic activity. The maintained competence of this embedded dentate granule cell network occurs despite the significant structural pathology which is unaffected by therapeutic interventions. In this proposal, we will build upon this foundation, and examine and manipulate dentate granule cells in both epileptic and control animals to generate a mechanistic understanding of how these epilepsy- associated disruptions to normal circuit functioning can be targeted to restore downstream, emergent properties of the hippocampus, such as learning and memory and emotional behaviors. The CENTRAL HYPOTHESIS of the present proposal is that the epilepsy-associated degradation in coding properties of dentate granule cells contributes significantly to both the cognitive and behavioral comorbidities that constitute key components of the core phenotypes of temporal lobe epilepsy. To test this Central Hypothesis, we propose to conduct a series of experiments centered on 3 SPECIFIC AIMS: Aim 1. Characterize the local circuit properties defining the active dentate granule cell network in epileptic and control mice. Aim 2. Determine the capacity, time course, and extent of long-term dentate gyrus circuit specific intervention strategies to rescue cognitive and behavioral function in epileptic mice. Aim 3. Assess the contribution of dentate granule cell hyperexcitability in epileptic mice to disrupted hippocampal spatial coding. We know little about the mechanisms that mediate the sparse yet deterministic firing properties of neuronal populations in the hippocampal dentate gyrus that are responsible for their role in information coding and plasticity. We know even less about how disease-associated degradation in these critical dentate granule cell properties develop, and in turn how this excitability disruption may erode cognitive and affective functions that the hippocampus normally supports. In addition to the enhanced excitability responsible for seizure generation, patients with epilepsy exhibit severe cognitive comorbidities, including deficits in emotion, mood, and learning and memory, processes typically thought of as limbic system functions. Understanding how epilepsy development alters the basic circuit properties within the limbic system may be important not only in targeting new therapies for seizure amelioration, but also in developing new treatments to reduce comorbid conditions accompanying epilepsy development, a largely unexplored area of therapy development.

NIH Research Projects · FY 2026 · 2011-07

Hereditary Multiple Exostoses (HME) is a rare autosomal dominant disorder that affects thousands of children worldwide. HME is characterized by cartilaginous-bony tumors called osteochondromas that form within perichondrium along growth plates and protrude into and collide with surrounding tissues. The tumors can thus cause skeletal deformities, compression of nerves and blood vessels and chronic pain, and become malignant in about 2-3% of patients. Current therapies are limited, and patients struggle with pain and limited mobility and undergo multiple surgeries through life. Most HME patients bear a heterozygous mutation in EXT1 or EXT2 that are responsible for heparan sulfate (HS) synthesis, thus causing a partial systemic HS deficiency. The HS chains -and the proteoglycans of which they are part- regulate and distinctly modulate many processes. Notably, they interact with signaling proteins including bone morphogenetic proteins (BMPs) and hedgehog family members and most often restrict and delimit protein distribution, availability and activity. However, it is not clear whether and which of these mechanisms may be deranged in HME and how it could lead to tumor formation. In the previous funding period, we found that conditional ablation of Ext1 caused an increase in pro-chondrogenic BMP signaling in perichondrium and a concurrent decrease in anti- chondrogenic pERK1/2 and Noggin, deranging normal homeostatic mechanisms that normally maintain the perichondrium phenotype. In preliminary studies, we have aimed to clarify how the osteochondromas acquire a growth plate-like organization, are able to grow unidirectionally against surrounding tissues and thus create damage and havoc. We have obtained evidence for the establishment of an IHH-PTHrP axis driving tumor outgrowth. Our central hypotheses is that osteochondroma development and outgrowth are driven by: (i) a steep local deficiency in HS; (ii) increased BMP signaling; and (iii) establishment of a neo IHH-PTHrP loop. As a result, we posit that osteochondroma development and growth are amenable to drug treatments directed against components of those regulatory circuits. We will use genetic, biochemical and cellular approaches and transgenic mouse models that closely mimic human disease progression and burden. The project will continue to provide fundamentally new insights into cellular and molecular mechanisms of tumor formation as well as normal functioning of those mechanisms in standard perichondrial and growth plate cells. It will also test possible therapies based on those insights and thus has major translational medicine value and implications. The number of HME patients is relatively small, but the community of their families is large. This project will thus provide a renewed sense of hope to patients and families alike that this disease will continue to be actively studied and a cure may one day be found.

NIH Research Projects · FY 2025 · 2010-05

PROJECT SUMMARY/ABSTRACT The Pediatric Hospital Epidemiology and Outcomes research Training (PHEOT) Program at Children’s Hospital of Philadelphia (CHOP) is a 2-year postdoctoral research fellowship designed to provide physicians with training in hospital epidemiology and outcomes research. The PHEOT program trains the next generation of physician scientists to best measure, investigate, and improve outcomes and patient safety for hospitalized children. Through a combination of formal coursework and mentored research projects, physician trainees develop expertise in comparative effectiveness research, exposure and outcomes measurement, severity adjustment, and decision analysis as they relate to pediatric hospital care. Five post-doctoral trainees simultaneously participate in the PHEOT program across the 2 years of training. Trainees benefit from the combined resources of two Centers at the CHOP Research Institute: The Center for Outcomes Research and the Center for Pediatric Clinical Effectiveness, as well as a rich array of “natural laboratories” for evaluating and improving health care processes and outcomes, including the Pediatric Advanced Care Service, the General Pediatrics Inpatient Service, the Center for Simulation, Advanced Education and Innovation, and the Center for Quality, Safety, and Analytics. All fellows complete Masters-level coursework in study design and biostatistics culminating in either the Master of Science in Clinical Epidemiology (MSCE) or the Master of Science in Health Policy Research (MSHP) degree at the University of Pennsylvania. Each fellow is assigned a mentorship team consisting of seasoned methodology, content, and biostatistics mentors who supervise the trainee in the successful completion and publication of at least one research project. PHEOT fellows also benefit from a host of professional development activities, including works-in-progress sessions, a weekly seminar series to teach academic medicine skills, and opportunities to present research at national meetings.

NIH Research Projects · FY 2024 · 2008-07

ABSTRACT In our original application for R01 HD056465 we hypothesized and proved that distillation of the genetic component contributing to obesity is easier to determine in children, where environmental exposure has had less of an impact due to a relatively short period of lifetime. During our first renewal, we successfully expanded this programmatic line of research by identifying additional variants. During the last funding cycles, we carried out a series of non-hypothesis-driven studies that were directed at uncovering loci that predispose to childhood obesity by conducting various genome-wide based approaches. For instance, employing a powered two-stage study design in a consortium setting, where cases were defined as being in the ≥95th percentile of BMI, we identified novel association signals which we subsequently replicated. This first NICHD funded study was ultimately published in Nature Genetics, with the subsequent larger trans-ethnic study being more recently reported. Intense genome-wide association studies (GWAS) efforts by the community have yielded key variants robustly associated with measures of adiposity, both in children and adults. Interestingly, while macro-level gene sets analysis of overall GWAS findings for adult waist-to-hip ratio (WHR) have implicated adipogenesis, comparable analyses of adult BMI GWAS datasets implicate central nervous system (CNS) processes. Despite these macro-level analyses, an important caveat of GWAS is that they only report genomic association signals and not necessarily the precise localization of culprit genes. As such, GWAS have not strictly represented an era of gene target discovery, rather it was simply a decade of signal discovery. One clear example of this is with progress in understanding the obesity GWAS signal that resides within an intronic region of FTO. Specifically, the signal is now known to influence the expression of nearby genes, IRX3, IRX5 and RFGRIP1L, rather than the `host' gene itself. These discoveries suggest that the FTO variant is actually an enhancer embedded in one gene that influences the expression of other causal genes. Since we already have dedicated infrastructure, namely the Center for Spatial and Functional Genomics at the Children's Hospital of Philadelphia (CHOP), to conduct such `variant to gene mapping' efforts, our team is poised to determine at scale how our reported childhood obesity loci affect the expression of specific genes in neuronal relevant cells, namely neural precursors, microglia, astrocytes and hypothalamic neurons. This involves the integration of high resolution `3D Genomics', `Assay for Transposase Accessible Chromatin sequencing' (ATAC-seq), RNA-seq and CRISPR. This final step will enable us to fully infer effector genes. Thus, the premise of this renewal is to uncover the correct functional context of the childhood obesity variants identified by our GWAS in order to translate these discoveries into meaningful benefits for pediatric care.

NIH Research Projects · FY 2026 · 2005-12

Project Summary Cytotoxic lymphocytes, including Natural Killer (NK) cells, are at the forefront of a therapeutic revolution in which the power of the cytotoxic cell is harnessed to kill tumor or virus-infected cells in patients with otherwise hopeless medical diagnoses. For nearly two decades, our laboratory has studied NK cell biology, learning that NK cells use a tightly controlled series of cell biological steps to precisely target their potent cytotoxic contents onto a diseased cell. This process is initiated and directed through the lytic immunological synapse, a specialized interface formed between the NK cell and its triggering target. In previous programs, we have shown that positioning of the NK cell’s destructive organelle, the lytic granule, is key to precise control of NK cell killing. When NK cells meet a target cell, lytic granules rapidly converge to the microtubule organizing center (MTOC); as activation proceeds, the MTOC and its granules move to the lytic synapse where they traverse through a dynamic actin meshwork to be secreted onto the target cell (degranulation). Lytic granule convergence is dynein- dependent and key to limiting killing to a single target cell. When convergence is disrupted, NK cells degranulate in all directions, killing “innocent” bystander cells. While convergence makes sense for surveillance cells who seek to destroy a single infected or malignant cell without causing damage to surrounding healthy tissue, it is limiting in the context of immunotherapy for an established tumor. We propose that blocking convergence without interfering with degranulation will convert NK cells into efficient non-directional destroyers, better suited for solid tumor cancer therapy. We predict that inhibitors and mutations that durably promote non-directional degranulation will promote killing efficiency and afford broader killing (escaping cells, stroma, and tumor- promoting immune cells) in complex pathogenic environments. In the proposed work, we test known and novel inhibitors of convergence, using high through-put small molecule inhibitor and genetic screens as well as a physical “synapsome mapping” approach to identify novel candidates. We ask whether we can achieve non- directional degranulation in a sustainable way (Aim 1A) without disrupting other critical NK cell functions (Aim 1B). In Aim 2, we confirm relevance of non-directional degranulation in a new, tumor-relevant, high-resolution, 3-dimensional model system we’ve called TheCOS (Thermal Collapse of Stroma). This hydrogel-based system allows us to generate instantaneous cell interactions in a manipulable complex environment suitable for high resolution live-cell imaging and functional assessment. We expect our insights to inform an immunotherapy strategy wherein a cytotoxic cell could be maximally efficient, destroying cells it normally wouldn’t, and potentially even avoiding exhaustion within the tumor environment. This would be of tremendous therapeutic value as broadly targeting the pathogenic environment with present cell-based immunotherapies has been a challenge. Equally important, the detailed “map” of synaptic function and novel tools we develop herein will provide critical resources for comprehending and harnessing the function of these powerful cytotoxic cells.

NIH Research Projects · FY 2025 · 2004-05

Lysosome-related organelles (LROs) are cell type-specific subcellular compartments that derive from the endosomal system but that serve unique and vital physiologic functions. They include melanosomes in which melanins are synthesized in skin melanocytes and in developing retinal pigment epithelial cells. The specific functions of LROs are conferred by cell type-specific resident transmembrane proteins, such as melanogenic enzymes and transporters, that are specifically targeted to newly forming LROs. Mutations in genes encoding the cellular machinery responsible for this intracellular targeting underlie the Hermansky-Pudlak syndromes (HPS), a group of genetic disorders in which defective LRO formation leads to several symptoms including oculocutaneous albinism with its associated defective vision and skin and eye hypopigmentation. The known isoforms of HPS result from mutations in genes that encode subunits of four obligate multisubunit complexes – AP-3 and BLOC-1, -2, and -3 – that facilitate protein sorting and membrane remodeling to promote cargo delivery from early endosomal intermediates to maturing melanosomes. We have shown that BLOC-1, together with AP-3, the microtubule motor KIF13A, and additional partners, functions to generate recycling endosome-like tubular transport carriers from early endosomes that extend along microtubules and transiently fuse with maturing melanosomes to deliver their cargoes. BLOC-2 and the lipid phosphatidylinositol-4- phosphate (PtdIns4P) function downstream of BLOC-1 to target the tubules to maturing melanosomes, but the molecular function of BLOC-2 is not known. Moreover, the spatiotemporal relationship between each of these complexes and PtdIns4P during tubule formation, extension, and targeting is not understood. Finally, it is not known how the ubiquitous AP-3 and BLOCs are adapted in specific cell types to target the tubules to LROs instead of for recycling. This competing renewal proposal seeks to fill these knowledge gaps using a combination of live cell imaging, biochemical and electron microscopy approaches, together with genetic manipulation in cultured melanocytes. If successful, our aims will establish new paradigms of tubular transport from endosomes to melanosomes that will extend to additional LROs and to ubiquitous endolysosomal compartments, identify potential targets of mutations in HPS patients who currently lack a molecular diagnosis, and provide potential therapeutic insights for the more debilitating HPS symptoms. The aims are: 1. Test whether BLOC-1 and its interacting partners associate with membrane tubules during their formation, extension, and/or tethering using live cell imaging coupled with electron microscopy analyses; 2. Test whether BLOC-2 and PtdIns4P regulate the dynamic association of tubular membrane transport carriers with microtubules using live cell imaging, biochemical, and unbiased proteomic approaches; 3. Test whether RAB30 confers cell-type specificity in BLOC-1-dependent membrane carrier targeting using knockout/ knockdown together with microscopy and biochemical analyses.

NIH Research Projects · FY 2025 · 2003-09

PROJECT SUMMARY/ABSTRACT For the Chronic Kidney Disease in Children (CKiD) Study renewal, we will continue to follow our existing cohort, and will recruit children between 14 and 17 years of age to enhance the power of the study to characterize the decline of kidney function and the development of cardiovascular disease as they transition to young adulthood. Remote data collection will be coordinated and conducted through national laboratory contractors who can assess vitals and obtain labs in the participant’s home. Data will be obtained between CKiD study visits by conducting data extraction through electronic health records. The Research Electronic Data Capture (REDCap) system will be leveraged to allow automated reporting for coordination and clinical activities and improve options for harmonization with external cohorts including CureGN and NEPTUNE. Most importantly, it will enable the study to collect data through web-based entry by computer or mobile phone directly by participants. Important research questions will include (a) methods to identify risk factors for decline in kidney function during childhood through young adulthood including acceleration of glomerular filtration rate (GFR) decline during puberty and the effect of acute kidney injury (AKI) on chronic kidney disease (CKD); (b) statistical-based machine learning prediction methodology to characterize the joint predictive value of clinical markers and social determinants of disease progression for individualized inference; (c) analytic strategies to describe risk factors for cardiovascular changes and outcomes via home blood pressure monitoring and assessing cardiac and vascular target organ damage, including characterizing changes in biomarkers like trajectories of indicators of metabolic bone health; (d) statistical methods to validate and refine GFR equations in young adulthood for consistent estimates during the transition from pediatric to adult care; and (e) methods to assess effects of therapies and exposures on outcomes including constructs of social function and emotional well-being.

NIH Research Projects · FY 2025 · 2002-09

ABSTRACT To address the severe shortage of academic pediatric endocrinologists, this Training Program will take advantage of the important new opportunities for advancing diabetes and endocrine research in children provided by such recent scientific advances as the Human Genome Project, islet transplantation and biomechanical and bio-engineered islets, and the NIH roadmap transformation of GCRC programs for patient- oriented research into Clinical Translational Science Awards. The Program will support Trainees during up to 2 years of research training at the fellowship level. The Training faculty includes 35 scientific mentors from the Children’s Hospital and the Perelman School of Medicine at University of Pennsylvania who have outstanding credentials and active funded research programs and well-established training records. These mentors will supervise Trainees in basic laboratory research and/or patient-oriented and translational research projects related to diabetes and endocrine disorders in children. Research opportunities will include several areas of basic research (ß-Cell Function, Hormone Action, Mechanisms of Disease, Endocrine Physiology, and Transcriptional Regulation). Patient-oriented research opportunities will include Translational Research, Disease Mechanisms, Pathophysiology, Diabetes Complications, Genetics, Clinical Trials, Metabolic Syndrome, Nutrition, and Epidemiology. The Program includes multiple interactions for Trainees with basic and clinical research and training in all aspects of research, including biostatistics, bioethics, molecular biology, etc. The Program is strongly supported by access to a superb range of institutional resources at Children’s Hospital and University of Pennsylvania, including the CTSA and the University of Pennsylvania DERC. The request is made to support 3 fellow slots in this Program each year. The long-term goal of this T32 Training Program renewal is to develop a new generation of pediatric endocrinologists who will be equipped to carry out innovative and scientifically rigorous patient- oriented and laboratory-based research in diabetes and endocrine disorders of children.

NIH Research Projects · FY 2025 · 2002-09

This proposal is in response to a request for applications for the Continuation of ChiLDReN, the Childhood Liver Disease Research Network. Over the past twenty years, through a coordinated effort, investigations of cholestatic pediatric disorders have been advanced and we have established a robust database and biorepository for further research. Little is known about the pathogenesis, natural history, and optimal treatment strategies for the rare pediatric liver diseases investigated by ChiLDReN. We at The Children’s Hospital of Philadelphia (CHOP) propose to continue to participate in this Consortium, and thereby advance the field through collaborative research. CHOP has been a highly productive member of ChiLDReN for the last 20 years. In this application, we propose to continue our participation in all aspects of the ChiLDReN consortium, including observational and interventional study protocols, genomics initiatives, ancillary studies and dissemination of research findings. Only through collaboration can we improve the quality and efficiency of care provided to all individuals diagnosed with one of the diseases studied by this network. We have assembled a team of physicians and scientists who contribute substantially to the intellectual and practical needs of the Consortium. The Core Staff includes the Principal Investigators, the Co-Investigators, the Program Manager and the Research Study Coordinators. Other interested scientists and clinicians will participate as determined by Consortium protocols. Our team will continue to collaborate fully with the SDCC and the other investigators to advance scientific discoveries in rare pediatric liver diseases. We will continue to follow patients in the active observational clinical protocols, including PROBE, BASIC, LOGIC, MITOHEP, PSC and Genetic Collection Study, and enroll new participants with biliary atresia and PSC. We will continue to collaborate with the SDCC to implement procedures for uniform data collection, handling and transmittal of data, as well as data audits and other data quality control procedures. We will continue leadership and committee membership in administrative roles, study protocols and disease working groups. The team at CHOP will continue to lead and actively participate in writing groups for Network studies, to promote the dissemination of new knowledge in these rare liver diseases. We will continue to develop new ancillary and translational studies leveraging the robust data and samples previously collected in the ongoing network-wide studies. With the continuation of the Childhood Liver Disease Research Network, robust collaboration among Centers will lead to advances in the field to improve outcomes for children with rare liver disease.

NIH Research Projects · FY 2025 · 2002-09

A major barrier to effective treatment of the central nervous system (CNS) in most inherited lysosomal storage diseases (LSD) is that the metabolic defect in all brain cells results in widespread pathology. The therapeutic principle for most LSDs is to transfer a normal enzyme cDNA into a subset of diseased cells, thereby correcting both vector-transduced cells and neighboring non-transduced cells that take up the secreted therapeutic enzyme. To achieve global correction, transduced cells must be dispersed 3-dimensionally throughout the brain so that secreted vector-encoded therapeutic enzyme can reach all non-transduced cells. Although intravascular injection of certain AAV vector serotypes can cross the blood-brain barrier and deliver genes widely in rodent brains, AAV vector distribution in large mammals occurs mostly in the lower brain and spinal cord, limiting the potential for treatment. Thus, optimization of vector distribution in large animal models of human diseases is critically needed to facilitate translation into clinical usage. In the prior grant period, we have shown that a novel AAV serotype (AAV.hu32) mediates widespread gene delivery in a cat model of alpha-mannosidosis (AMD) caused by a mutation in the gene encoding lysosomal a-mannosidase (MANB) that recapitulates the severe form of AMD. Notably, high doses of AAV.hu32 encoding MANB resulted in global correction of the storage lesions and improvements in disease parameters. Although this appears promising for translation into clinical usage, the vector doses required would be near the limits of production for clinical grade AAV vector when scaled up to human patients and there have been safety concerns raised in recent clinical trials for use of such high vector doses. Finally, new preliminary data shows that vector doses that are completely effective when AMD cats are treated in the early stages of the disease do not mediate complete brain correction when treatment is initiated at a more advanced stage of disease, providing additional impetus to improve global brain delivery at lower vector doses. In exciting new studies, we have found that injection via carotid artery was even more effective than intravenous injection, suggesting that passage of concentrated virus through the vascular bed of the brain improves uptake. Surprisingly, we also found that while direct injection of AAV.hu32 into brain parenchyma transduced oligodendrocytes, astrocytes and neurons, its vascular delivery resulted in the almost exclusive transduction of neurons. Thus, current studies will focus on reducing the required vector dose by determining the route the vector takes between blood and brain to specifically transduce neurons, increasing the levels of MANB produced by the genetically corrected neurons, and increasing the number of transduced cells by an alternative dosing regimen that will maximize vector uptake in the vascular bed of the brain. We will then determine if an optimized combination of these strategies mediates global correction when treatment is started at the advanced stage of disease of AMD cats, which will be the case for most human patients.

NIH Research Projects · FY 2026 · 2002-07

This application seeks to renew an NICHD-sponsored National Research Service Award T32 that supports fellowship training in the Department of Pediatrics at the Children's Hospital of Philadelphia (CHOP) and the Perelman School of Medicine at the University of Pennsylvania (Penn). A continuous commitment to pediatric research is necessary to ensure advances in the health of children, adolescents, and young adults. Establishing and maintaining a nurturing environment for pediatric subspecialty trainees to evolve into independent scientists is the foundation of our institution’s commitment to making advances in pediatric research. The long-term objective of our NRSA-T32 program is to provide an environment that promotes the maturation of pediatric subspecialty trainees into rigorous independent investigators who are addressing important questions relevant to the health and well-being of the pediatric population. The program leverages the robust research and training resources at CHOP and Penn, including a core of accomplished faculty mentors in the Department of Pediatrics and other departments at CHOP who are performing rigorous science in areas germane to pediatrics, have a strong record of successful mentoring and collaboration, and have a strong record of extramural funding. Four trainees will be supported each year and will be drawn from subspecialty fellows in the Department of Pediatrics, with a goal of recruiting trainees from a range of subspecialities and selected on the basis of potential for excellence in pediatric research. Trainees will typically receive support for two years of investigation and will emphasize the application of basic and translational science techniques, rigorous clinical and epidemiologic methods, and novel approaches to community focused research. Collectively, these approaches will serve to improve our understanding of pediatric diseases and to develop new and effective preventative and therapeutic interventions that optimize the health of children. Trainees will have access to the full array of research centers and cores at CHOP and Penn and will also benefit from extensive educational activities at CHOP and Penn, including a robust infrastructure for responsible conduct of research, master degree programs, certificate programs, and a range of seminars, among other research related resources. The Principal Investigator/Program Director and Training Director, will receive assistance from an advisory committee that includes internal and external research leaders who play a key role in selecting trainees, monitoring their scholarly progress, and ensuring optimal operation of the program. The renewal of this T32 program will continue the tradition established during the past 22 years, capitalizing on the tremendous strengths of CHOP and Penn, with outstanding subspecialty fellows, accomplished mentors, and numerous cutting edge research programs.

NIH Research Projects · FY 2024 · 1999-08

PROJECT SUMMARY Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycemia in infants and children. In the last 20+ years a total of 9 genetic loci have been associated with HI, however, in approximately 40-50% of cases a genetic cause is not identified. The elucidation of the molecular mechanisms responsible for HI is of great importance to guide the development of novel therapies and eventually, a cure. Building on the success of the previous cycles of this award, we propose a comprehensive approach to examine the mechanisms of disease for two novel forms of HI that we have previously mapped to specific genomic loci by linkage analysis and whole exome sequencing. In addition, we propose a broad discovery effort for novel mechanisms of disease by performing deep genotyping and phenotyping of both individuals and isolated islets in cases with negative genetic testing on constitutional DNA. Our overall hypothesis is that genetic testing negative HI cases can be explained by novel genetic loci encompassing factors important for ß-cell function and by somatic mutations of known HI genes. To test this hypothesis, we propose two aims: to examine the mechanisms responsible for dysregulated insulin secretion by novel genetic loci identified in families with dominantly-inherited HI: Hexokinase 1 and PASK; to identify the role of post-zigotic mutations in HI; and to continue our discovery efforts through a comprehensive approach to examine genomics and functionomics of HI by deep genotyping and functional evaluations of affected individuals and their pancreatic islets. This study will expand our understanding of the molecular genetics and pathophysiology of HI and will facilitate the identification of new genetic causes and potential new therapeutic targets for this devastating disease. The discovery of novel HI loci may lead to a better understanding of the mechanisms regulating insulin secretion and may benefit the development of new therapies not only for HI, but also for diabetes.

NIH Research Projects · FY 2025 · 1999-07

Summary Research advances in the field of Pediatric Cardiology have led to both improved clinical outcomes and major scientific insights. Research training through this NHLBI program has helped to achieve this success by constantly incorporating new discoveries into the individual training curricula of our mentees. Our NHLBI training program, now in its 24th year, has demonstrated that our trainees contribute strongly to advances in the field, while establishing their own independent, productive careers. The aims of our program are to: 1.To Identify, recruit and foster research trainees, both CHOP cardiology fellows and scientists best suited for a career focusing on the challenges facing pediatric cardiovascular medicine, who are willing to commit to training and career development in this field. 2.Match trainee interests and strengths with mentoring teams. 3.Provide mentor training and support. 4.Provide essential resources and novel opportunities for career development, with a goal of graduating trainees capable of independence. The objectives of this NHLBI-Postdoctoral Training Program are to:  Train a cadre of committed researchers to advance the field of pediatric cardiovascular research by addressing unmet needs.  Provide these individuals with the skill sets and foundation for career advancement.  Encourage leadership and innovation. Goals–to pursue the aims and objectives through a training program consisting of:  Mentored independent research projects, with a team approach, with requisite mentor training and scholarship oversight.  Didactic opportunities, including core requirements and individually selected courses designed to provide specific research skills  Seminars, workshops and a journal club focusing on research and progress in the field.  Career guidance including independent development plans (IDP), seminars and workshops focusing on professional development and survival skills.  Fostering a connection with mainstream pediatric cardiology research through participation in CHOP Cardiology research activities, CHOP Cardiac Center Research, the CHOP Cardiovascular Research Institute, and The Penn Cardiovascular Institute.

NIH Research Projects · FY 2026 · 1999-03

Abstract The spatial organization of chromosomes and gene expression are mutually influential processes. Perturbations of either can lead to developmental defects and disease. Studies on chromatin structure have been dominated by those focusing on the architectural transcription factors CTCF, YY1 and the cohesin complex. The central hypothesis of this application, supported by preliminary data, is that the non-DNA binding adapter protein Ldb1 presents a separate, equally important category of nuclear chromatin organizers. In prior work we demonstrated that artificial recruitment of Ldb1 to chromatin can be sufficient to forge long range enhancer- promoter contacts at the b-globin locus. This nominated Ldb1 as a key architectural protein in erythroid cells. Yet a global view of Ldb1 chromatin occupancy patterns during cell differentiation or cell cycle progression is lacking, and genome wide direct Ldb1 dependent chromatin contacts and corresponding transcriptional patterns are largely unknown. More broadly, the cause-effect relationships of architectural features and gene expression are still hotly debated in the field. Here we propose to carry out the following aims in collaboration with the laboratory of Dr. Ross Hardison. In Aim 1 we will assess in erythroblasts undergoing cell maturation the dynamic Ldb1 chromatin landscapes in relation to high resolution Hi-C chromosomal connectivity maps, and nascent transcription measurements (PRO- seq). In Aim 2 we will use an auxin-inducible acute Ldb1 degradation system to interrogate direct architectural and transcriptional Ldb1 dependencies. In Aim 3 we will exploit the massive and swift changes in chromatin architecture during the mitosis to G1-phase progression to assess the chromatin occupancy dynamics of Ldb1 and its role in establishing the multi-layered hierarchy of chromatin organization and gene expression. All aims are accompanied by perturbative and mechanistic experiments. We believe that the power of the proposed studies lies in the complementarity of highly dynamic natural transition states (cell maturation and cell cycle progression) as well as acute experimental perturbation (auxin degron), which to our knowledge has never been done before within the same cell system. Gains in knowledge are further enhanced by parallel studies in the same cellular system on CTCF, YY1 and cohesin with support from different funding sources. The forward looking view is that the end result of our studies is, for the first time, a unified view of the dynamics of major architectural components and gene expression. Therefore, the studies proposed here coupled with parallel studies in the lab generate a cost-effective synergy, such that the outcome will be greater than the sum of its parts.

NIH Research Projects · FY 2025 · 1998-07

PROJECT SUMMARY/ABSTRACT We seek continued funding for an interdisciplinary training program focused on Neurodevelopmental Disabilities (NDD). Based at The Children's Hospital of Philadelphia (CHOP) and University of Pennsylvania (Penn), this program is integrated into the CHOP/Penn Intellectual and Developmental Disabilities Research Center and broader CHOP/Penn neuroscience community. The goal of the Program is to train MD and PhD fellows in research focused on NDDs. There are three reasons for this focus. First, NDDs are common; ~10% of U.S. households live with an individual who has an NDD. These households bear significant and often lifelong financial cost and emotional impact. Second, NDDs have diverse causes — from genetic to acquired — that alter brain development, circuitry, and behavior. Understanding how these causes lead to the various NDDs will yield treatments for what are currently untreatable disorders. Third, the symptoms of the various NDDs are broad and impact all aspects of brain function, requiring interdisciplinary investigation. Training researchers to improve our understanding of NDDs and ultimately develop treatments will improve outcomes and quality of lives for individuals with NDDs and their families. Hence, we request continued support for 6 postdoctoral fellows/year who participate in a program designed to be three years in length. This number of trainees allows us to maintain a critical mass to support a diverse trainee pool that can learn from each other and is easily justified by the many outstanding applicants. In the labs of our 29 faculty mentors, trainees use state-of-the-art genetic, cellular/molecular, behavioral, physiologic, and structural/dynamic imaging techniques to pursue basic and translational research relevant to NDDs. While the main Program focus is this mentored research training, the trainees (and their mentors) also regularly meet for activities that develop oral and written communication skills and encourage exchange of scientific information. They also work with the Program Statistician to build a strong foundation in quantitative skills and fluency. The Program Directors help trainees develop customized plans to reach their research and career goals. Our past trainees are now leaders in NDD research across the country. In the last 15 years, 38 trainees came into the Program with a degree of MD (1), MD/PhD (5), DMV/PhD (1), or PhD (31). Twenty-four (63%) of these trainees are female and 10 (26%) of these trainees are from groups historically-excluded from biomedical science and research. Thirty-two different NDD T32 mentors have supervised these trainees. The 32 trainees who completed both T32 support and postdoc training since 2007 are now faculty members (14 Asst/Assoc Prof), scientists in academia (1 Instruct., 2 Res Assoc.), pharma/biotech (9), and at a non-profit (1), scientific writers (2) and clinicians (2), and one is the CHOP Assoc. Director of Diversity. This T32 combines the outstanding CHOP/Penn training environment, an exceptional cadre of trainees and mentors, an experienced multi-PI team, and rich institutional resources for NDD research and training to fuel a true ‘bench to bedside’ approach to translational research.

NIH Research Projects · FY 2025 · 1997-07

The field of medicine is experiencing a genomics revolution. The need to train physician scientists and investigators of varied academic backgrounds experienced in genomic and genetic methodologies and able to translate basic science discoveries in genomics into clinical care has never been greater. The major goal of this postdoctoral research training program is to train the future leaders of Medical Genetics and Genomics who will emerge from a variety of training pathways with varying amounts of research experience. Postdoctoral trainees with M.D. or M.D., Ph.D. degrees with clinical training in Medical Genetics, Pediatrics, Medicine, Psychiatry, Pathology, and other specialty areas will be eligible for support from this training grant. Funding for 5 training slots per year is sought. The clinical training years (typically year 1 for Categorical Medical Genetics trainees and years 1-3 for combined Pediatric and Medical Genetics trainees) is funded by the Perelman School of Medicine at the University of Pennsylvania (UPenn) and The Children’s Hospital of Philadelphia (CHOP), while funds for the research years of the overall training program are sought from this training grant. During the Medical Genetics Research Fellowship years supported by this Training Grant, the fellow devotes a minimum of 85% effort to research in a basic science laboratory. Research opportunities are abundant and extend across the entire field of genetics, with training in the laboratories of 42 Faculty from 6 core departments at CHOP/UPenn. Fields of research may encompass those areas that impact human genetics including, but not limited to: genomics, molecular genetics, cytogenomics, biochemical genetics, epigenetics, mitochondrial genetics, developmental biology, cellular biology, bioinformatics, systems biology, pharmacogenetics, gene therapy and others. During the research years, the trainee also takes seminar courses, attends journal clubs, research meetings, and departmental research retreats, and carries out minimal clinical activities, not to exceed 15% effort. Training stipends for the 2-3 years of research are requested in this application. The M.D. trainee will likely require further research training (not covered by this training grant), which might be acquired through an additional postdoctoral research experience or a protected faculty appointment with considerable mentoring from a senior faculty member. The training program outlined in this proposal is built on our past track record of success and is committed to recruiting a robust group of highly talented postdoctoral trainees with a multitude of genetics research interests to a rich and supportive research environment and through a rigorous program of training, mentorship and oversight develop the future physician-scientist leaders in Medical Genetics and Genomics.

NIH Research Projects · FY 2025 · 1976-07

The primary objective of the Pediatric Hematology Research Training Program has been to train individuals – mostly pediatric hematology/oncology fellows – for academic or industrial careers in pediatric-related, benign hematology. Pediatric benign hematology is a highly underserved area of research. Our Division, supported by this T32, has excelled at attracting, training, and retaining young, talented individuals to this field who have risen to leadership roles in pediatric benign hematology research. In this renewal, we propose to expand the scope of our T32, increasing our slots from 4 to 6 postdoctoral slots per year to pursue a new, more ambitious vision. The Specific Aims of this renewal are as follows: (1) This T32 renewal will offer a wide range of research training opportunities of particular import to pediatrics in hemoglobinopathies and other anemias, hemostasis/ thrombosis, megakaryocyte/platelet biology, transfusion medicine, stem cell development, bone marrow failure, and now, vasculogenesis. (2) It will support a wide pool of potential trainees. Besides the pediatric hematology/oncology fellows pool we will include: i. non-hematology pediatric fellows interested in pursuing research in benign hematology, and ii. candidates from a proposed sickle cell disease (SCD) subspecialty training program offering state-of-the-art research training related to SCD. (3) Training will continue to involve a “team” approach of a primary mentor plus advisory faculty with key ancillary skills (e.g., bioinformatics or gene therapy). (4) The T32 infrastructure will continue to carefully monitor individual trainee progress and oversee mentorship towards reaching the appropriate next career milestones using a novel “stepwise oversight” committee designed to transition pediatric hematology/oncology fellows from their clinical year(s) to their early assistant professor years. (5) Our program will continue to emphasize acquisition of skills critical for successful academic careers. Preparation of manuscripts, presentations and grant submissions, detailed bioethical training, laboratory or investigator leadership skills, and specific training in optimizing authentication of key reagents, in designing the laboratory and/or clinical study, and in rigorous data analysis. The training faculty is comprised of a closely-knit group of productive, well-funded, pediatric benign hematology physicians and scientists with strong records as mentors, supplemented by additional trainers that bring in skills key for the success of our trainees. Our program has been successful in the research training of highly talented individuals, and this effort to train such strong candidates remains a priority. Despite a national trend away from subspecialty research training in pediatrics, this T32 mechanism has enabled our Program to train outstanding candidates for academic careers to become national and international leaders in many areas of benign pediatric hematology. We believe that the expanded vision we propose for our T32 renewal shows the program’s continued vigor, and its ability to evolve and maintain relevance in our field of benign pediatric hematology.