Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2026 · 2022-05

Project Summary Recently, it was shown by Dr. Ben Boursi, Sheba Medical Center, that some metastatic melanoma patients who are refractory to anti-PD-1 immunotherapy can be converted to responders by fecal microbiota transfer (FMT) from a melanoma patient that had a complete response to immunotherapy. Unfortunately, other donor-recipient combinations were unsuccessful implying that an additional uncontrolled factor may determine the effects of microbiota modulation of immunotherapy. Concurrently, we have been using congenic C57BL/6 mice harboring different naturally occurring mitochondrial DNAs (mtDNAs) (mtDNAB6, mtDNA129, and mtDNANZB) to test melanoma sensitivity and anti-PD-L1 therapy. We discovered that the mtDNANZB mice are highly resistant to melanoma progression and strongly respond to anti-PD-L1 therapy, while mtDNA129 mice are permissive for melanoma growth and refractory to immunotherapy, with mtDNAB6 mice being in between. These mice also differ in their gut microbiota and metabolomic analysis of the mtDNANZB mice revealed impaired fatty acid oxidation of relevance to the elaboration of short chain fatty acids (SCFAs) by the gut microbiota. When we expressed the mitochondrially-targeted antioxidant enzyme catalase (mCAT) in the mitochondria of the mouse hematopoietic cells, we diminished the anti-tumor immune response of the mtDNANZB mice and changed the gut microbiota of both the mtDNAB6 and mtDNANZB mice. These observations led us to the hypothesis that: Both the gut microbiota and the immune system are modulated by the mitochondrial genome, in part through mitochondrial reactive oxygen species (mROS) production in immune cells linking the gut microbiota, tumor progression, and immunotherapy. To test this hypothesis, we propose three specific aims. First, we will evaluate mitochondrial function and mROS production in our three congenic strains and correlate this with their immune cell repertoire and function. Then, we will determine if these congenic strains show the same range of responses to other tumor types. Second, we will determine which subclass of hematopoietic cells are responsible for the anti-tumor and pro-immunotherapy response by using adoptive cell transfer (ACT) to replace mtDNA129 immune cells with mtDNANZB cells. We will then express mCAT in the functional immune cells to determine if this negates the anti- tumor and pro-immunotherapy response and changes their microbiota. Third, we will use FMT to replace the gut microbiota of the mtDNA129 and mtDNANZB mice with that of the three congenic strains to determine if mtDNANZB microbiota enhances the mtDNA129 anti-tumor and pro-immunotherapy phenotype and if mtDNA129 microbiota diminish the mtDNANZB phenotype. To confirm that this is mediated by mROS production, we will express mCAT in the responsible immune cells of the mtDNA129 mice and confirm that this blocks the induction of any anti-tumor and pro-immunotherapy phenotype induced by FMT from mtDNANZB mice. To expeditiously extend these findings to human mtDNA lineages and clinical service, Dr. Boursi has agreed to be a collaborator and Dr. Yardeni has arranged positions at both CMEM and Sheba.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Sepsis is broadly defined as life-threatening organ dysfunction caused by infection. However, the experience of sepsis varies widely between individuals and within individuals. Such inter- and intra-individual heterogeneity reflects complex, evolving pathobiology induced by host-pathogen interactions and modified by pre-existing patient characteristics and ongoing medical treatments. It is highly unlikely that a single—or even a small panel—of biomarkers will characterize an individual’s pathobiology with enough accuracy to unlock precision medicine approaches in sepsis. The challenge, therefore, is to identify and accurately characterize relevant phenotypes of sepsis and develop enrichment strategies to design and test novel targeted therapies. For this project, we propose testing the feasibility of novel approaches to collect, process, and analyze biologic data representing the immuno-inflammatory-metabolic response to infection early in the sepsis course and link this information to relevant organ dysfunction-based phenotype data from the electronic health record (EHR). We have assembled a multi-disciplinary team composed of experts in translational immunology, pediatric emergency medicine, critical care, and data science. We will test the feasibility of collecting and processing blood samples of different volumes for deep phenotyping at the pre-resuscitation phase, which is a critical timepoint, as well as 48 hours later to capture dynamic changes post-resuscitation. We will use these samples to characterize the functional immuno-inflammatory-metabolic biology using high dimensional flow cytometry to track surface and intracellular markers of lymphocyte proliferation, apoptosis, exhaustion, and cytokine production. In addition, we will extend flow cytometric techniques to perform bioenergetic evaluation, and will perform proteomic evaluation of the plasma immuno-inflammatory-metabolomic response using O-link. This project will take advantage of extensive clinical research infrastructure at the three study sites. In addition, the investigators will have access to innovative biologic assays at CHOP which will allow our collaborative team to glean key biologic phenotypes from critically ill and complex patients. In the R21 phase, we will complete a single-center pilot study to test the feasibility of collecting, processing, and analyzing the optimal blood specimens for deep phenotyping in the early phase of sepsis, linking them to the EHR registry data, and developing and testing a data pipeline to characterize the patients’ organ dysfunction-based clinical phenotypes. In the R33 phase, we will expand the sample collection to three sites and perform a proof-of- concept analysis to determine if there are differences in the immuno-inflammatory-metabolomic patterns of the children who develop either of the high-risk organ dysfunction-based sepsis phenotypes. Identifying relevant biological patterns in the R33 phase can then lead to future hypothesis-driven mechanistic studies and the development of targeted therapies.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY Progressive myoclonus epilepsy type 7 (EPM7) is due to a recurrent pathogenic variant in the gene KCNC1, which encodes the voltage-gated potassium (K+) channel subunit Kv3.1. EPM is a class of devastating conditions defined by onset of tremor, seizures, and ataxia in a previously normal child or young adult, with relentless deterioration to wheelchair dependency as well as epilepsy and myoclonus. Further research is required to clarify the functional role of Kv3 channels in normal brain function and how genetic variation in KCNC1 leads to EPM7 and other forms of neurological disease, so as to facilitate progress towards novel therapies, preventative measures, or cure. Such insights may prove generalizable to other forms of EPM, which remains a class of untreatable and incurable disorders. This 5-year collaborative application employs a comprehensive approach and newly-generated tools to test the hypothesis that the clinical phenotype of EPM7 is due to loss of Kv3.1 function, leading to the selective dysfunction of Kv3.1-expressing fast-spiking neurons in discrete locations throughout the brain. Targeted pharmacologic modulation of Kv3 channels with a potent, specific Kv3 activator will recover cellular and synaptic abnormalities of Kv3.1-expressing neurons, leading to decreased susceptibility to seizure and improvement in cerebellar dysfunction in an experimental model of EPM7. Proposed experiments will determine the relationship between specific KCNC1 variants, physiology, and clinical phenotype (mild intellectual disability with/without epilepsy; EPM7; or severe early-onset myoclonic epileptic encephalopathy) in a large cohort of human patients with KCNC1-related neurological disorders compiled by the applicant. To link KCNC1 variants to ion channel dysfunction, we will compare the biophysical properties of normal Kv3 K+ channels to channels containing variant Kv3.1 subunits, as well as the ability of a novel Kv3- specific pharmacological agent to normalize pathological channel activity (Aim 1). The impact of variant KCNC1 on the intrinsic excitability and synaptic and circuit function of Kv3.1-expressing neurons will be pursued using a new mouse model of EPM7 generated by the applicant (Kcnc1-R320H/+ mice, which recapitulate the core clinical phenotype seen in humans) (Aim 2). Then, we will attempt to ameliorate disease pathology via administration of targeted therapeutics in vivo (Aim 3). Results will provide novel information as to the role of Kv3.1 in cellular, synaptic, and circuit function and define the pathogenic mechanisms of KCNC1-related neurological disorders towards development and implementation of novel, targeted therapies in human patients.

NIH Research Projects · FY 2025 · 2022-04

PROJECT SUMMARY Neonatal hypoxic ischemic encephalopathy (HIE), a common brain injury from loss of oxygen and nutrients immediately prior to birth, results in long-term neurologic deficits even in the absence of significant perinatal cell death. A major gap in the perinatal injury field is the limited mechanistic understanding of how transient hypoxia results in persistent cellular deficits. A compelling mechanism linking prenatal hypoxia to persistent deficits is that prenatal hypoxia permanently alters the epigenome because the epigenome integrates development with response to the environment. To study the mechanisms underlying the pathology of HIE, I developed a novel mouse model of transient late gestation prenatal hypoxic injury that phenocopies mild HIE. Prenatal hypoxia leads to persistent behavioral and structural deficits in adult mice despite no significant increase in cell death in the fetal brain. My preliminary data showed decrease in dendritic spine density in corticothalamic neurons at postnatal day 28 (P28), weeks after hypoxia. Prenatal hypoxia was associated with upregulation of genes associated with the epigenome in single nucleus RNA and assay for transposase-accessible chromatin sequencing (snRNA/ATAC-seq) in the fetal neocortex within one hour of exposure. One of the most upregulated genes, the histone variant H3f3b, is a candidate for protecting the brain from more severe brain injury. However, the epigenetic trajectories of corticothalamic neurons seemed to be shifted even immediately after hypoxic exposure. Therefore, my central hypothesis is that H3f3b upregulation is necessary and sufficient to protect neurons from severe injury after prenatal hypoxia and distinct epigenetic regulators contribute to lasting neuronal injury. To test my hypothesis, I propose three aims. In Aim 1, I will use conditional knockout mice to test if H3f3b depletion increases cell death immediately after hypoxia and worsens the persistent deficits in spine density in P28 mice and behaviors in adult mice. In Aim 2, I will use in utero electroporation to test if overexpression of H3f3b improves deficits from hypoxia in spine density in P28 mice and behaviors in adult mice. Lastly, in Aim 3, I propose to use snRNA/ATAC-seq in the cortex of P28 mice to determine if prenatal hypoxia has a permanent effect on the epigenome of corticothalamic neurons. My long-term goal is to be a child neurologist who leverages my research expertise in molecular biology and clinical skills in perinatal brain injury both to develop targeted therapies towards this frequently devastating injury. My mentors, Drs. Eric Marsh and Michal Elovitz, are excellent physician scientists devoted to using translational research to improving outcomes in children with neurological disorders. Under their guidance for this K08 proposal, I will gain additional skills in bioinformatics, a deeper expertise in translational animal studies, and lab management skills necessary to become an expert in the epigenetics of neurological disorders and be prepared for a career as an independent R01-funded physician scientist.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY High expression of the transcriptional co-activator Meningioma-1 (MN1) is common in AML, and associated with a poor prognosis. Forced expression of MN1 in murine hematopoietic progenitors induces an aggressive leukemia. We recently discovered that the primary interaction partner of MN1 is the BAF nucleosome- positioning complex. MN1 stabilizes BAF on chromatin. MN1 binding is associated with sustained active enhancer chromatin at enhancers regulating a hematopoietic stem/progenitor program. We hypothesize that MN1 stabilizes promoter-enhancer contacts at these sites through a BAF dependent mechanism. The goal of this project is to uncover the molecular mechanism of MN1-mediated leukemic transformation. A better understanding of how MN1 causes leukemia may identify opportunities for targeted therapies in a patient population who is failing conventional AML therapy.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY High expression of the intrinsically disordered protein Meningioma-1 (MN1) is common in AML, and associated with a poor prognosis. Forced expression of MN1 in murine hematopoietic progenitors induces an aggressive leukemia. We recently discovered that the primary interaction partner of MN1 is the BAF nucleosome-positioning complex. MN1 stabilizes BAF on chromatin. MN1 binding is associated with sustained active enhancer chromatin at enhancers regulating a hematopoietic stem/progenitor program. Intriguingly, MN1’s entire coding frame is disordered. We hypothesize that MN1 causes AML by overstabilizing transcriptional hubs by increasing multi- valent, low affinity interactions that result in high local concentrations of BAF and early hematopoietic transcription factors. A better understanding of how MN1 causes leukemia may identify opportunities for targeted therapies in a patient population who is failing conventional AML therapy.

NIH Research Projects · FY 2026 · 2022-04

Summary Hematopoietic stem and progenitor cells (HSPCs) are regulated by a balanced signaling network, which is critical for HSPC homeostasis and prevention of malignant transformation. We previously showed that protein ubiquitination by CBL family E3 ubiquitin ligases controls JAK2 stability and activity that is important for curbing HSPC expansion and myeloid malignancies. Here we identified a novel signaling axis, where CBL/JAK2 upregulates RAB27B to enhance NRAS GTPase activity and ERK signaling. Importantly, aberrant activation of this pathway is critical for leukemia cell growth conferred by CBL and RAS mutations. Intracellular signaling can be dynamically modulated by post-translational modifications (PTMs) that regulate the temporal and spatial distribution of signaling proteins. RAB27B, a Rab GTPase that is resident in the Golgi and endosome membranes, regulates intracellular vesicle trafficking, docking, and fusion with plasma membrane (PM). Rab27b knockout mice display normal steady-state hematopoiesis. Strikingly, we found that Rab27b deficiency in primary HSPCs abrogates mutant but not wildtype NRAS-mediated signaling and cell growth. Mechanistically, we demonstrated that RAB27B regulates NRAS palmitoylation, GTPase activity, stability, and subsequent c- RAF/MEK/ERK activation. RAS proteins propagate signals only when associated with cellular membranes as a consequence of various PTMs that impact their trafficking between endomembranes and the PM. Therefore, a precise understanding of RAS’ interaction with membranes and trafficking is essential to understand RAS action and to intervene in RAS-driven cancers. The discovery of RAB27B as a novel regulator of RAS palmitoylation, a lipid modification for membrane anchors, propelled us to further define the molecular basis underlying the regulation of RAS/ERK signaling by CBL/JAK2/RAB27B and explore its functional significance in malignant HSPCs. In aim 1, we will investigate if Rab27b deficiency mitigates chronic myelomonocytic leukemia (CMML) development and malignant HSPC expansion induced by mutant Nras or Cbl deficient mice. More importantly we will study if Rab27b deficiency dampens NRAS signaling, stability, lipid modification and subcellular localization. In aim 2, we will use live-cell imaging and biochemical assays as well as genetic approaches, to dissect the dynamic regulation of NRAS trafficking, palmitoylation, and compartmentalized signaling by RAB27B. In aim 3, we will investigate the role of mutant CBL/JAK2 in regulating RAB27B level and explore the therapeutic potential of targeting RAB27B in primary HSPCs from human myeloid malignancies. RAS pathway mutations including NRAS and CBL define the proliferative CMML (pCMML) phenotype that is aggressive, predisposed to AML transformation and associated with dismal outcomes. This work uncovers novel RAB27B-mediated compartmentalized signaling dynamics that is crucial to key signaling proteins, and serves the basis for future therapeutic strategies for molecular targeting.

NIH Research Projects · FY 2025 · 2022-03

This project is a continuation of the original funded K99/R00 application. Having secured a tenure- track assistant professor role, jointly at the Children’s Hospital of Philadelphia and the University of Pennsylvania, the remaining aims will be pursued at these institutions. The aims proposed here have not been changed substantively from the ones originally proposed for the independent phase of the award. Real world decisions have to be made in environments that are noisy and uncertain. The frontal cortex—the brain’s principal center for cognitive control—must account for such uncertainty to guide adaptive decision-making. From a translational perspective, failure to resolve uncertainty is thought to represent a core computational deficit in schizophrenia, contributing to abnormal perceptions, distorted beliefs, and psychotic symptoms. While an extensive body of literature from humans as well as preclinical models have established a causal role of the frontal cortex in such decision making, recent work, including my own have also highlighted an important role of thalamocortical interactions between the frontal cortex and the medio dorsal (MD) thalamus in perceptual decision making under uncertainty. Building on this foundation, I will test whether MD–frontal cortex circuits also support value based decision making with uncertainty in the expected value of rewards/outcomes. First, we will train tree shrews on an object valuation task that introduces trial-by-trial uncertainty in the subjective value of two options, while recording neural activity from the frontal cortex to identify representations of subjective value. Second, we will employ multisite electrophysiological recordings and causal optogenetic perturbations to examine the mechanistic role and circuit substrates of MD-frontal cortex interactions in this paradigm. The insights developed here on the mechanistic basis of object valuation under conditions of uncertainty will form the basis of my future research program dissecting the circuit substrates of maladaptive beliefs and motivational anhedonia that underlie schizophrenia.

NIH Research Projects · FY 2025 · 2022-02

ABSTRACT Adeno-associated virus (AAV) vectors are in clinical development for delivery of genes to treat multiple genetic diseases including hemophilia. While progress has been made to optimize gene delivery, in some studies the required AAV vector doses were high, leading to toxicity and even fatal outcomes in one study. These findings highlight the need for novel approaches to reduce the AAV vector dose to minimize liver toxicity, anti-AAV immune responses, and genotoxicity. Our recent studies and work from others have identified an underappreciated limitation to efficient gene correction with AAV vectors. In a long term study of AAV gene delivery of FVIII in hemophilia A dogs, we found that most of the AAV vector genomes were highly rearranged in transduced liver tissues. These rearrangements typically disrupted the transgene, and so would compromise expression of the transgene product—unexpectedly, our data indicated that most of the AAV vector genomes present did not produce functional protein after transduction. These rearranged AAV genomes were present in integrated forms but also in AAV concatemers that may be episomal forms. It is unclear whether these rearrangements occurred during vector production or after transduction of the target cells, though data is accumulating that at least some of the rearrangements originate in vector producer cells. Our hemophilia A dog study also identified integration events in the canine genome within genes linked to cell growth and cancer that were associated with clonal expansions. Validation of integrated AAV DNA in these expanded clones by sequence analysis showed that in all cases integrated vectors were highly rearranged, with only one of five encoding an intact transgene. An extensive literature documents interactions of AAV with host DNA repair pathways in both vector producer and target cells, though the influence of host factors in AAV DNA rearrangements is mostly unstudied. We hypothesize that modulation of host cell pathways can suppress AAV DNA rearrangements, thereby allowing improved transgene expression per vector DNA copy. In this proposal, we will 1) implement a deep sequencing method to quantify rearrangement frequency in a statistically rigorous fashion, 2) identify cellular pathways that can be modulated with small molecules, siRNAs, or microRNAs that suppress vector rearrangements, and 3) devise novel delivery strategies that support efficient pathway modulation, suppress vector rearrangement, and boost transgene output per vector copy. These methods will be assessed during AAV vector production (Specific Aim 1) and after AAV delivery in the transduced target cells (Specific Aim 2). Our deliverables at the end of the project will be a greatly enhanced understanding of the interaction of AAV with host cell DNA handling pathways, and methods for modulating these pathways to allow safe and effective gene delivery at lower vector doses.

NIH Research Projects · FY 2026 · 2022-02

Organ transplantation remains the definitive treatment option for patients with end-stage organ failure. Maintenance of functional allografts requires organ recipients to stay on immune- suppressive drugs. However, most allografts have a limited lifespan because of the chronic rejection initiated by the host alloimmune responses. The majority of immunosuppressive treatments are targeted to the effector immune cells, such as T cells, leaving the root of alloimmune responses—alloantigen presentation—untouched and leading to an immune equilibrium which eventually is shifted toward graft rejection. Regulatory T cells (Tregs) with user- defined specificity could be harnessed to induce immune suppression at desired tissues. They also preserve the ability to tolerize antigen-presenting cells (APCs) through contact-dependent cellular crosstalk. Our vision is to develop a robust allospecific immune regulatory strategy that restricts alloimmune T cell responses at both the effector site (allograft) and the alloantigen presentation site(graft draining lymphoid tissue) to shift the immune equilibrium to long-term suppression in the allograft while keeping the remainder of the host immune system fully operational. By leveraging the ability of chimeric antigen receptor (CAR) to recognize any desired target and a lymph node targeting molecular vaccine to specifically deliver the target to lymph node APCs, we will engineer an orthogonal synthetic vaccine to bridge crosstalk between CAR Tregs and APCs via the CAR-directed interaction with its cognate bio-inert ligand synthetically displayed on APCs. This synthetic vaccine-mediated crosstalk will have two outcomes: 1) APC- to-CAR Treg signaling promotes CAR Treg expansion and migration to the allograft for targeted suppression with enhanced regulatory functions. 2) CAR Treg-to-APC signaling tolerizes APC to restrict alloreactive T cell priming and to promote the generation of induced regulatory T cells (iTregs), which enforces a self-sustaining immunosuppression cycle via “infectious tolerance”. We will evaluate the synthetic crosstalk in murine allotransplantation models. If successful, this platform technology could be implemented across a broad landscape for precision control of pathological conditions, including autoimmune diseases, graft-versus-host disease, and transplant rejection.

NIH Research Projects · FY 2026 · 2022-01

This career development award details a 5-year training plan to facilitate transition to an independent career as a hepatogeneticist focused on gene discovery and characterization for hepatobiliary disease. I completed my Pediatrics residency at St. Christopher’s Hospital for Children and my fellowship in Human Genetics at The Children’s Hospital of Philadelphia (CHOP). I am currently an attending physician and research fellow at CHOP in the Division Of Human Genetics. My clinical and research efforts focus on children with hepatobiliary disease. My goals for this proposal are to become more experienced with exome and genetic variant interpretation and to gain experience using zebrafish as a model to study hepatobiliary disease. I will also use this opportunity to develop my ability to design experiments, write successful grant applications, and lead a laboratory, to facilitate a smooth transition to academic faculty. My mentor for this proposal is Dr. Hakon Hakonarson, a Professor of Pediatrics and director of the Center for Applied Genomics (CAG) at CHOP. Dr. Hakonarson has mentored dozens of post-doctoral research fellows and K-awardees, and was the recipient of CHOP’s Research Mentor Award. I will be co-mentored by Dr. Michael Pack, a Professor of Medicine at the University of Pennsylvania (UPenn). Dr. Pack also has an extensive history of mentoring trainees and K-awardees and works closely with Dr. Hakonarson on novel gene characterization. I have also assembled a scientific advisory committee, consisting of Drs. Klaus Kaestner, Ben Stanger, Kirk Wangensteen, Tom Jongens, and Elizabeth Rand, all experts in the fields of hepatology, neurogenetics or genetics with extensive mentoring experience. I will also have the benefit of the outstanding resources at both CHOP and UPenn, which have facilitated career development for countless past trainees. My proposed research focusses on the discovery and characterization of novel genes implicated in hepatobiliary disease. We are assembling a cohort of individuals with unexplained hepatobiliary disease, and will apply a research pipeline to facilitate identification of novel genes. Our laboratory has already identified de novo nonsense and frameshift variants in MED12 as causal for Hardikar Syndrome, a syndromic form of biliary dysgenesis, of previously unknown genetic basis. Aim 1 of this proposal delineates how a patient cohort will be assembled and characterized, and Aim 2 details the characterization of the role of MED12 in biliary development. Completion of the proposed studies will improve our ability to genetically diagnose hepatobiliary disease, better characterize the genetic landscape of these poorly-understood conditions, and elucidate the mechanism by which nonsense and frameshift MED12 variants cause biliary disease. This proposal will also provide me with experience studying hepatobiliary disease in animal and cellular models, writing grants and scientific papers, and allow me to observe how academic laboratories are run. Furthermore, this patient cohort and the cellular and animal models developed as part of this proposal will be invaluable in my future career.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Traumatic brain injury (TBI) is the primary cause of death and disability in children and young adults1. TBI afflicts more than two million people annually in the United States, with an estimated 5.3 million TBI survivors living with lasting neurological impairments2,3. Mild TBI (mTBI) or concussion, accounts for nearly 90% of TBIs, with symptoms including deficits in learning and memory that profoundly affect the daily life and overall health of TBI survivors. Although TBI survivors suffer a range of cognitive impairments, deficits in learning and memory are most common4–6. The hippocampus is critically involved in both of these phenomena and highly susceptible to damage by TBI. Little is known about the precise mechanisms by which hippocampal damage produces memory deficits. Our preliminary data indicate that spatial memory (a type of episodic memory required for the discrimination of a spatially moved object), requires coordinated hippocampal theta and gamma rhythms in the local field potential and neuronal firing time-locked to those rhythms (Figures 7-9 and Innovation section below). Both of these required components are critically dependent on the activity of inhibitory neurons, and specific inhibitory neurons in hippocampal area CA1 and the dentate gyrus (DG) are significantly altered after TBI7,8. Based on these results we hypothesize that altered synaptic transmission in specific hippocampal inhibitory neuron populations alters the balance between excitation and inhibition (E/I balance), leading to local circuit dysfunction and significant weakening of the coordinated hippocampal oscillations and neuronal firing required for normal spatial memory. To test this hypothesis, in vivo and in vitro recordings together with chemogenetic manipulation of specific subpopulations of inhibitory neurons in area CA1 and DG will be used to determine whether restoring normal inhibitory neuron function will reinstate normal rhythms, time-locked action potential firing, and normal spatial memory.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY/ABSTRACT Survivors of pediatric brain tumors often experience significant problems with social connectedness during youth that have lasting effects as adults (e.g., reduced rates of marriage). However, the factors contributing to these difficulties are unclear and little is known about how domains of social connectedness influence health- related quality of life (HRQL) and psychological well-being over time in survivors of pediatric brain cancer. Further, intervention efforts to address these issues have been limited by a lack of research on the underlying risk and mechanistic factors of social connectedness. Candidate risk and mechanistic factors include age at diagnosis, treatment, and treatment-related morbidities across many domains (e.g., cognitive, neurologic, endocrine, metabolic). Additionally, brain cancer treatments disrupt neurodevelopmental processes that are essential to social behavior, such as brain connectivity, face processing, and social attention. Establishing the importance of social connectedness to overall health and the mechanistic processes contributing to social connectedness impairments in pediatric brain cancer survivors is important in order to develop appropriate interventions. The broad objectives of this proposal are to compare domains of social connectedness among survivors of malignant brain tumors to survivors of non-malignant brain tumors, evaluate the influence of social connectedness on HQRL and psychological well-being among survivors, and to evaluate risk and mechanistic factors for the trajectory of social connectedness. We propose an innovative study of youth treated for medulloblastoma (MB), cerebellar pilocytic astrocytoma (PA) or craniopharyngioma (CP) (N = 180; ages 8-16) using a 2-year accelerated longitudinal design with annual visits with cohorts stratified by time since diagnosis. Clinical differences between groups (e.g., malignant/non-malignant, use of craniospinal irradiation) allow for tests of their unique impacts on social connectedness and for identification of potential intervention targets. Participants will be recruited from the Children's Hospital of Philadelphia. At each study visit, participants will complete measures of social connectedness, HRQL, and psychological well-being, as well as assessments of body composition, neuroendocrine function, hearing, brain connectivity (e.g., MRI), social information processing (SIP) and social behavior. Neuroimaging markers of interest include structural connectivity, resting state functional connectivity, and functional connectivity in the social brain during social processing tasks. We hypothesize that social connectedness, and thus HRQL and psychological well-being, are uniquely impacted in MB survivors, and that risk (e.g., CSI) and mechanistic factors (e.g., hearing, social behavior) affect social connectedness over time. We expect to establish deficits in social connectedness as a notable late effect in survivors of medulloblastoma with significant impact on HRQL and well-being and to identify mechanisms of social connectedness. By identifying the mechanisms underlying social connectedness, we can then develop interventions that target key mechanistic factors and improve social connectedness, health, and well-being.

NIH Research Projects · FY 2026 · 2022-01

Project Summary Ovarian cancer is the most lethal female cancer. When the disease can be diagnosed at early stage, there is striking survival improvement (five year survival ≥ 90%), compared to late stages (≤ 40%). However, currently no early detection method for ovarian cancer has enough accuracy, and most tumors already progressed to advanced stages at diagnosis. Furthermore, over 70% of the adnexal masses detected on preoperative imaging are found to be benign after pelvic surgery. Current clinical tests rely on serum CA-125 and sonograms to diagnose the ovarian adnexal masses. However, CA-125 is elevated by many common benign conditions; and ultrasound imaging of ovary frequently misses small but malignant lesions. As a result, surgical removal of the lesion and histologic evaluation remains the only gold standard for diagnosis. These limitations dictate an urgent clinical need of a better preoperative diagnostic method with high detection accuracy, to lower the mortality rate, reduce unnecessary surgeries and preserve the life choices for many patients, especially young women at reproductive age planning for pregnancies. Here, we propose a completely different route to detect ovarian cancer signals from the blood T cell repertoire. This is feasible because the T lymphocytes recognize tumor antigens at initial stages, proliferate and alter the peripheral T cell repertoire. Therefore, detection of cancer-associated T cells (CAT) in the blood provides an exciting novel opportunity for non-invasive cancer diagnosis. However, no prior studies have achieved this goal because it is difficult to identify CAT in high-throughput, as most of the cancer antigens remain unknown. To prepare for this task, we developed the software TRUST and iSMART, to obtain antigen-specific TCRs from cancer datasets. These tools have enabled us to produce a large training set of CATs, which allowed us to identify diagnostic TCRs for the ovarian cancer patients. Following this result, we further developed DeepCAT, for pan-cancer prediction using blood TCR sequencing data, and demonstrated over 99% specificity and 86% sensitivity in a pilot study to predict ovarian cancer patients (n=14) from healthy donors (n=176). To develop this approach into a novel ovarian cancer specific biomarker, we have established a biorepository to prospectively collect specimens from patients with benign or malignant ovarian lesions and from healthy donors of similar age span, with related clinical information. In Aim 1, we will generate TCR sequencing data of the new patient samples to develop a novel, TCR-based ovarian cancer predictor using machine learning method. In Aim 2, we will combine this approach with existing clinical tests to obtain a multi-modality biomarker, and independently test it using the samples from the Uterine Lavage cohort led by Dr. Steven Skates. These Aims will be delivered by the PIs and co-investigators with complementary expertise covering gynecological oncology, clinical cohort recruitment, biostatistics, artificial intelligence, immunology and ovarian cancer biomarker development.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT This Mentored Patient-Oriented Research Career Development Award proposal will provide didactic and experiential training, expert mentorship, and an exceptional research environment to facilitate Dr. Caitlin Elgarten's development as an independent clinician scientist. Dr. Elgarten's long-term career goal is to develop strategies that use targeted measurement and manipulation of the microbiome to predict and prevent adverse outcomes of pediatric stem cell transplant (SCT). Although SCT offers a chance for cure for more than 1,500 children each year with life-threatening disorders of the hematologic and immune systems, the success of this treatment is limited by aberrant and/or delayed recovery of the new immune system leading to graft-versus-host disease (GVHD), serious infection and reduced survival. In this proposal, Dr. Elgarten will leverage data from three pediatric cohorts to characterize the disruption of the gastrointestinal tract microbiome that occurs during SCT. She will define the microbiome features that associate with acute GVHD and peripheral immune cell recovery, and identify the pharmacologic drivers of clinically relevant microbiome disruption. Through the adaptation of non-linear mixed effects models for repeated measures to model the multidimensional and highly dynamic nature of the microbiome, this study will deliver a greater understanding of the causal association of the microbiome and immune events after SCT. In doing so, this work will lay the foundation for identification of novel biomarkers and the rational design of interventions to improve SCT outcomes. Execution of these aims will also provide the candidate with essential experience in conducting observational microbiome research and with critical training in the advanced bioinformatics and statistical methods necessary to study the interaction of the microbiome, pharmacologic exposures and clinical factors that define risk for adverse outcomes after SCT. The candidate is a pediatric oncologist and SCT physician with formal training in pharmacoepidemiology and a background in clinical research focused on supportive care in SCT. She aspires to bring her perspective as a clinician and epidemiologist to the study of longitudinal microbiome change in this complex clinical scenario. Dr. Elgarten's goals for the K23 program are: 1) conduct a patient-oriented research project that will yield clinically meaningful results, 2) acquire bioinformatics skills for the analysis of microbiome sequencing data, and 3) develop a platform for analysis of longitudinal microbiome data in SCT based on non-linear mixed effects models for repeated measures. She has assembled a strong mentorship and advisory team, led by Dr. Brian Fisher, and inclusive of national experts in microbiome-host biology, deep sequencing methods and bioinformatics, quantitative model-based methods, and translational epidemiology. This guidance, combined with the extensive resources available at the Children's Hospital of Philadelphia and the University of Pennsylvania, will ensure the accomplishment of the proposed research and training goals and Dr. Elgarten's successful transition to independent investigator.

NIH Research Projects · FY 2025 · 2021-09

Neurodevelopmental processes are shaped by dynamic interactions between genes and environments. Maladaptive experiences early in life can alter developmental trajectories, leading to harmful and enduring developmental sequelae. Pre- and postnatal hazards include maternal substance exposure, toxicant exposures in pregnancy and early life, maternal health conditions, parental psychopathology, maltreatment, and excessive stress. To elucidate how various environmental hazards impact child development, it is imperative that a normative template of developmental trajectories over the first 10 years of life be established based on a sufficiently large and demographically heterogeneous sample of the US population. To accomplish this, the Healthy Brain and Child Development (HBCD) Consortium has been formed to deploy a harmonized, optimized, and innovative set of neuroimaging (MRI, EEG) measures complemented by an extensive battery of behavioral, physiological, and psychological tools, and biospecimens to understand neurodevelopmental trajectories in a sample of 7,200 mothers and infants enrolled at 27 sites across the United States (US). The HBCD Study will carry out a common research protocol under direction of the HBCD Consortium Administrative Core (HCAC) and will assemble and distribute a comprehensive and well-curated research dataset to the scientific community at large under the direction of the HBCD Data Coordinating Center (HDCC). The overarching goal of the HBCD Study is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize typical neurodevelopmental trajectories in US children and that will assess how biological and environmental exposures affect those trajectories. A special emphasis will be placed on understanding the impact of pre- and postnatal exposure to opioids, marijuana, alcohol, tobacco and/or other substances. To address these broad objectives, the sample of women enrolled will include: 1) a varied cohort that is representative of the US population; 2) pregnant woman with use of targeted substances (opioids, marijuana, alcohol, tobacco); and 3) demographically and behaviorally similar women without substance use in pregnancy to enable valid causal inferences. In addition, the HBCD Study will identify key developmental windows during which both harmful and protective environments have the most influence on later neurodevelopmental outcomes. The large, multi-modal, longitudinal, and generalizable dataset that will be produced for the first time by this study will provide novel insights into child development using state-of-the-art methods. The HBCD Study will inform public policy to improve the health and development of children across the nation.

NIH Research Projects · FY 2024 · 2021-09

Project Summary In order for the b-cell to precisely secrete insulin in response to glucose, glucose metabolism to ATP is tightly coupled to insulin secretion through the ATP-sensitive K (KATP) channel. Any disruption along this pathway leads to b-cell dysfunction and ensuing diabetes or, more rarely, congenital hyperinsulinism. While these diseases mostly arise from multiple factors, some cases are caused by single gene mutations, termed monogenic diseases. Interestingly, the same loss-of-function heterozygous mutations in the transcription factor HNF1a can result in both hyperinsulinism and diabetes, though presenting at different ages and through unknown mechanisms. In the proposed project, we will investigate the underlying causes of both HNF1a- related diabetes and hyperinsulinism. Our prior published work and additional preliminary data show that HNF1A-deficient human stem cell-derived β-cells exhibit increased basal and decreased glucose-stimulated insulin secretion, recapitulating the clinical disease. HNF1A-deficient β-cell models exhibited significantly decreased glycolysis, suggesting decreased ATP production in response to glucose. Expression analyses revealed broad defects in HNF1A-deficient β-cells in genes regulating cellular metabolism and decreased expression of KATP channel genes. Based on these previous findings, we hypothesize that HNF1a regulates both KATP channel expression and ATP production in response to glucose. Loss of HNF1a leads to uncoupling of glucose metabolism and insulin secretion at these two points in the insulin secretion cascade, impacting basal and stimulated insulin secretion in opposing directions and resulting in hyperinsulinism and diabetes, respectively. Using a novel and manipulatable human b-cell model system combined with a variety of analytical techniques, we will rigorously test our hypotheses and advance our knowledge of b-cell dysfunction in complex disease states. My long-term goal is to have a basic and translational research lab that studies the diseases resulting from pancreatic β-cell dysfunction. My proposed K08 project will help me learn new technical methods, develop a new intellectual foundation and generate preliminary data that will be instrumental in helping me start my own independent lab. I chose my co-mentors as they have complimentary research approaches and varied areas of expertise. I have a career development plan which relies on my extensive mentoring relationships, including regular interaction with my co-mentors and my advisory committee, which contains individuals within the local and national research community with varying areas of expertise. I have developed a plan of training which takes full advantage of the collaborative research environment at the Children’s Hospital of Philadelphia and University of Pennsylvania School of Medicine, including participation in trainings, seminars, and workshops to advance my knowledge base and professional skills.

NIH Research Projects · FY 2025 · 2021-09

Project Summary: Our ultimate goal is to find new ways to improve smooth muscle function in people with visceral myopathy, a disease defined by profound bowel, bladder and uterine smooth muscle dysfunction. Bowel dysfunction, called myopathic Chronic Intestinal Pseudo-Obstruction (CIPO), is often treated by intravenous nutrition. Bladder weakness often requires catheterization. When symptoms start in utero, colon growth is minimal, causing Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS). Only ~20% of people with MMIHS survive to adulthood. Current treatments may reduce symptoms but are not based on disease mechanisms. Recent data show that 44% of people with MMIHS/CIPO have heterozygous point mutations in gamma smooth muscle actin (ACTG2), one of 6 actin isoforms. Actin isoforms have distinct roles in cells, and while actin is well studied, ACTG2 is barely studied. Myopathy-causing ACTG2 mutations are spread throughout the actin structure. This suggests variant-specific disease mechanisms that could benefit from variant-specific therapies. To design such therapies, we need a deep understanding of how individual variants cause disease. We therefore pursue an integrated strategy, combining biochemical, structural, cellular and stem cell approaches to determine how ACTG2 mutations cause visceral myopathy. Technical breakthroughs and extensive preliminary data set the groundwork for success. In Aim 1, we develop new ways to express recombinant human actin in human cells, without tags and featuring natural post-translational modifications. This major innovation opens the way to biochemical studies of ACTG2, and should also facilitate studies of variants of other actin isoforms causing skeletal myopathy, cardiomyopathy, vascular disease, sensorineural hearing loss, and congenital malformations. Using recombinant ACTG2, we will study the biochemical-structural properties of disease-causing ACTG2 variants, and their interactions with key Actin- Binding Proteins (ABPs) that regulate actin assembly. To determine how mutations affect cell biology (Aim 2), we express wild-type or mutant ACTG2 in human Intestinal Smooth Muscle Cells (hISMC). We selected hISMC because disease-causing ACTG2 variants might alter interactions with ABPs or depend on cell-type specific post-translational modifications. Our innovative quantitative image analysis pipeline already revealed how the most common ACTG2 mutation (R257C) affects the actin cytoskeleton and cell biology. We will now use this strategy to study other ACTG2 mutations. Some mutations might also cause myopathy by preventing the MRTF-A transcription factor from entering the nucleus to induce contractile gene expression and smooth muscle differentiation. To test this hypothesis, we invented a new way to convert human Pluripotent Stem Cells (hPSCs) to visceral smooth muscle-like cells (Aim 3) and made cell lines expressing disease-causing ACTG2 variants. Our cross-disciplinary, integrated strategy should clarify mechanisms of ACTG2 mutation-induced visceral myopathy, leading to mutation-specific drug screening strategies and new therapies.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT The vast majority of human genes are multi-exonic and undergo alternative splicing to generate a several- fold increase in the variety of transcripts and proteins that are produced. In diseases like cancer, splicing is often globally aberrant and highly distinct from normal tissues, with increased levels of intron retention, alternative splicing and usage of de novo splicing junctions. A fundamental unanswered question in RNA biology is how the many layers that comprise the splicing code are interpreted to produce a given splicing outcome, and how these processes become dysregulated in human diseases. The extensive crosstalk between processes occurring on the DNA and those that act on the RNA suggest the chromatin state in particular may participate in orchestrating alternative splicing. We have observed that random genomic integration of a splicing reporter construct results in populations of cells corresponding to each possible splicing outcome, suggesting the genomic context at the site of integration exerts a powerful influence on splicing. However, due primarily to technological limitations, much remains unknown about the precise nature of the regulatory interplay between chromatin and RNA splicing and the potential factors that may be involved. The goal of this proposal is to address the current challenges impeding de novo discovery and develop unbiased experimental approaches to reveal hidden determinants of splicing regulation at the chromatin level. Our approach is designed to directly connect splicing outcome with unbiased proteomic profiling of the associated genomic region, in order to identify all of the potential chromatin features that are involved in enforcing a particular pattern of splicing. We also propose to engineer new systems to functionally validate a direct role for the epigenome in splicing regulation and to modulate splicing at will. Our findings have implications for both epigenetics and RNA biology, and in particular, how their interactions influence the various processes involved in gene expression. It also has the potential to shed insight into the causes and consequences of the widespread epigenetic and splicing dysregulation observed in human cancer. Importantly, our approach is not limited to the study of splicing and will also enable unbiased discovery of the mechanisms involved in other fundamental processes.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Multiple organ dysfunction syndrome (MODS) affects as many 57% of critically ill children, with mortality rates as high 67% in those infected. The long-term goal of this proposal, Antibiotics in MODS: PersonaLizing Exposures (AMPLE), is to leverage the well-established infrastructure from our PARADIGM study (R01HD095976) to identify optimal antibiotic dosing strategies for this highly understudied, high-risk population. Infection is a common occurrence in children with MODS, either as an inciting insult or as a result of a new, nosocomial infection. However, management of children with MODS and infection is complicated by the development of immune paralysis (IP), which has deleterious effects on immune function. Unfortunately, antibiotic management strategies and how they should be modified as a function of host immune status are key knowledge gaps in pediatric MODS. Timely attainment of target antibiotic concentrations is a crucially important, modifiable intervention to increase survival in these children, yet we currently have limited data on antibiotic pharmacokinetics (PK) in children with MODS with which to develop personalized dosing strategies. We will quantify antibiotic PK in 400 subjects enrolled in the PARADIGM study, an ongoing, NIHfunded, 22- center, prospective study of the epidemiology and risk factors for IP in 1,400 children with MODS. The objectives of this application are to use samples and clinical data from PARADIGM subjects to characterize the variability of concentrations for the antibiotics most commonly used in pediatric MODS; to investigate the relationships between antibiotic target attainment and outcomes in pediatric MODS with and without IP; and to develop model-based dosing approaches that rapidly achieve and maintain target antibiotic concentrations. The central hypothesis of this proposal is that precision, PK-driven antibiotic dosing strategies can be developed that adequately account for organ dysfunction and immune function in children with MODS. We propose to pursue the following AIMs: 1) To create and evaluate sophisticated population PK models for the 6 most commonly used antibiotics in pediatric MODS. 2) To define antibiotic target windows outside of which children with MODS (with and without immunoparalysis) who are being treated for infection are at increased risk for death and prolonged organ failure. 3) To use simulations to identify dosing strategies that achieve and maintain antibiotic concentrations within defined therapeutic windows. The proposed studies will answer the following key questions about the pharmacology of pediatric MODS: 1) What proportion of children are under- or over- exposed using the current standard dosing approaches?, 2) How does MODS impact antibiotic PK and outcomes in children?, 3) How does IP impact necessary target concentrations in pediatric MODS? This research is expected to be significant as it will result in first-of-its kind data that are a crucial and a necessary step toward developing Precision Antibiotic Dosing strategies for children with MODS.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY/ABSTRACT Regulating when and where genes are expressed is essential to the proper development, health, and viability of all living organisms. The processes that regulate gene expression are choreographed across a broad range of spatial and temporal scales spanning from molecular scales where regulatory proteins bind and unbind DNA at sub-second to second time scales, to the organization of the nucleus where proteins and DNA form dynamic sub-micrometer sized domains that fluctuate over seconds and minutes, to the coordination of these events across distinct tissue types over hours and across hundreds of micrometers to millimeters. Despite the dynamic nature of these processes, most of our knowledge about them comes from experiments on fixed samples that provide population and time-averaged data. Recently, the advent of high-resolution live imaging techniques have granted the ability to quantify the dynamics of gene regulation and have highlighted what has been missed by studies in fixed samples. Although these new imaging approaches have already provided remarkable insights, due to technical constraints they are generally applied to cells grown on glass coverslips and isolated from the tissue contexts in which they have evolved to function. The premise of this proposal is that in order to build a holistic and quantitative framework to understand gene regulation, we must develop and apply experimental approaches that access the broad range of spatial and temporal scales involved, and do so in endogenous contexts. To achieve this goal I propose to integrate cutting edge light-sheet microscopy, label-free interferometry, and molecular imaging tools that will allow quantification of single-molecule protein kinetics, transcriptional dynamics at individual gene loci, chromatin dynamics, and the compartmentalization of nuclei in actively developing animal embryos. I will apply these technologies to study the dynamics of gene regulation during early development in Drosophila Melanogaster embryos. These embryos provide an ideal context for studying fundamental aspects of gene regulation. They proceed from fertilization to differentiated tissue in around just 3 hours during which chromatin and nuclear organization is progressively established along with patterns of gene expression across the embryo. I propose experiments that leverage the new integrated technological approaches I will develop to ask: (1) How do the dynamics of transcription factor protein-protein and protein-DNA interactions affect their ability to find and bind their specific genomic targets and shape the nuclear environment? and (2) How are functional sub-nuclear compartments formed during embryonic development, and what is their role in shaping chromatin dynamics and gene expression patterns? Together this proposal will lead to new experimental capabilities that will provide fundamental insights on the dynamics of how gene expression is regulated from the molecular scale up to the organismal scale. These new types of integrated datasets will lay the foundations for developing a quantitative and predictive framework which may allow us to develop new therapeutic approaches for correcting aberrant gene expression in disease.

NIH Research Projects · FY 2025 · 2021-09

Abstract Human infancy is characterized by extraordinary maturation of brain function and structure with the highest change rate across the life span. These dynamic brain processes are supported by significant increases in regional cerebral blood flow (rCBF) to meet the metabolic demands of rapid brain growth during infancy. Devastating brain disorders such as cerebral ischemia and stroke also occur in infancy and their short- and long-term consequences are manifested by changes in rCBF and function around the injured brain site and connected areas. A standardized whole-brain population-averaged three-dimensional (3D) developmental perfusion atlas of rCBF would have a broad impact on understanding not only normal infant brain development but also brain disorders. Without a normal reference, it is not possible to detect disorders that alter rCBF. Despite its significant impact, to date, the rCBF atlas and trajectory of infant brain are not available. The goal is to establish the first age-specific whole-brain population-averaged 3D infant perfusion atlases and trajectories quantified by rCBF measures, as well as to delineate their functional and behavioral correlations. To adapt to relatively small infant brain, high-resolution (2.5x2.5x2.5mm3) rCBF will be obtained noninvasively using a novel arterial spin labeled (ASL) perfusion MRI technique (Aim 1-2), providing absolute quantification of a fundamental property of brain physiology. We will also systematically delineate the mechanistic relationship between infant rCBF dynamics and the maturation of corresponding brain function and behavior (Aim 3). These atlases will serve as a diagnostic screening tool by providing normal rCBF distribution and variability (e.g. z-sore maps) at multiple critical infant developmental stages as well as fill the neuroscientific knowledge gap of lacking fundamental brain physiological properties. ASL perfusion MRI uses magnetically labeled arterial blood water protons as an endogenous tracer. A recently developed 3D spiral ASL MRI method uses pseudo-continuous labeling (pCASL) with background suppression, a 3D stack-of-spiral readout and parallel imaging, making it possible to establish the high-quality infant rCBF atlas at resolution of 2.5x2.5x2.5mm3 or higher. This study will leverage two funded projects (R01HD093776 and R01MH092535) that will provide resources of recruitment and behavioral assessment of a relatively large infant cohort at the Children’s Hospital of Philadelphia. After optimizing the 3D spiral pCASL perfusion MRI sequence for infants, we will use the optimized sequence to acquire data from healthy infants at 0.5, 3, 6, 9, 12, 18 and 24 months (n=232 at initial time point) for making age-specific population-averaged atlases. The resulting high-quality and high-resolution age-specific whole brain perfusion atlases, the longitudinal maturational curves of rCBF, as well as individual dataset will be the first for infants, and will be valuable shared resources. The brain regions-of-interests where rCBF change is highly correlated with functional or behavioral maturation may also be used as prognostic biomarkers for predicting altered function and behavior in infants with brain disorders.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Across the world, spondyloarthritis (SpA) is the most common form of juvenile arthritis, accounting for as many as one-third of all cases. Since the introduction of biologic disease modifying agents such as tumor necrosis factor inhibitors (TNFi), inactive disease is a realistic goal for children with spondyloarthritis (SpA). However, side effects of TNFi include increased risk of infection, psoriasis, demyelinating disorders, and malignancy. Additionally, TNFi are expensive and may adversely impact patient’s and caregiver’s quality of life and anxiety. Recently, the COVID-19 pandemic prompted numerous inquiries regarding whether being on a TNFi increased the risk of becoming infected, and, if infected, whether being on a TNFi increased the risk of morbidity. In the absence of definitive answers many families’ next question was, “Can we stop the medication?” We must address two critical knowledge gaps to inform strategies for TNFi de-escalation in children with SpA. First, it is unknown if subclinical imaging represents a risk for flare or whether it represents benign residual activity. Presently, clinical equipoise exists in whether subclinical inflammation on pelvic MRI should impact treatment decisions about children with SpA and axial disease because we do not know the prevalence or the clinical relevance of these MRI findings. Second, the role of cellular biomarkers to predict flare after therapy de- escalation in juvenile SpA is unknown. Prior studies in polyarticular juvenile arthritis have identified cellular populations and biomarkers that can distinguish disease states that are highly associated with relapse. Similar studies have not been done in juvenile SpA. Our objective is to test the association of immunologic, serologic and imaging biomarkers with the risk of disease flare in children with SpA by leveraging the infrastructure and resources of the parent trial, Biologic Abatement and Capturing Kids’ Outcomes and Flare Frequency in Juvenile SpA (BACK-OFF JSpA) to perform mechanistic studies to address these critical knowledge gaps. This application has 2 specific aims: 1) to test the ability of imaging biomarkers at the time of medication de- escalation to predict subsequent flare in children with axial arthritis, and 2) to test the ability of cellular biomarkers at the time of medication de-escalation to predict subsequent flare in all children with SpA. The work proposed in this application will directly impact clinical care and significantly enhance the evidence base that clinicians, families, and patients use to make decisions about whether or not to de-escalate biologic therapy.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Our overall aim is to define the mechanisms underlying the circadian regulation of acute lung injury and subsequent recovery. Our published work shows that circadian rhythms confer a time of day specific protection from Influenza A Virus (IAV) infection. Mice infected at dawn had 3-fold better survival than those infected at dusk. While, we cannot clinically control the time of exposure to IAV, these data suggest that altering the circadian health of the host could affect outcomes. In fact, disrupting circadian rhythms genetically in mice, by deleting the core clock gene, Bmal1, worsened mortality from IAV. Further proof of the translational relevance of our mechanistic work came from our analyses of the UK biobank which revealed that disrupted circadian rhythms was an independent risk factor for Influenza related hospitalization. Severe influenza infection is characterized by extensive immunopathology and dysplastic lung repair and regeneration, often independent of viral burden. Both vaccines and anti-viral agents have limited efficacy. The current proposal addresses this need in the field by exploring a novel target—circadian rhythms as determinant of outcomes in IAV. Since the last submission, we have generated exciting preliminary data that shows that disruption of the AT2 clock is associated with (a) worse acute mortality, immunopathology and necroptosis and (b) delayed recovery in vivo and poor regeneration on organoid assays. Our overall goals are to: (1) Test the hypothesis that the disruption of the AT2 clock leads to a pro-inflammatory state at baseline that is further exacerbated by IAV infection, thereby worsening necroptosis. (2) To test the hypothesis that the circadian clock contributes to lung regeneration through Wnt- responsive regulation of the cell cycle via the Axin2+ epithelial niche. Our approach employs tissue specific circadian knock-out models induced in adulthood, circadian sampling throughout 24hrs, other genetic/environmental models of circadian disruption and tools form lung regenerative biology, customized to the circadian context. I have also gathered an outstanding team of collaborators and consultants with expertise in cell death, circadian bioinformatics, lung regeneration and virology. Elucidating these mechanisms is the critical next step towards modulating the host circadian rhythms for therapeutic purposes. While, we use influenza as our model, the principles uncovered thus, should be generalizable to other viral conditions of the lung.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Neonatal Opioid Withdrawal Syndrome (NOWS) is a major public health problem in the USA. About 100,000 American infants were exposed to opioids in 2017 and 25,000 of these infants were diagnosed with NOWS. Opioid-exposed infants have poorer outcomes compared to similar non-exposed infants including longer and more complicated initial hospital stays, higher likelihood of involvement in the child welfare system, higher rates of hospital readmission and emergency department visits, and lower use of recommended preventive services. One group of infants at particularly high risk for adverse outcomes are infants who receive pharmacological therapy to treat signs of withdrawal. Importantly, the care, treatment, and outcomes of these infants vary widely between birth hospitals and US states. Such variation suggests that the optimal pharmacological treatment for NOWS remains unknown, potentially exposing these infants to unnecessary or ineffective management. The NOWS Treatment Trial will assess the optimal pharmacological treatment for NOWS through a multicenter, comparative effectiveness trial comparing three commonly used medications. This application describes the CHOP/Penn Neonatal Clinical Trials Network, our outstanding team of investigators with vast clinical trial experience, our unparalleled research environment, and our large and diverse patient population which together ensure that we are ideally suited to participate in the design and execution of the NOWS Treatment Trial. We will contribute expertise in clinical trial methodology, substance abuse, and neonatal follow-up, providing rigorous scientific input into design and implementation of the multicenter NOWS Treatment Trial. We will then execute all aspects of the trial to the highest standard, including robust participant recruitment, rigorous adherence to all aspects of the study protocol, and neurodevelopmental follow-up to two years with minimal attrition.