Children'S Hosp Of Philadelphia

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The potential for the development of novel therapeutic modalities has energized the genome editing field since it first emerged in the 1990s and especially since the demonstration of programmable genome editing with CRISPR-Cas9 by multiple groups in 2012. There has been substantial progress with ex vivo therapeutic applications of genome editing in patients in the past few years, most notably with CAR-T immunotherapies for cancer and with durable treatment of hemoglobinopathies. Progress with in vivo therapeutic applications, i.e., somatic cell genome editing, has been slower due to the technical challenges inherent in the delivery of genome- editing tools into the body. As of the time of this writing, there are few published examples of successful genome editing performed in vivo in primates (including humans), with almost all examples involving somatic genome editing in the liver: TTR with Cas9 nuclease delivered by lipid nanoparticles (LNPs), PCSK9 and ANGPTL3 with adenine base editors delivered by LNPs, and PCSK9 with meganucleases delivered by adeno-associated virus (AAV) vectors. The prospects for genome-editing therapies extend to before birth, with in utero genome editing having the potential to treat genetic diseases that result in significant morbidity and mortality before or shortly after birth. Although restricted to small animal models so far, in utero genome editing has proven effective in the liver, lungs, heart, and other organs. Our Overall Program seeks to build on these early successes, pursuing goals that that would be of major impact in advancing the field of therapeutic genome editing. Our three Research Projects seek to develop base-editing therapies targeting the liver in order to treat three rare metabolic genetic diseases: phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI). Lead Project 1 will focus on LNP-based postnatal treatment of PKU, with the aim to file an IND application by the end of the five-year funding period and begin a phase 1/2 clinical trial soon afterwards. Project 2 will focus on LNP-based postnatal treatment of HT1, with the aim to file an IND application and begin a clinical trial, and prenatal treatment of HT1, with the aim of performing preclinical studies during the five-year funding period to enable an eventual IND application if the postnatal clinical trial proves successful. Project 3 will focus on AAV-based postnatal and prenatal treatment of MPSI, with similar aims as Project 2. Unique, specialized Resource Cores focused on off-target editing and in utero treatment of small and large animals will be indispensable in achieving these aims.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY This Multiple Principal Investigator (MPI) Project proposal for the Pediatric Immunotherapy Network is focused on high-risk neuroblastoma, a diverse and enigmatic malignancy arising from the developing sympathetic nervous system that remains lethal in 50% of patients despite intensive multi-modal therapy. There is an urgent unmet need for developing novel therapeutic interventions to decrease the incidence of relapse, increase overall survival, and reduce devastating toxicities associated with standard therapy. The primary goal of this Project is to achieve improved outcomes for patients with high-risk neuroblastoma through the development of a personalized vaccination strategy targeting individualized neoantigens. The central hypothesis is that high-risk neuroblastomas, despite a low tumor mutation burden (TMB), harbor a sufficient number of neoepitopes through canonical and non-canonical mutations to identify, predict, and validate optimal neoantigen peptides to engineer effective multivalent personalized neuroblastoma vaccines. The motivation for the proposed research is the urgent need to improve survival and to decrease treatment-related morbidities for patients with high-risk neuroblastoma. Indeed, the majority of high-risk neuroblastoma patients achieve a remission with standard therapy, and here we seek to engage the adaptive immune system to eradicate residual disease and prevent relapse. We will test our hypothesis through the two Specific Aims: 1) define the neoantigen landscape of high- risk neuroblastoma patient and genetically engineered mouse model (GEMM) tumors; 2) develop and test a readily translatable personalized vaccination strategy. In Aim 1 we will both provide the proof-of-concept that a multivalent vaccine consisting of both CD4+ and CD8+ epitopes is feasible for each high-risk patient and also credential our GEMM system for preclinical vaccination trials in Aim 2. We will use an integrative proteogenomic approach to identify up to eight immunogenic peptides for each personalized vaccine. In Aim 2, we will test both preventative and therapeutic efficacy of self-assembling nanoparticle multivalent peptide vaccines using our GEMM system on the CB57BL/6 background, and then compare this vaccine platform to a new lipid-peptide polymer vaccine system optimized to deliver peptides to dendritic cells. This Project proposes an innovative experimental strategy to identify, prioritize, and validate neoantigens in high-risk neuroblastoma, and a clinically relevant neuroblastoma GEMM system for preclinical evaluation of neuroblastoma vaccines. The significance of the proposed Project is the creation and validation of a novel immunotherapeutic approach that has the potential to revolutionize high-risk neuroblastoma standard of care by providing durable cures and decreased therapy- related morbidity. The expected outcome of this collaborative MPI Project is to establish the preclinical proof-of- concept for a personalized neuroblastoma clinical trial that we would seek to launch in the out years of this grant given the infrastructure we have in place. The successful completion of this Project will also enable personalized neoantigen-based vaccine development for other pediatric malignancies.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Our prior work using physical maps of systemic lupus erythematosus (SLE)-associated genetic variation in the context of the 3D structure of the genome in the nucleus of disease-relevant immune cell types have implicated the kinase HIPK1 as a factor controlling SLE susceptibility. This kinase has been studied in the context of cancer, but a role for HIPK1 in immunity, tolerance, or SLE has not been explored. In this exploratory, high-risk/high- impact application we will use genetic and pharmacologic targeting approaches to establish whether HIPK1 regulates T cell differentiation, T cell-dependent humoral immune responses, and SLE pathophysiology in powerful human and mouse models of follicular lymphocyte differentiation and function. The outcome of these studies is likely to forward basic understanding of the regulation of humoral immunity and suggest a completely novel immunomodulatory approach for the management of SLE disease.

NIH Research Projects · FY 2025 · 2023-07

Abstract: Leukodystrophies are a heterogeneous group of complex neurological conditions impacting the genesis and maintenance of myelin, or white matter. Next generation sequencing and emerging genomic technologies have improved the ability to identify molecular causes of disease, allowing for the improved characterization of disease processes and focused therapeutics. However, molecular characterization of leukodystrophies requires expert oversight as much of the current gene-disease validation and clinical actionability have not been formally characterized. Current diagnostic testing includes biochemical and genetic testing but reveals a diagnosis in only 50-80% of patients said to have a myelin disorder by brain magnetic resonance imaging. This leaves many patients with an uncertain diagnosis, which can limit treatment options, cause emotional distress, and limit access to clinical trials and, for some families, reproductive choices. The Global Leukodystrophy Initiative (GLIA) is a Rare Disease Clinical Research Network funded consortium of scientists and advocates that work collaboratively to advance the study of and clinical trials for leukodystrophies. The GLIA consortium has approached ClinGen to establish Gene and Variant Curation Expert Panels, using our disease-specific experts and bioinformaticians to curate and assess leukodystrophy-related genes and variants. There are at least 240 genes that currently fall in the purview of this group, which will be expertly curated and managed by this group. For the 25 most commonly diagnosed genes causing leukodystrophy, this group will provide comprehensive variant curation, including the most clinically actionable disease-causing variants. The overall impact of this application will be to clarify the disease-gene association in the leukodystrophies, improve understanding of clinical actionability in these disorders, and clarify the pathogenicity of variants in key high frequency genes. Together, these activities will provide the leukodystrophy community with diagnostic clarity for newborn screening, clinical trials and clinical care.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT. Complex V (CV, or ATP synthase) of the electron transport chain is the central enzyme of cellular energy capture. CV synthesizes ATP driven by the proton gradient generated by the electron transport chain. The advent of clinical gene sequencing has highlighted the devastating effects of deficiency of this critical enzyme. Pathogenic variants in CV subunits give rise to multi-system disease, including strokes, neuropathy, ataxia, retinopathy, and cardiomyopathy. Genetic variation in CV is frequent, and the inability to distinguish pathogenic mutations from the variants of unknown significance is a clinical challenge preventing understanding of prognosis and rational approach to management. However, no clinical test for CV function exists. This thwarts our ability to classify genetic variants. Our Goal is to develop a biochemical approach to evaluating CV function and ultimately predicting the clinical significance of CV variants. We previously demonstrated that basal ATP levels are normal with CV deficiency while the rate of ATP synthesis can be low, suggesting that clinical symptoms result from an inability of CV to accommodate an increased metabolic demand. Direct enzymatic testing of ATP synthesis by CV is impossible, as the substrate for CV is the proton motive force, which is dissipated when the enzyme is purified. We therefore propose to assay ATP flux in our human cell (fibroblast, transmitochondrial cybrid) models of diverse CV genetic variants, including novel candidate genes. We have observed that the biochemical effects of CV variants result in diverse biochemical sequelae; therefore, we will also assay oxygen consumption, mitochondrial membrane potential, matrix pH, CV assembly, and mitochondrial cristae structure and correlate results with clinical manifestations. We anticipate that this approach will furnish a biochemical and morphologic profile that informs the pathogenicity of the variant. We hypothesize that the observation that steady-state ATP levels are normal in CV deficient cell-lines despite low enzymatic flux implies that CV function is responsive to cellular metabolic state. We will introduce a series of provocative (stimulus-response) testing procedures that involve modulation of nutrient levels (glucose, αKG) and exposure to cell stress (galactose, lipopolysaccharide). By rigorously investigating the biochemical consequences of CV deficiency including in dynamic models of cellular stress, we will establish the foundation on which to develop clinical diagnostic assays to confirm CV mutation pathogenicity and treatment response. The Central Hypothesis of this proposal is: pathogenic variants in CV subunit genes evoke changes in CV bioenergic function resulting in diverse downstream biochemical defects that predict clinical presentation. Further, we propose that the clinical manifestations of Complex V deficiency severity are influenced by the biochemical and nutritional milieu in which the genetic deficiency finds itself. Experimental manipulation of this milieu may identify nutritional therapeutic approaches.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Training: The purpose of this K01 proposal is to prepare Dr. Katharine Press Callahan for a career as an independent physician-scientist focused on the ethical and social implications of genetic testing for critically ill neonates. Her long-term career objective is to conduct impactful research on the use of genetic testing for neonates using both quantitative and qualitative methods. To this end, Dr. Callahan and her mentorship team have devised a career development plan that integrates: (1) intensive mentorship from a team of mentors and advisors with whom Dr. Callahan has a track record of successful collaboration, (2) advanced training in genetic science and testing, qualitative methods, and medical simulation, and (3) an innovative research plan designed to investigate how neonatologists and parents of neonates manage the uncertainty of genetic information. Research: Genetic testing is increasingly used in ill neonates and holds promise to improve care. However, in practice, many genetic results contain substantial uncertainty, which can lead both clinicians and parents to misunderstand or misapply results, resulting in biased treatment plans. Little is known about how uncertainty affects neonatologists’ use and communication of genetic information or parents’ understanding of this information in practice. Dr. Callahan’s mentored, mixed-methods research will fill the critical need to identify and mediate potential risks that emerge as neonatologists and parents increasingly use uncertain genetic results in practice. Aim 1 will identify the types of uncertainty that neonatologists and parents perceive when they receive genetic results and examine how this uncertainty affects understanding and use of results. Aim 2 will assess the effect of uncertainty about prognosis on neonatologists’ counseling about genetic information and potential disability. Aim 3 will develop and preliminary test an information management tool that aims to improve understanding and standardize communication of genetic information and associated uncertainty between geneticists, neonatologists, and parents. Significance: Completing these aims within the context of a structured career development plan will prepare Dr. Callahan to be an independent investigator poised to execute the NHGRI vision of integrating complex genetic information into the clinical workflow in newborn medicine. Findings from this study will inform an R01 proposal to validate and disseminate the genetic information management tool developed in Aim 3.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT T cell trafficking is crucial for development, immune surveillance, and effector function. Migration is a complex process, choreographed by a host of chemoattractants that signal through GPCRs to direct T cell cytoskeletal responses. In vivo work shows that chemokines like CCL19 and CCL21 (CCR7 ligands) mediate naïve T cell migration within lymphoid tissues, while the lipid chemoattractant S1P regulates egress. A similar process occurs in peripheral tissues, where these signals control migration of migratory effector T cells out of inflamed tissues and into afferent lymphatics. Our lab recently overcame a longstanding technical problem that made it difficult to study S1P responses in ex vivo T cells. Using this advance, we discovered that these two chemotactic signals induce distinct modes of T cell motility. CCL19 induces long-duration lamellipodial migration while S1P induces a shorter burst of bleb-based motility. This work raises several questions: How do these chemoattractants elicit such different migratory responses? What do T cells do when confronted with competing cues? Why do T cells need multiple motile mechanisms? We hypothesize that CCR7 ligands and S1P activate different cytoskeletal signaling pathways that direct distinct modes of motility, which work alone or in combination to allow T cells to navigate complex environmental obstacles like those they encounter in vivo. To test this hypothesis, we will carry out two sets of studies. In Aim 1, we will pursue our preliminary data showing that CCL19 preferentially activates a Rac1-dependent pathway leading to lamellipodial protrusion, while S1P preferentially activates a pathway involving RhoA and phospholipase activity, which directs myosin-dependent contractility and bleb formation. To verify that that these signaling events are causally linked to the migratory responses we observe, we will treat cells with pharmacological inhibitors and assess motile responses and cytoskeletal remodeling using transwell assays and live cell imaging. To ask how cells integrate signals from multiple chemoattractants, cells will be exposed to S1P and CCL19 simultaneously and sequentially, and signaling responses and cell migration will be analyzed. In Aim 2, we will test the idea that CCL19-induced lamellipodial motility is optimized for long-distance migration in relatively unconfined settings, while S1P-induced bleb-based motility permits cells to pass through small, highly confined spaces. To achieve this, we will test chemotaxis within 3D collagen gels and passage through microfluidic channels with variable geometries that mimic in vivo challenges. As part of this analysis, we will ask how actin and myosin are redistributed in the cell as a function of chemoattractant stimulus and confinement. Finally, we will analyze T cell passage across lymphatic endothelial barriers using transwell assays and tissue explants derived from mouse ears. If successful, this project will complement existing in vivo studies of T cell trafficking by providing much needed mechanistic insights into the underlying molecular and cell biological mechanisms. In the long run, our findings will reveal valuable targets for the rational design of therapeutic approaches based on modulating T cell trafficking.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT This proposal describes a 4-year career mentored research project with the goal of defining the effects of intestinal Intelectin-1 (ITLN1) in microbial function and composition and its role in the genesis of obesity and early life weight gain. Successful completion of the research and training plan will enable the investigator to gain the skills necessary to secure independent funding and become an independent physician-scientist. The inves- tigator's long-term goal is to define how the host-microbiota interface modulates the risk of obesity early in life through multidisciplinary approaches and enable the design of preventative obesity strategies starting in the neonatal period. The research aims and career development plan will work towards mastery and independence in gnotobiology, gut anaerobic microbiology, and preclinical models of metabolism and proficiency in functional and bioinformatic analysis of the microbiota and translational research in early life. The primary co-mentors are Dr. Richard S. Blumberg – an expert in mucosal immunology and preclinical models to study gut-microbiota interactions – and Dr. Alessio Fasano – a leader in pediatric translational studies in the host-microbiome interface –at Mass General Brigham/Harvard Medical School. The collaborative and mentorship team comprises a local, multi-institutional group of experts in bioinformatics, intestinal microbial ecology, mucosal immunology, metabo- lism, and childhood obesity. The research proposal seeks to explore interactions between intestinal ITLN1 and microbes in the genesis of obesity. ITLN1 has been associated with obesity in humans, but the mechanism(s) behind(s) this association are unknown. The preliminary findings suggest that ITLN1 binds a particular subset of bacteria in vivo and modulates the metabolic activity of the microbiome. Furthermore, intestinal ITLN1 might protect against obesity in a microbiota-dependent manner. The overall hypothesis of this mentored research is that intestinal ITLN1 protects from obesity by modulating the metabolic activity of ITLN1-bound bacteria. In this proposal, we will use in vitro transcriptomic and phenotypic analysis of ITLN1-bound bacteria and bottom-up and bottom down microbiological approaches in conventional and gnotobiotic mice with and without ITLN1 to under- stand the effects of ITLN1 on intestinal microbes and seek to provide critical information about mechanisms by which ITLN1 in the intestine can influence the host-susceptibility to obesity. Furthermore, the applicant will ex- plore the translational implication of ITLN1 binding to microbes in infants with adequate and excessive weight gain during the first two years of life, as rapid weight gain in this critical window is associated with childhood obesity. Expected outcomes include direct evidence that loss of a single protein (ITLN1) in the intestinal epithe- lium leads to obesity and metabolic syndrome through its effects on the microbiota and the development of the investigator's expertise in the mechanistic study of the host-microbiota interface in obesity and translational stud- ies in early life. This career development award will set the stage for an independent research career focused on developing strategies to prevent and treat obesity targeting the host-microbiota interface in early life.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT An unmet need for psychiatric neuroimaging is a standard developmental frame of reference to benchmark studies of neurodevelopmental conditions such as schizophrenia the psychosis spectrum (PS). Using a modelling approach that has proven successful for non-imaging growth charts in clinical pediatrics, and in our preliminary work that was focused on a limited set of global brain MRI features, we propose to leverage data from our multi-site Brain Chart Consortium to produce computational charts of brain maturation for a far richer set of brain morphological features across anatomical scales. Our Consortium aims to be the largest and most inclusive possible, with preliminary data covering the entire lifespan, over 130,000 MRI scans, over 100,000 individuals and over 300 MR scanners. Using advanced, fully-reproducible pipelines for quality control, image processing and harmonization, multi-scale brain charts will define normative trends and milestones of growth which can be used to benchmark a new individual brain scan, or group of scans, while controlling for study- specific technical confounds. We will create and maintain an open resource to disseminate these charts for use by other researchers. We will use brain charts to identify clusters of developmental imaging phenotypes – brain profiles benchmarked by growth chart norms – with similarly-shaped maturational trajectories. Longitudinal analysis of twin datasets will specifically delineate heritable brain profiles, which we hypothesize will show genotype-by-age effects organized along the sensory-to-association (SA) axis of cortical maturation, in developmental epochs where risk for PS is hypothesized to emerge (Aim 1). We will perform genome- and transcriptome-wide association studies to identify brain profiles that are influenced by functionally active genetic variants associated with risk for PS (Aim 2). We will perform brain profile subtyping of individuals with PS diagnoses in our Consortium case-control studies (over 2000 MRIs), to characterize a PS subtype where deviations are most prominent along the SA axis in association cortices that undergo prolonged maturation through adolescence. Any individual’s chart-benchmarked brain profile, in a new study, can thus be characterized with a loading score that quantifies similarity to this PS subtype, and we will evaluate the association of the PS subtype loading score with the evolution of PS symptoms across multiple longitudinal follow-up studies of PS conducted at the University of Pennsylvania (over 2200 longitudinal MRIs, 800 participants, 450 with PS, age 8-35) that will be pooled and harmonized for this proposal (Aim 3). This proposal’s overarching goal is to create a practically useful brain chart resource and to demonstrate its transformative potential for studies of brain development in PS. This work capitalizes on the PI and assembled team’s expertise in psychiatric and developmental brain imaging, imaging-genetics and neuroinformatics. Cumulatively, the proposed research will provide a substantial advance in our understanding of typical brain development and altered neurodevelopment in PS.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT Uncertainty in genomic diagnostics creates a barrier to realizing the full potential of genomic medicine. Uncertainty is evident in: 1) our inability to determine if some DNA variants are pathogenic or benign and 2) our inability to predict to what extent a person with a disease-causing variant will be affected, due to variable expressivity. This proposal will study both phenomena for the autosomal dominant disorder Alagille syndrome, caused by mutations in one of two genes in the Notch signaling pathway, the ligand JAGGED1 (JAG1) or the receptor, NOTCH2. Alagille syndrome is characterized by pediatric liver, heart, vertebral, renal, ocular, and facial anomalies with highly variable expressivity. The mechanism of disease for JAG1-related Alagille syndrome is haploinsufficiency whereas the mechanism for NOTCH2-related Alagille syndrome is less clear, with fewer reported variants, less functional characterization, and a higher prevalence of missense variants (>50%). Missense variants are difficult to classify, often requiring functional validation to support or reject pathogenicity. In Alagille syndrome, functional characterization has been carried out for only 19/125 reported missense mutations, thus, despite a high detection rate, the diagnostic rate is lower due to this uncertainty. We propose to resolve uncertainty in the diagnostics of Alagille syndrome using assays designed to characterize the pathogenicity of JAG1 and NOTCH2 missense variants and analysis of gene expression data from patient liver samples to identify gene expression signatures that can be used for genotype-phenotype evaluations. In Aim 1, we will design a Site Saturation Variant Library of all possible nucleotide permutations at each nucleotide position across a region with high missense variant uncertainty in the JAG1 C-terminus and test this library by developing a Multiplexed Assay for Variant Effects (MAVEs) that will measure cellular localization of JAG1 as a readout of protein function. In Aim 2, we will use FFPE liver tissue samples to analyze gene expression differences between Alagille syndrome patients and controls, as well as between Alagille syndrome patients with mild versus severe liver disease. In Aim 3, we will study the molecular basis of NOTCH2 variants through functional, expression, and enzymatic assays using mutant cell lines. We hypothesize that these proposed assays will identify a high-throughput method to test missense pathogenicity (Aim 1), identify gene expression differences between Alagille syndrome patients and controls as well as gene expression signatures that are different between Alagille syndrome patients with mild versus severe liver disease (Aim 2), and determine the mechanism by which NOTCH2 variants cause Alagille syndrome through functional analysis (Aim 3). Ultimately, these data will improve variant analysis for Alagille syndrome, improve our understanding of the molecular basis of liver disease in Alagille syndrome, and establish a framework for scalable classification of missense variants, delivering diagnostic information that can directly help clinicians.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Small RNAs are ubiquitous regulators of eukaryotic gene expression in nearly all aspects of physiology from plants to humans. They function to regulate all facets of gene expression including transcription, RNA stability, and translation. An emerging class of small regulatory RNAs are tRNA-fragments (tRFs), produced from nucleolytic cleavage of tRNAs. tRFs have been implicated in cancer, neurodegenerative disease, viral infection, fertility, epigenetic inheritance, and aging. While the biogenesis of these RNAs is poorly understood, tRFs have been demonstrated to play roles in the regulation of transcription, posttranscriptional regulation of mRNA stability, and translation. Yet, the functions of tRFs throughout the different tissues of an organism in regulating cellular physiology are unknown. My lab is developing the roundworm C. elegans (worms) as a model to dissect the molecular mechanisms underlying the biogenesis tRF and cellular functions throughout the animal. The robust genetics, physiological assays, and molecular tools available in C. elegans provide a system ripe for the discovery of tRF biology. Importantly, we have developed small RNA-sequencing techniques to detect abundant tRFs in C. elegans, at levels much higher than previously published small RNA-seq datasets. Using these techniques, we will characterize tRFs in the different tissues of the fully developed adult using bulk and single-cell small RNA-seq. After determining the spatial and temporal expression of tRF species we will utilize both forward and reverse genetics to determine new factors required for their biogenesis. Further, we will employ biochemical enrichment strategies to determine factors that interact with and affect tRF function. Upon determining genes required for tRF biogenesis and function we will use mutant alleles of these factors to determine what roles these molecules have in regulating the physiology of the organism. Finally, we will use worm strains carrying loss-of-function mutations in RNA modifying enzymes to determine how RNA modifications affect tRF biogenesis, functions, and further regulate the physiology of the animal. The work on tRFs in C. elegans in my lab represents the first systematic dissections of all aspects of tRFs biology in a whole organism. What we learn about tRF biology in worms will be used to generate hypotheses about tRFs in other organisms. As tRFs have been implicated in many aspects of normal physiology in organisms ranging from yeast to humans, as well as in a wide range of diseases, comprehensively understanding how tRF are regulated and function represents a highly under-addressed aspect of biology.

NIH Research Projects · FY 2024 · 2023-06

Project Summary/Abstract Background: Structural racism, defined as all the ways in which society cultivates racial discrimination through reinforcing inequitable systems of education, healthcare, and employment, is manifested in macroaggressions, which are contained within the policies and practices within institutions serving the biomedical sciences. These macroaggressions foster and perpetuate discriminatory beliefs and practices that impair not only scientific productivity and new discoveries in biomedical research but also the career trajectories of those from ethnic minoritized groups and communities. Specifically, macroaggressions necessitate that individuals, especially those in positions of power and leadership change their prejudices and actions. Thus, interventions for macroaggressions and structural racism must recognize and address racial and ethnic prejudices and discrimination that arise on an individual level, known as, racial microaggressions. The harm of racial microaggressions is compounded when intersectional (relating to other marginalized backgrounds such as gender, nativity status etc.) and thus creates overlapping systems of oppression. In order to create a culture conducive to change within a range of biomedical research environments, trainees and their supervisors need to better understand and acknowledge their own backgrounds and how their perspectives have been shaped based upon a combination of their lived experiences interacting with a culture steeped in a history of structural racism. In order to facilitate institutional-level change in policies and procedures that disproportionately impact those from minoritized backgrounds, it is important that institutional leaders also be active change agents. Purpose: The primary objective of the proposal is to create a series of self-directed, self-paced learning modules that biomedical research trainees, supervisors, and organizational leaders can use to better understand ways that structural racism influences their own biases and behaviors as well as strategies to use when they are a target, bystander, or perpetrator of racial and intersectional microaggressions, and to use these strategies in addressing institutional level policies indicative of racial and intersectional macroaggressions. Additional objectives include collecting qualitative and quantitative data to evaluate the feasibility, acceptability, accessibility, and initial impact of the new modules on a range of participants. Methods and Design: A community-based participatory research (CBPR) approach will be used to ensure that the new educational modules are based upon empirical science, are theoretically-grounded, and include iterative feedback from a range of key stakeholder groups (e.g., biomedical trainees, supervisors, and leaders). A preliminary evaluation including random assignment of participants to intervention and comparator conditions will be used to determine whether the educational modules are associated with increases in knowledge and self-efficacy in appropriately handling racial and intersectional macroaggressions and microaggressions.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Morbidity and mortality in children with sepsis and multi-organ dysfunction syndrome (MODS) are substantial. Timely delivery of effective antibiotic concentrations to the site of infection dictates treatment outcomes, but antibiotic pharmacokinetics (PK) are highly variable in septic children, frequently leading to sub- or supra- therapeutic antibiotic concentrations. To ensure optimal clinical and microbiologic outcomes, attainment and maintenance of safe and effective antibiotic concentrations throughout the treatment course are paramount. Despite this, antibiotic dosing in children with sepsis is based primarily by a child’s weight and kidney function, without regard to other sources of PK variability, while clinical measurement of antibiotic concentrations is performed for very few drugs. The host response to infection is a major driver of organ dysfunction and antibiotic PK variability in pediatric sepsis: hyperinflammation, as well as sepsis-induced immune dysfunction (i.e. immunoparalysis), are both common and exacerbate outcomes. Further, in critically ill children with respiratory failure, bacterial burden and composition of the respiratory tract microbiome impact both host inflammation and clinical outcomes. Understanding how the host immune response, antibiotic PK, and the microbiome interrelate, and influence clinical outcomes, is imperative to optimize treatment in pediatric sepsis. The Collaborative Pediatric Critical Care Research Network (CPCCRN) will perform two concurrent, double- blind, placebo-controlled RCTs to evaluate the impact of individualized immunomodulation (anakinra for hyperinflammation; GM-CSF for immunoparalysis) on organ function outcomes in pediatric sepsis-induced MODS. These trials (named PRECISE) provide a unique framework for evaluating the interplay between host immunophenotype (hyperinflammation, immunoparalysis), immunomodulation, and antibiotic PK/PD through our proposal. We will leverage PRECISE trials and CPCCRN infrastructure to evaluate sources of PK variability in children with sepsis and MODS, investigate how host immune responses longitudinally modulate antibiotic concentrations, and study how antibiotic concentrations impact organ dysfunction duration and the respiratory tract microbiome. In Aim 1, we will determine the influence of host immunophenotype and response to immunomodulation on antibiotic PK early (1A) and throughout the course (1B) of pediatric sepsis-induced MODS. Aim 2 focuses on understanding how antibiotic concentrations impact organ function outcomes in the context of immunomodulation in pediatric sepsis-induced MODS. Lastly, Aim 3 will quantify how antibiotic concentrations, immunophenotype and immunomodulation impact the respiratory tract microbiome over time in septic children with respiratory failure. By quantifying antibiotic concentrations and evaluating the drivers of antibiotic PK in sepsis in the context of immunomodulation, our proposal will facilitate development of individualized treatment strategies during sepsis-induced MODS.

NIH Research Projects · FY 2026 · 2023-06

ABSTRACT Variability in the spatial layout of human brain functional networks on the anatomic cortex is a novel phenotype that can be extracted from functional magnetic resonance imaging (fMRI) with transformative potential for combined imaging-genetics studies of human brain function. Early results show that the individual-specific topography of Personalized Functional Networks (PFNs) is strongly associated with domains of psychopathology and cognition, including executive functioning (EF) which is impacted in multiple mental health conditions and undergoes profound changes during the period of adolescence. PFNs capture individualized aspects of brain function that have unique associations with clinical and developmental outcomes, compared to standard fMRI approaches, which can measure activity in these same functional networks but fail to incorporate the variation in functional network topography that exists among individuals. The over-arching hypothesis of this proposal is that targeting PFNs will rapidly accelerate the discovery of genetic contributions to the organization of brain function, leading to mechanistic insights into genetic risks for behavioral health conditions related to brain function. To this end, we will probe PFNs in genetically informative open fMRI datasets including the Adolescent Brain and Cognitive Development Study (ABCD, n=11,572, n=850 twin pairs, 5 longitudinal time points during study period) and the UK Biobank (UKBB, n>40,000), as well as locally acquired fMRI data on the 22q11.2 deletion syndrome (22qDS, n=100; controls n=500). Analyses will yield a cohesive investigation of inherited polygenic effects and rare, typically de novo copy number variants (CNVs), each of which are hypothesized to influence individualized functional network topography. First, we will use longitudinal twin models in ABCD to investigate the twin heritability of PFNs and their genetic correlation with behavioral domains such as EF, for example, testing the hypothesis that the genetic correlation between EF and association cortex PFNs will increase during adolescence (Aim 1). Second, we will perform genome- and transcriptome-wide association studies (GWAS and TWAS) of PFN topography in ABCD and UKBB to prioritize specific, functionally active genetic loci (Aim 2). Third, we will investigate the influence of rare CNVs on PFNs, using analysis of case-control Penn/CHOP 22q11.2DS data, in ABCD, CNV Risk Scores that we recently showed to correlate with deviations from the typical development of brain anatomy in a community cohort (Aim 3). Allanalyses will be conducted with fully reproducible, transparent imaging processing and genetic pipelines, capitalizing on the PI and assembled team's joint expertise in advanced fMRI methods, genomics, and informatics. Cumulatively, the completion of these aims will result in a major advance in our understanding of the genetic contributions to brain function and its relationship to psychiatric risk, leading to future experimental and clinical trials of targeted neuromodulation guided by individualized neurogenetics.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT The discrimination of highly similar episodes is termed behavioral pattern separation. This episodic memory process is altered by stress and decreased in humans with and rodent models for a range of brain disorders, including post-traumatic stress disorder (PTSD). Behavioral pattern separation is also susceptible to “load”; it is harder to discriminate episodes that are very similar (high load) vs. different (low load). Defining the underlying circuitry in memory stages and load-sensitivity is a key step to a future where poor behavioral pattern separation might be treated via circuit-based manipulations. The focus of this application is the role of the lateral entorhinal cortex (LEC) in behavioral pattern separation. The anatomical connections of the LEC suggest it is central to this stress-sensitive process. The LEC is innervated by polymodal-, emotion, and stress-linked brain regions. The LEC innervates downstream hippocampal regions critical for behavioral pattern separation, including the dentate gyrus (DG). In fact LEC layer IIa stellate fan cells (LECIIa fan cells) send glutamate directly to two key DG cells, DG granule cells and adult-generated neurons, which are both critical for “high load” pattern separation and are very sensitive to stress. Excellent human imaging and rodent lesion and neural recording studies also suggest the LEC has a role in behavioral pattern separation. However, the LEC’s causal role in orchestrating behavioral pattern separation and its memory stages is untested. The lack of data on LEC’s role is striking given that the LEC is vulnerable to stress, aging, and disease. A link between LEC and the poor pattern separation seen in age and disease — including in stress-induced cognitive disorders like PTSD — remains correlative. Direct evidence of the LEC’s role in behavioral pattern separation is paramount to clear understanding of cortical-hippocampal circuitry and its function in nonpathological and pathological states. In this revised R01 application, we propose three aims to provide fundamental understanding of how LECIIa fan cells are involved in behavioral pattern separation, during what memory stage and which memory load, and how the LEC-DG circuit activity could be manipulated to overcome stress-induced disruption of pattern separation. Aim 1. Test if the encoding and consolidation of behavioral pattern separation rely on the activity of LECIIa fan cell terminals in the DG. Aim 2. Test if behavioral pattern separation performance/retrieval is modulated by the activity of the LEC fan cell-DG circuit. Aim 3. Test if repeated stress disrupts behavioral pattern separation performance/retrieval in a way that can be reversed by LEC-DG circuit stimulation. The data from these Aims will fill major knowledge gaps in the existing models of the neural circuitry that supports behavioral pattern separation. They will provide essential behavioral and mechanistic insight to understand poor pattern separation and to fuel therapeutics to combat stress-induced cognitive dysfunction.

NIH Research Projects · FY 2026 · 2023-06

Approximately 1 million autistics will turn 18 in the next decade, many without the skills they need to achieve the quality-of-life that they and their families’ desire. Without effective supports, autistic youth struggle with daily living skills, regardless of their intellectual abilities. Daily living skills are fundamental to independence, paid employment, and better quality-of-life for autistic adults. Existing daily living skill interventions for this age group have only modest effects or show poor generalization to real world settings. Current treatments rely on explicit instruction of specific daily living skills (e.g., the steps for taking a shower), and they lack inclusion of mutable psychological factors that support the development and generalization of daily living skills. Treatments are further limited by inadequate knowledge of how social determinants of health (e.g., family income, community resources) contribute to daily living skills. Identification of mutable psychological factors and social determinants of health driving change in daily living skills for autistic youth exiting high school is a vital to improving public health. This knowledge will identify pivotal intervention and service delivery targets for improving daily living skills. Better executive function and self-determination skills are associated with more advanced daily living skills, and both factors improve with treatment in autism. Our central scientific premise is that interventions for daily living skills, and the service delivery systems that promote them, will be enhanced with greater knowledge of psychological and systemic factors that directly impact these skills. Further, enhanced daily living skills will result in downstream improvements in quality-of-life and productivity. This project will address gaps in our knowledge with a prospective longitudinal study that evaluates psychological factors that drive change in daily living skills during the time when autistic youth exit high school (AIM 1), as well as the impact of daily living skills on quality-of-life (AIM 2). We will also explore the influence of both individual- (e.g., family income) and neighborhood-level (childhood opportunity index) factors on daily living skills (AIM 3). Finally, there is a general need for large samples reflective of the autism population in the Mid-Atlantic region (i.e., IQ range, speaking/nonspeaking, sex, race, ethnicity). The proposed longitudinal study will contain 3 visits (T1: baseline, T2: +1 yr., T3: +2 yrs.; final N=170). Our recruitment strategy will ensure all participants have at least one timepoint pre- and post-high school exit. We predict: AIM 1, H1) executive function and self-determination will explain significant variance in concurrent daily living skills above covariates; AIM 1, H2a,b) executive functioning and self-determination at baseline will predict daily living skills at T3 and change in daily living skills over time above covariates; AIM 2, H3a,b) larger increases in daily living skills will predict better objective and subjective quality-of-life and better change in quality-of-life over time. In AIM 3, we explore both direct and indirect effects of social determinants of health. This project will generate critical knowledge for enhancing daily living skills interventions and delivery systems that will improve long-term outcomes for autistic adults and increase access to services.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT Bronchopulmonary dysplasia (BPD) is a severe chronic lung disease that develops over the first months of life in more than half of infants born less than 30wk of gestation. Infants who develop grade 3 BPD, the most severe disease form (defined as invasive ventilation at 36wk postmenstrual age), are at high risk for life-long deficits in health and cognition and poor quality of life. Unfortunately, rates of grade 3 BPD are increasing, and no therapies have been proven to treat this devastating disease. A key contributor to these care gaps is the nearly singular reliance on the prescribed respiratory support to define BPD presence and severity, select therapeutic interventions, and assess prognosis. Our data and others’ show that this subjective diagnostic approach masks significant heterogeneity in clinical presentation, treatment responsiveness, and outcomes. In other heterogenous chronic respiratory conditions such as COPD, cystic fibrosis, and asthma, evidence- based phenotyping (identification of patient subgroups based on shared characteristics) has become the new gold-standard to objectively classify distinct disease sub-types, improve patient counseling, identify novel disease mechanisms, and discover and implement more effective treatments. Our central hypothesis is that multidimensional phenotyping in grade 3 BPD is feasible, will accurately characterize disease heterogeneity, and will improve outcome prediction. Confirmation of this hypothesis will promote a frameshift towards an objective, systematic diagnostic approach in BPD and first-ever phenotype-specific trials in neonatology. Importantly, our motivating preliminary data support this hypothesis. Using retrospective results from chest CT, cardiac echo, and bronchoscopy, we showed that preterm infants with grade 3 BPD can be classified into phenotypes based on the presence or absence of severe parenchymal lung disease, abnormal large airways, and pulmonary arterial hypertension. This diagnostic approach correlated with pre-discharge outcomes and suggested possible phenotype-specific therapies. However, it relied on investigator chosen phenotypes rather than empirically derived subgroups, used qualitative results from only 3 diagnostic tests, and did not assess longitudinal phenotype stability or post-discharge outcomes. Our recent data indicate that serial quantitative cardiopulmonary imaging and evaluation of proven mechanistic contributors to BPD may improve disease phenotyping and outcome prediction. Our proposal builds on these preliminary data and will employ robust cluster analyses and longitudinal multidimensional imaging, biological, and clinical data in a large, prospective cohort of very preterm infants to accomplish the following Specific Aims: (1) define objective, evidence-based phenotypes in grade 3 BPD, and (2) define the strength of association between grade 3 BPD phenotypes and neurodevelopmental and pulmonary outcomes through 2 years’ corrected age using standardized development testing and validated outcome measures.

NIH Research Projects · FY 2025 · 2023-06

To facilitate the diagnosis of among 7000 rare genetic diseases, clinicians have developed diagnostic criteria that enumerate different elements that define disease. These include medical problems, physical exam findings, laboratory test results, and imaging findings. However, most clinical diagnostic criteria have unknown predictive value. Despite being critical for diagnosis and provision of genetic testing, they are typically proposed without rigorous evidence or estimates of performance such as sensitivity or specificity. Suboptimal criteria may cause faulty interpretations of genetic testing with variants of uncertain clinical significance or lead clinicians to overlook diagnosis, depriving patients of prognostication, reproductive planning, or targeted molecular therapies. Our previous work has delineated an approach to more evidence-based rare disease criteria. We developed novel clinical criteria for nevoid basal cell carcinoma syndrome using survey data and statistical optimization, and we estimate the novel criteria have improved sensitivity compared to the existing expert consensus criteria, particularly at early ages (53% versus 13% at 7 years). My central hypothesis is that diagnosis of rare pediatric genetic disease can be improved by utilizing evidence-based diagnostic approaches. Moreover, such approaches may be one avenue to address inequities in the provision of genetic referral and testing among individuals belonging to historically marginalized groups. Therefore, I will scale our previous work across the spectrum of rare genetic diseases using comprehensive, clinician-validated phenotype information to establish and test diagnostic methodologies. To address this hypothesis and progress towards my long-term career goal of becoming and independent physician-scientist that advances accurate and timely diagnosis for all children with a rare genetic disease, I have developed a comprehensive five-year career development plan. This plan delineates a strategy to gain knowledge and experience with natural language processing and machine learning, human-centered design and human factors, and electronic health record intervention. Using these new skills, I will create comprehensive, chronological phenotype histories for over 37,000 children with suspected or confirmed genetic disease. I will embed a tool in the clinical workflow that elicits clinician validation of these phenotypes. From these data, I will implement a framework to develop and validate diagnostic criteria in genetic disease. I will initially focus on 10 specific diseases. I will also develop computationally tractable machine learning algorithms to aid in diagnosis at scale. Next, I will develop a web-based user interface to empower other clinicians to develop and test their own diagnostic criteria. Finally, I will apply the same phenotyping and machine learning approaches at the health system level to predict which children are more likely to be diagnosed with a rare genetic disease. These endeavors will generate a foundation to establish my long-term research program that will implement clinical decision support for genetic diagnosis and prepare me to become an independent R01-funded investigator.

NIH Research Projects · FY 2025 · 2023-06

ABSTRACT a-Thalassemia (a-thal) is caused by insufficient production of the a-globin protein due to either deletional or non- deletional mutations of endogenous a-globin genes. In patients with severe a-thal (no or minimal synthesis of a- globin chains), a blood transfusion independent-state is achievable through allogeneic bone marrow transplantation, but this approach is limited to only some patients and is plagued by potential serious adverse effects, such as graft rejection or graft-versus-host disease. No mouse models of severe a-thal are available to study this disease and to test new therapies. Our proposed work will address these knowledge gaps by developing, characterizing, and validating mouse models and gene therapy vectors for treating severe a-thal. We hypothesize that new mouse models of a-thal will define the basic mechanism that governs RBC synthesis in the presence of excess ß-globin chains and how it affects erythropoiesis, iron metabolism and coagulation. In our first aim we will characterize these features in novel mouse models of severe a-thalassemia. As preliminary studies, we generated adult animals that do not produce a-globin chains (AG-KO) through transplantation of both AG-KO fetal liver and conditional AG-cKO hematopoietic stem cells into wild-type recipient mice. These animals demonstrate a worsening phenotype, with red blood cells (RBC) that express only b-globin chains. Due to severe limitation of these RBC to deliver oxygen, the mice eventually succumb to a condition resembling hypoxemia, showing splenomegaly, liver and kidney iron deposition, and vaso-occlusive events. We are now generating animals that only express one copy of the a-globin gene to characterize this disease in the context of minimal synthesis of a-globin chains. Most of the patients affected by a-thal carry large deletions of the a-globin genes. These deletions represent a serious challenge for gene therapy approaches based on genome editing. Therefore, we hypothesize that severe a-thal can be safely rescued by gene addition. In our second aim we will fully validate lentiviral vectors carrying the a-globin gene for their safety and ability to reverse the most severe forms of a-thal. We identified ALS20aI, in which a-globin is under control of the ß-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20αI yields exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. Indeed, ALS20aI rescues animals generated with AG-KO fetal liver or conditional AG-cKO hematopoietic stem cells, suggesting that a relatively low vector copy number could result in dramatic therapeutic benefits. We will test ALS20aI or its derivatives for their ability to express the safest and highest level of a-globin in mouse hematopoietic stem cells and human-derived erythroid cell lines that synthesize low or no a-globin chains. We will then evaluate the constructs for their ability to rescue the abnormal features observed in a-thal patient cells. Thus, the goals of this study are to develop novel adult mouse models of a-thal and an effective gene therapy approach for this disease.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Lower Urinary Tract Symptoms (LUTS) are among the most common reasons to see a urologist across the lifespan and are often accompanied by psychological consequences, such as anxiety and depression. Patients with LUTS may have a disrupted bladder–brain dialogue; they may say “I don’t even feel anything until I am wet”. Better therapeutic options can emerge from a better understanding of the fundamental brain circuits and cells that contribute to LUTS and that contribute to bladder–brain communication. A potential LUTS etiology that merits closer study is early life urinary tract infection (UTI). Clinical studies in females (who have UTIs 4x more often than males) show early life UTIs predict later life LUTS. Basic research on UTIs and other bladder insults in adulthood suggest inflammation underlies acute LUTS and psychological consequences. For example, adult systemic injection of cyclophosphamide (CYP) — the most common noninvasive (catheter-free) way to model UTI-linked inflammation — leads to bladder and psychobehavioral dysfunction. However, there is a striking absence of research on the role of early life UTIs in later life LUTS and psychological consequences. Here we show for the first time that female mice experiencing repeated, early life cystitis after CYP have bladder and psychobehavioral dysfunction in adulthood. These data fuel our hypotheses: Early life CYP-induced cystitis induces bladder and brain inflammation, which disrupts bladder-brain dialogue and leads to lifelong LUTS and psychobehavioral consequences; this later life bladder and brain dysfunction can be reversed by manipulation of key brain circuits in adulthood. In Aim 1, we will define early life CYP-induced inflammatory indices in bladder and brain across the lifespan. In Aim 2, we will determine early-life CYP-induced functional changes in bladder and brain across the lifespan, launching off from our pilot data: repeated, early-life CYP-induced cystitis leads to adulthood voiding dysfunction and anxiety-like behavior. We will use pharmacologic tools to address the mechanism and specificity of these functional changes, and test for anxiety- and depression-like phenotypes throughout life. In Aim 3, we will map and manipulate key components of the brain circuits that contribute to bladder-brain dialogue. Using our proven abilities to assess neuron activity in brain regions central to this dialogue (locus coeruleus, Barrington’s nucleus, frontal cortex), we will record the activity of sets of neurons in freely-moving mice before and after voiding events in our early life CYP model. We hypothesize that early life cystitis disrupts the ability of these neuron types to respond to sensation from the bladder and thus discriminate voiding cycle stages. We will then attempt to reverse the early life cystitis-induced bladder and psychobehavioral dysfunction in later life by optogenetically manipulating the activity of relevant neurons in adulthood, as we have already done in adult models. Given the prevalence of LUTS, the basic experiments proposed here have enormous potential to expand our understanding of how neonatal cystitis influences the bladder-brain dialogue throughout the lifespan.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Over 80% of children diagnosed with cancer become long-term survivors; however, 70% develop chronic or life-threatening late effects from treatment, and these often emerge during young adulthood. Guidelines recommend annual long-term follow-up care (LTFU) to manage and monitor for late effects, recurrence, or new cancer(s). Yet, as risk for late effects increases in young adulthood and survivors transition into adult-focused care, engagement in care plummets. Disengagement from LTFU leaves adolescent and young adult survivors (AYA) vulnerable to delayed or poorly managed diagnoses, and relates to lower knowledge and self- management abilities. Thus, interventions targeting re-engagement of AYA are critical for this vulnerable population. In order to address this critical intervention need, we propose REACH (Re-Engaging AYA survivors in Cancer-related Healthcare)—an innovative sequential multiple assignment randomized trial (SMART) with a two-stage design. We will leverage our research on transition to adult care, survivorship care plans, and digital health interventions. Stage 1 tests a low touch intervention (LTI), consisting of reminder “nudge” text messages and informational resources for up to 4 weeks compared to an enhanced usual care group that will receive written information (WI) only. Next, in Stage 2, AYA will be re-randomized based on responsiveness to Stage 1 (i.e., whether or not they made an appointment) into 16 weeks of intervention. Responders will either be re- randomized to receive a continued intervention (maintenance) or a stepped-up condition, while non-responders will only receive a stepped-up condition. Step-up may include expanded LTI (more text messages) or a high touch intervention (HTI). The HTI includes text messages and digital resources, including a personalized survivorship care plan (SCP), on a mobile-friendly platform with information tailored to variables such as barriers to care, treatment history and recommendations, and a personal health goal. Additionally, social worker and/or nurse support will be available to help overcome barriers (e.g., access care). The intervention options in Stage 2 are intended to enhance self-management beyond simply attending LTFU in order to sustain long-term engagement. Outcomes are measured after Stage 1 (T2), Stage 2 (T3) and at 9 months (T4). We expect those who start with LTI versus WI will be more likely to attend a LTFU appointment by T4. For Stage 2, those who receive the stepped-up condition compared to those with maintenance, and those who received HTI compared to LTI, will be more likely to attend an appointment by T4. Attending an appointment by T3 and indices of self-management are secondary outcomes. We will also assess moderators of intervention outcomes, intervention engagement, acceptability, feasibility, and cost. Qualitative interviews will assess multilevel barriers and facilitators of future implementation with stakeholders (AYA, parents/supports, providers, administrators). This trial responds to NOT-CA-22-029 and PAR-21-035 to promote optimal engagement in LTFU and self-management for AYA survivors, improving outcomes and reducing disparities.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Adverse outcomes for pregnant patients and infants associated with the hospitalization for childbirth are frequent and vary across hospitals in the United States. Organizational factors, such as organizational structure and culture, in birth hospitals are potential drivers of variation in care and outcomes. Maternal and neonatal levels of care, an organizational structure, are the risk-based services available at a hospital for mothers and infants, respectively. Organizational culture refers to the beliefs, perceptions, and values on obstetric and neonatal units in a hospital, a key component of which is the culture of patient-centered care. The care and health of the mother and infant are interrelated, yet studies have not formally assessed perinatal organizational factors, those created by the intersection of maternal and neonatal care at the hospital level. The overall objective of this study is to determine how organizational factors of obstetric and neonatal units, formalized by this proposal as perinatal profiles of organizational structure and culture, impact perinatal outcomes of mothers and infants and drive hospital variation. Dr. Handley will achieve this objective by 1) assessing the impact of perinatal level of care profiles, 2) assessing the impact of perinatal patient-centered care culture profiles, and 3) examining the interaction of perinatal structure and culture profiles on perinatal outcomes. Her analysis will leverage a nine-state, retrospective cohort of mother-infant pairs as well as primary data from her organizational culture survey of California birth hospitals, which will be linked with state data to produce a novel dataset. This proposal was designed to prepare Dr. Handley to become an independent investigator, specifically focused on integrating health services research and organizational science in order to understand how organizational factors on obstetric and neonatal units affect perinatal outcomes. Her research will be complemented by training in advanced statistical methods for observational data and primary organizational culture survey data as well as acquiring in-depth knowledge of organizational behavior, dynamics, and change. Working with a multidisciplinary team of mentors and advisors with experts from neonatology, health care management, obstetrics, and biostatistics will provide her valuable guidance in the completion of this work and planning of future studies. Understanding the implications of perinatal organizational structure and culture profiles on perinatal outcomes is critical to improving perinatal care. Through better understanding of drivers of outcome variation across hospitals and identification of organizational factors amenable to change, this work has the potential to shape perinatal level of care policies, inform initiatives to improve organizational culture and coordination of maternal and neonatal care, and guide future change at the hospital, state, and national levels, which are actionable strategies to improve outcomes of the mother-infant dyad.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Severe subglottic stenosis, the narrowing of the airway just below the vocal folds, develops as a response to intubation in close to 10% of the > 20,000 premature births per year in the United States. Severe cases require laryngotracheal reconstruction (LTR), in which surgeons split the cricoid and add a piece of autologous patient- derived cartilage to expand the airway and restore proper airflow. However, in children, the success rate is as low as 50% with a high incidence of restenosis requiring revision surgery. Graft failure is tied directly to the lack of sufficiently sized autologous cartilage in the child, and tissue engineering has been proposed to develop alterative grafting options for pediatric LTR. Some approaches, including some of our previous work, have been effective in producing functional cartilage, but the overall timeframe required for the construct to match the mechanical properties of native cartilage (>24 weeks) is not compatible with clinical translation (<8 weeks). Furthermore, current cell sources such as expanded autologous chondrocytes and mesenchymal stem cells frequently result in hypertrophic and calcified tissue. Our objective is to engineer a new type of cartilage implant that is populated with patients’ cells, mechanically viable and suitable for LTR within a clinically relevant timeframe. Our approach uses a microstructured polymeric scaffold with over 90% porosity and sufficient mechanical properties and with a pore structure uniquely capable of enhancing chondrogenesis of stem cells. Furthermore, cartilage progenitor cells have been proposed as a rapidly proliferating, highly chondrogenic cell source. To harness these cells, we have developed a minimally invasive biopsy procedure to harvest ear Cartilage Progenitor Cells (eCPCs). Our overarching hypothesis is that the microstructured polymeric scaffold combined with eCPCs will create cartilage implants with suitable mechanical strength, dimensions, and phenotypic stability for personalized, minimally invasive LTR. We propose to identify the microstructure that achieves the maximum chondrogenesis and the specific mechanism of action. The capacity of eCPCs to produce a robust cartilage phenotype, potentially better than that of ear chondrocytes and less prone to calcification, will also be studied. Finally, the engineered cartilage derived from the eCPCs seeded in the microporous scaffold will be test in a porcine LTR model. We expect that our findings will introduce a major innovation in the treatment of subglottic stenosis, laying the basis for long term pre-clinical safety studies and clinical translation in airway reconstructive surgery in children.

NIH Research Projects · FY 2026 · 2023-04

(<30 lines) Youth-onset type 2 diabetes (YO-T2D) is increasingly prevalent in parallel with the obesity epidemic, yet effective treatment and prevention strategies are limited. The physiologic increase in insulin resistance occurring during puberty, in combination with obesity-related insulin resistance, enhances the risk of T2D. Yet, it remains unclear why some youth progress through puberty with intact β-cell function, while others do not, despite similar phenotypic and metabolic characteristics. More information is needed regarding the unique events during puberty to better understand 1) the basic pathophysiology of glucose control, insulin sensitivity, β-cell function, and T2D risk in youth, 2) differences among girls and boys, populations at highest risk, and urban and rural geographies, and 3) the potential contribution of other risk factors including psychological, behavioral, and social and external contexts. Importantly, this research needs to address the timeline of pathophysiology and progression from normoglycemia or prediabetes to YO-T2D. The DISCOVERY of Risk Factors for Type 2 Diabetes in Youth (DISCOVERY) study provides a unique opportunity to characterize the risk progression profile and mechanisms underlying the development of YO-T2D, and evaluate the effects of modifiable and non-modifiable risk factors. Ultimately, the results of this study will establish a basic pathophysiology to inform future studies aimed at achieving target glycemia, improving insulin sensitivity, preserving β-cell function, and/or preventing YO-T2D. To address this goal, DISCOVERY will recruit, enroll, and follow a nationally-representative cohort of 3,600 at-risk obese youth in early puberty; extensively phenotype them as they transition through puberty; and characterize the course of decline and dysfunction in pathophysiological indicators that lead to YO-T2D. The expected duration of the DISCOVERY is 5 years, including planning, recruitment, follow-up, analysis, and reporting. In addition, DISCOVERY will store longitudinal biospecimens and genetic material with the intention of acquiring additional ancillary funding to pursue analysis of emerging indicators. Children’s Hospital of Philadelphia has experience in multicenter and diabetes-related investigations and will contribute to DISCOVERY through the recruitment of approximately 240 at-risk youth, implementation of the IRB-approved consensus protocol, participation on DISCOVERY committees, and collaboration on the analyses and dissemination of the findings from DISCOVERY.

NIH Research Projects · FY 2026 · 2023-04

Eye as a Window into Brain Health in Hydrocephalus Project Summary Hydrocephalus is a debilitating condition caused by excess buildup of cerebrospinal fluid (CSF) in the cerebral ventricles. The overall global prevalence of hydrocephalus in children is 88 out of 100,000, with the mortality rate of untreated hydrocephalus reaching up to 87%. Most pediatric hydrocephalus cases (>90%) are managed operatively, using a ventricular shunt to divert CSF. Unfortunately, timing of shunting is guided by gross measures of intracranial pressure (ICP) and brain health including ventricular size and clinical signs. Delaying CSF diversion can lead to elevated ICP and irreversible brain injury. Invasive ICP monitoring, while more precise, is not routinely adopted in children due to the risks of hemorrhage and brain injury. This proposal bridges a significant clinical gap in care by validating ocular blood flow as a precise biomarker of ICP and brain ischemia that can negate the need for invasive ICP monitoring. As a direct extension of the brain, the eye has served as a window into studying ICP, but to date none of the noninvasive approaches evaluating ocular hemodynamics has proven as reliable as invasive ICP monitoring. In our proposed study, ocular contrast-enhanced ultrasound (CEUS) using a high-speed ultrasound system is performed in a high-fidelity pediatric porcine model of hydrocephalus to validate ocular blood flow markers of ICP and brain ischemia. CEUS uses intravenously injected microbubbles of 2-3 µm in size, smaller than red blood cells, that can be tracked across multiple ultrasound frames using an advanced particle tracking method (called particle image and/or tracking velocimetry or PIV/PTV). As a result, spatial and temporal changes in ocular microcirculation can be quantified for assessment of elevated ICP and brain ischemia. While the CEUS technology is FDA-approved for pediatric applications, specifically for evaluation of focal liver lesions and vesicoureteral reflux, ocular CEUS is off-label. The investigative team stands ready for clinical translation following this proposal, as the PI currently leads the first FDA-regulated, Investigational New Drug (IND)-approved clinical trials applying CEUS in infants with brain injury and necrotizing enterocolitis. The central hypothesis of the proposal is that ocular CEUS will provide accurate biomarkers of ICP and brain ischemia. The overall goal of the proposal is therefore to 1) validate and refine the accuracy and reproducibility of the PIV/PTV for eye imaging using phantom models mimicking the complex ocular microvascular networks and spontaneous eye movement, 2) validate ocular CEUS indices of ICP and brain ischemia using an established pediatric porcine model of hydrocephalus and 3) assess in vivo safety of the optimized ocular CEUS protocol. Our work will set the stage for clinical translation of a new noninvasive tool for assessment of ICP and brain ischemia in pediatric hydrocephalus, which could ultimately impact survival and long-term outcomes of affected children.