Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2023-01

ABSTRACT Asthma is an inflammatory lung disease that affects >300 million people worldwide. Although mild asthma is driven by a Th2-associated, eosinophil-dominated immune response, factors such as obesity/metabolic dysfunction are associated with more severe asthma. Thus, the “obese asthma” endotype is associated with frequent asthma exacerbations, a shift away from Th2-dominated inflammation towards a Th17-dominated profile, and a marked female bias. A major gap in our understanding of mechanisms driving obese asthma is due to deficiencies in common mouse models of obesity; standard high-fat diet (HFD)-fed mouse models are unable to induce extensive weight gain and metabolic dysfunction (associated with obesity in humans) in female mice - a major shortcoming given the female bias observed in the obese asthma endotype. Importantly, our group has determined that HFD feeding of C57BL/6 mice housed at a temperature in which they are at metabolic homeostasis (thermoneutral temperature (TN); 30-33°C) promotes severe obesity and metabolic dysfunction in female mice. Preliminary data using this model demonstrate that allergen-challenged obese asthmatic mice demonstrate: (1) worse asthmatic outcomes compared to lean asthma controls, (2) a shift away from Th2 inflammation to a pro-inflammatory profile (Th17 or Th2/Th17), and (3) a pronounced female bias. Similar changes are NOT observed in male mice. These data suggest that TN housed obese female asthmatic mice model the human obese asthma endotype. Importantly, obese asthmatic female mice demonstrated a profound increase in the frequency of IL-17A-producing cells in the lungs, particularly mucosal-associated invariant T (MAIT) cells. MAIT cells are innate-like T cells abundant in lungs, liver, and adipose tissue. Although considered protective in models of lean asthma, in obesity, liver and adipose tissue MAIT switch to a pro-inflammatory state and contribute to metabolic dysfunction. In this application we hypothesize that, in obesity, liver and adipose tissue MAIT cells become pro-inflammatory and are licensed to migrate to the lung, where they drive severe asthma outcomes in female obese-asthmatic mice. This hypothesis will be tested in two Specific Aims. Aim 1: To define the functional and phenotypic differences of tissue resident MAIT cells in obese asthmatic females. We will examine patterns of MAIT cell accumulation and cytokine expression in lung and metabolic tissue (liver, white adipose tissue) over time, quantify differential MAIT cell responsiveness to TCR- dependent and independent signals, and perform non-biased transcriptional profiling of lung MAIT cells from lean and obese asthmatic female mice. Aim 2: To determine the role of MAIT cells in asthma outcomes in obese female mice. We will treat lean and obese asthmatic mice with a MAIT cell inhibitory ligand or adoptively transfer lung MAIT cells from lean or obese asthmatic mice and assess their necessity and sufficiency on the asthma phenotype. Completion of the proposed studies will facilitate a better understanding of the mechanisms underlying the unique clinical parameters associated with “obese asthma” and MAIT cell biology.

NIH Research Projects · FY 2026 · 2023-01

Abstract Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by dysfunctions in lysosomes, with cumulative frequency of 1 in 7000 live births. Over 2/3 of LSD patients present an involvement of the central nerve system (CNS) with a broad spectrum of severity (nLSD), which makes LSDs the most common cause of pediatric neuronopathic diseases. Allogeneic hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) are main treatment options for LSDs. However, they are largely unsuccessful in reversing neurological complications due to the poor penetration of the enzymes into the CNS, a major obstacle in treating nLSD. The impact of the proposed study is driven by the unmet medical need for efficient treatment of inherited nLSDs AND the major limitation of enzyme-delivery into the CNS. The cation-independent mannose-6-phosphate receptor (M6PR) plays a critical role in lysosomal enzyme trafficking and intercellular transfer for the majority of lysosomal enzymes, which is essential for metabolic cross- correction in treating LSDs. Developmental decline of M6PR on blood-brain-barrier (BBB) during early postnatal period in mouse and human is attributable to the lack of CNS enzyme delivery into adult brain. Using a dual luciferase reporter system with site-mutagenesis, we have recently identified microRNA-143 (miR143) as an epigenetic modulator to reduce M6PR protein levels on brain microvessels (BrMV). Using a mouse model of Hurler syndrome (severe mucopolysaccharidosis type I, MPS I), which is caused by the deficiency of α-L- iduronidase (IDUA), we demonstrated functional rescue of M6PR-mediated IDUA transfer in the brain of double- knockout (MPS/miR-143KO) mice with long-term CNS therapeutic benefits, as well as in human vascular endothelial cells by sequestration of miR-143 with miR-143-sponge sequences. The data provide strong scientific premise for the development of a novel approach that would selectively “open” BBB to systemic enzymes provided by any current treatment options or future enzyme/gene/cell therapies for synergistic CNS benefits in many nLSDs. In this proposal, we aim to develop an adeno-associated viral vector (AAV)-based translatable platform to “restore” M6PR pathway on mature BBB for advanced delivery of therapeutic enzymes into the CNS with 3 aims, including developing optimal artificial miR143 inhibitor (143in) and expression cassette(s) for robust and targeted reduction of miR143 on brain endothelia cells (Aim 1), in vivo examination of “on-target” and “off-target” expression and effects in mice with AAV/143in delivery (Aim 2), as well as preclinical evaluation of BrMV-targeted AAV/143in in correcting CNS abnormalities in MPS I mice by enzyme therapy derived from genetically modified erythroid/megakaryocytic lineages (Aim 3). The studies will provide a proof- of concept for a new in vivo miRNA-inhibitor mediated, brain-targeted approach that could be applicable for many other nLSDs involving M6PR pathway AND neurological diseases benefiting from advanced CNS delivery of therapeutics via adapting M6PR-mediated transport pathway by modification with M6P residues or IGF2-tag.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT Bronchopulmonary dysplasia (BPD) is the most frequent adverse outcome among infants born at <30 weeks gestation and is associated with adverse respiratory and neurodevelopmental outcomes. Early BPD includes a component of alveolar and interstitial edema resulting in reduced lung compliance. Some evidence suggests that the use of loop diuretics, such as furosemide, in preterm infants > 3 weeks of age improves pulmonary mechanics and oxygenation after 1 week of treatment; however, the evidence is weak because most studies were not conducted in the current treatment era characterized by widespread surfactant and antenatal corticosteroid use and data focuses mainly on short-term outcomes. Furthermore, diuretics have a range of side effects including electrolyte abnormalities, osteopenia, potential ototoxicity, and nephrocalcinosis. Despite weak evidence for their effectiveness and concerns over safety, diuretics are commonly used. There is a need to strengthen the evidence-base regarding diuretic use in BPD with data that answers the current, relevant clinical questions—whether an individual patient in the current treatment era will derive short- term benefits from the treatment and whether the treatment is safe and effective in the long-term. We designed a clinical trial that addresses prevailing clinical attitudes that some infants clinically benefit or are “responders” by including a run-in period that examines individual patient short-term response using an innovative N-of-1 trial design while also answering questions about the longer-term efficacy and safety of chronic diuretic use by following the run-in N-of-1 trial with a placebo-controlled, parallel group randomized controlled trial (RCT) of chronic diuretics among responders. The objective of this R34 application is to obtain necessary and sufficient information to enable final decisions about the approach of the larger clinical trial through the following specific aims: Aim 1: Estimate the number of responder infants available to enter the parallel group RCT phase of the trial after the N-of-1 trial run-in period and Aim 2: Evaluate provider and parent willingness to randomize responders to assess the potential dropout rate between run-in and randomization. Accomplishing these aims is particularly important because the use of a formal N-of-1 trial design as a run-in to a parallel group RCT is a unique and cutting-edge approach. Upon completion of these aims, this pilot will provide a detailed understanding of the extent to which participants are able to complete the N-of-1 trial and it will supply data on the percent of patients identified as “responders.” This will enable a more accurate assessment of the available sample size entering the parallel group RCT phase of the trial. The pilot will also provide the data necessary to determine the expected dropout rate between the run-in and parallel group RCT phases due to reluctance to randomize a “responder” to furosemide or placebo. Together this information will enable informed decisions about the larger trial sample size and the design of the N-of-1 run-in phase, and it will be of value for others planning studies of diuretics and BPD management strategies.

NIH Research Projects · FY 2026 · 2023-01

Abstract Myelodysplastic Syndromes (MDS) are heterogenous and poorly understood hematopoietic stem cell (HSC) failure syndromes common in individuals >60 years of age. With increased life expectancies, the incidence of MDS continues to rise, and will soon be the most prevalent hematologic disorder in elderly. There are no effective treatments for MDS patients, due to an insufficient understanding of the underlying pathobiology and a lack of faithful mouse models. Our research program is focused on filling these gaps, and then leveraging the resulting knowledge and tools to develop effective drug therapies. Already, we have discovered genetically-driven aberrant activation of innate immune pathways in MDS HSCs. We have also identified a critical function of innate immune pathways in normal HSCs, which has implications for chronic immune-related disorders, cardiovascular diseases, and hematopoiesis. We hypothesize that dysregulated innate immune signaling is a major contributor to the initiation and development of MDS, and is a feasible therapeutic target. Herein, we propose to test this hypothesis by carrying out the following complimentary 3-part research program: (1) Dissect the genetic and cellular underpinnings of MDS HSCs, with an emphasis on cell-intrinsic and cell-extrinsic immune-inflammatory factors. (2) Identify and characterize novel signaling pathways driving MDS phenotype in HSCs. (3) Develop novel therapeutic strategies for the treatment of MDS. The results of our research program will advance our paradigm-shifting model of the initiation, progression, and treatment of MDS.

NIH Research Projects · FY 2026 · 2023-01

Abstract Regulatory T cells (Tregs) mediate anti-inflammatory functions and have been most associated with self- immunity and autoimmune diseases. However, given the underlying role of subclinical or persistent low-level inflammation in metabolic and cardiovascular diseases, attention has recently focused on the potential role of Tregs in controlling the proinflammatory milieu in these pathologies. A subset of Tregs, the effector memory Tregs, are the most suppressive, but are also the most transient. We made the striking observation that high- density lipoproteins (HDL), but not other lipoproteins, significantly improved Treg survival. These findings offer an explanation for the positive correlation we found between HDL cholesterol and Treg abundance. Our preliminary data show that HDL preferentially bind and are internalized by Treg memory subsets, particularly by effector memory Tregs, promoting their survival by reducing effector caspase activation. Mechanistically, our new data also suggest that this HDL pro-survival effect may occur via activation of the AKT signaling pathway, which results in enhanced de novo fatty acid synthesis and reduced mitochondrial oxidative stress. Since HDL is a family of related particles with complex mixtures of lipids and proteins, we started exploring which HDL subspecies or components stimulate Treg survival. We found that the pro-survival effect was mediated by the HDL protein component, and particularly the relatively minor HDL protein constituent, apolipoprotein (APO)E, and/or other proteins that co-reside on APOE-containing HDL. In contrast, the major HDL scaffold proteins from the APOA family did not play a major role. Our overarching hypothesis is that APOE-rich HDL specifically interact with Tregs to trigger pathways that limit caspase-dependent apoptosis. Aim 1 will identify the intracellular signaling pathways involved in HDL-mediated survival of Treg, notably the receptor(s) involved in HDL binding/ internalization by memory Tregs and the mechanisms by which HDL promote Treg survival. We will also determine the effect of HDL on Treg suppression. Aim 2 will identify specific HDL subspecies and protein components that promote Treg survival. We will also perform deletional and site-directed mutagenesis experiments to identify the critical region(s) of APOE, to inform explorations of synthetic peptides that can mimic APOE’s pro-survival effects on memory Tregs. Aim 3 will assess the involvement of APOE-rich HDL in Treg survival in vivo. We will leverage the Cincinnati Pediatric Diabetes and Obesity Center cohort and probe the association between absolute numbers of Treg subsets, their functional characteristics, and HDL subspecies, before and after weight reduction surgery. The ability of HDL subspecies collected before and after surgery to promote memory and effector memory Treg survival will be studied. By identifying specific cellular pathways and specific lipoprotein species responsible for the Treg survival effects, we will open new avenues for potential therapeutic intervention aimed at modulating Treg function in a host of auto-immune and chronic metabolic diseases.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY The alveolar region of the mammalian lung is a complex, precisely structured tissue required for the primary functions of the respiratory system, gas exchange and tissue oxygenation. Damage to the alveolar epithelium plays a central role in human lung diseases including Acute Respiratory Distress Syndrome (ARDS), a prevalent, high impact clinical disorder that affects up to 5% of mechanically ventilated patients in the developed world. The mortality rate of ARDS approaches 40%, and the recovery for ARDS survivors is arduous, with a substantial burden of multi-system disability continuing 5 or more years following hospitalization. Critically, while many ARDS survivors recover lung function, a subset of patients develops persistently abnormal pulmonary function, imaging evidence of pulmonary scarring, and pulmonary symptoms even years after ARDS. To date, no data exists regarding the mechanisms that guide ARDS recovery. These challenges have been made more acute by the coronavirus pandemic, which has exposed a large proportion of the human population to acute lung injury. An enormous population of patients is at risk of both acute and chronic lung consequences of lung injury following coronavirus infection, emphasizing the clear and urgent need for new regenerative therapies to promote recovery from acute lung disease. Regeneration in many organs is driven by adult facultative progenitor cells. We recently discovered a facultative progenitor cell in the mouse and human lung which participates in regeneration after viral injury we call alveolar epithelial progenitors (AEPs). Progenitor cells control their chromatin carefully, as they must maintain more broad potential than fully differentiated cells, and so a hallmark of progenitor chromatin state is regions of active regulation between fully open and fully closed states, so called poised chromatin. Unique preliminary data from our laboratory and review of the literature support the idea that the chromatin modifying complex PRC2 is a critical regulator of the progenitor chromatin state of AEPs. In this application, using a combination of advanced lung organoids and genetic mouse injury models, we will identify the temporal and functional requirements for PRC2 function in lung progenitors, define the key binding partners and targets of the PRC2 complex in maintenance of AEP progenitor state, and evaluate the genomic loci regulated by PRC2 complex activity necessary for AEP-mediated alveolar regeneration. Understanding these fundamental mechanisms will provide the framework needed to understand alveolar regenerative biology at a granular level and develop therapeutic strategies to maintain and restore AT2 progenitor function to drive repair following infection and environmental stress.

NIH Research Projects · FY 2026 · 2022-12

Abstract Like skeletal muscle myofibers, cardiomyocytes in the heart constantly adjust their size based on perceived workload or disease stimulation, in which hypertrophic versus atrophic pathways are in balance to achieve an appropriate equilibrium matched to real-time workloads. In a less appreciated process, both heart and skeletal muscle can reduce size through molecular regulatory pathways that cause tissue catabolism. This reduction in size is referred to as atrophy and this process can underlie tissue remodeling and responses to disease stimulation or loss of sufficient nutrients (such as starvation) in which both tissues can serve as metabolic reservoirs. Here we uncovered a novel function for thrombospondin1 as a regulator of both cardiac and skeletal muscle atrophy. We have previously shown that the thrombospondin gene family (Thbs1- 5) plays a critical role in membrane stability through effects on the ER stress response and secretory pathways, as well as controlling the integrin and dystrophin-glycoprotein complexes present with the sarcolemma. However, more recently we have discovered that Thbs1 is uniquely induced by disease stimuli associated with cardiac remodeling and caloric restriction, and that Thbs1 uniquely regulates cellular atrophy and autophagy through an intracellular pathway within the ER/SR that functions at 2 levels. 1) Thbs1 directly binds and regulates the ER stress factor PERK and eIF2α to mediate cardiomyocyte atrophy through the transcription factor ATF4, and 2) Thbs1 selectively expands lysosomes and the vesicular pathway of autophagy. Hence, we hypothesize that Thbs1 is an ER-dependent chaperone that mediates cardiomyocyte size reduction, in part, by driving the catabolic process through autophagy. To investigate this hypothesis, we will interrogate 2 specific aims: 1) To examine the mechanisms of cardiac atrophy and autophagy through PERK/eIF2α/ATF4 signaling mediated by Thbs1 within the ER compartment. 2) To examine a mechanism whereby cardiac autophagy is mediated by Thbs1- dependent formation of lysosomes and associated catabolic vesicular activity. The proposed course of investigation will be conducted in both cultured cardiomyocytes and in genetically modified mouse models so that both reductionist and mechanistic approaches can be taken, as well as in vivo assessment in a physiologically relevant context. The proposed application is innovative as it will define for the first time what appears to be a novel cell biology pathway through Thbs1 that controls striated muscle remodeling through atrophy and autophagy.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT The COVID-19 pandemic led to unprecedented changes to healthcare delivery during the spring of 2020, including the rapid and wide implementation of telehealth. This expansion included areas in which telehealth had not previously been broadly applied, such as pediatric primary care. By its very nature, telehealth changes the communication and structure of the healthcare interaction, perhaps especially in pediatrics where both the parent and patient need to be engaged in the visit. Such changes may directly affect shared decision making, an essential component of high-quality care and a strategy to improve outcomes. Children with asthma and attention deficit hyperactivity disorder, the most common chronic conditions in pediatric primary care, are especially vulnerable to changes in health care delivery. These conditions are highly prevalent among urban, underserved children and while some barriers to care are alleviated by telehealth new ones may emerge. Understanding the impact of telehealth on an underserved, inner-city, primary care population is critical to prevent worsening disparities. Our long-term goal is to improve pediatric primary care by ensuring delivery of chronic disease care that meets families’ goals and improves children’s health and well-being. The overall objective of this proposal is to compare the decision-making processes and outcomes between telehealth and in-person pediatric primary care for children with chronic conditions. This proposal consists of three distinct aims that build upon one another by triangulating perceived and observed decision-making, parents’ and adolescent patients’ perceptions, and quantitative and qualitative data to achieve breadth and depth of understanding about shared decision making in pediatric primary care. In Aims 1 and 2 we will evaluate the quality of pediatric primary care delivered via telehealth compared to in-person care. Aim 1 will use rigorous survey methods to understand families’ perceptions of decision making that occurs during either a telehealth or in-person pediatric primary care visit. This will be coupled with chart review to understand the contribution of telehealth or in-person care to clinical outcomes. Aim 2 will build upon the first by video-recording healthcare visits so the extent of observed shared decision making can be assessed both quantitatively and qualitatively. Finally, in aim 3 we will use qualitative interviews to “feed forward” the data from aims 1 and 2 to parents, adolescents and healthcare providers to gain a more in-depth understanding regarding experiences of decision making in telehealth compared to in-person care. The expected outcome of this proposal is an understanding of the impact of telehealth delivery in pediatric primary care on decision making processes for children with chronic conditions. These results will have a positive impact on care delivery by facilitating the development of targeted approaches to supporting shared decision making as healthcare systems continue evolve and integrate telehealth.

NIH Research Projects · FY 2024 · 2022-09

Exocrine pancreatic insufficiency (EPI) occurs when the pancreas is unable to secrete enzymes and fluids adequate for digestion. Acute recurrent (ARP) and chronic pancreatitis (CP) are increasingly recognized in children and are an increasingly important cause of EPI. EPI results in malabsorption which can lead to malnutrition and growth failure in children. If EPI can be recognized or even predicted, its deleterious effects can be reversed with pancreatic enzyme replacement. Accepted diagnostic criteria for CP rely on subjective imaging findings which are limited by interobserver disagreement and accurate, non-invasive diagnosis of EPI is challenging, if not impossible. Currently diagnosis of EPI requires invasive testing in the form of endoscopy under anesthesia (ePFTs). Thus, there is an unmet need for validated, non-invasive measures of pancreatic health and function in children. Quantitative MRI techniques, including MR pancreatic function testing (MR-PFTs), pancreas parenchymal volume, and parenchymal signal mapping are showing promise as non-invasive markers of pancreatic health and function. The overall aim of this study is to define associations between non-invasive, non-contrast, quantitative MRI measures and established measures of pancreas health and function (including EPI diagnosed by a reference standard of endoscopic PFTs [ePFTs]) in children. We propose to test multiple non-invasive, non-contrast, quantitative MRI techniques for their ability to identify EPI and stage pancreatitis (acute pancreatitis to ARP to CP) in children. Our central hypothesis is that the quantitative MRI techniques under study will have diagnostic performance for changes in pancreatic health, allowing detection of EPI and quantification of changes of pancreatitis. Our preliminary studies show that we can successfully apply quantitative MRI techniques, inclusive of MR-PFTs, volumetric imaging, and parenchymal signal mapping in children. We have defined normal values for these MRI measures with preliminary data suggesting that threshold values derived from normal children can non-invasively identify EPI with excellent sensitivity. This application aims to: 1) Evaluate the diagnostic performance of MRI markers for EPI as an indicator of pancreas health in children, 2) Develop processing pipelines for quantitative MRI of the pancreas, and 3) Define quantitative MRI markers that can distinguish stages of pancreatitis and identify early CP. The successful completion of this study will generate unique, valuable data regarding non-invasive staging of pancreatitis and diagnosis of EPI. This study will generate protocols and processing pipelines to facilitate future clinical decision making and research use of MRI as a non-invasive test for EPI and pancreatic disease. Data from this study will set the stage for future studies of non-invasive imaging of pancreatitis and EPI in children.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY There is a fundamental gap in the availability of behavioral outcome measures that are appropriate, reliable, and valid for use with children with Down syndrome (DS). Lack of such outcome measures represents an important problem to identifying behavioral concerns, measuring treatment effects, and interpreting clinical trials aimed at improving the lives of individuals with DS. Without evidence-based behavioral outcome measures, interventions and treatment trials in this population will remain suboptimal due to poor study measures. Working groups convened by NICHD of leading experts in DS identified that no measure of behavioral concerns was evaluated as appropriate or validated for use with individuals with DS. The overall objective of this application is to develop and validate a novel measure of behavioral concerns, the Behavior Inventory for Down Syndrome (BIDS), that can be used in research, clinical practice, and treatment studies focused on children and adolescents with DS ages 2-17 years, in both English and Spanish. Our rationale for working with this population is that DS is associated with a distinct neurobehavioral phenotype, yet key behaviors of concern common in individuals with DS are omitted from currently available measures of behavioral concerns. Thus, proposed items on the BIDS will incorporate behavioral concerns raised by families and in clinical practice related to children with DS. We propose three specific aims: 1) demonstrate the psychometric properties of the BIDS among children and adolescents with DS in English and Spanish by identifying the BIDS measurement model, establishing reliability (test-retest and inter-rater reliability) and validity (internal and construct validity) 2) evaluating differences in the measure’s psychometric properties as a function of variation in selected demographic and clinical characteristics, and 3) characterize the trajectory of the BIDS over time among children and adolescents with DS to provide comparative data for clinical trials. To achieve these aims, we will conduct online, phone, and paper surveys in English and Spanish, and in-person assessments in English or with Spanish-speaking interpreters, targeting children ages 2-17 years with DS. Our research team is uniquely positioned to conduct this work, combining expertise in DS, measure development/evaluation, Latinx populations, psychological assessment, and comorbid medical conditions. We anticipate that this measurement study will provide critical guidance for future efficacy and effectiveness trials. Our goals are in line with the INCLUDE Project research priority to expand inclusion of individuals with DS in clinical trials by developing appropriate measures of behavior for this population. As our field continues to develop new pharmaceutical and clinical interventions, our project will support this work and have an impact on the 400,000 individuals with DS living in the United States.

NIH Research Projects · FY 2025 · 2022-09

Abstract The goal of this proposal is to determine how lineage-specific repressive epigenetic landscapes are established to safeguard cell identity. Current dogma is that pioneer TFs initiate lineage-specific activation processes by locally opening chromatin. In contrast, significantly less is known about how repressive epigenetic landscapes are dynamically reshaped during cell differentiation. This knowledge gap creates a major stumbling block in developing high-fidelity cell reprogramming technology. Hence, it is essential to define how alternative lineage programs are repressed and how this process can be manipulated. Intriguingly, our preliminary data unexpectedly revealed that pioneer TFs are involved in this process. We developed a new doxycycline (dox)- inducible CRISPR interference (CRISPRi) tool to target two pioneer TFs: OCT4 in human pluripotent stem cells (hPSCs) and FOXA1/A2/A3 (FOXA) in hPSC-derived endoderm differentiation. We found that OCT4 and FOXA play critical roles in repressing alternative-lineage programs. In fact, gene expression changes in OCT4/FOXA- CRISPRi cells correlate better with directional changes of the repressive H3K27me3 mark than with those of H3K4me1/3 active marks. To identify mechanisms underlying OCT4/FOXA-directed repression, we focused on the OCT4 and FOXA binding peaks that co-localize with the H3K27me3 domain. Interestingly, we found that these loci are highly enriched at key alternative-lineage genes, and unbiased motif analysis revealed significant enrichment for a PRDM14 (a key pluripotency TF) binding motif at OCT4 sites and a PRDM1 (a key germ-cell TF) binding motif at FOXA sites. This data provides the first indication that PRDM1 may play an important role in endoderm specification. Further, our ChIP-qPCR assays confirmed that OCT4-CRIPSRi hPSCs were impaired in recruiting PRDM14, PRC1/H2Aub, and PRC2 at OCT4-bound H3K27me3 sites. Intriguingly, our preliminary co-IP assay suggested that FOXA interacts with PRC1, and prior co-IP studies showed that OCT4 interacts with PRC1, and PRDM14 and PRDM1 interact with PRC25–9. Collectively, our findings led to our overarching novel hypothesis that pioneer TFs play a crucial role in establishing Polycomb repressive domains at key alternative- lineage genes by recruiting PRC1 and/or PRDM TFs, which in turn recruit PRC2. We will test this hypothesis in two Aims: Aim 1: Test the hypothesis that OCT4 and FOXA play instructive roles in recruiting PRCs and PRDM TFs to establish lineage-specific Polycomb repressive domains. Aim 2: Determine how PRDM1 impacts accurate lineage restriction during human endoderm specification. The successful completion of our Aims will provide valuable insights and reveal novel mechanisms underlying lineage-specific Polycomb repression to safeguard cell identity. In the long-term, this study will likely open new avenues to manipulate cell fate by selectively repressing alternative regulatory programs, with potential future therapeutic applications in regenerative medicine.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Abstract Rheumatic heart disease (RHD) is the most commonly acquired heart disease among children and young adults worldwide. Continuous antibiotic prophylaxis (secondary prevention) with monthly intramuscular benzathine penicillin G (BPG) is a cornerstone of RHD control programs and has been used successfully for decades in wealthy countries. Unfortunately, because of neglect in the global and national health agendas, most RHD- endemic countries (like Uganda) have no RHD programs, so most of the estimated 40 million people living with RHD worldwide remain undiagnosed and unprotected from further valvular damage. Considering these challenges, our objective is to partner with the Ugandan government to test a novel community-based program, ADUNU (Acholi for “heart”) that aims to find individuals with RHD and provide secondary prevention close to where they live. The program will be tested in two districts (one demonstration, one replication), building a foundation for national scale-up beyond the proposed research. ADUNU is based on two core, evidence-based technologies, (1) RHD testing using handheld echocardiography in community settings and (2) nurse-led, registry-based delivery of secondary prevention. ADUNU is a non-randomized experiment that will set up a new system in settings where RHD-related healthcare currently does not exist, with the goal of finding new cases and enrolling them in local registries that provide secondary prevention. ADUNU will be integrated with general primary healthcare and track outcomes over time, key factors in sustainability and scalability. In Aim 1, we will evaluate ADUNU using the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework, mapped to the RHD Care Cascade (an adaptation of the cascade used for HIV). Primary quantitative outcomes in each of RE-AIM’s five dimensions will be supplemented with qualitative data from in-depth interviews and administrative data. In Aim 2, we will evaluate the cost-effectiveness and budget impact of ADUNU in these two districts in order to justify investment and plan for scale-up of the program nationally in Uganda, beyond the proposed study. ADUNU will provide much-needed evidence to government stakeholders in RHD-endemic countries, demonstrating how a pragmatic, community-based strategy can deliver guideline-based care to people living with RHD. It will also provide critical, and currently absent, evidence on the cost-effectiveness, affordability, and sustainability of RHD programs in resource-limited settings.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT: A large percentage of children with Down Syndrome (DS) have obstructive sleep apnea (OSA) that is suboptimally treated by first-line surgical interventions. The persistence of OSA-related nightly intermittent hypoxemia and fragmented sleep may contribute to a cognitive impairment, as well as pulmonary vascular disease, myocardial dysfunction, reduced quality of life and daily functional impairment. While oxygen is sometimes used when other OSA therapies fail, its efficacy and safety have not been systematically studied. This R61/R33 is therefore designed to test the hypothesis that individuals with DS and moderate to severe OSA will have a safe and favorable response to low-flow oxygen treatment due to its effects on directly attenuating hypoxic episodes, with subsequent increased ventilatory stability and improvement in OSA. We further hypothesize that oxygen supplementation will lead to improvement in neurocognition, cardiac function, sleep, and quality of life. The R61 phase will actively engage families of patients with DS and our multi-stakeholder team to refine and pilot the protocol and recruitment strategies to ensure that we meet our recruitment/retention milestones in the R33 phase and we generate data most relevant to our patient population. In toto, we will screen approximately 328 children with DS and moderate to severe OSA, 5-17 yrs of age, who failed adenotonsillectomy, to identify oxygen responders, and then randomize 230 children to oxygen plus conservative therapy (OXT; administered using a patient-specific dose) or conservative therapy (CT) alone (weight management, sleep hygiene, nasal dilators) for 6 months. The primary outcome of the trial is working memory measured by co-primary endpoints that respectively assess caregiver-reported and objectively measured functions. Secondary outcomes include cardiac function and structure, right ventricular pressure, quality of life and sleep measures that will be collected under the supervision of experienced, central core laboratories. Six clinical sites experienced with pediatric clinical trials and established DS centers will participate in the trial. A Data Coordinating Center experienced with pediatric sleep trials with a strong history of working with these clinical sites will implement and monitor data quality control processes that addresses all stages of data handling. This study will fill a key knowledge gap in a potentially efficacious therapy for OSA, and provide evidence to support (or refute) the use of supplemental oxygen, as well as identify physiological markers to potentially identify patient subgroups most likely to experience benefit from this therapy.

NIH Research Projects · FY 2025 · 2022-09

Low-cost air sensors offer tremendous opportunities for researchers and community members to better understand air quality at neighborhood, indoor, and personal levels. Though these devices are often marketed as easy-to-use, users face multiple technical challenges including maintenance, calibration, data management, and data visualization. These issues often lead community members and researchers to form community-academic partnerships within a community-engaged research (CEnR) framework. However, successful community-academic partnerships require time and training to set expectations, identify team member roles, develop team processes and shared mental models, and design a project that balances the needs of a community with the expectations of academic researchers. In recognition of both the opportunities and challenges of using low-cost sensors in CEnR, we propose an innovative program to foster successful community-academic partnerships and equip research teams with the technical skills and knowledge to successfully utilize low-cost sensors in communities impacted by with air quality concerns. Our program, Research Innovations using Sensor Technology to Improve Community Health (RISE Communities), will accomplish three specific aims using a combination of in-person training, experiential learning, and a social learning community. We will recruit community-academic teams (5 per year) from throughout the US and provide dedicated time and training to build trust, set expectations, promote sustainability, and engage in evaluation (Aim 1). In addition, experts in the use of low-cost sensors and their application in communities impacted by air quality concerns will provide in-person courses, workshops, and hands-on training in their use. This training will equip teams with the requisite foundational knowledge in research methods employing low-cost sensors including how to select the appropriate sensor(s) for their research questions, deploy them in their own communities, and translate the data to action (Aim 2). Throughout the program we will cultivate a community of practice to facilitate continued interaction among participants and program faculty and extend the learning beyond the in- person training (Aim 3). Collectively, these aims address the urgent need in research for successful community-academic partnerships to address concerns regarding air quality. In addition, the RISE Communities program will promote the use of sensor technology to encourage data-driven action to improve public health.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Most children in foster care experience multiple forms of maltreatment, increasing risk for behavior problems (e.g., aggression, noncompliance, suspensions/expulsions from preschool or daycare). The foster care system is over-burdened and crisis-response oriented, limiting capacity to focus on prevention of behavior problems with foster caregivers and children. The Chicago Parent Program (CPP) is an evidence-based 12-session program (11 concurrent weeks, 1 one-month booster) with demonstrated significant and sustained improvements in consistent discipline, parenting self-efficacy, and child behavior problems 1 year following training. If available to foster caregivers, CPP could prevent or reduce behavior problems in foster youth, decrease caregiver stress, increase self-efficacy and consistent discipline with caregivers, and prevent unnecessary placement changes. However, adaptations to the existing program are necessary. The goal of this study is to conduct a clinical trial of CPP adapted for foster caregivers of young children. This will be accomplished through three aims: Aim 1: Adapt CPP to meet the unique needs of children ages 2-5 years who are placed with foster caregivers (CPP-FC) and pilot delivery coordinated with mandated healthcare visits (2 groups, 6-8 families). CPP content will be adapted (e.g., additional content targeting trauma and child maltreatment, modified discussion of vignettes, additional role-play targeting specific foster care scenarios). A stakeholder adaptation team, including 6 caregivers, has been established to guide development of enhanced content. Aim 2: Assess the impact of CPP-FC on caregiver stress & confidence in managing child behavior. Families (N = 300) will be randomized to CPP-FC or usual care, with assessments at baseline, 3 months (end of training) and 6 months. CPP-FC is expected to reduce caregiver stress and increase confidence. Aim 3: Assess the impact of CPP-FC on child behavior. Observations of structured interactions between caregivers and children at baseline and 3-months will be collected along with caregiver reports of child behaviors at baseline, 3 months, and 6 months. CPP-FC is expected to decrease child behavior problems and result in fewer placement changes. If hypotheses are confirmed, this study will provide an evidence-based model to prevent behavior problems in young foster children that could be spread to other institutions.

NIH Research Projects · FY 2025 · 2022-09

Abstract Bloodstream infections (BSI) caused by bacteria translocating across injured mucosa are a significant cause of morbidity and mortality in the 25,000 patients undergoing stem cell transplantation (SCT) in the United States each year. BSI secondary to translocation of oral organisms through damaged oral mucosaoccurs in nearly 16% of SCT recipients in the first three weeks after SCT despite current routine oral care. There are currently no known strategies to prevent post-SCT BSI from the translocation of bacteria through an injured mucosa. There is a critical need to identify preventive strategies to reduce BSI, and the oral mucosa as a specific route of entry has not been evaluated. In the absence of such information, SCT patients will continue to develop BSI from translocation of bacteria from mucosal barrier injury. Our long-term goal is to develop and disseminate clinically relevant and easily adoptable strategies to prevent BSI and improve outcomes after SCT. The overall objective of this proposal is to identify a clinically effective strategy to prevent or reduce BSI secondary to bacterial translocation through injured mucosa in the mouth. The rationale for the study is based on our baseline and pilot intervention data. The central hypothesis is that dental xylitol use, in addition to current oral care practice, are effective at reducing BSI from oral organisms, and decreasing the incidence of gingivitis, oral plaque, and oral ulcerations after SCT. Apart from our preliminary data, we are well-positioned to carry out the proposed work as we have a multicenter collaborative team consisting of pediatric bone marrow transplant physicians, dentists, and infectious disease specialists with focus and expertise in microbiome analyses. The following specific aims are proposed: AIM 1: Determine the effectiveness of twice-daily xylitol-wipe application on reducing BSI from oral organisms through a randomized, multicenter, double-blind, placebo-controlled study in pediatric SCT recipients. AIM 2: Determine the effectiveness of twice-daily xylitol-wipe application in reducing dental plaque, mucositis, oral GVHD, and gingival inflammation. AIM 3: Evaluate the influence of twice-daily xylitol-wipe application on oral microbiome diversity and saliva levels of pathogenic microbial species. The innovation of this proposal lies in the simplicity and affordability of the intervention. Xylitol is commercially available, inexpensive ($0.15 per application), non-toxic, and can be rapidly adopted into practice. If our hypotheses are proven, xylitol application could reduce the morbidity and mortality associated with BSI and decrease healthcare-associated costs of an estimated $40,000 per infection. At the successful completion of the proposed research, our expectation is to have demonstrated a significant reduction of bacteremia from oral organisms (Aim 1); decreased oral dental plaque, gingivitis, and oral ulceration (Aim 2); and preserved oral microbiome diversity and reduction of pathogenic microbial species in the oral cavity in those receiving daily dental xylitol wipe application (Aim 3) leading to substantial changes to the prevention of BSI in this population.

NIH Research Projects · FY 2025 · 2022-09

Combination antiretroviral therapy (cART) has led to dramatic increases in lifespan among HIV-infected individuals. Despite effective cART, however, HIV-associated morbidities exert a significant toll. HIV- associated neurocognitive disorders (HAND) occur in up to 50% of chronically infected individuals despite cART. The pathogenesis of HAND remains under investigation. Neuroinflammation is a hallmark of HAND, as established by clinical studies, autopsy studies, and animal models. Ongoing or intermittent replication of HIV in the CNS is likely to contribute to neuroinflammation through direct effects on the infected cells or through release of viral proteins and inflammatory mediators. Use of neural stimulants including methamphetamine can exacerbate the neurocognitive decline seen in HAND, but the mechanisms underlying this comorbidity are not understood. Microglia are the primary resident myeloid cells of the brain, are infected at early times following acute infection with HIV or SIV, can act as a CNS viral reservoir, and are thought to play a central role in the development of HAND. The pathways responsible for microglial activation and dysfunction following HIV infection remain incompletely defined. Microglia derived from induced pluripotent stem cells (iPSCs) provide a unique opportunity to examine the molecular mechanisms underlying microglial activation. iPSC-derived microglia will be introduced into cerebral organoids, providing the additional opportunity to define the effects of microglial activation on surrounding astrocytes, neurons, and other cells. Tetherin is a host restriction factor that captures HIV during the assembly process in infected cells and generates a proinflammatory signaling cascade within infected cells. Experiments in Aim 1 of this project will evaluate HIV-induced neuroinflammation both in an unbiased way and through a directed evaluation of the role of tetherin-mediated signaling as a trigger of microglial inflammation. RNAseq, cytokine production, and immunofluorescence microscopy will be employed to define microglial activation following HIV infection. In Aim 2, we will introduce HIV-infected microglia into cerebral organoids to define the molecular basis of HIV-induced neuroinflammation and neuronal dysfunction. Single-cell RNAseq and evaluation of neuronal health and electrophysiology will be performed in models representing acute infection and in ART-suppressed, chronic infection of the brain. The potential of methamphetamine to contribute to neuroinflammation and neuronal damage in the HIV-infected microglia/organoid model will then be defined, and the relevant pathways identified. Together, these studies will provide insights into the pathogenesis of HAND and the potential contribution of methamphetamine to neurocognitive decline.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract The primary objective of this R01 application is to evaluate the potential additive effect of animal-assisted intervention (AAI) on a manualized behavioral treatment targeting emotion dysregulation (ED) in children with autism spectrum disorder (ASD). ED in youth with ASD has been linked to significantly higher rates of psychiatric hospitalizations, suicidal ideation, use of psychotropic medications, school disciplinary actions, peer rejection, failed college and employment transitions, and poorer quality of life. Our group developed Regulating Together (RT), an intensive group-based treatment with a concurrent parent group that employs evidence-based intervention techniques of cognitive behavioral therapy, parent training, and mindfulness and acceptance-based therapy. Preliminary evidence for its feasibility and efficacy has been demonstrated, including reductions in emotional reactivity and dysphoria and increases in heart rate variability and cognitive flexibility at 10-weeks following intervention. Despite this, change did not occur immediately post intervention. Animal Assisted Interventions (AAI) have emerged as a promising approach for youth with ASD, demonstrating improvements in irritability, hyperactivity, positive affect, and quality of life. AAI also has shown to increase in-session engagement and learning. Yet, AAI has not yet been utilized to systematically target ED in ASD nor has it been examined in conjunction with a manualized intervention. In the current study, we propose a multi-site randomized clinical trial of RT with and without a canine present to evaluate its immediate and longer-term efficacy. In 240 youth (ages 8-12 years) with ASD, we will assess RT with AAI (RT-Canine) and without AAI (RT-Standard) on primary ED symptom of reactivity, and secondary outcomes, including dysphoria, caregiver stress, cognitive flexibility, mindfulness awareness, and emotion regulation skills knowledge. We hypothesize that the canines will increase therapeutic engagement measured through alliance, motivation, and improved behavior, which will then increase intervention session level learning, and ultimately immediate and long-term intervention outcomes. Furthermore, based on our preliminary evidence, we also will explore an objective outcomes measure of heart rate/heart rate variability and its relationship to engagement, learning, and ED symptom outcomes. This proposal is relevant to the NICHD Human-Animal Interaction funding opportunity by addressing the critical public health issue of ASD through examination of AAI efficacy by utilizing standardized data collection outcome methods. In addition, we closely address protection for both the canines and the youth with ASD throughout the proposal. Finally, we will make a concerted effort to include youth from minority groups, as emphasized in the FOA. Ultimately, findings from our study have the potential to expand both ASD and AAI research, with immense impact on the well-being and outcomes of youth with ASD as evidence-based interventions for ED in this population is a critical gap in mental health care.

NIH Research Projects · FY 2025 · 2022-09

Project Summary_Abstract The purpose of this K23 is to provide the training, mentorship, and research experiences needed for the applicant to become a successful independent clinical scientist with a research program focused on understanding mechanisms linking sensory anomalies, brain dysmaturation, and speech and language impairments in neurodevelopmental disorders. The training plan focuses on developing skills required to measure and understand the behavioral, auditory, and neurophysiological indices of early speech and hearing development in Fragile X Syndrome (FXS), with three specific areas of emphasis: 1) statistical methods and signal processing techniques required for cutting edge analysis of functional neurophysiology (fNIRS/EEG), 2) development of the auditory system and speech/hearing in infancy and 3) research methods and ethics for studying infants and very young children with FXS. Dr. Craig Erickson along with co-mentors Dr. John Sweeney and Dr. Lisa Hunter will provide the mentorship, training, and resources necessary to achieve the training objectives. This research program is relevant to several objectives listed in the NIH Research Plan on FXS and Associated Disorders, including but not limited to objectives 3.1 (Develop a standard battery of functional, objective measures to better characterize the emergence of the FXS phenotype across the life span and provide precise indicators of treatment effectiveness) and 3.4 (Conduct longitudinal studies of both humans and animal models to characterize the dynamic nature of the FXS phenotype across the life span and to identify moderators and mediators of the phenotype). FXS can be diagnosed in the infant and toddler years given its genetic/heritable etiology. However, current understanding of atypical maturation of brain function in FXS and its clinical manifestations is entirely based on studies of older children (5+), adolescents, and adults, at which point impairments associated with the disorder, including delays in speech and language, have been present for several years. Thus, understanding of neural mechanisms and timing of the early brain dysmaturation that lead to early delays in FXS remains limited, which in turn limits development of interventions. The proposed career development plan provides the PI with the skills to address this gap in brain-based markers of impairment in early FXS. The research plan uses EEG/fNIRS, auditory evaluations, and speech and language assessment to investigate auditory hypersensitivity and its relation to emergence of speech and language delays in FXS. This study occurs in two phases, with the first focusing on preschoolers (2-4 years) and the second, longitudinal phase focused on the infant years (0-2 years).Thus, the aim of this early career development program is to prepare the investigator to establish an independent research program focused on determining the timing and nature of brain dysmaturation that leads to early speech and communication impairment in FXS.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Premature infants have low nephron number (endowment) and are at high risk for chronic kidney disease (CKD) and end stage renal disease as adults. Most nephrons are added late in gestation through a poorly understood process called lateral branch nephrogenesis (LBN). As direct study of late gestation human kidney development is difficult, the non-human primate model (rhesus macaque) was recently identified as a suitable model to bridge this knowledge gap. The long-term goal is to apply the molecular findings of LBN in the non-human primate model towards development of therapeutic methods aimed at extending nephrogenesis in preterm infants. The central hypothesis is that a shift in the signaling milieu involving components of the developing kidney (nephron progenitor cells (NPC), ureteric bud (UB), and stroma) results in sustaining LBN over multiple weeks in late gestation. The rationale for this proposed research is that the genetically tractable non-human primate model system can be used to test hypotheses and apply therapeutic interventions aimed at improving human nephron endowment. The central hypothesis will be tested by using the latest molecular technologies to understand the molecular mechanism sustaining LBN in the non-human primate, including single-cell RNA sequencing, laser capture microdissection with RNA sequencing, single-nucleus RNA sequencing, and single-nucleus ATAC sequencing. Preliminary morphologic study on the postnatal day two marmoset kidney identified a single ureteric stalk with lateral branches consistent with LBN, suggesting the common marmoset could be used as a genetically tractable primate model. Expected outcomes include assembly of the largest primate late gestation developing kidney molecular dataset and identification of genes and pathways enriched and regulatory networks active during LBN, as well as identification of a genetically tractable model to study LBN to understand why prematurity leads to early cessation of nephrogenesis in humans, and how to extend it. These results are expected to have a positive impact on the current understanding of late gestation human nephrogenesis by identifying molecular pathways and potential therapeutic interventions for those born prematurely. In addition to the aims outlined in this proposal, career development plans include didactic training in molecular biology and bioinformatics through the Certificate program in Bioinformatics, wet-lab experience with the latest molecular technologies and multi- omic platforms, and career advancement through a selected advisory committee for transition to independence and submission of R01 during the K08 award period.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of genetic disorders characterized by ineffective erythropoiesis, hemolysis, and bi- or multi-nucleated erythroblasts in the bone marrow. The mechanisms of erythroid dysfunction in CDAs are incompletely understood; studying the molecular defects in these diseases can reveal critical pathways in terminal erythropoiesis. We have identified mutations in VPS4A as a novel cause of CDA in three unrelated patients with a syndrome of dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay, pointing to the importance of Vacuolar Protein Sorting 4A (VPS4A) for terminal erythropoiesis and normal red blood cell survival. However, the mechanisms of its action need further investigation. VPS4A is an ATPase that has been shown in yeast and in vitro cell cultures to participate with the Endosomal Sorting Complex Required for Transport (ESCRT)-III in endosomal vesicle trafficking, viral budding, and the abscission step of cytokinesis. It is required for budding of endosomal vesicles into multivesicular bodies, a critical step in the pathway for the sorting, recycling, and removal of transmembrane protein receptors. We have shown for the first time a human disease in patients with dominant negative mutations in the ATPase domain of VPS4A, resulting in dyserythropoiesis with erythroblasts connected by cytoplasmic bridges consistent with cytokinesis failure at the abscission step and reticulocytes with evidence of altered trafficking of the transferrin receptor (TfR1/CD71). We hypothesize that VPS4A is essential for terminal erythropoiesis and that loss of function results in dyserythropoiesis through a combination of cytokinesis failure and endolysosomal defects. The goal of this work is to define the molecular mechanisms by which these VPS4A variants disturb erythropoiesis, and to elucidate the roles of VPS4A and the ESCRT machinery in human and murine terminal erythropoiesis. We aim to model the erythroid defects due to VPS4 in vivo using a transgenic mouse model with erythroid-targeted expression of a known dominant-negative VPS4 mutant (VPS4EQ), enabling studies on the pathogenic mechanisms and natural history of the disease (Aim 1). Using human iPSCs, we will investigate the contributions of cytokinesis failure and iron trafficking defects to the dyserythropoiesis observed in these individuals using normal, patient-derived, and VPS4AEQ CRISPR gene-edited iPSCs (Aim 2). These studies will demonstrate the role of VPS4A and ESCRT-III machinery as an essential molecular pathway for erythropoiesis.

NIH Research Projects · FY 2025 · 2022-09

Pulmonary arterial hypertension (PAH) is a rare, fatal condition characterized by the gradual occlusion of the pulmonary arterioles leading to progressively increased pulmonary vascular resistance with worsening right heart failure and death. While rare mutations (e.g., in BMPR2) have been reported in a minority of patients, most patients carry no established mutations. We recently published on common genetic variation in 2,085 idiopathic/heritable (I/H) PAH cases and 9,659 controls of European ancestry using a genome-wide association (GWA) approach. Discovery and replication analyses were conducted in four independent cohorts with genotyping arrays from our US-based PAH Biobank (PAHB) study and three international cohorts with whole genome sequence data. We reported two novel loci, at HLA-DPA1/DPB1 and near SOX17 associated I/H PAH. HLA-DPA/DPB1 locus predicts a reduced annual mortality rate by 25-37% in I/H PAH. The lead SOX17 variant is located in a putative enhancer region in close spatial proximity to the SOX17 gene in endothelial cell (EC) precursors, which influences its expression based on our experimental validation. Our findings provide the first support for the contribution of common genetic variance to PAH risk and, combined with the recently reported data on rare mutations in SOX17 in PAH, highlight the causal role of SOX17 in PAH. Beyond PAH risk, we now hypothesize that PAH progression and outcomes are also genetically modified including from SOX17, a transcription factor, and HLA-DPA1/DPB1 as both novel candidate genes and possible therapeutic targets. To test this hypothesis, we have developed 3 specific aims (SAs) that will further expand the PAHB, the world’s largest PAH biobank, registry, and multi-omics dataset with whole exome sequencing (WES), RNAseq, whole genome genotyping, and non-targeted metabolomics data on nearly all subjects. SA #1 will collect serial longitudinal data in PAHB to interrogate associations between disease risk loci (SOX17, HLA-DPA/B1) with markers of PAH progression. We will also evaluate expression (eQTL) and metabolomics (mQTL) quantitative trait loci analyses for functional validation. Beyond disease risk SOX17/HLA loci, we generated additional preliminary data revealing genome-wide significance for seven novel genetic loci associated directly with survival in PAH in a second, independent PAH cohort. SA #2 will now replicate these new findings with outcomes (progression and survival) collected in PAHB from SA #1. As a unique feature of this proposal, we will interrogate our top loci in two other global PAH cohorts with available eQTL and mQTL data and perform a meta-analysis of all cohorts. We will also construct a risk stratification tool combining clinical risk factors and genetics (SOX17, HLA, 7 SNPs) for PAH outcomes. Finally, based on preliminary data on the protective role of SOX17 in EC function, SA #3 will validate the biological role of SOX17 pathway in the development of PAH using ECs/SMCs isolated from PAH patients as well as pre-clinical testing in murine PH models. Strong clinical association of these two new loci has implications for prediction of clinical outcomes, clinical trial design, and the development of novel drug targets.

NIH Research Projects · FY 2025 · 2022-08

R21 R33 Summary/Abstract Hearing loss is a major and growing public health concern, especially in low and middle income countries (LMICs) where about 80% of the world’s population with hearing loss reside. Our vision is to provide good quality, affordable, and sustainable hearing health care to underserved populations in LMICs and around the world. We propose to validate and then trial an innovative mHealth model for end-to-end hearing care facilitated by community health workers (CHWs). The smartphone-based solution uses hearing aids to first test hearing and then, if needed, programs the devices to provide gold standard rehabilitation. In the course of fifteen minutes, the user and their family should be able to experience a step change in their communicative ability. Following the fitting session, support will continue through innovative mHealth messaging support for their new hearing aids and communication with the CHW for troubleshooting. In the R21 phase of this funding, we will address three aims that validate key steps on the journey to better hearing for all. For Aim 1, we will perform clinical work in Cincinnati, Ohio and community work with CHWs in low-income South African communities to confirm that the hearing aids deliver hearing test precision to the highest standard. This audiometry will be part of a more comprehensive diagnostic package involving novel physical (smartphone video otoscopy) and behavioral (speech-in-noise) data. In Aim 2, we will evaluate the performance of the hearing aids to gold-standard and conventional clinical fitting standards. In a community setting, we will also determine usability by including CHWs and users. Training and supporting CHWs is critical for our approach and in Aim 3 we will work with our colleagues in the hearX Foundation to develop new training modules. Multiple techniques will be used to engage CHWs in each stage of the process, for example, adjusting existing modules found to be useful in previous hearing screening outreach. Freshly hired CHWs will test-drive and evaluate the training. Another part of this aim is to begin developing the mHealth user support system (apps) to be used in the clinical trial phase (Aims 4 and 5). Infographics and voice bytes will be translated to the local language (e.g. Xhosa) in an approach, using WhatsApp, we have shown to improve knowledge about hearing loss of preschool teachers. In the R33 phase of funding, the focus narrows to two clinical trials. In the first (Aim 4), we ask whether the smartphone-based, CHW-facilitated hearing aids successfully deliver benefit for their users. In the second trial (Aim 5) we ask whether an mHealth user support system, developed in Aim 3, is effective in delivering benefit. Both studies are randomized control trials, the highest level of clinical scrutiny. In Aim 6, we will model data from Aims 1-5 using AI towards a holistic hearing assessment. Positive, self-report outcomes and modeling should pave the way for an expansion to the next level, regional and national trials that, if successful, will be of huge personal importance for millions of individuals in LMICs. They will also open the gate to international adoption of this innovative, mHealth driven, community-based model of hearing care.

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract Candidate: I am a postdoctoral fellow in the lab of Dr. Ross Levine in the Human Oncology & Pathogenesis Program at Memorial Sloan Kettering Cancer Center (MSKCC). My PhD studies allowed me to hone the technical and experimental skills required for interrogating clinically tractable molecular dependencies in cancer cells. My current research focuses on the generation of models of acute myeloid leukemia (AML) evolution to be used in the discovery of novel molecular dependencies and potential therapeutic targets. To that end, I have broadened my capabilities with CRISPR editing to generate an inducible AML model that allows for temporal control of mutagenesis and optimized a single cell DNA sequencing technique to evaluate the clonal framework of AML. My proposed research will build upon these initial studies to develop a new suite of sequential mutagenesis models of AML. These models developed in the K99 phase of this grant will serve as tools for the discovery of essential proteins/pathways for leukemic cells. My long-term career goal is to lead an independent research group focused on the identification and characterization of molecular dependencies of AML using precise models of disease evolution through sequential mutagenesis. To accomplish these goals, I have outlined a career plan that will 1) expand of my technical skills and scientific capacity, 2) improve my scientific communications with the field, 3) advance my supervisory and leadership abilities, 4) develop and foster collaborative relationships and 5) prepare me for the transition to independence. Project: Molecular profiling studies of AML patients infer a progressive acquisition of mutations that drives leukemogenesis, but are unable to delineate the dominant clonal framework leading to disease or identify the precise mutational order for certain genes, such as NPM1. Current models of AML are unable to truly recapitulate the step-wise mutagenesis observed in patients. Our single cell sequencing studies have further resolved the clonal structure of AML at single cell resolution and with these studies, I aim to generate new models that accurately depict the sequential mutagenesis of AML evolution. The specific aims are: 1) examine mechanisms of co-mutational clonal dominance and mutation order in AML patients using single cell profiling, 2) determine the impact of mutational acquisition on disease development and progression of NPM1-mutant AML, and 3) elucidate molecular dependencies of disease derived from mutant NPM1 and co-occurring mutations. Environment: The Levine lab is part of the MSKCC Molecular Cancer Medicine Service, Human Oncology & Pathogenesis Program (HOPP), for which Dr. Levine is Chief. The Levine lab is a core member of the Center for Hematologic Malignancies and the Center for Epigenetics Research, directed by Dr. Abdel-Wahab and Dr. Kristian Helin, respectively. These affiliations at MSKCC, a state-of-the-art institution, provide a rich set of collaborative, technical and scientific resources to perform the research and career development proposed here.

NIH Research Projects · FY 2025 · 2022-08

Project Summary/Abstract Many pediatric and young adult patients require an allogeneic hematopoietic cell transplant (HCT) for treatment of deadly diseases besides cancer. Non-malignant disorders which are often treated with allogeneic HCT include severe inborn errors of immunity, inborn errors of metabolism, marrow failure disorders, and hematologic conditions such as thalassemia and sickle cell disease. Reduced intensity conditioning (RIC) and reduced toxicity conditioning (RTC) regimens are commonly used for patients with non-malignant disorders. RIC and RTC regimens usually contain alemtuzumab, a humanized monoclonal antibody that is directed against CD52. CD52 is expressed by the majority of lymphocytes and some other white blood cells. Alemtuzumab is included in RIC and RTC regimens for 2 main reasons. Alemtuzumab prevents graft rejection by depleting the recipient of lymphocytes including T cells which may recognize the allogeneic graft as foreign. Alemtuzumab also reduces graft versus host disease because alemtuzumab may linger at lytic levels through the administration of the hematopoietic stem cell graft and result in lymphocyte depletion of the graft. Adequate prevention of graft failure and graft versus host disease is essential to ensure successful outcomes and patient survival. We do not know the best way to dose alemtuzumab. We have previously reported that optimal peri- transplant alemtuzumab concentrations of 0.2-0.6mcg/mL on the day of graft administration (Day 0) reduce the risks of graft failure and graft versus host disease. Levels within this range also optimize early immune recovery. It is important to be able to dose alemtuzumab so that the majority of patients achieve Day 0 concentrations within this ideal target concentration window. We have performed detailed alemtuzumab pharmacokinetic (PK) studies and developed a population PK model to allow a Precision Dosing strategy to be developed. We applied this Precision Dosing strategy in a pilot feasibility study of 12 patients with good results. We are requesting funding in this current application to support a larger phase II study of Precision Alemtuzumab Dosing in pediatric and young adult patients with non-malignant disorders. We will evaluate the success of our approach in targeting patients to the ideal therapeutic concentration window of 0.2-0.6mcg/mL on Day 0 and the impact on the clinical outcomes of immune reconstitution, graft failure, and graft versus host disease.