Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Asthma is one of the most common non-communicable illness among children in the United States. This disease is comprised of multiple pathobiological subgroups, also known as endotypes, which have distinct pathologies and differential responses to treatment. Clinical evidence has identified an endotype of asthma comprised of individuals who are frequently (≥2 times per 12 months) hospitalized for an asthma exacerbation. While there have been previous studies examining the clinical characteristics of the frequent exacerbator (FE) endotype, the heterogeneity of the populations studied makes findings difficult to generalize to pediatric populations. Additionally, there have been no studies examining the underlying biological differences which may be responsible for recurrent exacerbations. To answer these questions, we have leveraged the Ohio Pediatric Asthma Repository (OPAR), the first statewide repository of patients admitted for an asthma exacerbation at any of the 6 major pediatric medical centers. In addition to clinical and epidemiologic data, biosamples (nasal epithelial cells, blood, etc) are stored in our laboratory at Cincinnati Children’s Hospital, allowing us a unique opportunity to explore the biologic underpinnings of the FE endotype. In our first study, we performed RNA- sequencing (RNA-seq) of upper airway epithelial cells and found: (a) FEs are a biologically distinct endotype of pediatric asthma; (b) the transcriptome of FEs is characterized by an increase in expression of gene modules enriched for nervous system processes; (c) non-FEs (nFEs) show an increase in gene modules enriched for allergic immunity as has been described previously in asthma; and (d) increased expression of modules enriched for nervous system processes in individuals with >2 annual exacerbations, regardless of whether it was the 2nd or the 7th. We also have preliminary data showing differences in methylation patterns between FEs and nFEs indicative of epigenetic changes coupled with our transcriptomic results. Together, our findings and existing literature suggest a durable molecular shift in the airways which predisposes an individual for frequent asthma exacerbations. Therefore, we hypothesize the unique transcriptional divergence exhibited in the airways of FEs is a durable change characterized by cell-specific epigenetic and gene expression changes. We propose to test this hypothesis by: (i) collecting samples from FEs during an exacerbation and a period of normal lung function to assess gene expression durability (Aim 1), and (iii) generate single cell RNA-seq and ATAC- seq datasets to determine cell-specific patterns of expression and gene regulatory networks associated with frequent asthma exacerbations (Aim 2). Our studies will further define the molecular etiology of this important pediatric asthma endotype and provide putative targets for therapeutic intervention.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Immigrants experience significant health disparities which are exacerbated by a heavy stress burden which in turn affects the epigenetic profiles of the immune system leading to chronic diseases. Cumulative stress burden for immigrant’s ranges from immigration-related stressors to unique psychosocial, environmental, and everyday challenges, all of which contribute to negative psychological and biological impacts on their health over the life- course. Although social and environmental conditions have been established as key factors driving disparities in health outcomes, the effects of stress and epigenetic change among immigrants remains poorly understood, impeding the development of novel and robust intervention approaches aimed at reducing health disparities. Epigenetic changes can act as surrogate markers for stress effect. However, very few studies have examined epigenetic marks associated with stress among African immigrants. Ethiopians form one of the largest groups of African immigrants in the US, yet this is the first study of this kind among Ethiopian American immigrants (EAIs). The objective of this proposal is to quantify cumulative stress burden and determine DNA methylation associated with stress among EAIs. We hypothesize that the DNA methylation profile in EAIs is associated with cumulative stress. To address cultural differences and experiences and for a wider translation to the community, our bilingual and bicultural multidisciplinary research team will work with relevant scientific partners connected to and/or part of EAI communities in Cincinnati and Columbus OH to achieve two Specific Aims: Aim 1) Measure cumulative stress burden and identify and define stress burden profiles among EAIs. Aim 2) Determine the association between cumulative stress and DNA methylation. Our proposed study, involving a comprehensive set of cumulative stress measures and genome- wide DNA methylation among an immigrant population, is the first study among Ethiopian American immigrants. The study will provide new insights to address health disparities among growing immigrant populations in the US informing novel and robust intervention approaches to reduce chronic illness and associated sequelae for vulnerable populations. Our long-term goal is to better understand how changes in psychological, social, environmental, and acculturative stressors affect the risk for chronic diseases through epigenomic mechanisms.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT/SUMMARY Leveraging the technologies and infrastructure in our previous LungMAP phases, we will construct a multi-scale map of developing acinar and alveolar structures during normal childhood and in the setting of childhood interstitial lung disease (chILD) characterized by aberrant lung morphogenesis. Linked to gene variants, chILD provides a unique opportunity to understand key genetic pathways and cellular interactions of human acinar and alveolar development, in turn informing injury-repair associated with lung diseases throughout the lifespan. Working in a collaborative LungMAP3 consortium, including other Research Centers, the Data Coordinating Center (DCC), the Human Tissue Core (HTC), our LungMAP3 project will use single-cell and spatial multiomics, high-resolution confocal and electron microscopy, patient-derived iPSCs, and bioinformatics including AI tools to define, model, and predict normal and diseased lung development. The resulting mechanistic insights and enabling technologies will be disseminated to the entire lung community via disease atlases and web portals, as we have done during LungMAP1 and 2, to accelerate lung biology discoveries and disease therapies.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Children with Autism Spectrum Disorders (ASD) present with numerous deficits in organization, planning, prioritizing, memory, and materials management (OPM). Yet OPMs are critical to successful academic performance. There is a particular demand for these OPM skills as children transition to the middle school environment which is associated with numerous challenges including increased expectations for achievement and behavior, copious homework assignments, increased demands on organization and planning/time management, learning that moves from rote tasks to abstract conceptual learning, etc. Not surprisingly, given their OPM problems and social challenges, children with ASD evidence high levels of academic problems in middle school. In fact, during middle schools years, the academic performance of youth with ASD is on average 5 years below their typical peers. Yet there are no evidence-based interventions for middle school youth with ASD. Using an iterative and collaborative design process, we developed an intervention, Achieving Independence & Mastery in School (AIMS), for middle-school youth with ASD without intellectual disability (ID). AIMS has been tailored to address the unique learning styles of youth with ASD. Preliminary evidence suggests that AIMS improves OPM and that improvements in OPM are associated with improved academic functioning. As a next step, an adequately powered randomized clinical trial comparing AIMS to an active comparison control (ACC) is needed to confirm that AIMS engages OPM and that change in OPM is associated with improvement in academic functioning. Fully included middle school youth with ASD (n=224) will be randomized to AIMS or ACC. At baseline, post, and two critical school year follow-ups, we will comprehensively assess OPM and academic outcomes. The aims are to examine the efficacy of AIMS on 1) improving OPM and 2) academic functioning. We hypothesize that the AIMS group will have higher OPM scores and improved academic functioning compared to the ACC group at post-intervention (primary endpoint) and follow-up. The third aim is to assess whether changes in OPM mediate changes in academic functioning. We hypothesize that the relation between AIMS and academic functioning improvement is mediated by OPM changes. We will also explore maintenance of OPM gains, determine the optimal time to deliver AIMS, and identify moderators of treatment success. AIMS has the potential to impact a large number of youths with ASD since over 50% of the population are fully included in the general education classroom. The lifetime cost for an individual with ASD is estimated to be $1.4 million/patient with the largest costs for special education and parental productivity loss. Thus, NICHD ASD research priorities include developing novel treatments delivered during pivotal transitions (e.g., middle school) to improve outcomes.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Maternal infections during pregnancy lead to adverse outcomes for the offspring, including short-term outcomes such as intrauterine fetal demise, preterm delivery and neonatal sepsis, and long-term sequelae such as neurodevelopmental impairment. A key factor contributing to fetal/neonatal susceptibility to infection is the failure to robustly produce innate immune cells such as neutrophils that form a first line of defense against infection. We recently made the novel observation that fetal hematopoietic stem and progenitor cells (HSPCs) are extrinsically restricted from activating the pathways that would lead to rapid production of neutrophils, termed emergency myelopoiesis (EM), by maternal interleukin-10 (IL-10), although how maternal IL-10 restricts fetal EM is unknown. Altered placental function is proposed to be the critical central mediator linking the effects of maternal inflammation on adverse outcomes in the offspring. In particular, maternal inflammation increases placental synthesis of serotonin that is released into the fetal circulation, reaching the fetal brain and disrupting the growth of serotonergic axons. We have found that fetal HSPCs express serotonin receptors and their proliferation is suppressed in vitro by serotonin, supporting the hypothesis that placental serotonin may also regulate fetal hematopoiesis. This proposal will test the hypothesis that maternal IL-10 restricts fetal emergency myelopoiesis via a multi-layered cascade of events involving a network of maternal decidual immune cells that trigger placental serotonin synthesis which is then released into the fetal circulation and reaches the fetal liver where it regulates fetal HSPCs in either a direct manner or indirectly by regulating the stromal and mature hematopoietic cells of the fetal liver niche. Our approach will be to 1) identify the IL-10-responsive cell in the maternal-fetal milieu and determine the function of that cell that results in restriction of fetal emergency myelopoiesis, and 2) determine how maternal inflammation is sensed in the fetal liver microenvironment and whether this signal, which we predict to be serotonin, regulates fetal HSPC function directly or indirectly. We predict that maternal IL-10 restricts fetal EM via its baseline function of establishing a tolerogenic profile in maternal decidual macrophages and that in its absence, the exaggerated inflammatory response of alternatively-primed macrophages negatively impacts placental function resulting in increased synthesis of serotonin that regulates fetal HSPCs directly and indirectly. This project will systematically identify the mechanisms by which maternal inflammation is translated across the placenta and sensed in the fetal liver to modulate fetal HSPC emergency myelopoiesis, uncovering potential nodes along the cascade that might be amenable to therapeutic targeting, and ultimately boosting neonatal immune function and improving neonatal outcomes.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT: Infections caused by double stranded DNA viruses are a common complication after allogeneic hematopoietic stem cell transplantation (HSCT), occurring in 82% of patients undergoing transplant at our institution. These infections are a significant source of both morbidity and mortality in the post-HSCT setting. Commercially available anti-viral medications have inadequate response rates, prolong hospitalizations, and have narrow therapeutic indexes with high toxicity rates. Additionally, there are no effective anti-viral medications for two of the more common viruses, BK polyomavirus (BKPyV) and adenovirus. An alternative approach for viral management is the use of virus-specific T-cells (VST); this cellular therapy approach uses peripheral blood from healthy donors to generate highly expanded and viral directed T-cell populations given as a simple intravenous infusion. This therapy has shown itself across multiple clinical trials to be safe and highly effective for the treatment of viremia and invasive viral disease caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), BKPyV, and adenovirus. The generalizability of this therapy has increased through use of partially-HLA matched, ‘off-the-shelf’ third-party VSTs where a product from a VST bank is chosen for a patient based off of anti-viral activity of the product and the degree of human leukocyte antigen (HLA) matching. However, while response rates are excellent (on the order of 70-90% depending on the virus), treatment failures occur even when a good and rationally chosen product is given. In this proposal we aim to improve understanding of the mechanisms underlying treatment success and failure following VST infusion. We hypothesize that both patient- and virus- specific factors facilitate the effective response to third-party VSTs and that treatment non-response is due to definable and non-mutually exclusive defects in one or both of these areas. We will use pre- and post-infusion samples collected from patients enrolled on an active third-party VST treatment study to complete these aims. From a recipient perspective, we believe that failure of VST persistence, inadequate antigen presentation, and failure of initial T-cell expansion can be detected in patients with poor response. From a viral perspective, we will use epitope mapping and NGS sequencing of viral genome to explore the role of both non-conservation of antigenic epitopes and antigen escape. We believe that these experiments will generate generalizable data on mechanisms behind VST treatment with potential implications for improving the therapy moving forward.

NIH Research Projects · FY 2025 · 2024-08

Project Summary / Abstract Chronic lung disease in children with systemic juvenile idiopathic arthritis (SJIA-LD) is a life-threatening disorder which is increasing in incidence and for which there are no proven effective treatments. SJIA-LD manifests as interstitial lung disease (ILD) with varying degrees of pulmonary alveolar proteinosis (PAP), fibrosis, and pulmonary artery hypertension, and frequently progresses to hypoxic respiratory failure. Although research is urgently needed to define optimal treatments for SJIA-LD, key knowledge gaps remain that are barriers to future research: a case definition, prospective evaluation of clinical disease progression, surrogate biomarkers of disease activity, and LD-specific patient-reported outcomes (PROs). Our objectives are to define SJIA-LD, its clinical disease progression, and surrogate biomarkers, to accelerate future research in this disease. We have developed and published preliminary outcome measures, identified serum inflammatory mediators and markers of lung injury in SJIA-LD patients, and piloted surveys to identify SJIA-LD specific symptoms that can be measured using validated PROs. To operationalize these preliminary findings, we have launched a pilot multicenter prospective cohort study of SJIA-LD, which to date has enrolled 45 patients through the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry. Our central hypothesis is that this proposed SJIA-LD study will result in the ability to accurately define SJIA-LD cases and characterize disease trajectories, and validate surrogate biomarkers of clinical disease progression and LD-specific PROs. In this study, we will enroll ~80 SJIA-LD patients across the CARRA network, and prospectively collect clinical data, biosamples and PROs over 2 years of follow-up. In Aim 1, we will determine the clinical SJIA-LD characteristics and progression by collecting LD features at baseline and longitudinally, and use this to support a data-derived expert consensus definition of SJIA-LD. In Aim 2, we will validate biomarkers of inflammation and lung injury in children with SJIA-LD by determining longitudinal (baseline, 6-months, and end-of-study) levels of cytokines, chemokines and lung injury markers, their responsiveness to change, and correlating them with clinical disease activity. In Aim 3, we will utilize PRO measures that reflect quality of life and lung disease symptoms in children with SJIA-LD by tracking changes in existing measures captured through the CARRA Registry, as well as in previously-validated lung disease-specific instruments which measure patient- reported SJIA-LD symptoms. We will leverage the CARRA Registry and infrastructure to longitudinally assess clinical disease features, surrogate biomarkers of disease activity, lung damage and PROs in a prospective cohort of children with SJIA-LD. Successful completion of the proposed aims are necessary to accelerate future clinical research, including interventional studies, and advance our long-term goals to treat and prevent SJIA- LD.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Neonates can now survive premature birth from as early as 22 weeks’ gestation, but often suffer from bron- chopulmonary dysplasia (BPD) – chronic lung disease of prematurity. BPD consists of several phenotypes – hyperdense lung consisting of fibrotic tissue or inflammation, hyperinflated airspaces, and airway collapse at various levels from the upper airway to the distal airways. However, even if a patient’s phenotypes are identified, there is no mechanism to calculate the relative contributions of the phenotypes to disease severity and symp- toms. Therefore, the goal of this proposal is to create and validate a tool to calculate the contribution of each BPD phenotype. Such a tool would allow patient-specific treatment based on an individual’s disease phenotypes. To address this clinical need, this proposal will create a tool to calculate the contribution of each phenotype of BPD to elevated breathing effort. Breathing effort, known as the work of breathing (WOB), is comprised of elastic work, which is used to expand the lungs and chest wall, and resistive work, which is used to move air through the airways. Resistive work can be further broken down into where in the airways the resistance occurs, e.g., upper airway, trachea, etc. Each component of WOB is related to a specific phenotype of BPD: hyperdense lung tissue will result in increased elastic WOB; hyperinflated airspaces indicate increased resistive WOB in the distal airways; obstruction or collapse in the bronchi, trachea, or upper airway will result in increased resistive WOB in each specified region. Specific Aim 1 of this proposal will validate each of these relationships between compo- nents of WOB and phenotypes of BPD and Specific Aims 2 and 3 will assess how they change in response to two common treatments for BPD – the bronchodilator albuterol and steroids, respectively. This proposal is innovative in its use of a novel, neonate-specific magnetic resonance imaging (MRI) scanner, which can be sited within a neonatal intensive care unit, novel MRI reconstruction techniques to assess lung parenchymal health, and computational fluid dynamics (CFD) simulations to assess respiratory airflow and re- sistance throughout the airway tract. We anticipate this study will result in more personalized diagnosis of premature neonates’ respiratory disease and more precise use of drugs used to treat them. Neither albuterol or steroids are effective in all neonates and may cause harm in some patients; for example, albuterol can exacerbate tracheal collapse by relaxing the pos- terior tracheal membrane, while steroids pose neurodevelopmental risks. This study will identify which patients will benefit from these treatments. In the long-term, the tools created for this study will be translated to other respiratory diseases with multiple levels of airway obstruction and lung issues, in which the main cause of res- piratory distress is challenging to identify, and may be used to titrate respiratory support, demonstrating the significance of this proposal to older pediatric and adult medicine, in addition to the target neonatal population.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMRAY Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus partially mediated by epithelial barrier breach, resulting in harmless antigens penetrating the underlying tissues, encountering immune cells, and triggering allergic responses. The surge in allergy prevalence is often attributed to shifts in environmental conditions and western life style. The molecular mechanisms involved in how the environment elicits allergic diseases is an area of active research that is yet to be fully understood. The aryl hydrocarbon receptor (AHR) is an intracellular receptor responsive to various external stimuli, including dietary compounds, air toxicants, pharmacologic agents, microbiota metabolites and tryptophan metabolites. Our research has uncovered that AHR acts as a sensor molecule, capable of either maintaining epithelial homeostasis and barrier integrity, or initiating an innate immune response when exposed to diverse AHR ligands from different environmental sources. As a result, AHR can play a dual role in EoE, both protecting against and exacerbating EoE. We have shown that select AHR ligands induced protective transcriptional responses in esophageal epithelial cells. These responses counteract pathways involved in the development of type 2 immunity, including restoring barrier function and the deficiency of SPINK7, a crucial endogenous protease inhibitor, normally essential for averting EoE. These anti-inflammatory reactions rely on the activation of the transcription factor Ovo Like Zinc (OVOL1). Conversely, environmental factors such as enriched tryptophan diets can stimulate AHR, exacerbating esophageal inflammation in mice. In this study we aim to elucidate the mechanism through which AHR mediates its protective and detrimental reactions in response to divergent AHR ligands. We have established experimental platforms including the generation of AHR and OVOL1 deficient esophageal epithelial cell lines, mice with conditional depletion of Ahr and Ovol1 in the esophageal epithelium, experimental EoE murine models and three-dimensional human organotypic esophageal raft cultures to test the hypothesis that AHR drives differential esophageal epithelial responses, and the nature of the AHR ligands dictates the AHR dependent protective (barrier function) and detrimental (innate inflammation) outcomes. Successful completion of this study holds immediate translational significance, as it could pave the way for dietary adjustments, probiotics, and newly-approved AHR agonists which will offer potential avenues for interventions against EoE.

NIH Research Projects · FY 2026 · 2024-08

Abstract: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the world’s most common life-threatening genetic diseases. Mutations in the genes PKD1 and PKD2 account for 85% and 15% of all ADPKD cases, respectively. ADPKD patients are heterozygous for either PKD1 or PKD2 deleterious mutations; homozygous mutations are considered incompatible with life. Interestingly, loss-of-heterozygosity studies and genome sequencing of patients’ cysts, have documented the clonal nature of the epithelial cysts in ADPKD. Altogether, these data support the two-hit model in which one mutation in either PKD1 or PKD2 is inherited, but a second (somatic) mutation is required for the clonal expansion of the epithelial cells and progression of cystic disease. While these data strongly support the two-hit model, there is scarce data reporting if, when, and how, a second mutation in an individual renal cell affects the behavior of that cell and its environment in vivo. We have generated a series of mouse lines that recapitulates the two-hit model. In an otherwise Pkd2 heterozygous animal, we can induce a second mutation during embryonic development but also at later timepoints. Our data confirms that single mutant cells are responsible for the generation of cysts and that all cells in the cyst arise from the originally mutated cell. Most importantly, our data reveals that not all mutant cells become cysts. We hypothesize that cyst inception is modulated by additional intrinsic and extrinsic factors affecting mutant cells. Specifically, we hypothesize that cyst inception and/or growth is driven by tubular identity, external sources of proliferation and the microenvironment created by the chimeric nature of the disease. We will test these hypotheses in three aims employing mouse models as well as kidney explants and iPSC-derived kidney organoids.

NIH Research Projects · FY 2025 · 2024-08

Most mental health (MH) disorders develop in early childhood but are not clinically identified or treated until later—delaying treatment services that could prevent the enduring effects of long-term MH problems. Moreover, children from low-income families and those with demographic differences exhibit higher rates of persistent MH disorders and are at greater risk for lags in identification and treatment creating a mental health gap in services. Head Start has shown early school success for low-income children, aged 3-5 years. Head Start monitors for early childhood MH symptoms, yet studies have found that when detected, only those most impaired are referred for treatment. In our research, we identified multiple barriers that preclude early treatment among Head Start preschoolers with developmental concern. We showed that caregivers encountered system barriers of Head Start teachers and primary care providers (PCPs) falling behind in referrals for intervention, and caregiver beliefs about stigma, their limited knowledge and distrust of healthcare hindered early engagement in services. Studies on MH treatment obstacles for low-income people, and demographic differences illustrate similar barriers to those found among Head Start preschoolers with developmental concern. Our team developed and tested a peer-based family navigator program for Head Start preschoolers with developmental concern. Navigators used trust and empowerment to increase caregiver advocacy thereby leading to improved professional alliances and treatment. A navigator program for those with primary MH concern has not been trialed. In this R34 pilot effectiveness trial, we propose to take this next critical step by tailoring and preliminary testing in a case series the Navigate-Train-Referral-Intervention Mental Health (NTRI-MH) intervention to promote access, engagement, coordination, and optimization of services for preschoolers with MH symptoms. In Aim 1, we will use focus group feedback from caregiver, navigator, Head Start teacher, and PCP stakeholders (n=30) to adapt NTRI-MH and create a web-based dashboard to monitor outcomes. Then, conduct a feasibility study for caregivers of Head Start preschoolers with MH symptoms, guided by family navigators and referrals by Head Start teachers and PCPs (n=20). In Aim 2, we will pilot test NTRI-MH for caregivers of preschoolers with MH symptoms compared to an active control group of caregivers who receive child behavior training (n=86). We will trial the effectiveness of the NTRI-MH mechanisms of caregiver beliefs on MH, empowerment, and professional alliances on family functioning and child emotion regulation. If the aims of the project are achieved, this study would have a large impact on early MH service use for low-income and those demographic differences with the potential to improve child MH outcomes.

NIH Research Projects · FY 2025 · 2024-08

Intestinal epithelial cells reside at the direct interface between the microbiota and mammalian host, and are thus uniquely poised to sense microbial signals that calibrate intestinal immunity and function. Tuft cells are a specialized epithelial cell that have recently been shown to respond to microbial stimuli and initiate and amplify type 2 immune responses in the intestine. However, despite a clear relationship between the microbiota and intestinal health, the mechanisms underlying how the microbiota instruct tuft cell homeostasis and function remain poorly understood. Epigenetics represent a central mechanism that can potentially link microbial triggers in the pathogenesis of intestinal disease. Consistent with this concept, we previously identified that epithelial loss of an epigenetic-modifying enzyme disrupted microbiota-sensitive intestinal homeostasis and increased susceptibility to intestinal damage. Our new preliminary data suggest that regulation of tuft cell development and homeostasis may be epigenetically regulated and dynamically controlled by distinct metabolites produced by the intestinal microbiota. Based on these findings, we hypothesize that (1) tuft cell responses in the intestine may be calibrated by distinct components of the microbiota through an epigenetic sensor and that (2) epigenetic mechanisms in stem cells may regulate tuft cell development and function. To investigate these hypotheses, we will (i) interrogate how distinct commensal bacterial-derived metabolites instruct intestinal stem cell differentiation to tuft cells through an epigenetic-modifying enzyme, and (ii) directly identify and investigate new mechanisms by which the stem cell epigenetic landscape can be modulated to control tuft cell differentiation in the intestine. This work will uncover novel epigenetically-regulated pathways that direct tuft cell biology in the intestine, and therefore guide personalized approaches for treating intestinal diseases.

NIH Research Projects · FY 2025 · 2024-08

A major focus of our laboratory is to broadly understand the molecular mechanisms by which cell wall- anchored (CWA) proteins on the surface of Staphylococcus epidermidis and S. aureus promote biofilm formation and virulence. Staphylococcal biofilms are highly adhesive and cohesive communities of surface- adherent bacteria that are highly resistant to antibiotic action and host immune responses, often resulting in recalcitrant infections. Specifically, the research will focus on the large, multi-domain CWA protein Aap from S. epidermidis, its ortholog SasG from S. aureus, and another large S. aureus CWA protein called SasC; each of these is known to mediate homophilic interactions that promote intercellular adhesion. Mechanisms of both reversible self-assembly and nucleation of functional amyloid fibrils will be studied in order to identify avenues for therapeutic intervention to prevent biofilm formation. In addition, the small, secreted S. epidermidis protein SBP and its ortholog from S. aureus will be investigated in terms of their ability to interact with Aap (and SasG) to facilitate assembly and potentially trigger liquid-liquid phase separation of macromolecular components in the biofilm matrix. Finally, the S. aureus protein SasX, implicated in the spread of a recent epidemic of methicillin-resistant S. aureus, is also cell wall-anchored but unlike the other CWA proteins is a small, intrinsically disordered protein. SasX will be assessed for its ability to interact with other staphylococcal surface proteins as well as keratinocyte ligands, given its demonstrated role in promoting biofilm formation as well as facilitating adhesion to epithelial cells and nasal colonization. In addition to structural and biophysical studies of these proteins, quantitative analysis of biofilm morphology and mechanical properties using confocal microscopy, rheometry, and force spectroscopy will provide complementary insights in a biological context. Over the next five years, our goal is to provide a structural and functional view of how the most important surface proteins in S. aureus and S. epidermidis physically hold the cells together in biofilms and to understand how those protein-protein interactions mechanically strengthen the biofilm. By understanding these interactions at the molecular level, our goal is to identify sites of vulnerability that will allow the design of therapeutic approaches to target the ability of these bacteria to form biofilms, rendering them susceptible to a broader range of antimicrobial agents.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Myelodysplastic Syndromes (MDS) are blood disorders caused by defective hematopoietic stem cells (HSC) that clonally expand and fail to produce blood cells of sufficient quality and quantity. Germline heterozygous mutations in DDX41, an essential RNA Helicase, are the most common cause of inherited predisposition to MDS. These mutations are typically frameshifts and cause loss of full-length protein. Patients with these mutations have normal health into adulthood but have an elevated risk of developing MDS with a median age of 69 years. The cellular and molecular mechanisms by which these mutations contribute to MDS pathogenesis remain poorly defined. The most common co-mutation in these patients is acquired missense mutation of the other DDX41 allele, often causing the amino acid substitution R525H. We developed two conditional mutant Ddx41 alleles to model the germline loss-of-expression mutation and the acquired R525H mutation in mice. We found that mice with hematopoietic-specific, heterozygous loss of Ddx41 live normal length lives and have predominantly normal hematopoiesis with only a modest reduction in red blood cell number. In contrast, the combination of one loss- of-expression allele and the R525H-mutant allele causes profound cell cycle arrest and apoptosis in proliferative hematopoietic progenitor cells. This observation calls into question why the R525H mutation arises and how the mutant clones that acquire it can expand to contribute to disease. In patients, the acquired mutation has a median variant allele frequency of just 10%, indicating it occurs in a non-dominant clone, consistent with reduced proliferative capacity. In this project, we plan to develop models of DDX41-mutated MDS that account for the cellular and temporal context of mutation acquisition, including DDX41-heterozygosity in hematopoietic and non- hematopoietic cells during natural aging and the acquisition of the R525H mutation in only a subset of HSC at an advanced age. To do this, we will age large cohorts of constitutive Ddx41+/- mice and Ddx41+/flox;Vav-Cre (hematopoietic-specific) mice to at least 24 months of age. We will also cross these models with a genetic model of accelerated aging to drive more rapid disease development. Finally, we will induce expression of the R525H mutation in a subset of HSC in the context of Ddx41+/- bone marrow to mimic the mixed clonality of MDS patient bone marrow. We hypothesize that these genetic models, which accurately follow the natural progression of the disease in vivo, will yield MDS-like disease states that can be further studied to elucidate the cellular and molecular mechanisms of MDS pathogenesis. The ultimate goal of these studies is to fully understand the cause of MDS predisposition in patients with DDX41 mutations such that we can rationally design strategies to prevent or treat the disease.

NIH Research Projects · FY 2024 · 2024-07

This project will include the development and evaluation of IMAAP, an Interactive Mobile Asthma Action Plan to support asthma self-management and adherence to ‘as needed’ rescue medications, which are a critical component of preventing exacerbations. An innovative tool that presents a patients’ physician-prescribed Asthma Action Plan recommendations in a way that all youth can understand is needed to improve the quality of asthma care. IMAAP will be an innovative, easy-to-use technology that leverages objective assessment of lung function (mobile spirometry), quantitative symptom report, and an individualized AAP to provide personalized, real-time treatment recommendation decision support to empower adolescents to improve their health. With this project, IMAAP will first be iteratively developed through discovery interviews and feedback from adolescents, their caregivers, and health care providers. Discovery interviews with health care providers, specifically, will be used to develop an innovative algorithm that will map asthma symptom and lung function presentations to patients’ individualized Asthma Action Plans. Prototype testing will be completed to ensure the safety of patients using IMAAP and to expeditiously obtain user feedback from patients and caregivers in a variety of situations prior to going live. Finally, a Phase IIb pilot randomized controlled trial will be conducted to compare a sample of 20 adolescents with moderate to severe persistent asthma (ages 12-18) using IMAAP to 20 adolescents in a treatment as usual group. The primary outcome for this trial will be feasibility of IMAAP. Feasibility will be assessed by comparing rates of participation, engagement, enrollment, retention, and reach to a priori criteria. User acceptability and usability will also be assessed as secondary outcomes, as well as electronic monitored rescue medication adherence and health outcomes. Results will lead to further refinements and an adapted version of IMAAP ready for full-scale efficacy testing in a future trial.

NIH Research Projects · FY 2024 · 2024-07

PROJECT SUMMARY Over 3.4 million patients in the U.S. have epilepsy, with no preventative treatments and approximately one- third of patients being refractory to drug treatment. This makes elucidating the basic mechanisms of epilepsy development a pressing priority so that more effective therapeutics can be developed. Abnormal excitatory neurons are a common pathological feature of childhood epilepsies, including tuberous sclerosis complex and focal cortical dysplasia, but it is unclear whether, and which, other cells are responsible for regulating these neurons. This proposal will address this deficit by evaluating the role of a potentially critical subpopulation of interneurons. The Danzer lab has developed a mouse model of epilepsy in which Pten, an mTOR pathway inhibitor, is deleted from a subset of hippocampal granule cells (DGCs) to introduce abnormal (DGC Pten knockout (KO)) DGCs into an otherwise normal brain. Epilepsy development in these mice provides evidence of granule cell involvement in epileptogenesis. Although not directly targeted in this model, animals develop impaired inhibition and interneuron loss. Findings suggest that epilepsy may develop via accumulation of aberrant DGCs followed by disruption of inhibitory restraint of those abnormal cells. This proposal will test the hypothesis that somatostatin-expressing (SST) interneurons provide critical inhibitory control over Pten KO DGCs. A key prediction of this hypothesis is that SST interneuron disruption will dramatically exacerbate epilepsy severity in these animals. The objective of this research is to evaluate the significance of hippocampal SST interneurons in the Pten KO model and determine whether ablation of these sprouted SST neurons induces an increase in disease severity. The hypothesis underlying this proposal is that SST interneurons restrain hyperexcitable granule cells in the Pten KO model and that loss of these sprouted SST interneurons will amplify epilepsy severity. To reveal SST interneurons, DGC Pten KO mice that express a fluorescent reporter in those interneurons have been generated. A viral strategy will be used to induce expression of a silencing archaerhodopsin in hippocampal SST neurons. Aim 1 will establish whether SST input to Pten KO cells is enhanced using morphological and patch clamp electrophysiology approaches. Aim 2 will functionally assess the importance of SST neuron-mediated inhibition by determining whether SST interneuron ablation in Pten KO mice exacerbates their epilepsy phenotype. To evaluate the impact of the loss of SST interneurons, SST interneurons will be removed either alone or in combination with Pten KO in DGCs to determine whether interneuron ablation and Pten KO in DGCs will result in more severe epilepsy than either insult alone. Virally-induced expression of diphtheria toxin receptor in SST interneurons will enable their ablation after treatment with diphtheria toxin. These studies will reveal how somatostatin interneurons are altered in the setting of epilepsy and the role they play in restraining mTOR hyperexcitable granule cells.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT: Chronic musculoskeletal (MSK) pain is leading contributor to disability. Many conditions that cause MSK pain have a higher prevalence in females. Females often report heightened sensitivity and lower pain tolerance in conditions where their male counterparts do not. Ischemia with reperfusion (I/R) injury is a common cause of MSK pain and exercise intolerance (through altered exercise pressor reflexes (EPRs)). Repeated I/R injury is often seen in complex regional pain syndrome and fibromyalgia, both of which have higher prevalence and prolonged pain in females compared to males. Clinical and preclinical research have linked sex hormones to pain. Prolactin (PRL) and the Prolactin receptor (Prlr) play a prominent role in inflammatory pain in females through dorsal root ganglia (DRGs) neuron sensitization. However, it is not yet known what roles PRL and Prlr play in ischemic myalgia. Pilot data demonstrated that prolonged behavioral hypersensitivity in females with repeated I/R correlates with upregulation in STAT3, TRPV1 and IL1R1 mRNA in the DRGs, while males show different transcriptional mechanisms. This may be due to sex-specific activation of AU-rich element RNA binding protein 1 (AUF1) in female DRGs. AUF1 regulates I/R-related behaviors and IL1r1 and TRPV1 expression and is known to be modulated by STAT3. Finally, Prlr antagonists not only inhibited female pain-like behaviors after I/R, but they also blocked the upregulation of STAT3, IL1r1, TRPV1 and AUF1. Therefore, we will test the central hypothesis that prolactin receptor modulates prolonged hypersensitivity following repeated ischemic injury in a sex- dependent manner. Transgenic and pharmacological approaches will be used to determine effects of Prlr on nociception and cardiovascular reflexes through specific neuronal subpopulations following repeated ischemic injury in males and females. Pain-related behavioral assays, EPR tests, ex vivo forepaw muscle/median and ulnar nerves/DRG/spinal cord single unit electrophysiological recording preparations, and molecular analyses (realtime PCR and RNA-Seq) of DRGs will be conducted. Aim 1 will determine the neuronal subtypes containing Prlr that modulate hypersensitivity after repeated I/R injury through knockout (KO) of Prlr in TRPV1+ sensory neurons. Aim 2 will determine the transcriptional mechanisms within sensory neurons that modulate prolonged hypersensitivity following repeated I/R injury and test the working hypothesis that neuronal (TRPV1+ vs all sensory neurons) STAT3, downstream of Prlr, regulates pain-like behaviors and EPRs after repeated I/R injury in females through activation of AUF1. This research will be the first to assess the sex-dependent role of Prlr in MSK pain and will provide some of the first evidence to support development of sex-based, treatment strategies for ischemic myalgia.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Correction of genetic muscle diseases, such as the muscular dystrophies, is a unique problem due to the distribution of muscle throughout the body in inaccessible locations. Current clinical trials for Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, involve gene therapy strategies that utilize AAV serotypes to deliver micro-Dystrophin (µDys). While AAV gene therapies are promising, use of lentiviruses that have some desirable characteristics are not considered for skeletal muscle gene therapy. AAVs contain protein capsids that mediate entry into cells, in contrast to lentiviruses that have a membrane envelope derived from budding off from host cells. Enveloped viruses utilize membrane fusion to enter cells, which is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We tested if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We used a pseudotyping platform, a general process where envelope proteins are altered to change the tropism of the virus, to engineer Myomaker and Myomerger on the membrane of lentiviruses. We found that these muscle fusogenic lentiviruses leads to specific transduction of skeletal muscle and that locally and systemically injected virions can deliver micro- Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. In addition to lentiviruses, extracellular vesicles are another type of membrane vehicle being considered for gene therapies. Extracellular vesicles are a heterogenous group of membrane particles released from most cell types and can contain factors important for homing and entry. We have also uncovered a system where Myomaker and Myomerger are present on extracellular vesicles and these vehicles exhibit delivery of material to muscle cells. We will explore these novel membrane vehicles that harness the intrinsic properties of myogenic membranes. Specifically, we propose to: 1) identify an optimal dosing strategy for lentiviruses pseudotyped with Myomaker and Myomerger 2) characterize these novel lentiviruses and molecularly dissect their fusion mechanism with muscle cells 3) develop extracellular vesicles engineered with Myomaker and Myomerger that target skeletal muscle. Successful completion of these studies will provide unique insight into these novel vectors specific for skeletal muscle, which have the potential to complement limitations of current viral-based gene therapies.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT. Patients with juvenile-onset recurrent respiratory papillomatosis (JoRRP) suffer from recurring epithelial papillomas along the respiratory tract caused by low-risk H PV viruses, predominantly H PV6. This most common benign neoplasm in children varies significantly in clinical course. Patients undergo an average of 4-5 surgeries in the first year post-diagnosis, and severe cases can result in hundreds of surgeries and progression to cancer. The lack of effective medications significantly lowers the quality of life for children with RRP, and highlights the need for new biomarkers and therapies. To develop therapeutics and prognostic markers requires overcoming a deep gap in knowledge of viral and host cell factors that produce papillomas (ie, candidate targets and markers). This is now possible thanks to the development of models of normal and JoRRP 3D stratified squamous epithelium (SE). Using fresh tumor and matched nondiseased (ND) tissue from children with JoRRP, we generated primary monolayer keratinocytes, which we then engineered into organotypic epithelial rafts. With this established pipeline of internally controlled 2D/3D models, we have made significant strides. Our global transcriptome analyses of 2D JoRRP/ND keratinocytes defined a JoRRP-specific EMT-like disease signature, which we validated in rafts and patient specimens. Single-cell RNA sequencing (scRNAseq) of HPV6+ RRP specimens (vs matched HPV- ND controls) further uncovered 16 transcriptionally distinct keratinocyte subpopulations (clusters C0-15). CS bears striking similarity to our recently published cancerassociated "HI ODEN" subpopulation induced by high-risk HPV16. Both are located in superficial layers where H PV amplification occurs, and both share biomarkers including the ELF3 transcription factor, which is required for HIDDEN formation. Our new data show that the HPV16 E6/E7 oncogenes are sufficient to induce HI ODEN, suggesting that conserved HPV6 E6/E7 activities might induce analogous subpopulations at the epithelial surface. In Aim1, we test whether and how HPV6 E6 and/or E? induce CS, and whether CS is required for viral progeny production. Our scRNAseq data also revealed striking abundance of HPV6 E5γ and δ transcripts across clusters, indicating importance for viral processes and/or keratinocyte phenotypes. While functions of low-risk HPV E5γ/δ proteins are unstudied in translational disease models, HPV6 E5γ was reported to bind the EGFR and ERBB2 receptor tyrosine kinases. In Aim2, we test whether HPV6 E5γ and/or E5δ sustain JoRRP phenotypes, and whether they do so, at least in part, via EGFR/ERBB2 signaling.

NIH Research Projects · FY 2024 · 2024-07

Project Summary Ambulatory children with cerebral palsy (CP) can have profound impairments of the lower extremities including physical deformities, loss of motor function, and resultant poor quality of life. Orthopedic multi-level surgery (MLS) is standard of care to address lower extremity impairments and children experience a decrease in function requiring extensive physical therapy (PT) for up to two years to regain function. Evidence for post-surgical PT best practices is vastly unknown. This application proposes a pilot project to obtain preliminary data examining the variation in PT frequency and type of intervention delivered during usual care across settings to ambulatory children with CP following MLS, and describe the relationship between type of intervention and recovery of gait at 6 months. Participants will be recruited from two large academic centers where MLS surgery is performed. Sixteen ambulatory children with CP that undergo MLS, consisting of at least one bony and any number of soft tissue procedures, will be recruited and evaluated at baseline before surgery and 6 months post-surgery. Using a published path model for studying dose in CP, comprehensive details about therapy frequency and type will be captured via our successful EHR implemented flowsheet for children treated at the two academic sites, and via REDCap surveys, with identical fields to the flowsheet, from outpatient community therapists. This study will capture details on all types of PT intervention delivered with specific interest in the five types delivered to children with CP following MLS, with the most recent evidence (in children that have not undergone surgery), to improve gait. These five types include mobility training, treadmill training, partial body weight supported treadmill training, fitness training and ankle foot orthoses (AFOs). We will count the total number of PT Interventions to improve Gait (PTIG) delivered and describe the relationship between PITGs to the recovery of gait at six months post-surgery. We hypothesize that children seen by academic therapists for post-surgical PT will have greater frequency of therapy sessions and higher count of PTIGS than children seen by community outpatient therapists. We also hypothesize that the count of PTIGs delivered will have a positive relationship with gait measured in two ways; self- selected walking speed and walking performance (steps/day). The proposed research is innovative and will guide development of precision rehabilitation approaches that are translatable to clinical practice. The proposed research is significant because findings will inform future work to determine the most effective strategies to improve health and motor outcomes for children with CP after MLS.

NIH Research Projects · FY 2025 · 2024-07

This project will investigate the neural mechanisms and clinical antecedents underlying listening difficulty (LiD) in school-age children born very preterm using a prospective cohort design. The long-term goal is to enhance auditory and language outcomes for at-risk children. The central hypothesis is that extended high-frequency (EHF) hearing loss stemming from exposures like ototoxic medications disrupts the integration of spatial and talker cues during competing speech processing, thereby being a mechanism underlying LiD in preterm children. The project will leverage an existing cohort of over 300 very preterm infants with prospectively collected perinatal and neonatal clinical data, including audiological, behavioral, and neuroimaging results. Antecedent factors of LiD and EHF hearing loss will be determined by testing associations between neonatal clinical variables (e.g., ototoxic medication exposure, abnormal MRI at term, social risk factors) and later emergence of LiD and EHF hearing loss. Identifying early clinical antecedents will allow targeted monitoring of high-risk infants and guide neuroprotective management in the NICU to mitigate exposures leading to impairments. A subset of 115 preterm children and 35 controls aged 6-8 years will undergo questionnaires, audiological testing (including EHF thresholds), and neuropsychological assessments. EHF hearing loss will be defined as >20 dB HL from 10-16 kHz. Listening difficulty will be quantified using the Evaluation of Children’s Listening and Processing Skills (ECLiPS) caregiver questionnaire. A subset of 35 preterm children and 35 controls will also undergo magnetoencephalography (MEG) during a competing speech paradigm manipulating talker and spatial cues. Neural tracking of the speech envelope will be evaluated using inter-event phase coherence. The specific aims are: 1) Evaluate neural mechanisms underlying LiD in preterm children and 2) Determine clinical antecedents of LiD and EHF hearing loss in preterm children. The hypotheses are: 1a) EHF hearing loss will be associated with greater listening difficulty, 1b) EHF hearing loss will disrupt the integration of spatial and talker cues as evidenced by reduced synergistic effects on neural speech tracking, 2a) Antecedents of LiD will include abnormal MRI at term and high social risk status, and 2b) Antecedents of EHF hearing loss will include ototoxic medication exposure and otitis media history. Linear regression models will evaluate associations between EHF hearing loss and ECLiPS scores. Mixed effects models will test for interactions between EHF hearing loss, spatial cues, and talker cues on neural tracking. Clinical antecedents of EHF hearing loss and LiD will also be determined using regression models. By clarifying the neural mechanisms and clinical antecedents of LiD in preterm children, this project will lay the groundwork for developing targeted therapeutic interventions and early identification of high-risk children.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Attention Deficit Hyperactivity Disorder (ADHD), the most common childhood behavioral diagnosis, is a heterogeneous disorder linked to poor adult outcomes. The challenge of ADHD treatment and the limitations of current approaches are exemplified in the results of the most comprehensive, NIH-funded study (Multimodal Treatment of ADHD) in which, at 8-year follow up, individuals in all four treatment arms showed the same high rates of psychiatric hospitalizations, traffic citations, illicit drug use, and arrests. These results call for a need to explore innovative therapeutic option such as circuit-based noninvasive neuromodulation. Here we propose to use accelerated (i.e., >1 session/day) intermittent theta-burst transcranial magnetic stimulation (iTBS) to target the pre-supplementary motor area (pre-SMA), a brain region affected in ADHD and involved in the abnormal response inhibition observed in this disorder. The proposed target engagement will focus on neurophysiologic and behavioral measures. The rationale of the proposal is based on preliminary data showing that transcranial magnetic stimulation (TMS) and electroencephalography (EEG) inhibitory network biomarkers can be modulated by pre-SMA iTBS. We hypothesize that pre-SMA iTBS can modulate inhibitory network biomarkers (R61 phase) and behavioral response inhibition (R33) in ADHD. In the R61 phase, we will deliver sham- controlled accelerated pre-SMA iTBS (2 iTBS sessions per day) to 40 ADHD adolescents (12-17 years old) to detect whether TMS-quantified (cortical silent period, short-interval intracortical inhibition; Aim 1) and EEG- based (alpha and beta-band power; Aim 2) inhibitory biomarkers can be modulated. For Aim 3 (R33 phase), we propose to recruit 50 ADHD adolescents and deliver 5 consecutive days of sham vs active accelerated pre- SMA iTBS (total 10 iTBS sessions) and quantify iTBS effects on stop-signal reaction time. Completion of this project will provide the basis for designing a large sample clinical trial for the treatment of ADHD. Furthermore, this study will provide crucial tolerability and safety data for accelerated iTBS in the pediatric population and promote repetitive TMS research in other pediatric neuropsychiatric disorders.

NIH Research Projects · FY 2026 · 2024-07

Translational approaches to unravel organ-specific microvascular endothelial responses in sepsis. PROGRAM SUMMARY: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to an infection and is associated with high mortality. Systemic inflammation and endothelial activation with resultant microvascular leak, thromboses, and hypoxic tissue injury are hallmarks of sepsis. Yet, in over 100 trials, drugs aimed at modulating cascades of inflammation and coagulation have not proven to be efficacious. The primary reason for this failure is attributed to clinical and biological variability among critically ill patients. More recently, high-throughput approaches including gene-expression profiling have shown promise in disentangling patient- level heterogeneity in the host immune response. In contrast, sampling challenges coupled with cell and organ level heterogeneity have impeded a similar understanding of the host endothelial response. It follows that translational approaches that shed light on human microvascular pathobiology may lead to the discovery of targeted therapies that restore tissue homeostasis and shift sepsis care paradigms toward organ recovery. My research program seeks to address key knowledge gaps that currently impede scientific progress through projects spanning 3 domains. 1) DISCOVERY: The endothelium in patients remains inaccessible. Moreover, while endothelial heterogeneity and organ-specificity are increasingly recognized, their contribution to the evolution of organ dysfunctions remains poorly understood. To address this, we will enrich circulating endothelial cells from whole blood of children with septic shock with the primary objective of developing a transcriptomic atlas at single-cell resolution. By comparing signatures of circulating endothelial subsets from patients, relative to published datasets of tissue-resident endothelial cells, we will identify organ-specific targets for future hypotheses testing. 2) DISEASE MODELS: The reductionist nature of current preclinical models has impeded translation of basic science discoveries into improved sepsis outcomes. Thus, there is a significant need for advanced disease models that recapitulate human sepsis pathobiology. My laboratory will develop human induced pluripotent stem cell derived microvascular organoids and stimulate them with biomarker risk- stratified plasma from patients to model endothelial dysfunction in human sepsis. Our models are expected to serve as robust testbeds for hypotheses testing. 3) DISEASE MECHANISMS: Finally, we will establish cutting- edge CRISPR-Cas9 gene-editing tools in vitro to efficiently study the mechanistic basis of candidate genes identified through our studies. This transdisciplinary proposal is made feasible by a long-standing study of pediatric septic shock with unparalleled access to biospecimens, institutional shared resources, and ongoing collaborations with a rich network of scientists with technical expertise. The approaches established through this proposal are expected to enhance our understanding of organ-specific microvascular endothelial responses and hold potential to inform development of precision therapies. Funding through the NIGMS ESI- MIRA will thus maximize my laboratory’s research efforts to improve the outcomes of children with sepsis.

NIH Research Projects · FY 2025 · 2024-07

Summary Gaucher disease (GD) is a common lysosomal storage disease, occurring at a rate of 1 in 500 in Ashkenazi Jews and 1/60,000 in the general population. Defective acid β-glucosidase (GCase) in GD results in progressive accumulation of glycolipid substrates in tissues leading to hepatosplenomegaly, weakened bone, impaired blood clotting and neurological impairments. Despite many advances in GD therapy, treatments are only partially effective for the more common visceral forms and are completely inadequate for a highly symptomatic and frequently lethal subtype of the disease, neuronopathic GD (nGD), affecting the central nervous system (CNS). Current enzyme replacement treatments (ERT) for nGD are limited by two major challenges: limited penetration of the blood-brain barrier (BBB) and instability of GCase of existing ERT resulting in short half-lives in circulations and organs. We have developed a novel Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanocarrier that can penetrate the BBB. When SapC-DOPS is combined with a novel stable enzyme, named fGCase, allows for efficient transport into the brain with sustained bioactivity. Our intravenously delivered, long-acting SapC-DOPS- fGCase retains kinetic stability in mouse plasma and cells, penetrates through the BBB into the CNS, and displays prolonged activity leading to reduction of brain-accumulated glycolipid substrates. Preliminary results suggest that interplay between SapC, phosphatidyl serine (PS), a receptor for SapC, and the lymphatic system influences SapC-DOPS’s ability to transport the enzyme across the BBB and process in the brain. These preliminary findings strongly suggest that the highly brain-stable SapC-DOPS-fGCase will maintain adequate and sustainable activities to restore GCase function in GD brains. Based on these promising features of SapC- DOPS and fGCase, our overall hypothesis is that SapC-DOPS-fGCase will reestablish GCase function in GD brains and improve brain disease outcomes to advance enzyme treatment for nGD. In Aim 1, we will 1) determine the pharmacokinetics and biodistribution of SapC-DOPS-fGCase, 2) evaluate the therapeutic impact in nGD mouse models and human nGD iPSC-derived midbrain-like organoid model, and 3) investigate the underlying mechanism(s) of this novel CNS-ERT approach in protecting neuronal cell functions. In Aim 2, we will define the pathways involved in the transport of SapC-DOPS-fGCase across the BBB. Our focus will be to investigate the interplay of cell surface PS and SapC-DOPS and the role of CNS-lymphatics for SapC-DOPS-fGCase processing in the brain. Completing this preclinical study will provide proof of concept for SapC-DOPS-fGCase to be a transformative ERT for human nGD. Additionally, we will gain a deeper understanding of PS-dependent, CNS-targeting of SapC-DOPS through the CNS-lymphatic system.

NIH Research Projects · FY 2025 · 2024-07

Abstract/Project Summary Mice and human physiology are deeply affected by brown fat through its control of metabolic homeostasis and production of signaling molecules. As such, brown fat specific genetic modifications in mice have arisen as a truly relevant tool to understand brown fat role in controlling physiology with applications in an array of organ systems. For this, Cre-recombinase drivers under control of the UCP1 promoter have been used. In this proposal, we show that the most commonly used mouse model of brown fat targeting is the source of some previously unnoticed negative effects. Thus, our preliminary data supports an unmet critical need to generate a new mouse model to target mature brown adipocytes in an efficient and safe manner. For these reasons, the main goals of this application are to generate (Aim 1), validate (Aim 2) and make widely available (Aim 3) a new model of brown fat targeting. For this, we will use an innovative but validated CRISPR/Cas9 targeting technique to insert Cre-recombinase driven by a defined ucp1 promoter into a safe harbor location. Next, we will use Cre-mediated lineage tracing techniques to address its cellular specificity. Due to the broad interest in brown fat biology, this mouse model is anticipated to be broadly used by scientists in an array of fields of interest to multiple NIH institutes. This new mouse model of brown fat targeting is expected to have a positive impact as a step forward towards developing better preclinical mouse models. We will make our new model available to the community through a mouse repository. For these reasons, this proposal is directed to the FOA PAR-21-167, “Development of Animal Models and Related Materials for Research”.