Cincinnati Childrens Hosp Med Ctr

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT This patient-centric collaborative translational research project will define cellular and viral biomarkers and mechanisms dictating recurrent respiratory papillomatosis (RRP) patient response to the HPV therapeutic vaccine PRGN-2012. RRP is a debilitating neoplastic disease caused by chronic infection with HPV types 6 and 11 that has no approved medical therapies. A recently published phase 1 clinical trial led by Dr. Clint Allen at the NIH Clinical Center demonstrates that treatment with the gorilla adenovirus-based HPV vaccine PRGN-2012 results in a 50% response rate in patients with severe, aggressive RRP. Our long-term goal is to maximize vaccine treatment effectiveness and confer response in patients who are currently non-responsive. Correlative data limited to pre-treatment papillomas linked the state of the tumor microenvironment to vaccine response. Specifically, response was associated with a) low HPV gene expression, b) low stress keratin 17 (K17) expression, c) high expression of the CD8 T cell chemokines CXCL9-11, and d) prominent CD8 T cell infiltration. Conversely, non-response was associated with a) high HPV gene expression, b) high K17 expression, c) low CXCL9-11 and high neutrophil chemokine CXCL8 expression, and d) prominent neutrophil infiltration with diminished T cell activation. The current proposal greatly expands this correlative clinical data by defining response-related reprogramming in specific epithelial and immune cell subpopulations, extends analysis to paired pre- and post-treatment clinical samples to define phenotypic response outcomes as goals for effective therapy, and tests the hypothesis that high HPV load and gene expression in RRP papilloma epithelial cells attenuates CXCL9-11/CD8 T cell responder phenotypes by inducing K17. We will comprehensively define epithelial and immune cell subpopulations and intercellular signaling using single cell and spatial transcriptomics on pre-treatment and post-treatment papillomas from responders and non-responders collected as a component of the planned prospective phase 3 clinical trial that will power the trial to validate clinical response rates (Aim 1). Complementary mechanistic studies will elucidate the functional role of high vs low HPV gene expression in dictating epithelial phenotypes, cytokine signaling, and immune cell activation and infiltration using isogenic patient-derived organotypic epithelial rafts with low vs high HPV load (Aim 2). Epithelial and immune cell subpopulations and intercellular signaling will be profiled by single cell and spatial transcriptomics for integration with responder and non-responder clinical tissue data. Candidate molecules mediating responder vs non- responder phenotypes, including K17, will be mechanistically tested using pharmacologic, and loss and gain-of- function approaches. These studies unite the epithelial/HPV biology, immunology, pathology, and clinical expertise of an established partnership between Drs. Wells and Wikenheiser-Brokamp at Cincinnati Children’s Hospital and intramural NIH/NCI collaborator Dr. Allen to advance fundamental understanding of RRP pathobiology for development of new treatments that overcome mechanisms of resistance to effective therapies.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY/ABSTRACT Sickle cell disease (SCD) affects millions globally and results in recurrent vascular occlusions, chronic organ damage and early death. While childhood mortality is reduced and patients are surviving to adulthood with current therapies, an increased risk of hematological malignancy (HM) in SCD is becoming evident. Risk of HM is higher following gene therapy. Gene therapy involves hematopoietic stem cell transplant (HCT) of gene- modified autologous/sickle hematopoietic stem and progenitor cells (HSPC), and has had remarkable success and FDA approval. A similar high risk is observed in allogeneic-HCT SCD patients with suboptimal engraftment. However, the specific pathobiology that predisposes to increased HM is unknown, and necessary to address, since curative therapies (gene therapy and HCT) are on the forefront. We hypothesize that increased oxidative stress induced DNA damage results in HSPC clones with deleterious mutations; these clones amplify due to persistent HSPC replicative stress from SCD, and when the replicative stress is further magnified in gene therapy, where autologous gene-modified HSPC have to regenerate to reconstitute the entire hematopoiesis following myeloablative conditioning. We will: (1) Perform gain and loss of function experiments to increase or reduce the oxidative stress-induced DNA damage to HSPC, and study the development of clonal hematopoiesis and HM in SCD mice versus their normal counterparts. (2) Impose HCT-mediated regenerative stress on autologous young and old HSPC and assess whether development of HM occurs de novo, or from existing clones with deleterious mutations in putative oncogenes by high-depth next generation sequencing. Modeling the increased risk of HM in SCD will help identify underlying mechanisms, which can then be targeted to mitigate this risk.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY/ABSTRACT Cerebral visual impairment (CVI) is the leading cause of visual impairment occurring early in life; however, it often remains undiagnosed until preschool age or later. This is problematic because vision is the primary sense used for incidental and intentional learning to build foundational motor, language, and cognitive skills leaving undiagnosed children with a disadvantage. The proposed research aims to (1) develop a screening algorithm for early detection of CVI and (2) establish neurodevelopmental profiles that will inform intervention for children diagnosed with CVI. Our overarching hypothesis is that early detection of CVI and understanding the relationship between visual impairment and neurodevelopment are imperative for informing interventions to optimize a child’s abilities. In our first aim, we will test a novel comprehensive screening algorithm for CVI detection in infants at three developmental time points between 4- 12 months corrected age. In our second aim, we will utilize an interdisciplinary standardized assessment protocol including visual and developmental assessments at two-time points to understand neurodevelopmental subtypes in young children with CVI. This proposal will be carried out by an interdisciplinary research team with a well-established track record of clinical practice and research in CVI. This ground-breaking proposal will lead to a paradigm shift in the current approach to CVI management in young children. The findings from this study will lead to the development of a screening protocol for early detection of CVI that can be easily incorporated into practice by medical providers who routinely interact with young infants. Additionally, identifying neurodevelopmental profiles will be the foundation for the establishment of clinical guidelines for the care of young children with CVI.

NIH Research Projects · FY 2026 · 2025-03

Project Summary There has been considerable progress in identifying subsets in effector, memory, and exhausted CD8 T cells, and our understanding of the role of each subset in response to infection has greatly improved. However, much less is known about the heterogeneity of naïve CD8 T cells, the predecessors of these antigen-experienced T cells. We have recently tackled this important issue, and our preliminary data show that there is a naïve CD8 T cell population with a superior ability to generate more effector CD8 T cells after infection. A deeper understanding of the naïve CD8 T cell heterogeneity will help to develop strategies to produce just such a better naïve CD8 T cell population. Since naïve T cells have an enormous TCR repertoire diversity capable of recognizing virtually any non-self antigens including pathogens and tumor neoantigens, making a high-quality naïve T cell population that gives rise to potent effector and memory T cell responses would provide better protection against known and unknown pathogens, enhance responsiveness to existing vaccines, and even prevent the development of cancer. Our long-term objective is: To elucidate how naïve CD8 T cell heterogeneity is generated and maintained and to understand qualitative differences among naïve CD8 T cell subsets; Ultimately, to use this information to establish novel strategies to induce protective naïve CD8 T cell immunity against pathogens and cancer. Toward this objective, we will investigate both mouse and human naïve CD8 T cells. Our proposed studies in this application represent a new direction of research in the area of naïve CD8 T cell heterogeneity. The following specific aims are proposed: Specific Aim 1: To elucidate how naïve CD8 T cell heterogeneity contributes to immune response and homeostasis of T cells in mice. Specific Aim 2. To define human naïve CD8 T cell atlas by examining antigen specific CD8 T cells in the young and aged adults as well as newborns.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Primary sclerosing cholangitis (PSC) causes progressive destruction of the biliary tree, is associated with inflammatory bowel disease, and lacks medical therapies to prolong transplant-free survival. In children, it commonly overlaps with autoimmune hepatitis (AIH). Development of effective therapies is hampered by incomplete understanding of the pathogenesis of this disease. Intestinal dysbiosis, exposure to toxins, retention of bile acids, and variants in susceptibility genes are entertained as etiologic factors. It is possible that inherited risk for PSC is particularly relevant for disease onset and progression in children. Genome wide association studies (GWAS) identified 30 PSC-risk loci, including loci near TNF superfamily (SF) genes, TNFRSF14 and TNFAIP3, and near GPBAR1 encoding the bile acid receptor TGRS. Many PSC risk variants are within noncoding DNA which complicates inference on their biological relevance. If causal, they likely alter transcription factor binding to regulatory elements and expression of target genes in particular cell types. Recent reports suggest that hepatic macrophages (MP), including TGRS+ ones accumulating in patients with PSC, are critical for control of bile duct epithelial injury, repair, and fibrosis. In preliminary studies, we performed multiome-seq (tandem single nuclear [sn]ATAC and snRNAseq) on liver tissue samples from patients with pediatric onset PSC and/or AIH which revealed significant overlap of 11 PSC-risk loci with accessible chromatin in MP populations. These loci were predicted to control 20 genes, including TNFRSF14, involved in cell-cell-communication (CCC). Liver bulk RNAseq of a cross-sectional cohort of patients with pediatric PSC identified upregulation of several TNFSF members in patients with advanced fibrosis. Single cell spatially resolved transcriptomics (SRT) provided preliminary insights into the cellular composition of the periductal niche in PSC. In vitro, deletion of GPBAR1 in bone marrow derived MP repressed polarization of CD206+ profibrogenic MP. Multicellular, human liver organoids (HLO) differentiated from induced pluripotent stem cells (iPSC) of a patient with AIH and deleterious mutation in TNFAIP3 upregulated expression of TNFa and smooth muscle actin upon stimulation with specific danger signals compared with HLOs from a healthy control. We hypothesize that genetic and environmental factors, i.e. toxic bile acids, control expression of TNFSF proteins by periductal MP, which mediate CCC with neighboring cells and drive biliary fibrosis in PSC. To test this hypothesis, we will perform multiome-seq on liver tissue samples from patients with pediatric PSC/AIH to predict how GWAS-PSC risk loci impact gene regulation in hepatic MP subsets across disease types (PSC vs PSC/AIH overlap) and fibrosis stages (Aim 1), describe CCC of MP with cells comprising the periductal niche in patients with PSC of various fibrosis stages using SRT (Aim 2), and validate the role of genetic variants on responses of MP to danger signals, including bile acids, in iPSC-derived cell-lines and HLOs. Therapeutic potential of TNFSF as targets for control of hepatic MP responses and fibrosis progression will be tested in mouse models of PSC (Aim 3).

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Calcium is the most stringently regulated ion in multicellular organisms and an essential component of cellular signaling pathways. A major influx of calcium from the external environment is required to initiate bone mineralization in vertebrate embryos. Though the major transcellular routes of calcium entry at embryonic stages have been determined for both mammalian and aquatic species, how the amount of calcium uptake is calibrated remains uncertain. The Barske lab has identified an unexpected regulatory role for the transcription factor Sox10 in acquiring calcium for larval bone mineralization in zebrafish. sox10 mutants are known to lack or be severely deficient in many neural crest-derived cell lineages, including glia, pigment, and sympathetic, sensory, and enteric neurons. The lab’s preliminary studies revealed that in the absence of sox10, a kidney-associated gland makes excess amounts of an anti-hypercalcemic hormone, stanniocalcin, which blocks most calcium uptake and thus bone mineralization. sox10+ crest-derived cells were observed in close contact with this endocrine gland in control but not mutant fish. Neither Sox10 nor neural crest have previously been linked to embryonic mineral regulation, marking this a notable advance for the field. The objectives of this proposal are to determine the identity of the sox10+ crest-derived lineage that interacts with the endocrine gland as well as the molecular and cellular pathways linking the two. The hypothesis tested in Aim 1 is that these sox10+ cells are the precursors of the sympathetic nerves that will regulate function of this gland in adults. If supported, this would have the broader implication that cells destined to become an organ’s sympathetic ganglia may make contact early and begin regulating organ function while still in the progenitor state, presumably through non-neuronal mechanisms. The hypothesis tested in Aim 2 is that the regulatory interaction between crest and gland involves deranged signaling of the Calcium Sensing Receptor, a key factor for adult calcium homeostasis that may also be involved in calibrating embryonic calcium uptake. Completion of this aim will add an embryonic dimension to the abundant literature on adult calcium homeostasis and bone mineralization, emphasizing that hormone production must be kept in balance even at early stages when calcium content is climbing rapidly. This is relevant for human gestation as well: developmental endocrine disruptions analogous to those of this fish model could interfere with calcium uptake through the placenta, even with maternal dietary calcium supplementation, and contribute to low bone mineral density at birth.

NIH Research Projects · FY 2026 · 2025-02

Project Summary/Abstract Blood transcriptional modules (BTMs), consisting of functionally related gene sets developed through dimensional reduction computational methods, have emerged as a significant tool for the biological interpretation of bulk transcriptomic data. However, current BTMs, primarily derived from North American and European adult data, fail to capture the immunological characteristics of pediatric populations across diverse geographical settings. To address this significant gap, we aim to develop pediatric-specific BTMs (pBTMs) that capture the broad range of heterogeneous physiological states during childhood and include underrepresented pediatric geographical cohorts through public data mining. For this purpose, we will construct modules utilizing transcriptome data from children aged birth to 12 years. We have gathered 13,388 samples from 76 studies across various physiological conditions, enabling the construction of robust and widely applicable pediatric- specific modules. This work will be the first to establish pediatric-specific BTMs, serving as a valuable resource for pediatric immunology research and empowering the development of vaccines and therapies to reduce the burden of infections and other immune-mediated diseases in childhood. This project also aims to address several limitations of previous frameworks by incorporating an improved a priori knowledge base using pediatric single- cell datasets and an assessment framework for the biological relevance of the pBTMs using genome-wide association studies (GWAS). Our state-of-the-art and novel computational framework will be delivered as a portable, version-controlled, publicly available pipeline that can be replicated in other disease and tissue type settings. To demonstrate the utility of pBTMs and as a proof of concept, we will utilize these modules to define immunological differences across geographically disparate pediatric populations, informing research into variability in childhood immune responses.

NIH Research Projects · FY 2026 · 2025-02

During the early phase of infection, Mycobacterium tuberculosis proliferates in macrophages and other cells, preventing apoptosis by the induction of proteins such as from the Bcl-2 family, leading to necrosis of the infected cells. Necrosis increases tuberculosis (TB)-associated morbidity by causing tissue destruction, inducing inflammation, promoting fibrosis, and impairing vascular supply, thereby reducing the penetration of antimicrobials and immune cells to the areas where they are needed most. We have recently demonstrated that adjunctive use of navitoclax, an orally bioavailable, pro-apoptotic small molecule Bcl-2 inhibitor (in clinical trials for cancer treatments, with excellent safety profile), improves bacterial elimination and decreases lung damage in animal models of TB. Additionally, navitoclax has anti-fibrotic effects, which can reverse and prevent lung fibrosis and may promote antibiotic penetration into TB lesions. In fact, post-TB lung disease is a recognized consequence of pulmonary TB, with associated chronic adverse outcomes beyond the TB treatments, including bronchiectasis, poor lung function and respiratory symptoms. Our central hypothesis is that navitoclax (or similar pro-apoptotic drugs) could be used as a cell death mechanism (CDM)-based small molecule, host-directed therapy (HDT) approach to shorten TB treatments, and prevent post-TB lung disease. To understand how these novel therapeutics impact the lung immune landscape, remodeling and bacterial clearance, we have developed several novel, clinically translatable positron emission tomography (PET)-based imaging biomarkers to longitudinally profile lesional characteristics in live animals: 18F-ICMT-11 for apoptosis, 18F-FAPI-74 for fibrosis, 11C-rifampin and 18F-pretomanid (both chemically identical to the parent antibiotic) for tissue antibiotic exposures as well as advanced magnetic resonance and computed tomography imaging to visualize pulmonary damage and necrosis. Finally, we have developed complementary high-dimensional immunophenotyping by flow cytometry to assess the lung immune landscape, remodeling and fibrosis. We will develop CDM-based small molecule HDT approaches for TB treatments, which brings together cutting-edge technologies and cross-disciplinary expertise in TB pathogenesis (Jain), pulmonary immunology (D'Alessio) and oncology (Carroll). There are currently no HDTs approved for clinical use for the treatment of pulmonary TB. Therefore, in this proposal, we will leverage our expertise in animal models of TB, advanced whole-body in vivo imaging and high-dimensional immunophenotyping to gain mechanistic insights on the role of pro- apoptotic drugs to shorten treatments for drug-susceptible and multi-drug resistant (MDR) pulmonary TB as well as to prevent post-TB lung disease. Our goals are to utilize novel pro-apoptotic HDTs to develop short (2-3 months versus current 4-6 months or longer), and efficacious TB treatment regimens which also prevent / improve post-TB lung disease, as well as develop novel clinically-translatable imaging approaches to expedite the development of pro-apoptotic drugs for shortening TB treatments.

NIH Research Projects · FY 2026 · 2025-02

Project Summary Abstract Lymphangioleiomyomatosis (LAM) is a destructive lung disease associated with cystic lung remodeling and progressive respiratory failure. LAM is caused by TSC1 or TSC2 germline mutations that activate the mechanistic target of rapamycin complex 1 (mTORC1). Sirolimus, an mTORC1 inhibitor, is the only FDA-approved drug for LAM, stabilizes lung function and improves symptoms in LAM patients, but disease progression resumes when sirolimus is discontinued and some patients do not respond to therapy. Despite the important clinical observations of distinct therapeutic responses to sirolimus in LAM, the cellular mechanisms underlying sirolimus insensitivity are not understood. The objective of this proposal is to elucidate the mechanisms underlying sirolimus insensitivity to improve LAM treatment. This proposal is supported by novel preliminary data derived from both bioinformatics and laboratory findings. Our single cell RNA-sequencing analysis identified a sub- population of cells that are insensitive to sirolimus in heterogenous cells from Tsc2-null xenografts on sirolimus. The sub-population of cells was characterized by the increased expression of YAP-WNT pathway genes and stemness cell markers. The findings were validated using ELT3-245, a Tsc2-null sirolimus-resistant cell line. In addition, we discovered that YAP-WNT activated downstream target gene expression independently of mTORC1 in LAM lung explants and LAM cells in vitro. Our central hypothesis is that the activation of YAP-WNT crosstalk in LAM lung cells promotes the “LAM cell stemness state” that drives sirolimus insensitivity. We postulate the combination of sirolimus and YAP/WNT antagonists will overcome the primary cytostatic effects and enhance sensitivity to sirolimus. Three specific aims are proposed: Aim 1. Identify the molecular mechanisms underlying sirolimus insensitivity in LAM. Aim 2. Evaluate the role of YAP-WNT pathways on genes and processes underlying sirolimus insensitivity in vitro. Aim 3. Determine the effect of YAP-WNT inhibition on restoration of sirolimus sensitivity in LAM cell survival and lung remodeling in vivo. The completion of this proposal will provide for the first time: 1) a full spectrum of genomic/epigenomic resources of differential sirolimus responses in LAM. 2) molecular determinants of acquired sirolimus insensitivity and unique LAM cell subtype insensitive to sirolimus, 3) molecular insights into the role of YAP-WNT crosstalk in sirolimus insensitivity, and 4) preclinical proof-of-principle evidence for a novel regimen of YAP-WNT suppression in restoration of sirolimus sensitivity and inducing remission. Innovative concepts of direct clinical relevance on YAP-WNT crosstalk leading to LAM cell stemness state and sirolimus insensitivity will be assessed at single cell resolution coupled with preclinical models using non-invasive optical imaging to longitudinally monitor treatment responses in mouse lungs. Our work is highly significant and has positive impact as a comprehensive understanding of the molecular mechanisms underlying sirolimus insensitivity will reveal novel targets and inform remission-inducing therapeutic strategies for LAM patients and other mTORC1-hyperactive diseases.

NIH Research Projects · FY 2026 · 2025-01

ABSTRACT The genome is transmitted from one generation to the next by two highly specialized cell types, sperm and egg. In the adult testis, spermatogonial stem cells continuously produce progenitors that rapidly expand their population (transit-amplify) before undergoing terminal differentiation into sperm. Across tissues, cell state transitions from stem cell to progenitor to differentiated cell require regulated gene expression programs. Post- transcriptional mechanisms that regulate RNA, from splicing through translation, play a major role in shaping these gene expression programs. My research program seeks to define how post-transcriptional regulation shapes gene expression programs to support the function of spermatogonial stem and progenitor cells, using mouse as our model. We leverage single cell methods as well as approaches unique to spermatogenesis to provide enhanced stage-specific insights into progenitor function. We will focus on two Areas: Area 1: During spermatogenesis, retinoic acid (RA) signaling induces early progenitors to form committed progenitors via canonical regulation of transcription and noncanonical regulation of translation. We have demonstrated that RNA-binding protein and translational regulator DAZL supports the formation of committed progenitors, but whether DAZL’s activity affects RA-induced molecular changes remains unclear. Here, we will test the hypothesis that DAZL enhances RA-mediated changes in transcription and translation to induce formation of committed progenitors. Area 2: Committed spermatogonial progenitors enter terminal differentiation when they initiate meiosis in response to RA signaling. RA activates gene expression of Stra8 and Meiosin, which encode a heterodimeric transcription factor that drives the initiation of meiosis. We recently discovered that the RA-dependent upregulation of Meiosin gene expression requires the RNA-binding complex MEIOC-YTHDC2-RBM46, which mediates mRNA degradation of transcriptional repressors of Meiosin. Based on these data, we proposed the conceptually novel model that committed progenitors must acquire the molecular competence to initiate meiosis in response to RA. Furthermore, this competence is regulated by MEIOC-YTHDC2-RBM46 destabilizing mRNA. Here, we propose to expand on this model. We will define the mechanism by which MEIOC-YTHDC2-RBM46-bound mRNAs are degraded, identify MEIOC’s molecular function within the complex, and determine whether genetic ablation of transcriptional repressors targeted by MEIOC-YTHDC2- RBM46 is sufficient to rescue the competence of Meioc knockout committed progenitors. Our proposed research directions will define how post-transcriptional regulation interacts with transcriptional networks to support spermatogonial stem and progenitor cell function, with implications for human reproductive health. More broadly, our studies will inform how post-transcriptional mechanisms may be implemented in other contexts of normal cellular differentiation and co-opted in cancer.

NIH Research Projects · FY 2026 · 2025-01

Chronic inflammation of the placental membranes (chronic chorioamnionitis (CCA)) occurs frequently and involves excessive chorionic trophoblast death and infiltration of maternal CD8 T cells to the chorio-amnion membrane. While the etiology of CCA remains unclear, CCA has been found in the presence and absence of an infective etiology. Increased clarity on the inflammatory factors and fetal or microbial antigens that drive CD8 T cell cytotoxicity and trophoblast death will have profound implications for how CCA is treated. The proposed research investigates how Human Leukocyte Antigen-C (HLA-C) expressed by chorionic trophoblasts promotes cytotoxic CD8 T cell responses during CCA. HLA-C is the only classical Major Histocompatibility Complex (MHC) molecule expressed on chorionic trophoblasts that can present microbial and fetal antigens and directly activate cytotoxic CD8 T cells through the T cell receptor (TCR). HLA-C is unique to humans, and HLA-C orthologues do not exist in rodents or macaques. The proposal builds on our discovery that CD8 T cells in healthy membranes are differentiated memory T cells, which compared to blood memory T cells have vastly reduced expression of the cytotoxic molecule perforin. Moreover, CD8 T cells from healthy membranes respond to TCR stimulation, but do not release cytotoxic granules nor kill sample matched chorionic trophoblasts during co-culture. This suggests that control of CD8 T cell cytotoxicity is critical to avoid trophoblast killing in healthy pregnancy. Preliminary analysis of CD8 T cells in membranes with CCA shows they infiltrate the chorion, upregulate perforin and form direct contacts with chorionic trophoblasts. In many of the trophoblast-T cell contacts perforin is present indicating that these are immune synapses promoting CD8 T cell degranulation and trophoblast killing. Thus, CD8 T cells in CCA acquire higher cytotoxic capabilities that they likely use to kill chorionic trophoblasts. We hypothesize that tight control of CD8 T cell cytotoxicity is required to avoid trophoblast killing in healthy pregnancy. Increased immunogenicity of chorionic trophoblasts, including high expression of HLA-C and lack of inhibitory signaling, elicits CD8 T cell cytotoxicity and trophoblast killing during CCA. To test this, we will i) determine how cytotoxic CD8 T cells contribute to trophoblast death during CCA using state-of-the-art high dimensional flow cytometry (HDFC)-based cytotoxicity assays of primary CD8 T cells and trophoblasts; and ii) Define the cell-cell communication networks that control CD8 T cell killing of chorionic trophoblasts. Cutting-edge machine learning approaches of integrated single cell RNA-seq, HDFC, cytotoxicity and clinical data will be used to identify pathological CD8 T cell and chorionic trophoblast types as well as the disrupted communication networks between them, that contribute to cytotoxicity and trophoblast death. Loss and gain of function interventions using in vitro expanded primary chorionic trophoblast lines will be used to test strategies to protect trophoblasts and reduce CD8 T cell cytotoxicity. Discovering pathological cellular networks and trophoblast antigens that drive CD8 T cell killing of trophoblasts offers transformative insights in the etiology of CCA.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Ischemic heart diseases, which include heart failure, are linked to systolic dysfunction resulting from the loss of cardiomyocytes (CMs). The ultimate therapeutic approach for this condition is the regeneration of new CMs. Recent studies on heart regeneration suggested that stimulating the natural division of CMs, known as mitosis, is a promising strategy for myocardium regeneration. YAP is a transcriptional cofactor that shuttles in and out of the nucleus in response to physiologic inputs. The Hippo pathway is a kinase cascade that suppresses YAP by phosphorylation, which prevents YAP from entering the nucleus. Our previous studies and others have shown that inhibition of the Hippo pathway causes YAP nuclear localization, promoting CM proliferation and suppressing pathologic remodeling in mice and pigs, suggesting that targeting the Hippo pathway could be a potential therapeutic approach for treating ischemic heart diseases. However, CM proliferation induced by the inhibition of the Hippo pathway is self-limiting, suggesting other mechanisms can regulate the function of YAP. In the pathophysiological context after myocardial infarction (MI), we found that YAP is transiently localized to the nucleus of CMs, which is associated with acetylation. YAP is acetylated by CBP/p300, but YAP deacetylation is regulated by SIRTs and its cofactor NAD+. Here, we propose that metabolic stresses regulate YAP acetylation through NAD+, which affects YAP activity and subcellular localization. We will test how the NAD+/SIRT1/2 axis regulates YAP acetylation in mouse models with myocardial infarction and investigate whether combined targeting the Hippo pathway and YAP acetylation can enhance cardiac regeneration more effectively than targeting either pathway alone. This mechanism involves a Hippo-independent, post- translational regulation of YAP activity called acetylation. Unlike the Hippo pathway-mediated YAP phosphorylation that occurs in the cytoplasm, we found YAP to be acetylated in the nucleus, representing an additional mechanism for YAP regulation. This comprehensive understanding of the mechanism of YAP in regulating cardiac regeneration and remodeling can lead to the development of more effective therapeutic strategies for treating ischemic heart diseases.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Nationally, 1 in 7 families with children are food insecure. Childhood hunger is associated with worse health and developmental outcomes; children in some families also experience more urgent healthcare utilization. During pediatric hospitalization, some parents/ caregivers (hereafter referred to as parents) experience hunger due to an inability to afford enough food in the hospital. This hunger may increase parent’s mental “fog” and reduce their ability to be present during the hospitalization. The hospitalization itself causes financial strain due to missed work and increased medical and non-medical expenses during the hospitalization. Pediatric hospitalization is an opportunity to intervene and support families that go hungry in the hospital and lessen the burden going home after hospitalization. At our institution, we instituted an in-hospital food support intervention on one inpatient unit to provide meal cards for our cafeteria for all parents of children insured by Medicaid or uninsured, which reduced parental hunger rates from 86% to 16%. Hospital leadership has asked our study team to spread it throughout the hospital. Additionally, we partnered with parents to design a post-discharge food support intervention that provides grocery gift cards and frozen meals to families who identified household food insecurity. We tested this intervention in the clinical system with high ratings of feasibility, acceptability, and appropriateness from a representative group of family participants. We will compare these interventions to usual care, as-needed social work referral. In our first aim, we will determine the effect of the in-hospital food support intervention on parent-reported and clinical outcomes, such as hunger, parent presence in the hospital, post-discharge urgent healthcare use, and time to return to normal routines after hospitalization. We will evaluate this intervention using a stepped-wedge randomized trial, where the unit of randomization is the clinical unit. Our second aim will determine the effect of the post-discharge food support intervention on parent-reported and clinical outcomes, including sooner return to work and school and urgent health care reutilization. This aim will be evaluated through a nested randomized control trial within the stepped wedge design. Our parent-centered and experienced study team is well-poised to complete the proposed project. Our three parent co-investigators bring different experiences to our study team, and our co-investigator team has worked together on many successful projects. Our Stakeholder Advisory Board is composed of both local and national stakeholders who are committed to this project and the dissemination of our findings.

NIH Research Projects · FY 2026 · 2025-01

Abstract: Blood cell production takes place via stepwise differentiation from hematopoietic stem cells into multipotent progenitors that give rise to unipotent progenitors responsible for producing each of the major blood cell lineages (lymphocytes, monocytes, neutrophils, and red blood cells). This process takes place inside the marrow of the bone where a unique microenvironment provides supportive and regulatory signals that regulate stepwise blood cell differentiation. We and others have shown that the bone marrow microenvironment is spatially and functionally heterogeneous and contains local structures with diverse and unique functions in homeostatic, inflammatory, and regenerative hematopoiesis. Our ability to manipulate hematopoiesis to treat disease has been hampered by our lack of understanding of these anatomical cues. Our long-term research objective is to define the physiology of blood cell production in the bone marrow tissue in homeostasis and stress. My short- and medium-term research program (the focus of this proposal) will test the hypothesis that the bone marrow has a high degree of spatial organization; it contains discrete specialized vascular microenvironments -discovered by us using new imaging approaches- that recruit and regulate myeloid and erythroid progenitors and dictate how the bone marrow functions in homeostasis, inflammation, or regeneration. We will carry out the following complementary 3-part research program in the following areas: (1) Dissect the function of specialized microenvironments that function as niches for each major blood lineage during homeostasis, with a focus on understanding how hematopoietic cells move through different specialized microenvironments as they differentiate. (2) Determine how specialized sinusoids function as “antennas” to sense inflammation and decide which -and how- hematopoietic progenitors respond to inflammation. (3) Discover how hematopoietic stem cells and progenitors engraft in the bone marrow after transplantation and define how two types of specialized microenvironments regulate short-term and long-term hematopoietic recovery after transplantation. Together these studies will provide a new framework for understanding blood differentiation, dissecting hematopoiesis during disease, and designing systems for multilineage blood cell production ex vivo

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Obesity is a widespread health concern impacting millions of individuals and is strongly associated with increased risk of cardiovascular disease (CVD). Despite advancements in understanding the metabolic implications of obesity, the connection between lipid droplet (LD) dynamics and CVD remains relatively unexplored. This project aims to address this gap by focusing on the functional characterization of a novel microprotein, Nutritionally regulated adipose and cardiac-enriched gene (Nrac), and its involvement in LD biogenesis and lipid metabolism within the cardiovascular system. Microproteins, a class of proteins often overlooked due to their small size, are increasingly recognized for their crucial roles in cellular functions. Nrac, encoded by the previously annotated noncoding RNA A530016L24Rik, has emerged as a significant player in lipid metabolism. Prior studies have shown that Nrac is highly expressed in cardiac and adipose tissues, with dynamic regulation in response to nutritional states. Our preliminary data indicates that a global gene deletion of Nrac leads to cardiac stress and altered lipid metabolism, emphasizing its potential significance in cardiovascular health. Furthermore, our data suggests Nrac localizes to the sarcoplasmic reticulum (SR) membrane, the primary site of intracellular LD biogenesis, and protein interaction data predicts its direct interaction with proteins involved in this process. Therefore, we hypothesize that Nrac localizes to the SR membrane in cardiomyocytes to coordinate LD biogenesis and subsequently contribute to lipid metabolism. Through two specific aims, this research seeks to understand the mechanisms by which Nrac influences LD dynamics and lipid metabolism. Aim 1 will investigate Nrac's localization and interaction with proteins involved in LD biogenesis using human induced pluripotent stem cells (hiPSCs) differentiated into cardiomyocytes (hiPSC-CMs). This aim will examine the endogenous expression dynamics and localization of Nrac, along with functional assays to assess its potential role in LD biogenesis. Aim 2 will evaluate Nrac's impact on lipid metabolism in a diet-induced obesity model utilizing Nrac knockout mice. By subjecting these mice to a high-fat diet challenge, this research aims to determine whether Nrac plays a regulatory role in lipid metabolism, contributing to obesity progression. Echocardiography, metabolic assessments, lipidomic analyses, and functional studies will be conducted to evaluate the effects of Nrac deficiency and overexpression on adiposity and cardiac health. Upon completion of these aims, the project aims to provide a comprehensive understanding of Nrac's role in LD dynamics and lipid metabolism, particularly in cardiomyocytes. This knowledge could potentially identify Nrac as a therapeutic target for mitigating lipid abnormalities associated with obesity and reducing the risk of CVD.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY While most infants acquire language with ease, congenital hearing loss commonly causes deficits in speech- language development. Additionally, about 40% of infants with hearing loss have a physical or developmental disability that prevents reliable behavioral responses. Therefore, there is a critical need for an objective measure (i.e., neural biomarker) of speech processing to improve the clinical management of infants and children with hearing loss. Neural speech tracking (NST) is a novel electrophysiological (EEG) technique that quantifies hierarchical encoding of speech from low-level acoustic features (fundamental frequency, spectrogram, speech envelope, acoustic onset) to high-level content (phonetic, prosodic, and semantic). However, little is known about the developmental trajectory of NST while infants are acquiring language and the impact of hearing and environmental factors. To address these gaps, we will conduct a prospective longitudinal study of NST in full- term infants at 1, 6, and 12 months of age. Testing will include assessment of hearing and the environment (social risk, shared reading, home and language environment) as they relate to language outcomes. The specific aims of the study are to evaluate hearing, environmental factors, and language outcomes (Aim 1), examine the developmental trajectory of NST and spontaneous EEG (Aim 2), and examine relationships among hearing, environment, NST and spontaneous activity, and language outcomes (Aim 3). The central hypothesis is that NST will be modulated by age, hearing, and environmental factors and that NST will be correlated with later language outcomes. The expected outcomes are that hearing and environmental factors will have a measurable impact on NST and that NST will prove to be a neural biomarker of language acquisition. The proposed research will advance the field of hearing research by providing a strong foundational knowledge of the developmental trajectory of NST and the impact of hearing and environment as the infant brain assembles functional networks that support speech processing. To improve clinical management of infants and children with hearing loss – a key mission of the NIDCD – a neural biomarker of how speech is encoded in the brain is essential. The K23 award will provide structured training and experience in three core areas: 1) Impact of the Environment on Language Acquisition, 2) Advanced Neurophysiological Techniques, and 3) Scientific Dissemination, Grantsmanship, and Biostatistics. The proposed study will take place at Cincinnati Children's Hospital under primary mentorship of Dr. Hunter along with an accomplished team of experts in early language development and assessment, socioenvironmental risk, NST and spontaneous EEG, auditory neurodevelopment, computational neuroscience, and biostatistics. The training will build upon Dr. Blankenship’s clinical and research experience and ensure that she achieves her long-term career goal to become an independent clinician-scientist using translational methods to improve the diagnosis and treatment of infants and children with hearing loss.

NIH Research Projects · FY 2026 · 2024-12

Summary of Proposed Research Medulloblastoma (MB) represents the most common malignant childhood brain tumor. Despite aggressive multimodal therapy, the prognosis for many MB patients is grim: nearly half succumb to the disease following tumor recurrence, generally a fatal event for these patients. This presents an urgent need for novel therapeutic modalities that target the relapse-initiating cells and their growth at the root of tumor recurrence, and improve patient survival while minimizing adverse side effects. By using single-cell analysis of MB tumor tissues, we identify a population of transit-amplifying progenitors expressing Olig2, which marks glial progenitors and oligodendroglial precursor cells during neural development, as a prominent tumor-initiating/propagating population during the early phase of MB tumorigenesis. Strikingly, OLIG2 expression is upregulated in relapsed tumors in animal models of sonic hedgehog (SHH)-MB after chemotherapy and human recurrent MBs, suggesting a potential role for glial progenitors and their underlying fate determinants in MB tumor recurrence. Although acquisition of the neuronal progenitor identity is critical for SHH-driven MB formation, which could reflect growth or progression, the contribution of glial progenitor cells during tumor recurrence remains elusive. We hypothesize that a population of glial progenitors are intermediate progenitors predisposed to the transformation before transition to neuronal progenitors in MB initiation and recurrence, and that glial specification factors-mediated core regulatory pathway are a hitherto undiscovered MB tumor-promoting cues that may serve as a potential therapeutic target for MB recurrence. Given that glial progenitors may represent a novel tumor-initiating cell in malignant and recurrent MB, in this proposal, we will dissect tumor heterogeneity, evolution, and microenvironment during tumor recurrence and test the hypothesis that glial progenitors are critical transit-amplifying cells that initiate tumor recurrence. In addition, we will define signaling mechanisms and characterize glial fate determinants-mediated regulatory circuits that drive MB recurrence through genetic and pharmacological approaches. Thus, these proposed studies should advance our understanding of the cells of origin for tumor recurrence and underlying pathogenesis at molecular and cellular levels, and facilitate devising new, effective strategies to treat these deadly pediatric brain cancers.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Noroviruses are a group of highly infectious viruses responsible for over 600 million cases of gastroenteritis worldwide annually. Despite their widespread prevalence, currently there is no licensed vaccine against norovirus infection and disease. Very little is known about the nature of innate immune responses to norovirus infection in humans. In recent decades, advances in high-throughput “omics” technologies, along with the computational algorithms developed to analyze and interpret such data, have created the ability to profile immune responses at unprecedented depth and to better understand the complex interactions between the numerous components of the immune system. Here we propose to delineate the transcriptomic responses in human peripheral blood and stool collected from volunteers early after controlled infection with varying doses of two different strains of Norovirus and to use them as early innate transcriptional predictors of the effective adaptive anti-norovirus humoral and cellular immunity that develops later. We will perform transcriptomic, cytokine, and innate (NK and CD14+) cell profiling of peripheral blood and transcriptomic profiling of stool collected as part of two parallel DMID trials of healthy adults challenged with different strains and doses of Norovirus (clinicaltrials.gov ID NCT02473224, GII.2 strain, n=38, and NCT04174560 GII.4 strain, n=45). Samples will be collected pre challenge and on days 1, 3, 5, and 14 post-challenge, enabling profiling of the response kinetics during the acute phase of infection. Integration of transcriptomic data with our recently published atlas of human transcriptional responses across 13 vaccines, including many live attenuated vaccines, will enable a comparative analysis that will provide deep insight into how immune responses to gastrointestinal viruses, like Norovirus, may differ from those against respiratory or other types of viruses. Additionally, we will utilize norovirus-specific antibody titer (IgA, IgG, and blocking antibodies) and antibody-secreting cell and memory B cell frequency data, together with measurements of activated and cytokine (IFN-g, TNF-a, IL-2, IL-13 etc.) secreting Norovirus-specific T cells and combined single cell RNA and immune repertoire sequencing to comprehensively evaluate the humoral and cellular immune responses to Norovirus infection. We will incorporate the transcriptomic response data with machine learning approaches to identify transcriptional signatures of innate immune activity that predict each parameter of anti-norovirus humoral and cellular immunity, leveraging the independent trials as discovery/validation datasets to identify robust predictors. Together, these analyses will provide deeper insight into the immune responses to wild-type norovirus infection in humans and help understand mechanisms leading to the induction of effective anti-norovirus immunity. This information will lay important groundwork and provide a unique reference to compare immune responses to potential Norovirus vaccine candidates.

NIH Research Projects · FY 2026 · 2024-11

PROJECT SUMMARY CD8+ T cells play a pivotal role in protective immune responses against intracellular pathogens and cancer progression. Most CD8+ T cells activated during a primary immune response will contract and die, however, a small percentage will persist and form long-lived memory populations (CD8+ Tmem). CD8+ Tmem can occupy various anatomical niches and rapidly respond to subsequent antigen encounters, leading to effective secondary responses. Despite the importance of cytotoxic functions, much work has shed light on the ability of CD8+ T cells to confer protection from pathogens in the absence of cytotoxic machinery. However, the underlying mechanisms responsible for such protection remain ill-defined. Our lab has demonstrated that memory CD4+ T cells are able to engage innate immune cells and drive inflammatory responses, a process dependent on MHC-TCR and Tumor Necrosis Factor Superfamily (TNFSF) ligand-receptor interactions. Here, ligands expressed by memory CD4+ T cells engage receptors on myeloid cells, leading to NFκB-dependent cytokine production. We have generated preliminary data suggesting that CD8+ Tmem, but not effector CD8+ T cells, can also activate myeloid proinflammatory responses during cognate interactions, albeit through distinct mechanisms from those of memory CD4+ T cells. CD8+ T cell-induced myeloid proinflammatory responses result in production of several key innate cytokines, including IL-1β and IL-6, both of which are critical to anti-microbial responses. Additionally, antigen-presenting cells cultured with CD8+ Tmem display increased transcript levels of Type I Interferons (IFN) and Interferon-Stimulated Genes (ISGs), both of which are critical to antiviral and anti-cancer responses. Similar increases in ISG responses were also observed in vivo within various tissues after selective reactivation of CD8+ Tmem. These data suggest an evolutionary mechanism wherein reactivation of CD8+ Tmem results not only in rapid effector function and cell killing, but also innate activation and induction of a systemic anti-microbial response. The objective of this proposal is to test the overall hypothesis that induction of innate inflammation is a critical, non-canonical function of CD8+ Tmem necessary for optimal immune responses. In Aim 1, we plan to discern the molecular mechanisms underlying CD8+ Tmem-induced innate inflammatory responses and the importance of this axis in microbial immunity. We will identify key ligand-receptor interactions necessary for proinflammatory cytokine production and initiation of Type I IFN responses. In Aim 2, we will investigate the mechanisms that promote or attenuate CD8+ T cell-induced innate responses, particularly focusing on the role of immune checkpoint molecule signaling during cognate interactions. Successful completion of these aims will uncover a novel function of CD8+ T cells in modulation of innate inflammatory responses, with profound implications for therapeutic development.

NIH Research Projects · FY 2024 · 2024-09

Retinopathy of prematurity (ROP) disrupts postnatal retinal vascular development within the incompletely vascularized retina of preterm infants causing up to 40% of all childhood blindness worldwide. ROP cannot be cured or prevented; treatment stops disease progression but is associated with significant ocular, visual and potentially systemic morbidity. While, postnatal oxygen supplementation underlies ROP pathogenesis, it cannot be safely modified to prevent ROP. Preeclampsia and chorioamnionitis are placental conditions which significantly correlate with postnatal vasoprotection despite oxygen stress (protection paradigms), evidenced by reduced ROP development. Mechanisms underlying placentally-mediated postnatal ROP vasoprotection are not clear; however, our group has identified the LIM homeobox 5 (LHX5) transcription factor as a placental factor which may underlie a “final common pathway” of postnatal infant vasoprotection. We have shown that preeclampsia and chorioamnionitis demonstrate placental LHX5 silencing possibly mediated by promotor methylation. LHX5 inhibits WNT signaling through direct transcriptional activation of the canonical WNT antagonist Secreted frizzled related protein 1 (sFRP1) during development and consistent with this, sFRP1 is decreased in placental tissues associated with protection paradigms. WNT signaling mediates retinal vasoprotection and reduced ROP in animal models, which is inhibited by sFRP1. Our preliminary data support a role for LHX5-sFRP1 silencing in postnatal vasoprotection via WNT signaling, finding 1) reduced plasma sFRP1 and 2) WNT signaling pathway enrichment in the systemic circulation from infants with gestational exposure to protection paradigms compared to ROP development. Our central hypothesis postulates that placental LHX5 silencing is mediated by oxygen and contributes to postnatal vasoprotection via WNT pathway enrichment in the placental, feto-placental, systemic and ocular microenvironments. We will test our central hypothesis using three specific aims designed to: 1) Determine if LHX5 mediates a vasoprotective WNT response to oxygen stress via direct transcriptional regulation of sFRP1 in retinal pigment epithelial cell- differentiated induced pluripotent stem cell models 2) Determine if LHX5 promotor methylation localizes to histologic regions typifying protection paradigms and if it is modified by oxygen stress and 3) Determine how LHX5 placental protein expression relates to protection paradigm or ROP clinical outcomes and if it correlates with WNT signaling changes within the proteome or feto-placental circulation. The rationale for the proposed research is that a mechanistic understanding of placentally-mediated postnatal vasoprotection, will provide new opportunities for the identification and development of targets for disease prevention. This proposal seeks to be the first to characterize patho-mechanisms underlying a final common pathway of placentally-mediated postnatal ROP vasoprotection within both placental and ocular microenvironments. This is important as may allow for mitigation of deleterious ocular effects from oxygen therapy in preterm infants.

NIH Research Projects · FY 2025 · 2024-09

Abstract: Defining how the bone marrow functions under stress is indispensable to understand hematopoiesis during disease. A major limitation in the field is that most studies (including those from our group) have ignored the fact that hematopoiesis takes place in multiple bones. Instead, the field has largely focused on investigating hematopoiesis in long bones – as these are readily accessible and yield large amounts of hematopoietic cells for analyses- and assumed that the rest of the skeleton behaved in a similar manner. We have demonstrated that the bone marrow response to a hematopoietic insult is dramatically different depending on the bone examined. In this proposal we want to understand the cellular mechanisms driving these heterogeneous responses. We have found that -after treatment with G-CSF- the sternum shows reduced numbers of neutrophils and neutrophil progenitors while long bones show expansions in these populations. In response to hemorrhage both the sternum and the tibia increase erythrocyte production, however, the skull fails to increase erythropoiesis. Based on this we hypothesize: a) the bone marrow response to stress is variable across the skeleton; b) some bones have specialized to preferentially respond to specific insults; c) this is non-autonomously regulated by the unique composition -and anatomy- of the microenvironment in each bone. We will test this hypothesis in two aims. In Aim 1 we will determine how bone marrow macrophages control the differential response to G-CSF in sternum vs long-bones. In Aim 2 we will whether competition between erythroid progenitors and trabecular bone controls stress erythropoiesis.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Studies have identified a lower fatality risk in households that secure their firearms locked and unloaded when compared to those where firearms are stored unlocked and loaded. Such findings are consistent across populations, firearm types, and injury outcomes. Though clinician-provided storage counseling on firearms has high acceptability with patients and is efficacious in enhancing secure storage practices , most pediatric clinicians are not providing firearm safety counseling despite firearm related injury being the leading cause of death among children and adolescents. Reported barriers to counseling include a lack of knowledge related to storage strategies and counseling techniques. Thus, a screen-based virtual reality (VR) intervention grounded in education and behavior change theory was developed to support mastery of firearm safety counseling behaviors among pediatric clinicians. Within VR, a user can interact with graphical characters in a seemingly realistic way to deliberately practice skills through receipt of immediate feedback. Dr. Real led the development, usability testing, and preliminary efficacy testing of a human-facilitated version of the VR intervention. Given the promising preliminary data, Dr. Real has developed a prototype for an automated version of the intervention to promote scale and spread. Dr. Real’s long-term goal is to establish an independent research career in the field of firearm injury and mortality prevention. The overall objective of the application is to adapt the automated VR intervention and assess preliminary outcomes including acceptability, feasibility, and usability to prepare for a future effectiveness-implementation trial. The rationale for the proposed research is that an evaluation of an automated VR intervention will inform scalable strategies to train clinicians to provide storage counseling, resulting in increased secure storage practices by patients and caregivers, resulting in decreased access to firearms among children and adolescents, and ultimately lower rates of firearm related injuries and deaths. To achieve our objective, we will accomplish these specific aims: (1) adapt the automated VR intervention by conducting formative evaluations with stakeholders and (2) assess implementation outcomes at multiple sites. To accomplish these aims and his long-term career goals, Dr. Real requires training in (1) firearm injury prevention research, (2) community engagement, and (3) implementation science. This training will occur through a combination of didactic, writing, and experiential research activities . He will receive mentorship from leading researchers at University of Michigan and Harvard Medical School, with Dr. Patrick Carter as the primary mentor. The scientific environment at Cincinnati Children’s Hospital Medical Center strongly supports experienced investigators such as Dr. Real with an abundance of resources. The expected outcomes of this work include foundational data to inform a technology-based intervention easily accessible to clinicians to curb the leading cause of death among children and adolescents.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Surgical treatment via ventriculoperitoneal shunt or endoscopic 3rd ventriculostomy is currently the most commonly used strategy to treat pediatric hydrocephalus. However, shunt-treated hydrocephalic patients continue to experience significant lifelong neurological problems and high malfunctioning shunt replacement surgery rates. The endoscopic choroid plexus (ChP) coagulation helps manage cerebrospinal fluid (CSF) volume by surgically removing the primary CSF production organ in the brain ventricles. However, this surgical procedure requires neurosurgeons with extensive training and still achieves only partial ChP removal. This study aims to evaluate a novel, potent, and less invasive ChP ablation tool in human ChP organoids and rodent brains that can support new ways of treating neonatal hydrocephalus. The NIH Funding Opportunity program announcement PAR-21-122 recognizes the unmet need for translational studies for neurotherapeutics agents for treating neurological or neuromuscular disorders and new therapeutics. In response to this opportunity, our proposal focuses on developing a new ChP ablation tool via ChP-specific delivery of a clinically validated suicide gene. By utilizing cutting-edge techniques such as recombinant adeno-associated virus (AAV) vector targeting ChP, human ChP organoids, robust rodent models of neonatal hydrocephalus, and advanced MRI, including DTI and animal behavior assays, our project aims to achieve rigorous efficacy and safety studies of this new tool. The completion of this study will determine (1) pharmacokinetics and off-target effects and (2) the therapeutic efficacy of this AAV-mediated ChP ablation tool, which has high potential to provide a new strategy for treating pediatric hydrocephalus.

NIH Research Projects · FY 2025 · 2024-09

Abstract Neurofibromatosis type 1 (NF1) is a genetic disorder that occurs in about 1:3000 individuals. NF1 is caused by inheritance or de novo mutation/loss of the NF1 gene. Individuals with NF1 are predisposed to numerous manifestations, including the development of plexiform neurofibromas (PNF) and/or optic pathway glioma (OPG). Unlike in more aggressive tumors, apart from changes at NF1, PNF cells do not show many recurrent somatic changes; somatic changes in OPG are rare. Twin studies suggest that germline modifiers of NF1 disease exist, but these remain largely unstudied. Because the identification of NF1 modifiers should enable risk stratification and identification of targetable therapeutic pathways in individuals with NF1 we developed a multidisciplinary team of geneticists, bioinformaticians, statisticians and animal (mouse and fish) modelers. Relying on close collaboration among team members, we will continue identifying genes showing increased numbers of potentially disruptive variants to study. In Aim 1, we will expand our number of individuals with PNF and/or OPG and test if variants are associated with tumor number, tumor burden, or presence of OPG. In Aim 2, we will test candidates in a well-characterized mouse model of plexiform neurofibroma, using the power of mouse genetics. To enhance rapid screening of relevant genes and variants we generated a zebrafish model of PNF and OPG, which will be exploited in Aim 3, by screening for effects of larger numbers of genes predicted to act as NF1 modifiers. The proposed research will provide a basis for precision medicine in NF1. If risk variants in genes are associated with disease severity and/or correlate with tumor burden, then the development of genomic risk assessment tools will be possible at diagnosis.

NIH Research Projects · FY 2025 · 2024-09

Abstract On average, a human genome harbors ~27,000 structural variations (SVs), including deletions, duplications, and other genomic changes over 50 bp in size, which contribute significantly to phenotypic variations. Recent studies have shown that SVs can disrupt local genome organization known as topologically associating domains (TADs), resulting in misexpression of neighboring genes and causing developmental disorders and other diseases. In this project, we study genomic disruptions associated with frontonasal dysplasia (FND), a congenital craniofacial disorder that profoundly affects the structure and function of the orofacial complex, as a model for understanding genome organization and molecular mechanisms underlying craniofacial development and malformations. Several independent studies have associated FND with partly overlapping heterozygous microdeletions at Chromosome 2p21 in which SIX2 is the only protein-coding gene. SIX2 is a member of the SIX- and homeo-domain containing DNA-binding transcription factors. In all vertebrate genomes, Six2 is physically linked to Six3 in a tail-to-tail configuration, with these two genes organized into separate TADs flanking a conserved TAD boundary. Six2, but not Six3, is abundantly expressed in the cranial neural crest cell (CNCC) derived frontonasal mesenchyme and in nephrogenic mesenchyme during mouse embryogenesis. Whereas Six2+/- mice are phenotypically normal and Six2-/- mice exhibit kidney hypoplasia with normal frontonasal structures, our preliminary study found that deleting Six2 together with part of the Six2/Six3 intergenic region, but not including the Six3 gene or Six2 distal enhancers, caused midline facial clefting in heterozygous mice. On the other hand, mice carrying a heterozygous deletion including Six2, Six3, and their intergenic region in between, could survive with no frontonasal defects. We hypothesize that SIX2-related FND is caused by gain of SIX3 expression in developing frontonasal mesenchyme due to TAD boundary disruption and enhancer adoption rather than by SIX2 haploinsufficiency as previously believed. This project will test this novel hypothesis and unravel the genomic and molecular developmental mechanisms underlying SIX2-related FND. Data from these studies will provide novel insights into mechanisms of craniofacial development and functional divergence of the SIX family transcription factors, and lead to improvements in molecular diagnosis, medical assessment and interpretation of clinical genomics data, and treatment/care of SV-associated developmental disorders.