Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2021-04

Aging in humans is associated with a host of brain diseases, including tumors of glial progenitor cells and degeneration of neuronal cells. However, the mechanisms by which age and disease risk interact are poorly understood. Recent studies from our group have shown that somatic single nucleotide variants (sSNV) accumulate even in nondividing neurons in the human cortex, resulting in thousands of sSNV per neuronal genome by old age. Further, the patterns of sSNV that are found can be classified, and normal brains appear to have somatic variants that were present at birth, variants that accumulate over time, and variants caused by oxidative damage. Our studies also find a significantly higher rate of sSNV accumulation in neurons from Alzheimer’s disease (AD) brain, likely related to increased oxidative damage. These studies relied on new techniques that allow deep whole genome sequencing of DNA isolated from a single neuron taken from frozen postmortem brain. This new study aims to further characterize the rates and patters of somatic variants that accumulate in single neurons and glia as an individual ages, and determine how this accumulation of mutations is related to AD as well as the formation of glial tumors. The first aim will examine neurons form different regions of normal brain at different ages. This will give us a better understanding of how these mutations accumulate with age, and the specific mutational forces at work in different brain areas. The second aim will perform a similar analysis, this time focused on glial cells instead of neurons. In the final aim, we will look at neurons in the brains of individuals who had AD, and analyze how the rate and patter of mutations differ compared to aged- matched normal brain. This will provide valuable insight into the causes of somatic mutations in AD. There is compelling evidence to suggest that somatic mutations in individual neurons are an important factor in at least some neurodegenerative disorders, and our data implicate them in normal cognitive aging. For the first time, the tools exist to examine these questions, and this study is designed to determine how somatic SNV impact normal aging, brain tumor formation, and AD. This is a crucial step in understanding the molecular cause of AD, and a prerequisite to the development of treatments and cures.

NIH Research Projects · FY 2025 · 2021-04

Project Summary The long-term goals of our research program, which has been supported by NIGMS since 2007, has been to understand how host-microbe interactions influence the physiology and behavior of Caenorhabditis elegans, with the anticipation that studies of the simple animal host will provide insights into interactions between microbes and more complex animal hosts. We have brought a broad interdisciplinary perspective, with an experimental approach grounded in the molecular genetics of C. elegans, to studies that have spanned evolutionarily conserved pathways of innate immunity, the integrative physiology that connects infection and immunity with cellular and organismal responses to stress, and how interactions with microbes influence neuronal signaling and behavior of C. elegans. Our most recent focus, and the principal goal of this project over the next five years, is to understand how bacteria influence nervous system signaling and behavior of C. elegans. We have described how specific virulence-associated secondary metabolites produced by the pathogenic bacteria Pseudomonas aeruginosa can modulate expression of a TGF-beta ligand in a pair of sensory neurons of C. elegans to promote avoidance behavior, defining a genetic, neuronal, and chemical basis for the molecular mechanisms by which microbial metabolites can modulate host organism behavior. We have further determined how environmental and endogenous cues converge on the regulation of neuroendocrine gene expression, revealing insight into the hierarchical regulation of inputs that control decision-making behavior of C. elegans. Having defined the molecular pathways involved the innate recognition of P. aeruginosa by the sensory nervous system, we will continue to take a systematic genetic approach to turn our attention to the question of how infection and changes in internal state can modify neuroendocrine gene expression and behavior. We also plan to expand the scope of our studies in a more exploratory manner, to identify additional genetic and neuronal pathways that are modulated by host interactions with not only pathogenic bacteria such as P. aeruginosa, but also bacterial species that have been identified in close association with C. elegans in its natural environment. We expect that the genetic and overall experimental tractability of the simple C. elegans host will enable us to work towards a comprehensive analysis of how microbial metabolites act on the nervous system to modulate neuroendocrine physiology and behavior. The microbiota and its metabolites have been increasingly implicated in diverse aspects of homeostasis and the pathogenesis of disease in host animals. We anticipate our studies of C. elegans will have implications for the understanding of host-microbe interactions in other hosts organisms.

NIH Research Projects · FY 2025 · 2021-04

Project Summary: Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract that has a prevalence of over 800,000 in the US alone. The economic impact is disproportionally high because it affects primarily young individuals. The characteristic periods of remission and relapse necessitate frequent hospitalizations. There has been a recent shift in clinical practice from a reactive to a proactive CD treatment strategy, recently coined as “treat-to-target”, where patients are treated to achieve not only an initial response-to-therapy, but also longer clinical remissions and improved mucosal healing. However, this approach requires the regular assessment of disease activity using objective markers enabling treatments to be tailored to the individual patient. Therefore, there is an unmet need for new tools to improve diagnostic accuracy, to quantify disease burden, and to monitor treatment efficacy. Our application in response to PAR-19-056, “Robust quantitative MR imaging markers of response to therapy in Crohn's disease” is aimed at developing and evaluating noninvasive, contrast and radiation-free quantitative imaging markers for assessing disease activity and for monitoring response to therapy. Currently available non-contrast MR imaging (MRI) sequences such as diffusion-weighted MRI (DW-MRI) and the calculated apparent diffusion coefficient (ADC) maps are attractive but limited in providing robust and reproducible markers. Different imaging protocols or different scanners result in different ADC values. Our primary goal is to develop robust and reliable quantitative DW-MR imaging markers for the non-contrast and non-invasive assessment of CD. The proposed novel MRI acquisition and model fitting techniques will provide measurements of slow and fast diffusion as well as fraction of fast diffusion, as highly accurate, quantitative biomarkers for cell proliferation, density and size, and tissue perfusion—all indices that characterize the extent of disease activity (i.e., inflammation) in the tissue micro-structure of the bowel. To achieve this goal, we will first develop and implement an advanced distortion and motion corrected (DiMoCo) DW-MRI technique and a spatially constrained probabilistic intravoxel incoherent motion (SPIM) model to compute robust and reproducible markers. Next, we will reduce the imaging time with estimated x4 acceleration with an accelerated image acquisition and a new, advanced deep learning-based parameter estimation technique. The proposed imaging techniques and software tools will provide robust quantitative markers, thereby enabling the accurate assessment of CD activity and response to therapy. They will also reduce the need for invasive endoscopy procedures as well as the total number of other tests typically ordered for monitoring disease, effectively reducing both the disease burden and the overall cost of healthcare. Another important goal is to develop and broadly disseminate open source software that will enable the standardized evaluation of other diseases presently evaluated with DW-MRI that would benefit from the advanced diagnostic and assessment capabilities of the proposed DiMoCo SPIM-DW-MRI technique.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract In Alzheimer's disease (AD), the accumulation of intracellular posttranslationally modified Tau aggregates, is a hallmark of AD pathology and a strong correlate of cognitive impairment. While the dozen or so Posttranslational Modifications (PTMs) on Tau in pathological aggregates have been studied for decades, our new quantitative and qualitative mass spectrometry approaches have discovered ~100 PTMs in the angular gyrus from 98 human Alzheimer's Disease patients in these aggregates, i.e. Braak stages V/VI, and age matched control subjects without pathological changes in the frontal lobe. In this proposal, we aim to develop a large-scale proteomics platform to map the temporal occurrence of PTMs as disease progresses. We will examine a valuable new set of cases, selected to encompass all Braak stages from 0 to VI. All cases in this new cohort will have detailed premortem clinical data, which will allow us to detect and assess correlations between clinical and pathophysiological findings and any tau PTM that is identified in this study. For this analysis, we will study 3 brain regions (entorhinal/amygdala, and temporal and visual cortex) in 20 Braak 0 subjects; 40 early stage Braak I/II subjects; 40 mid stage Braak III/IV subjects; and 40 end stage Braak V/VI subjects. We will also map the global proteomes of the brain specimens from this well-characterized patient cohort to identify the enzymes associated with these Braak stage dependent PTMs. The findings of the tau characterization and mapping of the proteomes will be validated using widely accepted Tau sensor assays used to study Tau aggregation in AD. In summary, this Tau PTM specific dataset, the deep proteome maps and the subsequent validation experiments in seeding assays will be used to test our overarching hypothesis: Understanding how the progressive accumulation of Tau PTMs results in the toxicity and seeding competence of tau, and mapping the disease progression dependence of these PTMs and the enzymes responsible for them will allow us to develop interventional strategies at earlier and prodromal stages of disease.

NIH Research Projects · FY 2025 · 2021-03

Congenital heart disease (CHD) affects ~1% of all live births in the United States. Over 85% of individuals with CHD now live well into adulthood1–4, exposing a burden of non-cardiac disabilities, such as neurodevelopmental disabilities. In fact, over half of all children with moderate or complex CHD suffer from neuropsychological deficits, with impaired executive functions (EF) the most common. EF are critical higher-order neurocognitive functions important for independent living and mental health. However, predicting who will be more impaired and in need of intervention is challenging, as routinely measured patient and medical factors explain only one-third of the variance in outcomes. Because impaired EF is particularly amenable to treatment, better predictors of EF are needed to appropriately allocate services and improve outcomes. To develop such methods, we first focus on dextro-transposition of the great arteries (d-TGA). Among the severe forms of CHD, d-TGA is the more common, occurring in 3/10,000 live births. d-TGA leads to severe in utero hypoxia that is corrected soon after birth with an arterial switch operation. Additional surgery and cardiovascular sequelae are rare. Thus d-TGA patients have the most uniform postnatal course of all CHDs but, like other CHDs, is associated with hypoxia and has significant yet variable impairment in EF. This project leverages adult d-TGA subjects being studied under R01HL135061 and d-TGA patients involved in prior Boston trials to create the largest, best characterized d-TGA cohort to date. We propose to perform sophisticated image analysis on brain MRI data and add genetic testing focused on neuroresilience and hypoxia response genes. First, we will employ our sulcal pattern analysis to determine the extent of in utero alterations in brain development, as sulcal patterns are determined prenatally and remain stable into adult life. Second, we will explore the rich club structural and functional networks to separate highly connected central hubs (rich club) that form early in life from less connected peripheral regions which are thought to be adaptive. The overarching goal of this study is to use novel MRI analyses to determine the brain organizational changes associated with altered EF and the modulating role of neuroresilience and hypoxia response genes in adults with d-TGA. Toward these ends, we propose the following specific aims: Aim 1. Determine the relationship between sulcal patterns and executive function in adults with d-TGA and if this relationship is modified by (a) presence of neuro-resilience gene ApoE ε2 or ε4 alleles, or (b) variants in hypoxia response genes. Aim 2/3. Determine the relationship between structural/functional connectivity using rich club and executive function in adults with d-TGA and if this relationship is modified by (a) presence of neuro-resilience gene ApoE ε2 or ε4 alleles or (b) variants in hypoxia response genes. Successful completion would help determine brain changes associated with altered EF and the potential modulating role of neuroresilience and hypoxia response genes as well as inform the balance of in utero versus adaptive changes. This knowledge is relevant to the larger CHD group and will inform the need for prenatal versus postnatal interventions.

NIH Research Projects · FY 2025 · 2021-03

Project Summary How the same genome is parsed by the transcriptional machinery to yield hundreds of distinct cell types remains one of biology’s enduring mysteries. Within the heart, atrial and ventricular cardiomyocytes (aCM and vCMs) are examples of distinct but related cell types with unique expression profiles. These cell-type-specific expression profiles are essential to normal heart function, as their perturbation leads to arrhythmias and contractile dysfunction. Our studies in the prior funding period identified three transcriptional regulators, TBX5, CHD4, and ESRRA/G that are essential to maintain aCM or vCM identity and whose inactivation causes atrial fibrillation and cardiomyopathy. Here we continue the analysis of chamber-selective gene expression by investigating two key questions: (1) what are the rules that govern enhancer and promoter compatibility and how do these rules contribute to tissue-specific expression, specifically to aCM and vCM identity? (2) what are the trans-activing factors responsible for chamber-selective expression? We propose the following Specific Aims: (1) To evaluate the compatibility between tissue-selective enhancers and promoters. Using massively parallel reporter assays performed within intact animals to assess combinatorial interaction of hundreds of thousands of tissue-selective enhancers and promoters, and to dissect the sequences that encode enhancer- promoter compatibility. Based on these data, we will generate novel chamber-selective regulatory elements for research and clinical gene therapy applications. (2) To identify trans-acting factors responsible for chamber- selective expression. We will use computational prediction of aCM and vCM identity regulators and a novel in vivo forward genetic screen to identify candidate transcriptional regulators that govern chamber-selective gene expression. We will characterize the functional role of selected novel regulators on heart morphology, gene expression, and chromatin landscape. Together, these studies will yield fundamental insights into cardiomyocyte gene regulatory mechanisms that will inform our understanding of atrial fibrillation, cardiomyopathy, and other cardiac pathologies.

NIH Research Projects · FY 2025 · 2021-02

ABSTRACT Food allergy (FA) has become a public health concern, affecting a sizeable segment of the population. Despite the alarming increase in its prevalence, efforts to contain the FA epidemic have been stymied by the limited understanding of disease pathogenesis, especially the role in this process of early life gut dysbiosis. In that regard, our studies have established a critical role for immunomodulatory Clostridiales and Bacteroidales species in enforcing immune tolerance to FA by inducing the differentiation of RORgt+ Treg cells, which act to suppress FA. Early life expansion of RORgt+ Treg cells is induced by the bloom in Clostridiales and Bacteroidales species during weaning from maternal milk to solid food. This expansion is counter-regulated by resistin-like molecule beta (RELMb), which is elevated in FA subjects and mice. Accordingly, the focus of this proposal is to elucidate the mechanisms by which early life dysbiosis promotes FA and its long-term implications in terms of disease persistence and response to microbial therapy. We hypothesize that the microbial and immunological changes ushered by weaning early in life provide a window of opportunity for tolerance induction to solid food in a process regulated by RELMb, whose dysregulation by dysbiosis promotes FA (Aim 1). We also hypothesize that the ineffective differentiation of RORgt+ Treg cell populations and the reciprocal emergence of Th2 cell-like Treg cells, an imbalance we have identified to play a fundamental role in FA, acts to promote disease pathogenesis by licensing IgE anti-food and anti-bacterial antibody responses (Aim 2). Finally, we hypothesize that products and metabolites of individual immunomodulatory bacterial strains, including Toll-like receptors activators, aryl hydrocarbon receptor ligands and secondary bile acids, act to enforce oral tolerance in FA by promoting RORgt+ Treg cell differentiation (Aim 3). The proposed studies will provide fundamental new insights relevant to the pathogenesis of FA and offers novel opportunities in early life disease intervention and therapy.

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Recent evidence suggests that environmental factors causing somatic mutations during the lifetime have a more crucial role, not only in cancer but also in other common diseases, including heart failure. Recent studies have also shown that somatic single nucleotide variants (sSNV) accumulate even in nondividing cells, such as neurons in the human cortex, resulting in thousands of sSNV per neuronal genome by old age. However, genomic DNA changes in aging cardiomyocytes (CM) remain poorly understood. The accumulation of somatic DNA mutations over time has recently been demonstrated to be a hallmark of aging in many human cell types. The current study aims to determine the landscape and role of somatic mutations in aging and cardiac disease by adopting a new technique that allow deep whole-genome sequencing of DNA isolated from single CM taken from the frozen postmortem heart. The first Aim of this study is to evaluate the somatic mutational burden (sSNVs) in aging CM genome. We will also compare CM mutational burden with postmitotic cells from another organ (neurons) to define differences in accumulation rate during aging. In the second Aim, we will ask what are the mutational signature and the mechanisms of mutation formation in the aging human heart and if the heart mutational signature is different than the brain mutational signature. Further to recapitulate the mutational signature and related phenotype in the heart we will directly induce oxidative stress in an in vitro culture model of primary CMs. The final Aim will focus on evaluating the genotoxic effect of radiation in CMs after childhood radiation therapy and the role of radiation in premature aging. The proposed research is significant for the comprehensive, results-based development of strategies for understanding natural aging and disease progression in the human heart. Together with the planned characterization of mutational signatures, the anticipated results may provide knowledge to develop new strategies for preventing the heart disease associated with aging. The proposed study is only possible because of a series of innovations that are, at this time, uniquely available to our research team, 1) a novel method to isolate single CM nuclei from frozen myocardium based on CM ploidy and nuclei cardiac troponin T expression. 2) A major breakthrough by developing “LiRA” and “PhaseDel” algorithm to call sSNV and sSV confidently from tetraploid cells that considers cell-specific depth distributions of DNA sequencing and allele-dropout rates in scWGS data. For the first time, our study will reveal the landscape of somatic mutations, genomic changes during aging and after radiation therapy in human heart muscle cells in a single-cell resolution. In the long term, this study will provide insights that might allow blocking some of the mutational processes ameliorating age-related myocardial dysfunction.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT Pulmonary arterial hypertension (PAH) is a life-threatening disorder characterized by elevated lung pressures, right heart failure, and premature death. Current therapies fail to prevent disease progression due to their inability to suppress and/or reverse pulmonary arterial smooth muscle cells (PASMCs) driven muscularization of distal microvessels. The origin of these highly proliferative PASMCs remains incompletely understood, but may be closely related to the maladaptive behavior of contiguous pericyte (PC) populations. In addition to providing mural support to capillaries, PCs can differentiate into other cell types in response to stress. We recently reported that human PAH lung PCs share lineage markers and functional properties with PASMCs, such as morphology and contractility. We thus hypothesize that PASMCs in PAH vascular lesions originate from capillary PCs. Fate-mapping of PCs in chronic hypoxia mice revealed that PCs dissociate from capillaries and relocate to precapillary arterioles, where they co-express markers of mature SMCs and contribute to muscularization. Through single cell and bulk RNA-seq analysis, we discovered that the HIF2A/SDF1 signaling pathway is a master regulator of differentiation of PCs into SMC and a major modifier of PC dysfunction in PAH. We propose to: 1) demonstrate that HIF2a/SDF1 activation causes PC dissociation from pulmonary capillaries, 2) define the molecular mechanism by which HIF2a/SDF1 signaling drives PC differentiation into PASMC-like in human and mice, and 3) determine whether manipulation of HIF2a/SDF1 in PCs can alter the severity of vascular remodeling in animal models of PH. This project will provide novel insight into pericyte pathobiology and establish HIF2a/SDF1 as a potential therapeutic target in PAH, for which the first drugs to reverse muscularization and improve outcomes in PAH may be found.

NIH Research Projects · FY 2026 · 2020-12

PROJECT SUMMARY/ABSTRACT Ischemic diseases, including critical limb ischemia and myocardial infarction, affect millions in the U.S. While surgical interventions remain the standard of care, their inability to restore microvascular networks presents a major limitation. Our lab has advanced two key areas to address this challenge: (1) generating endothelial cells (iECs) from human induced pluripotent stem cells (iPSCs) using ETV2, providing a scalable autologous vascular cell source, and (2) demonstrating that mitochondrial transplantation enhances EC engraftment via PINK1-Parkin-mediated mitophagy. However, our preliminary data show that iECs exhibit a less robust bioenergetic response to mitochondrial transplantation than primary ECs, necessitating optimization. We previously demonstrated that mitochondria from donor cells lacking mtDNA (ρ0 cells) still efficiently trigger mitophagy and that PINK1 accumulation on donor mitochondria is essential for this process. Building on these insights, we will engineer donor ρ0 cells to generate mitochondria enriched with stable, membrane-bound PINK1, optimizing their ability to activate mitophagy and enhance iEC bioenergetics. Additionally, our work revealed that transferred mitochondria act as transient mitophagy triggers rather than integrating into recipient cells. This suggests that nanoparticles (NPs) coated with mitochondrial membranes could replicate this effect while offering enhanced stability and scalability. To pursue this, we partnered with Dr. Liangfang Zhang (UCSD), a leader in NP technology, whose group developed outer mitochondrial membrane (OMM)-coated NPs. We have successfully fabricated and characterized OMM-NPs and will now develop PINK1-enriched OMM-NPs to enhance mitophagy activation in iECs. Our overarching hypothesis is that optimizing mitochondrial products— either through genetically engineered donor mitochondria or PINK1-enriched OMM-NPs—will enhance iEC functionality and revascularization potential. We propose three aims: (1) genetically engineer mitochondrial donor cells to enhance mitophagy post-transplantation, (2) develop PINK1-enriched OMM-NPs for mitophagy induction, and (3) evaluate the efficacy of iECs enhanced with mitochondrial products in ischemic tissue treatment. These studies will provide mechanistic insights into mitochondrial transfer and establish a translatable strategy to enhance iEC engraftment, advancing vascular regenerative medicine.

NIH Research Projects · FY 2025 · 2020-12

Project Summary Immunotherapy using checkpoint blockade has revolutionized cancer treatment. The outcome of therapy directly results from changes imposed on the tumor microenvironment (TME) by checkpoint blockade. However, only a subset of patients respond. What controls this disparity is poorly understood. We propose to develop and apply tools that can help differentiate responders from non-responders in different pre-clinical tumor models soon after the start of treatment. We have shown that immuno-positron emission tomography (Immuno-PET) can be used to monitor infiltration status of specific subsets of immune cells, namely T cells and myeloid cells. We use small (~15 kDa) camelid-derived single domain antibodies (nanobodies) that have nM to pM affinity for their targets to perform immuno-PET imaging. Our unique chemical approaches provide imaging agents of unprecedented quality and sensitivity. Even cells that display proteins of relatively low abundance such as CTLA-4 can be clealrly imaged. We have shown in several (syngeneic) tumor models that monitoring the dynamics of cytotoxic T cells in the TME can be used to distinguish early responders from non-responders. This observation has allowed us to stratify animals into responders and non-responders, then excise their tumors, isolate the immune infiltrating cells, and subject these to single-cell RNA sequencing. These data show that the myeloid compartment and the cytokines and chemokines it produces, plays a major role in determining the outcome of anti PD-1 treatment. We propose to expand these initial findings to additional mouse tumor models, given the distinct cells of origin that give rise to them and their differences in susceptibility to immune intervention. This project is aimed at bringing to light key changes that take place in the TME early on, using immuno-PET. Our complementary molecular analyses will help design more effective therapies. Macrophages and DCs in the TME of responders produce CXCL9, a chemoattractant for cytotoxic T cells that helps maintain their activated state. This chemokine is therefore a key player in the outcome of anti-PD-1 therapy. We thus propose to re-engineer the TME by using chemistry to make novel CXCL9-fusion proteins and deliver them to the TME. Single-domain antibodies are perfect candidates for such fusions. Their small size allows excellent tissue penetration and their high affinity ensures efficient delivery to, and retention in, the TME. Imaging the distribution of CXCL9 or its receptor will shed further light on the anti-tumor immune status. We will therefore generate nanobodies as imaging agents specific for such cytokines and their receptors.

NIH Research Projects · FY 2025 · 2020-12

Our long-term goal is to determine the molecular mechanisms that control metabolic homeostasis and there by identify therapies for metabolic diseases. While metabolic regulation is vital for an organism’s function and survival, metabolic dysregulation associated with insulin resistance gives rise to diabetes, non-alcoholic fatty liver disease, dyslipidemia, and cardiovascular disease. The goal of this project is to define the role of hepatic TAZ (transcriptional co-activator with PDZ binding motif) in the regulation of glucose and lipid metabolism in normal and insulin resistant states. Proteins that regulate cell growth overlap with those that control metabolic homeostasis. Therefore, we determined whether TAZ, which is known to control proliferation, regulates hepatic metabolism. Using molecular, biochemical, and genetic approaches, we obtained preliminary data which reveal that TAZ, independent of the Hippo pathway, is a unique regulator of energy homeostasis in the liver. Hepatic TAZ protein is dynamically altered by fasting and feeding, and TAZ regulates the differential expression of gluconeogenic and lipogenic genes in response to fasting and feeding. However, in insulin resistant states, the dysregulation of TAZ leads to perturbations of both glucose and lipid homeostasis. To build on this preliminary work, we propose a series of molecular and mouse studies to delineate the molecular mechanisms whereby hepatic TAZ regulates glucose and lipid metabolism in physiologic and pathologic insulin resistant states. Our aims are listed below. Specific Aim 1 is to define the role of hepatic TAZ in the regulation of gluconeogenic gene expression and glucose homeostasis. Specific Aim 2 is to define the role of hepatic TAZ in the regulation of de novo lipogenic gene expression and triglyceride homeostasis. We expect that our studies will define a new role for TAZ in metabolic regulation and will identify molecular mechanisms whereby glucose and lipid homeostasis are physiologically regulated. We also expect that our studies will provide mechanistic insights into the pathogenesis of insulin resistance, and thereby enable the development of therapies for insulin resistance-associated metabolic diseases, including diabetes, hepatosteatosis, dyslipidemia, and cardiovascular disease.

NIH Research Projects · FY 2025 · 2020-12

eCD4-Ig is a potent and exceptionally broad fusion of the first two domains of CD4 to an antibody Fc domain and a short tyrosine-sulfated coreceptor-mimetic peptide. In rhesus macaques, adeno-associated virus (AAV)-expressed eCD4-Ig mediates consistent and very effective long-term protection against SHIV- AD8 and SIVmac239. eCD4-Ig also has properties that make it especially useful for establishing a functional cure in rhesus macaques and perhaps in humans. These include its potency, breadth, difficulty- of-escape, low immunogenicity when expressed by AAV, consistent expression by AAV, potent intrinsic ADCC activity, and collaboration with serum antibodies to mediate ADCC. These properties allow eCD4- Ig to circumvent two major problems associated with using AAV-expressed antibodies to establish functional cures, namely immune clearance and viral escape. In the forthcoming award period, we will improve the technologies allowing AAV-mediated delivery of eCD4-Ig by optimizing its expression as an AAV transgene, by eliminating residual antibody responses to AAV-delivered eCD4-Ig, and by assessing the role of the eCD4-Ig Fc domain in anti-eCD4-Ig antibody responses and in control an established SIVmac239 infection. These efforts will develop technologies that can be applied to the ongoing efforts by many laboratories to prevent new infections, to provide long- acting alternatives to combined antiretroviral therapies, and to reduce the scale of the viral reservoir.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT The gastrointestinal tract is composed of a single layer of epithelial cells that is in equipoise with immune cells and a vast number of microorganisms. Inappropriate responses to these microorganisms, either through genetic predisposition, altered immune or epithelial responses, or yet to be defined environmental influences, are postulated to lead to inflammatory bowel diseases (IBD). The immune signals that recognize and respond to bacterial and viral components of the microbiome remain incompletely understood. Interferons (IFNs) play a major role in antiviral immune defense in the intestinal epithelium, and are also important in regulating proliferation, differentiation, survival and effector functions of immune and non-immune cells. There are three classes of IFNs: type I IFNs (IFNα, β, and others), type II IFN (IFNγ) and type III IFNs, or IFNλs. To date, most studies investigating the use of IFNs on IBD have focused on type I IFNs and were not found to be effective. Dr. Ivan Zanoni recently reported that IFNλ decreases oxidative stress and intestinal damage in a murine model of colitis and that exogenous IFNλ can suppress intestinal inflammation. Importantly, we identified two unrelated patients with infantile-onset IBD with rare and functionally deleterious mutations in IFNλ2 and IFNλ3. Of note, each patient's disease improved significantly with age. We have preliminary data that IFNλ2 and IFNλ3 may be more important in the first months of life than FNλ1, and data illustrating more severe murine colitis in Ifnλ3-/- mice as compared to wild type mice. Taken together, we hypothesize that INFλs are essential modulators of mucosal homeostasis, prevent development of IBD, and hold therapeutic potential. Current therapeutics available for the management of IBD fail to treat a large number of patients. This work will provide a better understanding of the role of IFNλ in mucosal homeostasis, and may provide the groundwork to implement novel strategies to treat IBD by manipulating IFNλ signaling. Unraveling the role of IFNλ in maintaining mucosal homeostasis will be achieved through the following aims: (1) Establishing the developmental expression of IFNλs, related cytokines and receptors in humans at different ages using bulk and single cell RNA sequencing technologies (2) Determining the role of IFNλ signaling in predisposition to development of colitis in vivo using various murine models of colitis; (3) Characterizing the functional consequences of patient-encoded IFNλ variants in vitro using T84 cells as well as human control and patient- derived intestinal organoids. This award will enable Dr. Ouahed to acquire necessary structured training to become proficient in critical skills: RNAseq analysis, murine models of colitis and immune analyses, and generation/manipulation of intestinal organoids and epithelial analyses, complimented with didactic coursework, assisting her path to independence as a successful physician-scientist.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Poly ADP-ribose (PAR) is an RNA-like protein modification whose dysregulation is linked to age- dependent neurodegenerative diseases including ALS, Parkinson’s and Alzheimer’s disease. PAR is a core component of stress granules, a type of membraneless ribonucleoprotein (RNP) granules that can seed aberrant protein aggregation leading to neuropathology. Unlike RNA, PAR can form into both linear and branched structures. Strikingly, inhibitors of PAR polymerase, which is a class of FDA-approved anticancer drugs, were shown to mitigate neurotoxicity in cell models of neurodegeneration, reflecting a potential role of PAR in neurotoxicity. Two laboratories at Johns Hopkins University led by Sua Myong (Biophysics department) and Anthony K. L. Leung (Department of Biochemistry and Molecular Biology ) bring together orthogonal expertise in molecular imaging, chemical and proteomic methods to investigate the molecular basis of PAR- driven protein condensation and aggregation mechanism responsible for neurodegenerative diseases, which may pave new ways of developing therapy. Our recent discovery of a PAR modifying method (Leung et al, Mol Cell, 2019) and mechanism of FUS liquid-liquid phase separation in ALS/FTLD-linked cases (Myong et al, Mol Cell, 2019) places us in an ideal position for tackling the poorly understood role of PAR in biomolecular condensation implicated in neurodegenerative diseases. In Preliminary Studies, we discovered that (i) PAR is extremely potent in condensing FUS (fused in sarcoma), an RNA binding protein localized in stress granules and implicated in ALS/FTLD and (ii) PAR targeted proteome is enriched in stress granule components. Building on these exciting results, we propose to uncover the role of PAR in driving biomolecular condensation by employing single molecule, biochemical, meso-scale, biophysical and cellular platforms.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY Aberrant changes in mature neural connections and their function can be induced by infection and inflammation within the brain. The intracellular protozoan parasite, Toxoplasma gondii, is one pathogen that infects the brain, evokes a prolonged inflammatory response and can cause seizures. Persistent infection by this parasite is also associated with behavioral alterations and is a considerable risk factor for developing psychiatric illness, includ- ing schizophrenia. Over 30% of Americans are presently living with an incurable long-term infection of Toxo- plasma gondii, yet, despite its prevalence and implications for serious neurological disorders, we lack sufficient understanding of the effect of life-long parasitic infection on the brain – a major organ of the human body in which the parasite invades cells. This study seeks to elucidate the underlying mechanisms of brain circuitry dysregu- lation in long-term infection in response to this unmet need. Our recent studies have revealed that Toxoplasma gondii brain infection causes changes in inhibitory circuitry (specifically the loss of inhibitory synapses) along with changes in inhibitory neurotransmission, leading to the onset of seizures. Moreover, preliminary studies demonstrating increased expression of classical complement cascade components, elevated microglial activa- tion, and substantial microglial ensheathment of neurons and inhibitory nerve terminals, propose dynamic inter- active roles for the innate immune system and resident-macrophages of the brain in altering functionally mature neural circuits during parasitic infection. In this proposal, we test the hypotheses that molecular components of the innate complement pathway mediate inhibitory synapse loss and seizures, and that microglia remove and phagocytose these synapses following chronic Toxoplasma gondii infection. By capitalizing on multi-scale imag- ing modalities, the aims in this proposal will deliver novel ultrastructural and real-time insight into the impact of chronic parasitic infection on mature neural circuits and will offer a novel mechanism as to how persistent Toxo- plasma gondii infection may contribute to both seizures and psychiatric illness. This study is directly aligned with the NIH's Blueprint Program's mission to expand our understanding of the neuroimmune dynamic interactions that give way to neurological disorders.

NIH Research Projects · FY 2025 · 2020-09

Summary Cancer patients accumulate a wealth of electronic medical record (EMR) data during the diagnostic, decision- making, treatment, and follow-up processes of their care; most of these data are found in unstructured narrative form that remains dormant for secondary research purposes. Even when patients enroll in clinical trials that gather detailed case report forms, a holistic picture of their cancer journey is the exception, not the rule. Answering seemingly simple questions requires intensive manual review of patient records, a tedious process that can take hours per patient case, limiting researchers’ ability to construct large observational cohorts. With the exponential growth in the quantity of EMR data, it is not tenable for even a very large team of manual curators to thoroughly and exhaustively evaluate records at scale. Understanding the “deep phenotype” of a cancer patient requires a complete picture of both tumor and host. Critical cancer phenotypic variables include morphology, tumor location, extent of invasion, predictive and prognostic biomarkers, treatment exposure history, and response to treatment. Host phenotypic variables include fitness (eg performance status and comorbidities), adverse effects of treatment, and non-medical determinants of health (eg global distress, financial toxicity, and behavioral habits). Phenotypic profiles are typically constructed from multiple data sources and temporality is critically important. As many phenotypic variables are available only in EMR free text created over time, the cancer research community needs new, openly-available natural language processing (NLP) methods and systems to transform phenotypic detail from EMRs to data for advancing translational research. We have been developing DeepPhe, a platform for turning this rich data into computable longitudinal summaries of cancer diagnostic, prognostic, and treatment information. Since our last submission in 2019, there has been an unprecedented speed of developments within the Artificial Intelligence field, mainly in its subfield of text processing as exemplified by the advent of large language models (LLMs) and then very large language models. In this renewal, we will build on our and community’s methodology advancements, including the use of LLMs for EMR processing, to deliver a state-of-the-art, comprehensive, modern open-source tool for extracting deep phenotype information and provide novel visual analytics approaches. Our case studies will demonstrate the utility of our tools and drive the development of a vibrant community of cancer researchers using DeepPhe. Our community development efforts are aligned with the mission of the NCI Cancer Research Data Commons to advance methods of extracting and representing precision medicine phenotypes.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Influenza virus is a persistent global menace that every year infects an estimated 5-10% of adults and 20-30% of children worldwide causing over 500,000 influenza-related deaths. Most annual influenza infections are in the very young, the elderly, and in individuals with chronic health conditions such as asthma. However, recurrent influenza pandemics caused by the emergence and spread of highly pathogenic novel influenza A strains, such as occurred in 2009, disproportionately causes severe illness in healthy children and younger adults. This study of life-threatening influenza virus lower respiratory tract infection (LRTI) in young hosts is designed by an established multidisciplinary group of investigators to better understand how host innate and adaptive immunity to influenza virus is associated with disease susceptibility, severity and clinical outcomes. In the Pediatric Intensive Care Influenza (PICFLU1) study, we hypothesized that infection with the influenza virus triggers hypercytokinemia and immune dysregulation in a genetically susceptible host resulting in severe life-threatening infection. Confirming our hypothesis, in PICFLU1 (AI084011, enrolling 2008-2016), we identified a hyperinflammatory phenotype coexisting with innate immunosuppression; both were associated with mortality. We also identified associations between functional variants in IFITM3 and MBL2 with pediatric influenza-related death. In the Immunobiology of Influenza Virus-Related Critical Illness in Young Hosts study (PICFLU2, enrolling 2020-2025), we test the hypothesis that distinct severe influenza LRTI phenotypes – defined by host immunobiology – can be identified for targeted preventive and therapeutic interventions. In this study we aim to: 1. Identify biomarkers in children with severe influenza LRTI that can be used for prognostic stratification and predictive enrichment in future immunomodulatory clinical trials; 2. Determine if pre-existing strain specific immunity to influenza virus protects against life-threatening disease and influences viral shedding and disease severity; and 3. Identify genes essential for anti-viral immunity and/or containment that explain host susceptibility to severe influenza infection or its outcome. To achieve these aims, we will enroll 600 additional children and young adults with confirmed influenza infection (300 intensive care unit and 300 ward or outpatient) across 35 PICFLU sites. Across PICFLU studies (2008-2025) we will have DNA on ~1,000 young hosts infected with influenza virus to identify important endophenotypes for risk stratification and predictive enrichment in future clinical trials targeting prevention of and more rapid recovery from severe influenza-related disease. Identified influenza virus susceptibility and severity genes are potential “druggable targets” for immune modulation. Our findings could personalize the care of young individuals with severe influenza infection based on distinct immunobiologic host phenotypes based on patient age, influenza strain, clinical presentation, innate and adaptive immune biomarkers and host genetics.

NIH Research Projects · FY 2024 · 2020-09

Summary Eukaryotic genomes are packaged in chromatin, linear arrays of nucleosomes in association with nonhistone proteins performing structural, enzymatic, and regulatory functions. This proposal aims to elucidate the interplay between chromatin organization, remodeling and modification and two key nuclear functions: gene transcription and DNA repair, using single molecule imaging in living cells to obtain comprehensive datasets on the real-time dynamics of transcription and DNA repair proteins and chromatin motions, and their integration with theory and modeling with predictive power. We will apply single molecule tracking (SMT) to image at high spatiotemporal resolution the organization, dynamics, regulation and function of a prototypical pioneer transcription factor, GAGA factor (GAF) in Drosophila. We will image the global and local nuclear organization and dynamics of wild-type and mutant GAF binding to cognate DNA elements genome-wide, and at Hsp70 promoters in live hemocytes. We will image the global and local dynamics of eight prominent chromatin and transcription protein effectors linked to GAF functions. SMT datasets from the factors imaged above are used to construct theoretical models for GAF interactions with chromatin targets and test models by experimental manipulation. Studies will be extended to human NF-Y, a distinct pioneer factor that makes accessible chromatin at the Hsp70 promoter in human cells. We will examine the interplay between chromatin organization and dynamics and DNA repair, using very fast (vf) CRISPR that can generate a double strand break (DSB) anywhere in the genome with high spatiotemporal resolution. We will determine DSB repair kinetics and chromatin reorganization through time- resolved chromatin analysis and real-time imaging of repair factors after generating DSB. We will determine the impact of topologically associated domains and loop extrusion on chromatin modifications and relaxations that accompany DNA repair, and integrate chromatin and DNA repair kinetics datasets to construct theoretical models for 4D chromatin reorganization during DSB repair. We will employ a series of chromatin remodeler and DNA damage response mutants to document causal relationships, and expand the reach of vfCRISPR to other DNA repair processes including base excision repair and mismatch repair. We will merge the above approaches to explore how DNA repair-mediated chromatin alterations affect transcription in human cells, and reciprocally, how transcription and associated chromatin changes influence DNA repair dynamics. We will image dynamics of pioneer and non-pioneer factors and key DNA repair enzymes at the active Hsp70 gene in living human cells, varying the timing of DSB and heat shock to evaluate the influence of DSB on different stages of transcription. Simultaneous imaging of labeled locus and nascent Hsp70 mRNA will reveal how transcription affects dynamics of the damaged locus.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Our goal is to build a comprehensive atlas of intestinal epithelial, stromal, and immune cells from both monogenic and polygenic very early onset inflammatory bowel disease (VEOIBD) patients (0<6 years of age) at the single cell level by leveraging the diverse expertise of our VEOIBD Consortium (www.veoibd.org). We hypothesize that developing this atlas will inform upon disease pathogenesis and enable targeted therapeutics for VEOIBD. Moreover, identification and characterization of novel VEOIBD causative genetic variants will inform upon novel IBD pathogenic networks extending beyond VEOIBD. We selected key members of our Consortium to facilitate these goals, including three core patient recruitment sites (Dr. Scott Snapper, Boston; Dr. Christoph Klein, Munich; Dr. Aleixo Muise Toronto), each with combined expertise in immune phenotyping (Snapper), transcriptomics (Dr. Snapper, Dr. Alex Shalek, Dr. Ordovas-Montanes, Kean), proteomics (Dr. Mathias Mann), functional genomics (Drs. Muise, Dr. Klein, Dr. Snapper) as well as expertise in intestinal organoid generation and functional assessments (Dr. Hans Clevers/Dr. Edward Nieuwenhuis), data analysis/network development (Dr. Eric Schadt) and data sharing (Dr. Larsson Omberg/Sage Bionetwoks). To generate a VEOIBD cellular and molecular atlas, our goal is to pursue in VEOIBD patients a multi-omic approach to generate in-depth patient- specific libraries of data including: 1) single cell and bulk transcriptomic and proteomic data generation from biopsies; 2) paired single cell and bulk transcriptomics and proteomics from patient-derived organoids; 3) paired transcriptomics, and proteomics from patient blood. To further inform upon this data set, we will couple these data with deep immune phenotyping (through mass cytometry) and functional characterization of VEOIBD patient-derived organoids. In this application, we propose to study 150-200 VEOIBD patients (< 6 years) and 30- 40 age-matched controls employing bulk and single-cell RNA sequencing and proteomics from biopsies and paired peripheral blood samples. In addition, we will perform deep immune phenotyping on intestinal biopsies (75-100 patients; 15-20 controls) with paired peripheral blood, as well as organoid development coupled with bulk and single cell RNA sequencing and functional assessments on 100-150 VEOIBD patients and 20-30 controls. The multitude of cell types in the intestine contributing to disease pathology has to date been mostly studied in aggregate form. In order to deconvolute the cell specific signal within the tissue and ‘assign’ disease relevance, we will apply our multiscale network (MultiNet) approaches in combination with single cell omics technology. Based on the above data sets, we will validate novel VEOIBD causal gene variants which will not only lead to a better understanding of the disease for these patients but also enhance the development of the VEOIBD immune atlas and epithelial signature and associated networks. Importantly, we will share these libraries of data and organoids with the scientific community to accelerate our mission of identifying therapeutic targets, and cures for VEOIBD.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Genetic disorders and congenital malformations, which may be genetic, are the leading cause of infant mortality in the United States. However, we still do not fully understand which genetic disorders are responsible for infant deaths and the full scope of their impact. This NIH K23 research proposal represents a multidisciplinary effort to gain further understanding into the genetic contributions to infant mortality, leveraging the expertise that Dr. Wojcik has already gained through her dual training in clinical genetics and in neonatal-perinatal medicine and providing further training in genomic analysis, epidemiology and biostatistics, and clinical research study design. Building off of Dr. Wojcik's prior research on the implications of genetic diagnoses in the infant and neonatal period and her experience in exome analysis for novel disease gene discovery, the objective of this study is to determine the prevalence of Mendelian genetic disorders within a cohort of deceased infants (Aim 1) and to evaluate the public health impact of these diagnoses using parental survey data (Aim 2) and data obtained from the National Center for Health Statistics (Aim 3). Under the mentorship of internationally-recognized experts in neonatology and genomic medicine (Pankaj Agrawal, MD, MSSc), human genetics and rare disease gene discovery (Heidi Rehm, PhD), the ethical/legal/social implications of clinical genetics (Ingrid Holm, MD, MPH) and in collaboration with experts in parental grief after the loss of an infant (Richard Goldstein, MD), clinical genetics (Wen-Han Tann, MBBS), perinatal mortality/epidemiology (Dominique Heinke, ScD), with additional research and career mentoring from successful researchers in human genomics (Alan Beggs, PhD and Robert Green, MD, MPH), Dr. Wojcik will strive to provide answers to bereaved families. Concurrently, she will gain the training necessary to build her own career as an independent clinician- researcher with a focus on the intersection of clinical genetics and neonatology towards a better understanding of infant mortality and ultimately its prevention.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Inflammatory bowel disease (IBD), comprised of ulcerative colitis (UC) and Crohn's disease (CD) are chronic disorders of the GI tract with rapidly increasing prevalence. Despite recent advances in treatment, a significant proportion of patients have suboptimal responses to medical therapy, leaving an urgent need to identify new therapies. One promising new approach to treat IBD is through the manipulation of regulatory T cells (Tregs). Tregs are an immune modulating subset of CD4+ lymphocytes that antagonize the activation and effector function of multiple immune cell types and promote tolerance to self-antigens. Adoptively transferred Tregs are effective in murine models of IBD. An alternative approach to disease management through Treg manipulation is to increase Treg numbers in vivo. Interleukin-2 (IL-2, Proleukin®) is a T cell growth factor. IL-2 is currently licensed for the treatment of metastatic renal cell carcinoma and metastatic melanoma. At low doses, IL-2 promotes the selective activation and expansion of Tregs in humans. Tregs constitutively express CD25, a component of the high-affinity IL-2R, while CD25 is only transiently expressed by activated conventional T effector cells. Low-dose (LD) IL-2 selectively expands Tregs in humans and is safe in chronic GvHD and other phase 1 and 2 clinical trials. We recently published that LD IL-2 is protective in a humanized mouse model of IBD. Based on this preclinical data, we initiated and have almost completed a Phase 1b/2a clinical trial of LD IL-2 in 24 patients with UC. Subcutaneous (sc) LD IL-2 is well tolerated and associated with a biological response and pTreg expansion in UC: overall, 41.6% of patients have achieved either response or remission including 60% of patients treated with the maximum effective dose (MED). With this exciting data, we have developed a Phase 1b/2a clinical trial to assess the safety and the efficacy of LD SC IL-2 for the treatment of CD and to study the immunoregulatory effects of IL2 in the peripheral and mucosal immune compartments. To date, we have obtained: 1) provisional IRB approval; 2) an Investigational New Drug (IND) approval from the FDA; 3) support from commercial entities to supply drug free of charge to patients. We have designed a comprehensive immunophenotyping strategy to assess the biological effects of LD IL-2 and to correlate these findings with clinical outcomes. In this study we propose: Aim 1: To determine the safety of sc LD IL-2 in the treatment of moderate-to-severe CD. We propose a phase 1b/2a clinical trial of daily sc LD IL-2 for 8 weeks in CD patients to determine the maximum effective dose (MED) and safety profile, and to assess a signal of efficacy. Aim 2: To determine in CD patients whether sc LD IL-2 modulates peripheral blood and lamina propria Tregs in vivo and correlates with clinical outcome. We will perform deep immunophenotyping in CD patients treated with LD IL-2 and comprehensively assess the effects of LD IL-2 on CD4+ Tregs and other immune cells in both peripheral and mucosal compartments, and correlate changes in immune phenotype with clinical outcome. Overall this trial is designed to determine the MED and safety profile of LD IL-2 in CD, to obtain a signal of efficacy, and to assess mechanistic underpinnings.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Pediatric chronic widespread pain (CWP) is a serious public health problem resulting in high levels of healthcare utilization and disability. Youth with CWP also frequently report exposure to adverse childhood experiences (ACEs; abuse/neglect, violent/conflictual home environment, etc.) and a significant subset continue to experience physical and psychosocial impairment long-term. Certain mind-body interventions such as mindfulness-based stress reduction (MBSR) or meditation may be particularly appropriate for youth with CWP as they have been shown to modulate stress-induced maladaptation of the HPA-axis, autonomic nervous system, cardiovascular system, and brain structure (e.g., hippocampus). However, it is currently unknown if these targets are affected in youth with CWP. Preliminary research indicates that allostatic load (AL), or “wear and tear” on the nervous system due to stress, may contribute to pain chronicity. Similarly, evidence suggests that the hippocampus, a brain structure that is among the most deleteriously affected by stress, plays a role in pain perception. However, no study to-date has examined AL and hippocampal functioning in relation to stress exposure in youth with CWP. Mind-body interventions such as MBSR or meditation are an important and safe therapy option for both pain and stress reduction in youth with CWP and may modulate the negative impact of ACEs, so there is a critical need to know if these mechanisms are engaged in this population. The proposed research project utilizes multifactorial physiological and neuroimaging measurement techniques to enhance our understanding of the potential role of these mechanisms in pain-related impairment and responsiveness to mind-body interventions over time. The aims of this submission are to better characterize AL, assessed via a multifactorial composite, and hippocampal functioning via fMRI in pediatric CWP as specific targets for mind-body interventions that can lead to treatment optimization and improved compliance. The long-term goal of this K23 award is for the candidate to establish an independent research career aimed at carrying out mechanistically informed mind-body interventions. Chronic widespread pain was selected as a model condition because of its commonality within pediatric pain clinics and strong association with central sensitization, high stress, psychological impairment, and functional disability. The primary training objectives are to acquire expertise in neurobiological measurement and interpretation and to leverage current research activities in complement to this proposal that will lay the foundation for future mechanistically driven clinical trial grant submissions as an independent investigator. The candidate will accomplish this through: 1) mentorship in a clinical/research environment, 2) hands-on training in the neurobiological measurement of the physiological effects of stress, complemented by didactics in neuroendocrine and fMRI measurement, 3) mentorship and coursework in advanced data analytic techniques, 4) leveraging psychosocial practice activities and clinical trial involvement in conjunction with effort on the proposed award, and 5) execution of the proposed research plan and submission of an independent investigator award application. These studies and training will provide the necessary data to inform the development of an R01 clinical trial designed to test the physiological mechanism(s) of response to MBSR, meditation, and other non-pharmacological techniques in youth with CWP.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Therapeutic trials in rare diseases are challenging, particularly those that involve children and therapeutic choices with potentially life or death consequences. Patient-specific tissue-chip approaches have the potential to demonstrate therapeutic efficacy without exposing patients to risks associated with experimental therapy or randomization to the control arm. Moreover, patient-specific tissue-chip approaches may de-risk clinical trials by optimizing patient selection and inform future clinical trials by elucidating mechanisms that underlie the vari- ation in patients' therapeutic responses. Achieving these long range goals requires demonstration that patient- specific tissue-chip platforms accurately predict the therapeutic responses of individual patients. Here we pro- pose to test the hypothesis that tissue-chips predict therapeutic responses in catecholaminergic polymorphic ventricular tachycardia (CPVT), a rare inherited arrhythmia and to gather information critical for the design of future therapeutic trials. CPVT is among the most malignant and difficult to treat of the inherited cardiac arrhythmias. A hallmark of CPVT is ventricular arrhythmia induced by exercise and emotional stress. Despite standard-of-care therapy, in- cluding β-blockers, implantable cardiac defibrillators (ICDs), or surgical sympathetic cardiac denervation, the estimated 8 year fatal or near-fatal event rate is ~15%, with death occurring in ~6%. Over the past decade, fle- cainide has proven to be effective therapy for many CPVT patients, either in combination with β-blocker or as monotherapy. However, some patients do not respond to flecainide. Mechanisms of non-responsiveness and predictors of response have not been identified. We have recently reported that CaMKII inhibition is a promis- ing therapeutic strategy for CPVT, and future therapeutic trials of CaMKII inhibition will likely be performed in CPVT. In the UG3 phase of this proposal, we will recruit patients whose clinical response to flecainide is known, and generate iPSCs from these patients. At the same time, we will optimize tissue chip platforms to assess ar- rhythmia risk using patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). In the UH3 phase, we will per- form two "clinical trials" in a dish: First, in a "retrospective clinical trial" in a dish, we will compare patients' known flecainide responses to the responses of their iPSC-CMs. Second, we will assess the spectrum of genotypes where CPVT inhibition is effective, and determine if there are favorable or unfavorable interactions between CaMKII inhibition and flecainide. Together these studies will rigorously test the hypothesis that personalized disease models can predict indi- vidual patient therapeutic responses and can be used to help plan future clinical trials.

NIH Research Projects · FY 2024 · 2020-08

In mammals, hematopoietic stem cells (HSCs) first arise from a specialized hemogenic endothelium that lines the developing embryonic aorta, migrate to and expand in the fetal liver, and ultimately colonize the bone marrow, which supports hematopoiesis throughout adult life. These distinct anatomic locations harbor specialized microenvironments that support the developmental maturation, expansion, and ultimately the balance of self-renewal and differentiation of HSCs. The transcriptional programs that promote formation and differentiation of hematopoietic stem and progenitor cells (HSPCs) have been widely interrogated, but much remains to be learned about the supportive niche cells of the hematopoietic microenvironment and the mechanisms of cell-cell interaction that specify HSC emergence during development, HSC migration, lodging, and expansion in fetal niches, and the ultimate quiescence, self-renewal, and differentiation in the bone marrow. In our preliminary data, we have gathered evidence for number of cell types, including endothelial cells, mesenchymal cells, macrophages, neural crest derivatives, and somites as components of the hematopoietic niche. We will gather comprehensive “omics” data to catalogue the gene expression programs within the distinct hematopoietic niche cells that occur during development in the aorta-gonad-mesonephros (AGM), fetal liver, bone marrow, and placenta (aim 1). Our approach begins with tomo-seq, which enables us to discover gene expression patterns unique to cell populations like endothelium that have region-specific specialization. We will validate cell-specific expression in FACS purified cells by single cell RNA-seq and in situ hybridization, and will document functionality using morpholino and CRISPR knock-down in the experimentally tractable zebrafish model. We then use ATAC-seq to define functional open chromatin around these genes, and motif-finding software to identify DNA-binding regulatory factors that are candidate drivers of hematopoietic cell fate. We will employ a computational pipeline and develop novel algorithms to analyze these data (aim 2). Hypotheses emerging from aims 1 and 2 will be tested by constructing novel reporter strains of zebrafish and mice, as well as engineered pluripotent stem cells carrying synthetic reporters and drivers (aim 3). Our goal is to define the molecular circuitry that specifies niche cells during the critical periods of HSC emergence and expansion, and to probe cross-talk between niche elements and HSPCs. We hope to glean unique insights into the molecular mechanisms that drive hematopoietic formation and maturation during embryonic development, and to enhance our understanding of HSC maintenance, quiescence, self-renewal and differentiation.