Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2021-08

Motor neuron diseases (MNDs) such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are a class of progressive neurodegeneration disorders, often caused by minor genetic abnormalities to which motor neurons are particularly vulnerable. Because each of these diseases is rare and the molecular pathologies so diverse and unresolved, it is hard to envision a therapeutic intervention tailored to each individually, or a single treatment to effectively treat them all. Current genome editing tools are easily programmable to target discrete genomic loci and affected tissues of MNDs are largely similar. Thus, a general genome editing therapeutic strategy for MNDs fitted to each mutation could meet this urgent need. Base editing tools can theoretically correct any C:G>T:A and A:T>G:C transition, yet the factors that determine efficiency and precision of base editing are not fully understood, and editing outcomes at a given locus are frequently unpredictable. For the development of base editing correction strategies at a great number of loci, screening through a multitude of base editor variants – currently any permutation of >10 deaminase enzymes and >15 Cas proteins, and counting – and sgRNA combinations for every target is prohibitive. A clear understanding of Cas protein, deaminase, and sequence determinants of editing outcomes is needed to facilitate the design of base editing strategies. We intend to develop a general workflow to design effective base editing strategies for causal MND SNPs and deliver these tools to MND affected tissues. SMA is a monogenic MND with a well-defined genetic cause, and animal models harboring the human causal gene that faithfully recapitulate disease phenotypes of SMA patients. Successful development of an effective and safe SMA genome editing treatment will assist the development of similar genome editing therapeutics for other genetic MNDs, including some forms of familial ALS. In this project we will (1) create a computational predictive model of base editing to facilitate the design of effective base editing strategies; (2) develop protocols to efficiently deliver base editing therapeutics to disease relevant tissues in mice to enable genome editing; and (3) use this pipeline to optimize a base editing therapeutic to rescue SMA in mice, and develop genome editing therapeutics for other causal MND mutations, focusing first on the most common single point mutation causal to ALS in North America, SOD1A4V. I will take advantage of the world-class genomics environment of the Broad Institute and the expertise in AAV-delivery at the Stanley Center for Psychiatric Research to realize these goals and develop skills that will help me to continue this work as an independent investigator. Through mentorship meetings and courses on grant-writing and data visualization I will improve my science-communication skills so that I may compete successfully for additional NIH R01 and R21 funding as a faculty member. By attending conferences and publishing my work I intend to establish myself as leader in the field of therapeutic genome editing for MND.

NIH Research Projects · FY 2024 · 2021-08

Abstract: Intrinsically disordered proteins (IDPs) are found in over 50% of human proteins where they play essential roles in a wide range of cellular functions including transcriptional regulation, DNA repair, cell signaling, and apoptosis. As a result of their importance in key processes associated with cellular growth, proliferation, and death, proteins containing IDPs are often associated with cancer. The ability of IDPs to adopt a wide range of conformations raises a number of key challenges to standard biochemical, biophysical, and computational techniques. Despite these challenges, our ability to treat many cancers depends on an understanding of the molecular basis for diseases. This, in turn, presents a pressing need to understand the mechanistic basis of IDP function and dysfunction. This proposal will study protein-nucleic acid interactions driven by intrinsically disordered proteins in two pressing diseases: COVID-19 and cancer. For the F99 phase (Aim 1) of the award, I will build upon my computational and experimental biophysics training to continue investigating the SARS- CoV-2 nucleocapsid protein and its ability to package its viral genome. The COVID-19 pandemic, preceded by previous coronavirus outbreaks caused by SARS and MERS, necessitates study of these viruses in order to better combat them. Coronaviruses contain large RNA genomes that are packaged into a relatively small virion, mediated by the nucleocapsid protein, a highly disordered multidomain RNA binding protein. A current outstanding question is how SARS-CoV-2 package their 30 kb genomes into a relatively small (<100 nm) virion. The conserved structural motifs in coronavirus genomes known as packaging signals has been shown to confer genome specificity, yet the relationship between packaging signals and genome compaction are opaque. My thesis work combines single-molecule fluorescence spectroscopy with all- atom and coarse-grained simulations to construct a mechanistic understanding of how N protein drives RNA packaging. Success of this project will reveal the role of IDP-encoded multivalency in selective genome packaging. Since the architecture of the nucleocapsid protein is conserved throughout coronaviruses it will also present new insight into mechanisms that can be broadly targeted for therapeutic intervention. The K00 phase (Aim 2) of this proposal will study the contribution of IDPs in transcriptional regulation, genome organization and cancer development. Fusion-oncogenes are a common genetic translocation event which often involve a DNA binding domain becoming fused to an IDP. During the post-doctoral phase I will obtain training in super-resolution microscopy to investigate the effects of transcriptionally active fusion-oncogenes. Several studies have shown that IDPs from transcription factors drive the formation of transcriptional assemblies (transcriptional condensates) at sites of gene expression. I will test the hypothesis that fusion-oncoproteins lead to the formation of long-lived aberrant transcriptional condensates that drive the expression of proliferative genes. This will provide direct mechanistic insight into the molecular basis of fusion-oncogene driven cancers. These combined training plans will prepare me for a successful research career using quantitative biophysical and single-molecule techniques in the field of mechanistic cancer biology.

NIH Research Projects · FY 2025 · 2021-08

PROJECT ABSTRACT Congenital hearing loss affects 1 in 500 newborns, making it the most common sensory disorder in humans. Children with hearing loss are at risk for poor speech, language, and social development with noted negative effects on quality of life. The most effective treatment for severe-to-profound hearing loss in children is cochlear implantation. The cochlear implant (CI) is the most successful and widely used sensory prosthesis in humans and cochlear implantation has restored hearing to hundreds of thousands around the world. While the majority of CI users experience significant improvement in speech perception, a significant portion do not. There is a critical need to identify individuals at-risk for poor outcomes prior to cochlear implantation in order to: (1) provide accurate pre-operative counseling, (2) tailor post-operative care, and (3) develop new treatment strategies for these types of hearing loss. To date, the best predictors of CI speech perception outcomes rely on complex statistical modeling of clinical factors associated with hearing loss or intra-operative electrocochleography (ECoG), neither of which can be routinely used pre-operatively. There is increasing evidence that specific genetic variations that negatively affect the health of spiral ganglion neurons (SGNs) are associated with worse postoperative CI speech perception outcomes. The primary goal of this grant proposal is to better understand genetic contributors to postoperative speech perception outcomes in children. We recently showed that variations in the gene TMPRSS3 are associated with worse CI speech perception outcomes. Although TMPRSS3 is one of the most common causes of genetic hearing loss, the function of the TMPRSS3 protein and the mechanism by which it causes hearing loss are not known. TMPRSS3 is involved in expression of calcium- sensitive potassium channels in inner hair cells. A knock-out mouse model shows rapid hair cell degeneration soon after the onset of hearing. However, deafness-causing mutations in TMPRSS3 are notable for causing not only a severe-to-profound congenital hearing loss (DFNB10) but also a later onset post-lingual hearing loss (DFNB8). In addition, the expression of TMPRSS3 includes hair cells and also SGNs. We hypothesize that TMPRSS3 has functions in the inner hair cells as well as in the SGN. The aims of this project are to: (1) examine the complex interplay between genetics, ECoG, and post-operative speech perception scores in children with CIs, (2) improve our understanding of TMPRSS3 through development of a new mouse model for late onset DFNB8 hearing loss, and (3) develop a new gene therapy for TMPRSS3 hearing loss. The expected results of this study will be: (1) a genetic risk index for poor CI outcomes in children, (2) a better understanding of the function of TMPRSS3 in hearing and hearing loss, and (3) a novel gene therapy for TMPRSS3 hearing loss. The results of this study will have direct clinical impact as well as pave the way for future gene therapy trials in humans.

NIH Research Projects · FY 2025 · 2021-08

This application requests support for a training program that prepares highly qualified pediatricians to assume leadership positions as investigators in Pediatric Infectious Diseases. Based in the Division of Infectious Diseases at Boston Children’s Hospital, the program takes advantage of the rich academic and research resources of Harvard Medical School, Harvard T.H. Chan School of Public Health, and affiliated teaching hospitals to bring together outstanding didactic experiences and research opportunities relevant to infectious diseases of children. Support for three positions per year is requested. Pediatricians enter the program for a minimum of three years of integrated clinical and research training that includes at least two years of supervised research under the mentorship of a member of the teaching faculty. The 31 members of the teaching faculty are accomplished investigators and experienced mentors chosen from the Division of Infectious Diseases at Boston Children’s Hospital and affiliated units at other Harvard institutions. Under the guidance of the research mentor and the Program Steering Committee, an individualized curriculum is designed for each trainee in one of three broad areas of investigation: microbial pathogenesis, host response and vaccines, or epidemiology and health outcomes. Trainees in the epidemiology and health outcomes pathway have the opportunity to earn a Master of Public Health degree from the Harvard School of Public Health as an integral part of the training program, providing training in epidemiology, biostatistics, clinical trial design, and analytical methods. Additional specialized training in infection control, antimicrobial stewardship, or transplant infectious diseases is also offered. All trainees have seminars and tutorials in grant preparation and scientific writing that supplement the mentored research experience. An individual Scholarship Oversight Committee is appointed for each trainee to monitor progress and to provide career guidance. During the second or third year, trainees are expected to present their work at national meetings, to prepare one or more manuscripts for publication, and, in most cases, to apply for a K08, K23, or other career development award to support the transition to independence as an investigator. Over the past ten years of the Pediatric Infectious Diseases fellowship program, 27 of 30 graduates have remained in academic positions, with 15 involved in research-based investigation, and 12 involved in primarily clinical academic activities.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT Our overarching vision of the Boston Children’s Hospital and Harvard Medical School Intellectual and Developmental Disorders Research Center (IDDRC) is to improve the lives of individuals with IDD with timely and efficient translation of scientific research through collaboration among our institutions’ exceptional investigators and clinicians in partnership with the external IDD community. To achieve our broad vision, we organize the Center’s research around four clearly defined themes: 1) Discovery of genetic and non-genetic causes of IDD; 2) Determination of the cellular bases of IDDs using advanced imaging and analysis tools; 3) Identification of translational phenotypes in animal models of IDD to validate therapeutics; 4) Accelerated translation of research discoveries into new prevention and treatment strategies for IDDs. The Center currently supports 106 research projects and 68 investigators through the Administrative Core and the four scientific Cores. The Administrative Core is the hub of the Center as it provides both scientific and administrative leadership which promotes synergistic, interdisciplinary interactions that address IDD-related issues at multiple levels, trains the next generation of young investigators and facilitates outreach and dissemination of IDD research to diverse audiences. The Genetic Analysis and Editing Core (GAEC) provides access to the latest technological advances both in genetic analysis and in gene editing. The Cellular Imaging Core (CIC) facilitates the study of cellular and circuit biology through state-of-the-art imaging and image analysis services which enable visualization of fixed tissue, in vitro organ explants and in vivo awake behaving model organisms. The Animal Behavior and Physiology (AB&P) Core provides investigators with access to a wide range of validated technologies and scientific expertise for in vivo rodent behavioral, biochemical and physiological measures in a well-controlled and rigorous preclinical setting. Finally, the Clinical Translational Core (CTC) provides full access for IDDRC PIs to all services required for translation of research discoveries into clinical innovation; this ranges from biosample collection and storage, generation of patient-derived stem cell models of diseases, drug screening platforms on the preclinical side to neurobehavioral and electrophysiological assessment of IDD patients of different age groups as well as statistical and regulatory support of clinical trials. The Cores all interact through shared projects, providing complementary expertise and tools to address unique aspects of central scientific questions, and the Core directors meet to exchange ideas and optimize resource utilization in the monthly Executive Committee meetings. This integrated approach aims to enhance the translational potential of basic research in IDD by putting patients at the center of the drug discovery cycle starting with genetic and molecular screens through clinical trials. The Center engages frequently with patient- advocacy groups and the IDD community in a bi-directional manner to ensure that their needs and concerns steer the Center’s efforts.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Title:Delineation of pathogenic mechanisms of NOS1AP and TRIM8 mutations in monogenic SRNS/FSGS Steroid resistant nephrotic syndrome (SRNS) is a leading cause of childhood chronic kidney disease1, marked by proteinuria and edema. Renal biopsy typically reveals focal segmental glomerulosclerosis (FSGS). 59% of children with SRNS are unresponsive to standard therapy1,2. A majority of them progress to end-stage renal disease with loss of the kidney glomerular filtering cells, podocytes1,2. Mendelian genetic causes of SRNS/FSGS have been detected in ~11-30% of pediatric cases3–6. SRNS/FSGS disease genes encode critical pathway components in podocyte biology2,7–10. Human mutations impair these SRNS/FSGS pathways, causing podocytopathies2,7–10. The proposed research will explore the pathogenic mechanisms underlying two novel monogenic causes of SRNS/FSGS in human NOS1AP and TRIM8 mutations, which were discovered by the applicant. The applicant’s preliminary data generated the hypothesis that human NOS1AP and TRIM8 mutations cause SRNS/FSGS through dysregulation of the CDC42 pathway and TRIM8 E3 ligase functions, respectively. The applicant, thus, proposes the following specific aims (SAs) using innovative cell biological, proteomics, and mouse models approaches: (SA1) Define the mechanism of CDC42 dysregulation caused by NOS1AP SRNS mutations; (SA2) Dissect the pathogenesis of TRIM8 SRNS/FSGS mutations in podocytes; (SA3) Delineate the pathogenesis of NOS1AP and TRIM8 mutations in SRNS in mice. The applicant has created a comprehensive career development plan supported by his mentor to (1) ensure his progress and success in carrying out this research proposal and (2) to facilitate his transition to an independent research career focused on disease modeling of nephrotic syndrome. This plan begins with regular meetings with his mentor and advisory committee—national and global academic leaders in medicine and science—to provide research and career guidance. The plan additionally includes (i) research and career development seminars, (ii) proteomics and microscopy methodology courses and (iii) activities for career growth including conference presentations and publications, mentoring of junior trainees, and application for independent research funding. The applicant and mentor have, also, agreed upon a transition plan to distinguish himself from the mentor’s laboratory. His training will be carried out in an unparalleled academic environment at Boston Children’s Hospital and Harvard Medical School, which provides dedicated career development programs and all necessary research support and supplies through his mentor and institutional core services. Collectively, this research and career development proposal is a product of the applicant’s ambition and capacity to transition to an independent research career in nephrology.

NIH Research Projects · FY 2025 · 2021-07

The hereditary spastic paraplegias (HSP) are a group of over 80 neurodegenerative conditions and the most common cause of inherited spasticity and associated disability. This K08 proposal focuses on prototypical forms of HSP in children caused by biallelic loss-of-function variants in four genes that encode subunits of the adaptor protein complex 4 (AP-4): AP4B1, AP4M1, AP4E1, and AP4S1. Progressive degeneration of the cortico-spinal tracts renders most children with AP-4-associated HSP wheelchair-dependent by the age of 10 years. Currently, there are no therapies that halt disease progression, and few patients are known to have survived into adulthood, highlighting the urgency for research into the fundamental biology of HSP. The AP-4 complex is crucial for the intracellular trafficking of transmembrane proteins, including the autophagy-related protein ATG9A. How altered trafficking of ATG9A leads to impaired neurodevelopment and axonal degeneration and how ATG9A distribution can be restored is currently unknown. In this proposal, I will address this unmet question by developing neuronal models of AP-4 deficiency and testing novel modulators of AP-4-dependent protein trafficking. In preliminary experiments, we have systematically screened small molecule modulators of ATG9A trafficking using a cell-based phenotypic assay that measures ATG9A distribution as a surrogate of AP-4 function. We identified several modulators of ATG9A distribution. I will test the hypothesis that these restore trafficking and function of ATG9A in vitro in neurons derived from AP-4-HSP patients and in vivo in an ap4b1-/- zebrafish model. This proposal presents a five-year research career development program focused on the study of AP-4 in HSP to expand the breadth and depth of understanding the role of protein trafficking and autophagy in this group of diseases. The goal is the establishment of a cross-organismal screening platform to identify and develop novel modulators of protein trafficking for the treatment of HSP. The candidate is currently a resident in Child Neurology at the Department of Neurology at Boston Children's Hospital and Harvard Medical School. The outlined proposal builds on the candidate's previous research on protein trafficking, autophagy and neurodegeneration and integrates new domains of expertise in cell biology, advanced microscopy, and iPSC-derived neurons and genetically-engineered zebrafish to model human diseases. These skills are reflected in his mentoring team consisting of primary mentor, Dr. Mustafa Sahin, and a scientific advisory committee consisting of Dr. Craig Blackstone, Dr. Thomas Schwarz, Dr. Annapurna Poduri and Dr. Leonard Zon. The proposed experiments and didactic work will position the candidate with a unique set of cross-disciplinary skills that will enable his transition to independence as a physician-scientist in the field of translational neuroscience in childhood-onset neurological diseases.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Dopamine (DA) is important for many behaviors such as motivation, learning, and movement. Malfunction of DA signaling is related to various psychiatric and motor symptoms, and DA-related drugs are commonly used to treat schizophrenia, ADHD, OCD, autism, personality disorders, and mood disorders. Although DA regulates various behaviors, it had been believed that the role of DA neurons is uniform: to signal "reward prediction error" (RPE), the discrepancy between actual and predicted reward value. Recently however, we and others showed that DA neurons projecting to different regions in the striatum exhibit distinct properties and serve distinct functions. We found that DA in the anterior striatum (AS), central and posterior striatum (CS/PS), and `tail' of the striatum (TS) signal canonical RPE, regulate the execution of skills, or signal threat prediction error, respectively. Therefore, dopaminergic projections from the midbrain to the AS, CS/PS, and TS must be differentially and precisely established for them to regulate our brain functions properly. However, the manner and molecular mechanisms by which specific dopaminergic connections are established in the striatum are unknown. To address this question, we have searched for synaptic, homophilic cell-adhesion molecules that are differentially expressed in the AS, CS/PS, and TS. We identified that three Protocadherins (PCDHs), PCDH17, PCDH10, and PCDH19, are selectively expressed in the AS, CS/PS, or TS, respectively, during development and in adults. Furthermore, in the midbrain, PCDH17, 10, and 19 are expressed by DA neurons projecting to the AS, CS/PS, or TS, respectively. Based on these expression patterns, we hypothesize that PCDH17, 10, and 19 are the molecular codes for the AS-, CS/PS-, and TS-projecting DA neurons, respectively, and that they play critical roles for the establishment of functionally segregated DA circuits. To test these ideas we have generated novel mouse lines in which Cre is expressed under the Pcdh promoters, and constitutive (null) and conditional knockout (KO) mice for each of the three PCDHs. Using these mouse lines, we propose to: Aim 1: Determine whether PCDH17, 10, and 19 are the molecular codes for functionally segregated DA neurons in adults. Aim 2: Investigate the effects of inactivation and activation of PCDH-expressing DA neurons during various stages of development. Aim 3: Examine the role of PCDH proteins in the establishment of specific DA connections. We will use interdisciplinary approaches with molecular/cell biological, histological, mouse genetic, electron microscopic, electrophysiological, in vivo recording/imaging, and behavioral techniques to address these aims. Our work will molecularly define functionally distinct DA circuits and reveal how specific DA circuits establish in the mammalian brain. PCDH17/10/19 are implicated in different disorders: PCDH17 in mood disorders and schizophrenia, PCDH10 in autism and OCD, and PCDH19 in epilepsy and personality disorders. Thus, our study may also provide a link between specific DA circuits to certain disorders and suggest novel strategies to treat these devastating disorders.

NIH Research Projects · FY 2024 · 2021-07

PROJECT SUMMARY Despite the known roles of chromatin remodeling complexes in driving more than 20% of human cancer, the role of chromatin remodeling complexes in conferring therapeutic response in pediatric solid tumors is much less understood. Rhabdoid tumor requires residual SWI/SNF activity for transformation and progression. However, it is not known if SWI/SNF and its effect on the underlying epigenome is therapeutically targetable and if a compound targeting this complex will be successful. Furthermore, SWI/SNF has been implicated in epigenetic mechanisms of resistance suggesting this complex may be able to both confer sensitivity and resistance depending on the cancer context. Fusion positive alveolar rhabdomyosarcoma (ARMS) gains chemo-resistance without the simultaneous gain of mutations to drive this resistance. These data indicate ARMS relapse may be driven by epigenetic mechanisms. Therefore, the overall objective of this study is to define the role of chromatin structure in conferring therapeutic sensitivity in rhabdoid tumor (Aim 1) and resistance in rhabdomyosarcoma (Aim 2). I have identified mithramycin as a SWI/SNF inhibitor that induces epigenetic reprogramming and durable tumor regression in rhabdoid tumor. A consequence of mithramycin treatment is amplification of H3K27me3, a novel therapeutic vulnerability as well as the restoration of chemosensitivity. The overall goal of the F99 phase (Aim 1) is to identify synthetic lethalities that arise from SWI/SNF inhibition. Specifically, aim 1.1 will define inhibition of H3K27me3 histone demethylases KDM6A/6B as a therapeutic vulnerability in rhabdoid tumor. Aim 1.2 will define the mechanism of mithramycin-dependent chemosensitivity. These goals will build advanced expertise in mechanistic pharmacology, high-throughput sequencing, and in vivo modeling of combination therapies. In contrast to RT which is known to be chemo-refractory, alveolar rhabdomyosarcoma is initially responsive to chemotherapy before gaining resistance. Therefore, the K00 phase of this fellowship (Aim 2) will define the role of chromatin remodeling in fusion positive alveolar rhabdomyosarcoma therapeutic resistance (ARMS). Aim 2.1 will identify the chromatin remodeler that coordinates with PAX3/7-FOXO1, the oncogenic transcription factor that drives ARMS transformation and progression. Aim 2.2 will profile chromatin remodeling during the establishment of chemoresistance in an established ARMS mouse model. This phase will expand expertise in genomic approaches to include single-cell genomics and in vivo modeling to include transgenic models. In summary, this study addresses the need for a mechanistic investigation into the role of chromatin remodeling in driving therapeutic response in pediatric solid tumors. Data and training acquired in this phase will prepare me for a career exploring epigenetic mechanisms of therapeutic resistance in pediatric sarcomas.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY/ABSTRACT Cerebral palsy (CP) is the most common childhood-onset motor disability, affecting 764,000 individuals in the United States alone. The lifetime medical costs for a single individual with CP are estimated at $1.4M, which represents a substantial healthcare and economic impact. A diverse set of risk factors contributes to CP, including prematurity, intrauterine infection, and hypoxic ischemic encephalopathy. In approximately 20% of cases, there are no clear perinatal risk factors (“cryptogenic CP”). There is accumulating evidence from rare familial cases and a growing number of isolated cases suggesting that cryptogenic CP may result in part from single gene disorders, including over 50 treatable inborn errors of metabolism. However, these studies have involved small numbers of participants, with patient populations characterized using administrative data with limited attention to precise clinical characterization. The full breadth of the genetic landscape of CP is unknown. We hypothesize that a substantial portion of individuals with cryptogenic CP will have a pathogenic or likely pathogenic variant in a single gene providing an explanation for their symptoms. We propose rigorously phenotyping a large prospective cohort of individuals with both cryptogenic and non-cryptogenic CP who have undergone exome sequencing through an institutional genomics pilot study, and then analyzing exome sequencing data to determine the presence of single gene disorders in each subgroup. To accomplish these goals, we will classify patients as cryptogenic CP or non-cryptogenic CP. We will systematically, rigorously, and longitudinally characterize neurological, motor, communication, and neuroimaging phenotypes using research measures validated for CP. Next, we will analyze exome data using an institutional pipeline for variant interpretation. Finally, we will build a statistical model that correlates the presence of a genetic disorder with phenotypic measures in order to help predict which individuals with CP are most likely to have a single gene disorder. If applied to the population at large, the proposed work could lead to identification of single gene disorders in thousands of individuals with CP, including treatable conditions where a molecular diagnosis may positively alter a child's developmental trajectory. Determining etiology represents a first step in understanding the biological substrates of CP needed for developing rational therapeutics for this highly prevalent condition.

NIH Research Projects · FY 2026 · 2021-07

PROJECT SUMMARY Schizophrenia is a debilitating neurodevelopmental disorder and a leading cause of global disability. Both genetic and environmental factors increase the risk of developing the disease. Recent genome-wide association studies identified over 200 genetic loci with associations with schizophrenia. Two of the strongest associations involve mutations in complement component 4 (C4) genes (C4A and C4B) and SLC39A8. Work from the Stevens lab showed that overexpression of C4A, but not C4B, increased schizophrenia risk and promoted micro glial dependent synaptic pruning. These studies highlight the role of complement-mediated neuron-glia interactions in schizophrenia pathology. Although great strides have been made towards understanding the genetic risk conferred by C4, less is known about the risk associated with SLC39A8. SLC39A8 encodes a manganese influx transporter and loss-of-function mutations cause a congenital glycosylation disorder. Evidence from human postmortem tissue and rodent models points to a role of dysglycosylation in schizophrenia pathology. The schizophrenia-associated SLC39A8 variant is associated with aberrant glycosylation in the brain. Neuronal glycosylation also regulates complement-microglia interactions, raising the possibility that dysglycosylation could play a role in complement-associated schizophrenia risk as well. This study aims to investigate the role of glycosylation in both environmental and genetic risk for schizophrenia by testing the hypothesis that aberrant glycosylation of complement proteins is involved in genetic and environmental schizophrenia risk. Experiments in Aim 1 will characterize the impact of environmental stress on glycosylation and inflammation in the brain. Experiments in Aim 2 will investigate the role of glycosylation in complement-mediated neuron-glia interactions and determine if differential glycosylation confers increased schizophrenia risk through in vitro assays performed using C4A and C4B overexpressing mice. Finally, experiments in Aim 3 will examine complement-mediated neuron-glia interactions in mice expressing the schizophrenia-associated SLC39A8 variant. The proposed study uses a multidisciplinary approach to understanding the mechanisms underlying schizophrenia risk. Proposed studies will be performed under the supervision of Dr. Beth A. Stevens (Boston Children’s Hospital), an expert in neuron-glia interactions and complement pathways. The expertise available in the Stevens lab along with the highly collaborative environment at Boston Children’s Hospital will ensure the successful completion of this proposal and provide the training and mentorship necessary for the applicant to achieve her goal of becoming an independent investigator.

NIH Research Projects · FY 2025 · 2021-07

ABSTRACT. Transforming growth factor beta2 (TGF-β2) is an important therapeutic target for renal fibrosis, the principal cause of end-stage renal failure in chronic kidney disease (CKD). TGF-β2 triggers renal fibrosis in vivo and, in response to kidney injury, is upregulated in renal myofibroblasts, pericytes, and proximal tubule epithelial cells—cell types that mediate kidney fibrosis. Earlier studies showed that an antibody to mature TGF-β2 arrested renal fibrosis in a rat model of diabetic kidney disease, but further therapeutic development was not followed up. In vivo, TGF-β2 exists mainly as a latent pro-complex (proTGF-β2) in which prodomains are noncovalently bound to the growth factor. Secreted proTGF-β2 is stored in different extracellular milieus where it undergoes activation, i.e. release of the growth factor (mature TGF-β2), to initiate signaling. Preliminary data point to αVβ6-dependent and -independent mechanisms of proTGF-β2 activation as different modalities that can be therapeutically targeted for renal fibrosis. Aim 1 of this proposal is to develop new antibodies that specifically target the prodomain and block proTGF-β2 activation as a novel therapeutic strategy for renal fibrosis. Antibodies will be selected from an innovative yeast display antibody library, screened for activation-blocking activity in vitro, and tested for therapeutic efficacy in mouse models of acute kidney injury. Aim 2 is to determine high-resolution crystal structures of proTGF-β2 to define the mechanism underlying latency and facilitate drug development by uncovering new strategies to prevent activation. The candidate has assembled an exceptional team of mentors and advisors with expertise in renal pathophysiology, drug discovery, and structural biology to ensure the success of the project. The team will provide career guidance and training in techniques essential for the candidate’s future independent career at the interface of structural biology, drug discovery, renal fibrosis, and CKD. The candidate will receive extensive training in 1) X-ray crystallography, 2) antibody discovery, 3) renal pathophysiology, 4) immunofluorescence microscopy, and 5) mouse models of acute kidney injury and renal fibrosis. These skills will extend the candidate’s already versatile foundation in genetics, molecular biology, protein biochemistry, and structural biology. Boston Children’s Hospital and surrounding institutions (e.g., Harvard Medical School) constitute a robust training environment with unparalleled intellectual capital and remarkable infrastructure, which include cutting-edge yeast display platforms for antibody discovery at the Institute for Protein Innovation and unparalleled resources and expertise in the Renal Division at Brigham and Women’s Hospital, that will enhance the candidate’s growth and support his proposed research. Career development will be accomplished through direct mentorship, education through fellowship training offices, and attendance of conferences. The results of this proposal will establish the foundation of the candidate’s future research programs as an independent investigator in renal biology. The candidate plans to apply for the NIDDK Small Grant Program if available to K awardees and an R01 to facilitate his transition to independence.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY/ABSTRACT: Research: Food allergy is a potentially life-threatening health condition that affects an increasing proportion of individuals in the US alone. Current daily management of patients with food allergies is limited by the unavailability of reliable biomarkers to accurately predict onset, severity and resolution of disease, and by the lack of curative treatments to replace strict avoidance of the culprit food. A deeper understanding of additional mechanisms - other than IgE - acting in food allergy pathogenesis will be key in filling these gaps. This proposal details a five-year plan to provide Dr. Crestani with the training and expertise to evaluate the role of RELMb as a novel biomarker and molecular player in food allergy building and expanding on strong preliminary observations both in a murine model of food allergy and in food allergic children. The comprehensive evaluation of the role of RELMb in food allergy holds the promise of identifying strongly needed biomarker(s) of disease and possible targets for therapeutic interventions which will be the focus of future longitudinal investigations and may significantly impact the daily care and quality of life of food allergic patients. Candidate: Dr. Crestani long-term goal is to become an independent, NIH-funded investigator focused on disease mechanisms and biomarkers of food allergy, with the goal of improving the currently suboptimal care of food-allergic individuals. There is an urgent and unmet need for novel biomarkers and therapeutic approaches in food allergies. To achieve this goal, her short-term objectives are to obtain further training in biostatistics, data analysis, laboratory techniques, as well as practical skills in cohort building and management. This will be accomplished with formal classes, collaborative work, attendance at conferences, and guidance from established mentors/scientific advisors with relevant expertise. This will result in a unique combination of practical skills and scientific knowledge that will successfully position her for an R01 application and an independent career as a physician-scientist in the field of food allergy. Environment: Dr. Crestani will be mentored by Dr. Chatila (Primary Mentor), a leader investigator in mechanisms of food allergy and head of a fully NIH-funded laboratory, and Dr. Phipatanakul (Co-Mentor), an expert in epidemiology, human cohorts, and clinical investigation in asthma and allergic diseases. Dr. Crestani has assembled an extraordinary team of advisors, including Drs. Lynda Schneider, Kari Nadeau and Rima Kaddurah-Daouk, who have committed their time, resources, and expertise to facilitate Dr. Crestani’s career development and successful completion of the proposed project. During this award period, Dr. Crestani will obtain a Master of Public Health in Clinical Effectiveness through the Harvard School of Public Health (HSPH), and complete additional complementary coursework. The academic environment created by the mentors, institution, and Harvard community at large will provide the ideal milieu for learning and research implementation that will guarantee Dr. Crestani transition into a highly successful independent investigator.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Hematopoietic Stem/Progenitor Cells (HSPC) gene therapy has provided clinical benefits in several patients affected by a variety of genetic diseases, some of which already reached market authorization for selected indications. However, the use of semi-randomly integrating vectors poses the risk of insertional mutagenesis and ectopic/unregulated transgene expression. These issues become even more relevant when the affected gene needs to be highly express to exert its function and when its activity directly impacts genome stability, such as the case for Recombination-Activating 1 (RAG1) gene. RAG1 is express in a high but tightly regulated manner in differentiating lymphocyte precursors, where it directs the VDJ recombination process required for assembly the T- and B-cell receptors, and its inactivating mutations are one of the most frequent causes of severe- combined immunodeficiency (SCID). While the high risk of genomic damage due to unregulated RAG1 expression has so far hampered the use of viral vectors to treat RAG1 deficiencies, there is a need to develop novel and effective therapeutic approaches, especially for patients who lacks a compatible HSPC donor or are not eligible for allogeneic transplant. The long-term goal of our proposal is to address this unmet medical need and develop an effective novel treatment directed at restoring both function and expression control of the RAG1 gene on autologous patient derived HSC. Our central hypothesis is that gene repair strategies that preserve physiologic expression control represent a safe and effective approach for treating RAG1 deficiencies. We reported that by tailoring culture conditions and gene delivery vehicles, it is possible to partially overcome the biologic barriers that constrain gene editing in the most primitive and clinically relevant HSPC subsets (Genovese, Nature 2014; Schiroli, Science-Translational-Medicine 2017). Within this project we will capitalize our previous achievements to i) directly fix RAG1 mutations, ii) improve efficiency of current HSPC gene editing protocols and iii) investigate non-genotoxic conditioning on suitable mouse models. Functional correction of the engineered RAG1 gene will be stringently assessed on patient derived cells, by exploiting state-of-the-art in vitro T cell differentiation assay and in vivo xenotransplantation experiments. We will take advantage of our recently optimized gene editing procedure and barcoding technology (BAR-seq, Ferrari et al, Nat. Biotech. 2020) to maximize editing efficiency while reducing cellular toxicity on the treated HSPC, thus increasing the yield of long- term engrafting lymphoid cells. To support the rational for clinical testing, we will assess correction of the disease phenotype by limiting amounts of functional HSPC in two RAG1 murine models and test efficacy of emerging immunotoxin conditioning regimens to reduce transplant toxicity and increase lymphoid reconstitution. Overall, this project will contribute to the development of an innovative treatment approach for RAG1 deficiencies and position homology-based gene editing as a standard for precise HSC engineering, providing for safer and more efficacious therapeutic strategies with broad applicability in hematology.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract This proposal details a 5-yr plan to prepare the candidate, Ruobing Wang, MD, for a career as an independent physician-scientist positioned to impact our understanding of pediatric lung diseases, particularly Cystic Fibrosis (CF). As a clinician taking care of CF patients, she has identified rare individuals with homozygous F508del CFTR mutations who met clinical criteria for inclusion as: 1) CF Long-term non-progressors (LTNP), with preserved lung function 2) CF Rapid progressors (RP), whose lung function declines rapidly. Whole exome sequencing uncovered rare missense polymorphisms in i) SCNN1 (which encodes epithelial sodium channel ENaC) in LTNPs, and ii) two genes in epithelial alternative chloride channels (ANO1 and SCL26A9) in RPs. The central hypothesis of the proposal is that the extreme phenotypes of CF are due to the alterations in epithelial ion and fluid transport driven by these gene defects, and that the extreme-phenotype disease severity and mechanism can be modeled and studied in vitro with a reprogrammed cell-based platform. Using a novel protocol, Dr. Wang generated airway basal-like cells from induced pluripotent stem cells (iPSCs) which can then differentiate into basal, multi-ciliated, and secretory lineages on air-liquid interface, forming a functioning airway epithelium with intact barrier function and aberrant trans-epithelial chloride transport. Normalization of chloride transport in these cells by CFTR gene editing confirms the reliability of her model to recapitulate CF phenotype. The central goals of the project are to establish this novel iPSC-platform for CF disease modeling and study the epithelial function of the extreme-phenotype patients. She is now uniquely poised to complete the aims to 1) To test whether iPSC-derived airway epithelia can serve as a platform to model airway epithelial ion and fluid transport and muco-ciliary transport in CF, 2) to model extreme-phenotype CF patient and interrogate the role of candidate modifier genes and their impact on ion and fluid transport, and 3) To establish the iPSC-platform for personalized drug response, and test the therapeutic role of pharmacologic targeting of alternative ion channel candidates. Dr. Wang has 80% protected time from Boston Children's Hospital (BCH) Division of Respiratory Diseases and the Department of Medicine. Her co-sponsors are 1) Dr. Darrell Kotton at the Center for Regenerative Medicine (CReM) at Boston University (BU) with whom she has trained for the past 1.5 years, and 2) Dr. Benjamin Raby, the chief of BCH Division of Respiratory Diseases. Furthermore, Dr. Wang has assembled a team of extraordinary scientific advisory members, each bringing their specific expertise, to assist her career development and scientific research. A detailed training plan is presented that includes mentored research, didactic coursework, presentations at meetings, and a timeline for completion of the research aims, preparation of manuscripts, and future R01 application. The proposed research, training plan, mentorship committee, and scientific-clinical environment at BCH and BU will position the candidate to transition to independence by the end of the award.

NIH Research Projects · FY 2025 · 2021-06

Abstract/Project Summary Vibrio cholerae is a human diarrheal pathogen and an environmental organism that persists in the arthropod intestine. Our preliminary results suggest that the V. cholerae high cell density quorum sensing regulator HapR orchestrates the metabolic transition of this microbe from intestinal pathogen to intestinal symbiont by extending host life span, activating host innate immunity and epithelial repair mechanisms, and promoting host metabolic homeostasis. Here we use the powerful genetics of the model arthropod Drosophila melanogaster to understand the lines of communication between host and pathogen that underlie this transition. We will identify bacterial metabolites that activate host innate immunity and trace the host pathways that are co-opted by the bacterium for this purpose. We will determine whether the arthropod intestinal mucus or peritrophic membrane supports growth of V. cholerae and whether digestion by HapR-activated degradative enzymes activates peritrophic membrane synthesis. Finally, we will explore the role of V. cholerae-derived tryptophan in promoting host metabolic homeostasis. These studies not only expand our appreciation of the role of V. cholerae HapR in the host-pathogen interaction but also provide a new paradigm of quorum sensing control of the host-microbe symbiosis.

NIH Research Projects · FY 2025 · 2021-05

SUMMARY This K99/R00 proposal describes a five-year mentored research and training plan that will facilitate the transition of Dr. Patricia Davenport to an independent academic researcher in neonatal hematology. Dr. Davenport is a productive and dedicated young physician-scientist working in a clinically relevant and understudied field. PLTs are active participants in both hemostasis and inflammation, yet clinical awareness of these dual roles has lagged behind research discoveries, particularly in neonatology. Preterm infants are at high risk of spontaneous bleeding (particularly intracranial) and thus are typically transfused at higher PLT counts than children or adults, in the hope of preventing bleeding. However, a recent large randomized trial in preterm neonates found that liberal PLT transfusions increased mortality and risk of chronic lung disease, without decreasing bleeding. The mechanisms mediating these findings are unknown, but we hypothesize that they are at least partly related to the developmental differences between neonatal and adult PLTs. The overarching aim of this proposal is to improve the management of neonatal thrombocytopenia through a better understanding of the consequences of PLT transfusions in neonates with various underlying pathologies. Our overall hypothesis is that the effects of PLT transfusions increasing neonatal mortality and severity of chronic lung disease are mediated by an amplification of the neonatal inflammatory responses. To test this hypothesis, we designed the following Specific Aims: 1. Determine the effects of PLT transfusions on newborn mice with and without endotoxemia; 2. Determine the effects of PLT transfusions on mortality in a neonatal model of sepsis; and 3. Characterize the effects of PLT transfusions in a neonatal murine model of chronic lung disease. This research is highly significant, as knowledge gained from it will inform clinical practice and research. Dr. Davenport will receive mentorship from her co- mentors, Dr. Martha Sola-Visner, an NIH-funded researcher in the field of neonatal PLT biology, and Dr. Stella Kourembanas, a world-renowned investigator in neonatal pulmonary physiology and chronic lung disease. In addition, Dr. Davenport will have the guidance of her Scientific Advisory Committee, composed of distinguished scientists with expertise in transfusion medicine and immunology, neonatal sepsis and inflammation, infectious diseases, and study design/statistics. The training opportunities and resources at Boston Children’s Hospital (BCH) and Harvard Medical School provide an ideal environment for the candidate’s career development. The Division of Newborn Medicine at BCH is committed to Dr. Davenport’s success and has assured at least 75% protected time to devote to the activities described in this proposal. A detailed career development and training plan is presented, including mentored research, didactic coursework, seminars and presentations at scientific meetings, and a plan for manuscript writing and R00 submission. The expertise and knowledge gained from this Award will enable Dr. Davenport to obtain future R01 funding and transition to an independent research career focusing on neonatal PLT biology, and particularly the interactions of PLTs with the neonatal lung.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Retinal vascular dysfunction leads to visual impairment and loss of vision, a phenomenon that occurs in retinopathy of prematurity (ROP), diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Injury of the retinal endothelial cell (REC) initiates a series of pathogenetic events that ultimately lead to accelerated progression of retinal diseases. How REC injury leads to transcriptional changes that determine whether the retinal vascular function is restored or leads to pathological changes is not known. Sphingosine 1-phosphate (S1P), a blood-borne lipid mediator that signals via G protein-coupled S1P receptors (S1PR1-5). The applicant’s laboratory discovered the first S1PR and worked out its functional roles in vascular barrier maintenance, development/ maturation, anti-inflammatory processes, cell survival and endothelial/ pericyte interactions. Although two FDA-approved S1PR-targeted drugs are efficacious in the treatment of multiple sclerosis, retinal blistering and macular edema are dose-limiting adverse effects due to the impairment of retinal barriers. We recently showed that S1PR signaling suppresses vascular endothelial growth factor (VEGF)-induced AP-1 transcription factor activity and permits Norrin/Wnt/ß-catenin-dependent REC gene expression, thus leading to retinal REC specialization. Among the AP-1 factors, JunB protein expression is most prominently regulated by S1PR signaling, an event needed for optimal vascular network expansion and formation of deep retinal vascular plexus. The central hypothesis of the proposal is that REC S1PR signaling establishes JunB transcription factor gradients and permits the REC organotypic specialization mechanisms. In this manner, attenuated S1PR signaling axis drives poorly functional retinal vascular network and vasoproliferative ROP. In this proposal, the first specific aim will elucidate mechanisms and consequences of S1PR sculpting of JunB transcription factor gradients in REC. Second, how S1PR signaling in the REC promotes organotypic specialization by enabling efficient Norrin/Wnt/ß-catenin-dependent signal transduction and gene expression will be conducted. Specific focus will be on omega-3 fatty acid transporter (MFSD2A) and iron transporter (TFRC). The relevance of these mechanisms in the mouse models of ROP will be addressed in specific aim 3. These studies are anticipated to enhance our understanding of basic mechanisms of retinal vascular development, specialization and disease in the retina and ultimately lead to approaches that tame retinal disorders by targeting the S1P lipid signaling axis and to provide S1PR inhibitors with fewer side effects.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY / ABSTRACT Candidate: Dr. Marissa Hauptman is an Instructor of Pediatrics at Harvard Medical School (HMS), a pediatrician and environmental medical toxicologist, and Assistant Director of the Pediatric Environmental Health Center at Boston Children’s Hospital (BCH). At BCH, she completed her pediatrics residency and a joint pediatric environmental medicine and health services research fellowship. Building on her early work, her proposed career development plan focuses on 2 areas of career development—geospatial and biomarker environmental exposure science, clinical biomedical informatics—that will add to her research toolkit, increase the rigor of her work, and enhance her ability to develop, evaluate, and integrate environmental exposure science and epidemiology into clinical medicine. She will gain these skills through coursework, experiential learning, mentorship, and participation in seminars and national and international meetings. Environment: Dr. Hauptman is supported by extensive research, professional, and academic resources at BCH, HMS, and Harvard T.H. Chan School of Public Health, including the BCH Institutional Centers for Clinical and Translational Research, Harvard-Chan NIEHS Center on Environmental Health, and the Harvard Catalyst Program. Her committed team of mentors and advisors include national experts in geospatial, air pollution and biomarker environmental exposure science and epidemiology, biostatistics/bioinformatics and clinical prediction methodology. Research: Despite increasing evidence of its adverse effects, air pollution has not been effectively addressed by healthcare providers in clinical medicine. Further knowledge is clearly needed, as elucidating robust environmental air pollutant biomarkers and readily available geospatial data as well as underlying mechanisms may lead to new therapeutic options and strategies for clinicians to better address a patient’s environment at the bedside. Dr. Hauptman proposes to build on prior work to rigorously evaluate and develop—through geospatial and biomarker environmental exposure science, epidemiology, and biomedical informatics—a feasible innovative Asthma Integrated Risk (AIR) Clinical Prediction model that relies on readily available data through patient report, the electronic medical record system, public available data sources and biomarkers that can assist clinicians in predicting high risk pediatric patients with asthma and potentially tailoring environmental and therapeutic interventions. At the end of this project, Dr. Hauptman will be well- positioned to apply for an R01 to further test whether implementation of this clinical decision support intervention improves asthma morbidity by more comprehensively identifying and addressing environmental air pollutants in children with chronic health conditions. The successful completion of this project will position Dr. Hauptman for the next stage of her career as an independent investigator addressing environmental exposures in clinical medicine.

NIH Research Projects · FY 2025 · 2021-05

Retinopathy of prematurity (ROP), a leading cause of blindness in children, afflicts ~14,000 premature infants yearly in the US. About 1,500 of those develop severe ROP, requiring treatment. ROP has increased in the last decade due to (1) increased multiple (and more preterm) births after in vitro fertilization; (2) increased survival at low gestational ages at high ROP risk; and (3) higher levels of supplemental oxygen with more ROP incidence. Current treatments (laser photocoagulation and anti-vascular endothelial growth factor (VEGF) drugs) target late-phase retinal neovascularization and have adverse effects. We need to find new ways to treat ROP. Nutrient deficiency occurs in preterm infants and is associated with ROP development. Early full amino acid supplementation, starting the first day of life, improves weight gain, which in turn reduces ROP risk. However, specific amino acid requirements are unknown. Circulating L-serine levels are lower in premature infants with lower gestational age and higher risk for ROP. We preliminarily found that L-serine supplementation prevents retinal neovascularization in a mouse model of ROP, and retinal glia might be the primary retinal cells in response to L-serine. Therefore, we propose that: L-serine affects retinal neovascularization by controlling glial cell angiogenic factors. In the mouse model of ROP, we will examine if (1) L-serine supplements inhibits retinal neovascularization; (2) retinal glial cells (which control neovascularization) mediate L-serine inhibitory effect on OIR; and (3) L-serine decreases OIR by regulating glial pro-angiogenic factors via lactate. This study will determine (1) if oral or i.p. L-serine inhibits neovascularization in OIR, modeling ROP and (2) the role of glial cell L-serine synthesis and key mechanistic pathways in controlling pathologic retinal vessel growth. Successful completion of our study will likely establish a critical role of L-serine in ROP prevention. There is high translational value in this work, as oral or i.v. delivery of L-serine to preterm infants is very feasible. Systemic L-serine supplementation may prevent ROP and might possibly prevent other complications of preterm birth (intraventricular hemorrhage or bronchopulmonary dysplasia).

NIH Research Projects · FY 2026 · 2021-05

Non-alcoholic fatty liver disease (NAFLD) has reached epidemic proportions in our society, and yet our understanding of the pathogenesis of this disorder remains rudimentary 7. Clinical studies show a close correlation between insulin resistance and the development and progression of NAFLD 8,9. To understand mechanistically the changes in triglyceride, cholesterol, and bile acid metabolism that occur with the development of NAFLD, a clear understanding of how insulin regulates these processes is necessary. Until now, studies of insulin action in the liver have been done with the assumption that all hepatocytes are equivalent. This assumption was made out of practicality, as our ability to isolate and analyze different populations of hepatocytes individually was limited. Yet, hepatocytes clearly vary in terms of the metabolic functions they perform, and their susceptibility to different insults 10. For example, the perivenous hepatocytes are the predominant site of bile acid synthesis and the most common site of triglyceride accumulation in NAFLD 11,12. Here, we will determine how insulin modulates gene expression in the perivenous hepatocytes to maintain homeostasis. Our novel, unpublished preliminary data reveal a striking example of zone-specific transcriptional regulation by insulin. We find that insulin suppresses Cyp8b1 only in the perivenous hepatocytes. Cyp8b1 encodes the sole enzyme capable of catalyzing the 12a-hydroxylation of bile acids 13; 12a-hydroxylated bile acids increase hepatic cholesterol and promote the progression to non-alcoholic steatohepatitis (NASH) 14-16. In the absence of insulin, the de-repression of Cyp8b1 in the perivenous hepatocytes is associated with increased 12a- hydroxylated bile acids, increased hepatic cholesterol, and severe inflammation. The fact that NAFLD progression in humans is also associated with an increase in 12a-hydroxylated bile acids and hepatic cholesterol, and the fact that inflammation marks the development of non-alcoholic steatohepatitis, a more severe and progressive form of disease, highlight the importance of studying this pathway 17-19. Based on these and other preliminary data, we hypothesize that insulin modulates the activity of b-catenin, a master transcriptional regulator that is activated only in the perivenous hepatocytes 20, to maintain normal lipid homeostasis and prevent inflammation. To test this hypothesis, we aim to (1) define the insulin-regulated cellular transcriptional programs in the liver using single-nuclei sequencing; and (2) dissect the role of b-catenin in producing the transcriptional and physiological response to insulin. We expect that insulin can reprogram the perivenous hepatocytes by modulating b-catenin driven transcription, and that this is required for normal homeostasis. Such results may ultimately lead to the development of precise interventions that reverse the effects of insulin resistance in the perivenous hepatocytes, preventing NASH.

NIH Research Projects · FY 2025 · 2021-04

The Biogenesis of Platelet-Derived Extracellular Vesicles and their Impact on Megakaryocyte Maturation Abstract Thrombocytopenia is a major clinical problem encountered in multiple conditions, and severe thrombocytopenia (platelet counts <50 x 10^9/L) can lead to life threatening bleeding. Current treatment options have severe side effects, are in limited supply, involve blood products, and the platelet response typically takes up to 12 days. Therefore, there is an urgent need to identify new thrombopoietic agents that increase platelet counts for patients. In many inflammatory conditions platelet counts rise, resulting in thrombocytosis, but what initiates this platelet up-regulation is not well understood. Our lab uses inflammation as a model of exacerbated thrombopoiesis that results in differences in platelet quality and quantity in order to 1) gain a better understanding of the basic biology of megakaryocyte (MK) maturation their production of platelets, 2) identify thrombopoietin (TPO) independent pathways of MK maturation, and 3) determine ways to reduce platelet-related morbidity and mortality in inflammation. We have discovered a novel regulator of MK maturation during inflammation: platelet-derived extracellular vesicles (PEVs) in the bone marrow. Our preliminary data indicate that platelets package and shed MVs in an agonist-specific mechanism dependent on Rho GTPase signaling; the mechanism of Rho-mediated regulation of PEV formation and packaging will be explored in Aim 1. We also found that PEVs enter the bone marrow from the plasma, and bind to and are endocytosed by MKs both in vitro and in vivo. In Aim 2, we will examine how platelet-derived MVs interact with MKs. Specifically, we will determine the mechanisms by which they bind to and are internalized by MKs and how their cargo transferred. In inflammatory conditions such as SLE, ongoing platelet activation increases levels of circulating PEVs. These PEVs deliver disease-related changes from the plasma milieu directly to MKs in the bone marrow, reprogramming the MKs to make more pathogenic platelets. In Aim 3, we will identify the PEV factors that alter MK gene expression and platelet content in SLE. Successful completion of the proposed experiments will, for the first time, provide a detailed roadmap of how PEVs alter the hematopoietic environment in the setting of inflammatory disease. The insights gained may identify novel therapeutic targets that (i) alter PEV poduction independent of platelet activation (Aim 1), (ii) hijack the PEV/MK interaction to alter MK maturation (Aim 2), and (iii) inhibit pathologic MK reprogramming during SLE and other inflammatory diseases (Aim 3).

NIH Research Projects · FY 2026 · 2021-04

PROJECT SUMMARY This CAUSE application brings together seasoned clinical and laboratory investigators in inner-city asthma, with expertise in clinical studies and clinical trials, immunology, genetics, environmental exposures, bioinformatics, data management, and statistics. The investigators have long track records in implementing multi-center and single-center clinical trials and observational studies in allergic diseases, including asthma, to the standards of NIH funded clinical research networks, in conducting NIH fundamental research on disease mechanisms in asthma and in training generations of investigators in asthma research In part A we demonstrate that we have the personnel and facilities to conduct asthma network-wide and Clinical Research center-specific research on inner-city children with asthma populations recruited from the allergy and asthma clinics at Boston Children's Hospital and from our just completed, as well as ongoing, NIH-funded studies of inner-city schoolchildren with asthma, allergic diseases and healthy controls. We have a highly experienced team, IRB-approved protocols for recruitment and clinical characterization of asthma patients and healthy controls and an infrastructure which includes clinical research facilities, investigational pharmacy services, a laboratory facility capable of processing, storing and shipping human samples, a state-of-the-art immunology research laboratory with a 25 year focus on asthma and a data management facility with quality control plans, and capability to upload data into the NIAID designated repositories and biostatistical support. In part B our Center specific project draws from previous work on the novel NOTCH4 pathway and airway inflammation and will draw on an already well-characterized urban school population of asthma patients and healthy controls. Our overall hypothesis is that NOTCH4 signaling acts to regulate airway inflammation and increases asthma severity and loss of control in inner-city school children. Our aims are to 1) test the hypothesis that elevated peripheral blood NOTCH4+ Tregs defines a population of patients whose asthma is driven by an IL-6 dependent mechanism that confers a more severe or poorly controlled phenotype 2) determine the environmental determinants of the NOTCH4+ Tregs and how they mediate disease severity and control and 3) investigate whether regulatory variants that increase NOTCH4 protein expression are associated with more severe asthma phenotypes and endotypes. This project will confirm the role of environmental exposures we have found important in urban schools and homes of children with asthma and that regulatory variants that impact signaling may be modified by novel mechanistic gene by environment pathways. We will elucidate novel mechanisms fundamental to the biology of airway inflammation and pave the way for future biomarker driven approaches to inform future precision therapy. We address a critical knowledge gap in reducing disproportionate asthma burden in vulnerable individuals. We will contribute extensively to the CAUSE as a CAUSE-Clinical Research Center, with our infrastructure and expertise.

NIH Research Projects · FY 2025 · 2021-04

Understanding neuronal subtype-specific function of NAc in cocaine addiction Abstract Drug addiction is a chronic, relapsing brain disorder characterized by compulsive drug seeking and use despite harmful consequences. It is an urgent social and health problem contributing to more than 90,000 deaths and incurs a yearly cost of over $700 billion in the United States (see NIDA website). It is believed that long-term maladaptive changes in the brain reward system play a central role in the development of addictive disorders. However, the underlying mechanism remains largely unknown. The long-lasting effect of drugs on animal behavior and the risk of relapse in human addicts indicate that some stable changes in the brain reward system induced by drugs of abuse mediate these long-term behavioral adaptions. Accumulating evidence suggests that drug-induced molecular, cellular and circuitry changes, especially those in the nucleus accumbens (NAc), play important roles in drug addiction. However, due to the cellular heterogeneity of the mammalian brain, the cell type-specific mechanism of addition is unknown. To overcome the cell heterogeneity issue and to advance our understanding of the cell subtype- specific mechanisms of drug addiction, we propose to identify the neuronal subtypes in NAc involved in addiction by comprehensively analyzing the transcriptional profiles of this brain region in a neuron subtype-specific manner, using a clinically relevant intravenous cocaine self-administration (IVSA) mouse model. Furthermore, cell type-specific profiling/manipulation approaches will be used to understand the function and mechanism of specific neuron subtypes during addictive process. To achieve this goal, we have the following specific aims: 1) Profile cell type-specific transcriptome of different neuron subtypes of NAc using a mouse model of cocaine IVSA; 2) The function and circuitry mechanisms of Tac2+ D1 MSN subtype in cocaine addiction; 3) Understand the epigenetic mechanism of the neuron subtype-specific functions in cocaine addiction. Completion of the proposed study will not only advance our understanding on how different NAc neuron subtypes contribute to drug addiction, but also reveal novel therapeutic targets for treating this disorder.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY Brief resolved unexplained events (BRUE) are frightening episodes characterized by appearance of life-threatening choking, cyanosis, and limpness in infants. These common events are resource-intensive and current management inadequately addresses persistent symptoms. Infants with BRUE commonly have oropharyngeal dysphagia with aspiration, which is a modifiable risk factor for persistent symptoms, but there are no studies determining the mechanism behind this swallow dysfunction and if swallow interventions reduce morbidity. Systematic investigation of the contribution of oropharyngeal dysphagia to disease burden in this population is urgently needed, as this approach has vast potential to optimize clinical outcomes, improve quality of life and reduce healthcare utilization. Daniel Duncan, MD MPH is an Instructor at Harvard Medical School (HMS) and a subspecialist within the Aerodigestive Center at Boston Children’s Hospital (BCH). He has gained substantial clinical research experience during his medical training and has demonstrated commitment to an academic career in patient oriented research. His career goal is to direct a clinical research program focused on identifying evidenced-based, state of the art interventions for oropharyngeal dysphagia and aerodigestive disorders that transform clinical care for this vulnerable pediatric population. This Career Development Award will provide additional mentored training and research opportunities in high resolution impedance-manometry and decision analysis for Dr. Duncan to advance his quantitative research skills while addressing the current knowledge gap related to mechanisms of swallow dysfunction in BRUE. The proposed innovative studies will systematically determine mechanisms by which thickened feeds modulate swallow function and confirm these findings in a larger cohort, which will allow for derivation of an improved algorithm for BRUE care. He will study effects of alterations in liquid viscosity on upper esophageal motility using pharyngeal impedance-manometry, follow a prospective cohort to determine predictors of response to thickening, and use decision analysis to identify patients that could receive empiric thickening. The mentorship and scientific training afforded by this career development award will be critical for Dr. Duncan’s academic development. His primary mentor, Dr. Rosen, is an aerodigestive expert. His co-mentors, Drs. Jadcherla and Omari, are experts in neonatal swallow dysfunction and impedance-manometry; all are outstanding clinical researchers with deep commitment to mentorship. He will be supported by his Scholarship Advisory Committee consisting of Drs. Snapper, Nurko, Landrigan, and Stamoulis, who lend content-area expertise. His formal training includes advanced coursework in decision analysis at Harvard School of Public Health, personal instruction on esophageal motility and decision analysis, and professional development courses. His training and research activities will be conducted in the unparalleled academic environments of BCH and HMS, which are firmly committed to Dr. Duncan’s successful transition to independence.