Boston Children'S Hospital

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY/ABSTRACT T cell-B cell interactions that result in B cell maturation and antibody production were once thought to occur exclusively in lymphoid organs. It is now appreciated that T peripheral helper (Tph) cells provide excessive help to B cells resulting in autoantibody production in inflamed peripheral tissues. Such disordered Tph cell-B cell responses have been implicated in some forms of chronic inflammatory arthritis in both adults and children. The pathways that regulate the Tph cell-B cell axis in non-lymphoid tissues are not understood. By studying joint fluid from patients with juvenile idiopathic arthritis (JIA), we found evidence of dysregulated Tph cell-B cell responses in children with early-onset and anti-nuclear antibody (ANA) positive forms of the disease. Our preliminary studies also identified a novel T cell subset, T peripheral regulatory (Tpr) cells, in the joints of patients with JIA. These Tpr cells co-express regulatory T (Treg) and B cell-helper T cell factors. Further, Tpr cells have the ability to inhibit Tph cell-B cell interactions that result in plasmablast generation in our T cell-B cell co-culture system. The central hypothesis of this application is that Tpr cells restrain Tph cell-B cell interactions in the joints of patients with inflammatory arthritis. The central hypothesis in this research proposal will be tested with two Specific Aims that will be conducted with biosamples (peripheral blood, synovial fluid, tonsil tissue) from children with JIA as well as healthy controls. Through our established T cell-B cell co-culture system, Aim1 will assess the mechanisms by which Tpr cells regulate Tph cell-B cell interactions. We will test the hypothesis that Tpr cells are uniquely equipped to inhibit proliferation and cytokine production in B cell-helper T cells as well as antibody production, class switching, and maturation in B cells by inducing alterations in gene expression and metabolism. Using a multiomics approach coupled with functional assays, Aim 2 will define the developmental pathways and transcriptional program required for Tpr functionality. We will test the hypothesis that Tpr cells differentiate from Treg cells in the joint and have a unique transcriptional program that is distinct from other Treg subsets and ensures Tpr functionality in tissues. The research is innovative because we will use a systems immunology approach coupled with synovial fluid samples from children with JIA to study T cell-B cell responses directly from affected tissues. Completion of this research will be significant because Tpr cells represent a novel regulatory pathway with the capacity to restrain Tph cell-B cell responses and ameliorate tissue-specific inflammation in arthritis and other autoimmune diseases.

NIH Research Projects · FY 2026 · 2023-12

Proper functions of all immune systems rely on maintenance of immune homeostasis. One family of immune cells that play key roles in immune homeostasis are regulatory T cells (Tregs), a subset of T cells that prevent excessive inflammation and autoimmunity. Tregs differentiate from naïve T cells in a manner that depends on the transcription factor FoxP3. Loss-of-function mutations in FoxP3 lead to fatal multi-organ autoimmune and inflammatory conditions. Despite the importance, the molecular functions and mechanisms of FoxP3 remain poorly understood. We have recently made a significant progress in our structural approach to understand the versatile mode of DNA recognition by FoxP3. We determined the crystal structure of FoxP3 in complex with DNA (Leng et al, Immunity, 2022), breaking the decades-old dogma that FoxP3 forms a domain-swap dimer. More recently, we found that FoxP3 recognizes a new motif composed of TnG repeats by forming a novel multimeric assembly and that this is accompanied by bridging of two DNA molecules (unpublished). Additional preliminary data suggest that this mode of DNA binding also occurs in Treg cells and is important for FoxP3 functions. These findings suggest an exciting new model: unique functions of FoxP3 in Treg development is mediated by its ability to recognize TnG repeats and to form or stabilize DNA loops. We here propose three aims to test and further develop this model. Aim 1 is to determine the cryo-EM structure of the FoxP3 multimers in complex with TnG repeats and to validate the structural mechanism using a combination of biochemistry and cellular immunology. Aim 2 is to investigate functional consequence of such assemblies in T cells using genomic approaches, such as ChIP-seq and HiChIP. Aim 3 is to identify and characterize the direct interacting partners of FoxP3 that mediate its transcriptional function, and to investigate the role of FoxP3 multimerization on its interactome. Together, the proposed research builds upon a strong set of exciting findings from my lab and will have a broad impact on TFs beyond FoxP3. That is, it will help understand how TFs can utilize a multimeric scaffold to recognize new sequence motifs and alter chromatin conformation. Additionally, TnG repeats belong to a group of genetic elements called microsatellites, which are often thought as “junk” or pathogenic. Thus, our work also implicates a novel role for microsatellites in transcriptional regulation and, at the same time, FoxP3 as one of the first exemplary TFs that exploit microsatellites for their functions.

NIH Research Projects · FY 2025 · 2023-12

Project Summary/Abstract: Capillary Malformation (CM) is one of the most common vascular malformations. CM comprises enlarged vessels lined with sprouting endothelial cells (EC) and disorganized mural cells. In Sturge Weber Syndrome (SWS), a neurocutaneous disorder, CM occurs in the skin, brain, and eyes. Patients may suffer from seizures, strokes, glaucoma, and only symptomatic treatment is available. CM is caused by a somatic mutation in GNAQ, the gene that encodes the a-subunit of the G-protein – Gaq. The GNAQ mutation – the arginine (R) at the amino acid position of 183 replaced by the glutamine (Q) – is enriched in ECs isolated from CM in the skin, brain, and choroidal vessels of the eye. To date, little is known about how the R183Q point mutation in the Gaq activation domain leads to abnormal capillaries. Moreover, nothing is known about the interactions between the mutant EC and neighboring mural cells surrounding CM vessels. Our preliminary data suggest that lentiviral transduced human EC expressing the R183Q allele (EC-R183Q) have high levels of pro-inflammatory markers, a weaker junctional barrier, and exhibit heightened response to laminar shear stress. Immunostaining of CM tissue sections also reveal disorganization in mural cell environment labeled with Desmin, Calponin, NG2 and alpha-SMA. Moreover, our histological analyses of CM tissue sections revealed MRC1pos, LYVE1pos and CD68pos macrophage-like cells surrounding the CM vessels of the brain and skin suggesting a pro-inflammatory environment. The goal of this fellowship proposal and training plan is to examine how the EC-R183Q interact with both the mural cells and macrophages. I hypothesize that EC- R183Q triggers the disorganized assembly of mural cells and subsequent macrophage attraction to the abnormal vessels. The proposed study will test this hypothesis through the following aims: (1) to identify paracrine interactions between EC-R183Q and mural cells (2) to identify and characterize mural cell and macrophage interactions with EC-R183Q in vivo. Completing these aims will provide new insights into the relationship between EC and the surrounding mural cells and macrophages in the CM. This fellowship training will provide me with technical, communication, and mentoring skills to leverage my next step to independence. Working with Drs. Joyce Bischoff and Arin Greene (co-sponsor) at Boston Children’s Hospital will provide me with an excellent training environment from both the basic science and clinical aspect of the proposed work.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY The sensory thalamus serves as a hub for incoming information encoding different features of the outside world. This information is parsed and modified as it is transmitted to the cortex. In the visual thalamus (the dorsal lateral geniculate nucleus), retinal ganglion cell axons converge onto proximal dendrites of thalamocortical neurons, forming clusters of boutons that stud the dendrites. Many of these bouton clusters are ensheathed by astrocytes along with synaptic terminals from neuromodulatory projections and inhibitory dendrodendritic presynaptic terminals. Collectively, this synaptic motif is called the glomerulus, described beautifully by electron microscopy in many thalamic nuclei and across many species. While the glomerulus is quite striking, the functional relevance of this structural motif is still debated. To address this critical question, here, we propose to establish and characterize mice in which the glomerular astrocytic sheath is disrupted by genetic deletion of a member of the family of fibroblast growth factor receptors. Our preliminary data shows that deletion of this receptor from astrocytes results in reduced astrocytic sheaths around clusters of retinal ganglion cell boutons and aberrant retinogeniculate functional refinement. We will extend these findings to elucidate the role of this receptor in the development of astrocytic morphology, glomerular structure and the elimination and strengthening of retinogeniculate synapses in the visual thalamus. We will also examine whether and how defects in astrocytic ensheathment of the glomerulus may contribute to aberrant retinogeniculate synapse refinement. Finally, we will take advantage of the disrupted glomerular ensheathment in mutant mice to test the hypothesis that the glomerular structure enhances crosstalk between synapses within the astrocytic sheath that is mediated by glutamate and GABA spillover. If these proposed experiments are successful, results from these studies will advance our understanding of how information is organized in the thalamus and the functional sequela of disruption of this organization.

NIH Research Projects · FY 2026 · 2023-12

Abstract/Project Summary Glaucoma and other optic neuropathies are characterized by retinal ganglion cell (RGC) non-regenerative axon damage and eventual cell death. Optic nerve crush (ONC), which transects all RGC axons, is often used to model this process, and to seek interventions that preserve RGCs and promote regeneration of their axons. Following ONC in mice, ~80% of the RGCs die within 2 weeks, and virtually none of the survivors regenerate axons. We and others have used ONC to identify interventions that lead to increased survival, increased regeneration or both. However, these treatments are only partially effective and many relevant targets are not well-suited for the clinic. It is therefore important to identify additional and improved promoters of survival and regeneration. Kinases are key regulators of cellular responses to insults and stresses. Furthermore, because of their discrete enzymatic activity, kinases are druggable targets. Thus, we performed a preliminary in vivo CRISPR-assisted kinase screen paired with ONC in adult mice to determine which kinases prevent RGC survival and axon regeneration. To our excitement, the first round of screening through 750 kinases identified 17 anti-survival and 5 anti-regenerative hits. Many of these hits represent new mechanistic and translational insights. In the proposed study, we will first examine the mechanistic underpinnings of two novel target kinase pathways (namely LKB1/SNRK and Mkk4/Mkk7) that we have already verified the efficacy of with larger experimental cohorts beyond our preliminary screen. These experiments will allow us to more completely understand their mechanisms of action to formulate ideal therapeutic interventions. Additionally, we will complete the validation of our remaining target kinases with full experimental cohorts in the ONC model. Finally, we will choose the most promising gRNAs with robust neuroprotective effects and test their ability to protect RGCs in our recently developed glaucoma model. At the completion of these studies, we expect to be well-positioned to leverage the knowledge generated here into druggable targets to treat multiple conditions causing blindness.

NIH Research Projects · FY 2025 · 2023-12

Abstract The long-term goal of this proposal is developed to uncover the molecular mechanisms and identify critical regulators governing lymphatic vascular function in health and disease in hopes of offering potential new therapeutic targets to combat cardiovascular and metabolic disorders. At the third year of postdoctoral training, the PI has published his first postdoctoral project and presented his studies in several prestigious conferences that pave the way for his transition to an independent investigator. The lymphatic system is responsible for maintaining interstitial fluid homeostasis, immune cell trafficking and lipid absorption. Lymphatic function participates largely in the pathogenesis of metabolic and cardiovascular diseases such as obesity and atherosclerosis. How lymphatic dysfunction leads to abnormal lipid transport and fat deposition, and conversely, how these metabolic diseases impair lymphatic function are poorly understood but highly medically relevant questions. Embryonic mice lacking Foxc2, a key transcription factor, prevents the formation and maturation of collecting lymphatic vessels. Despite the crucial role of Foxc2 in lymphatic development and remodeling, little is known about the role of Foxc2 in adult physiological and pathological lymphatic function; limitation is largely due to a lack of appropriate animal models. In this proposed application, the PI recently has generated novel inducible lymphatic endothelial cell (LEC)-specific Foxc2 gain-of-function and Foxc2 loss-of- function mice. Utilizing these powerful genetic tools, the PI observed that deficiency of LEC Foxc2 in adult mice dramatically increased the lymphangiogensis, improves impaired lymphatic drainage and lipid absorption in metabolic disorders (unpublished data). In the K99 phase, the PI will continue the current project to determine the molecular mechanism by which Foxc2 regulates adult lymphangiogenesis and lymphatic function in obese model. While in R00 phase, the PI will investigate the role of Foxc2 in modulating macrophage cholesterol efflux and reverse cholesterol transport in atherosclerotic model, as well as how Foxc2 will affect atherogenesis, which is diverging from his mentor’s work on Epsin, a family of endocytic adaptor proteins. Moreover, other than the unique mouse models and cutting-edge techniques, the PI will develop a novel LEC-specific nanoparticle delivery system based on his recent published platform to specifically target LEC Foxc2, thus may halt inflammation and inhibit atheroma progression. This proposal will be valuable for restoring impaired lymphatics to treat cardiovascular and metabolic diseases.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT Prolonged sedation treatment is currently considered standard practice in the safe and compassionate care of critically ill neonates and infants despite leading to opioid tolerance and a high incidence (35-57%) of physical dependence. A unique cohort of infants with congenital long-gap esophageal atresia (EA) undergoes complex perioperative critical care necessitating extraordinarily prolonged sedation (on the scale of weeks). Although our group recently reported decreased brain size and delayed brain growth in term-born infants following long-gap EA repair with prolonged postoperative sedation, there is a fundamental gap in our knowledge of (i) underlying mechanisms and (ii) long-term neurodevelopmental outcomes. Guided by strong preliminary data, this proposal will address 3 key gaps in our knowledge: (1) timing of brain findings (pre vs. during perioperative repair); (2) regional specificity (gray vs. white matter) of (mal)adaptations; as well as (3) infant brain and clinical correlates to early neurodevelopmental outcomes at 1-year of age. The study will employ structural MRI techniques to address these aims. Selected term-born and premature infants with short-gap (brief pain/sedation treatment) and long-gap EA (prolonged sedation treatment) will be scanned twice (AIM 1): before (as neonates), and after complex perioperative critical care (at 4(±1) months of age). Early neurodevelopmental outcomes will be evaluated using standard approaches (AIM 2). The findings will inform (I) mechanisms of brain (mal)adaptations associated with delayed vs. abnormal brain development in infants exposed to prolonged sedation; (II) identify early diagnostic and prognostic indicators for longitudinal neurocognitive correlates; and (III) inform future development of neonatal/infant therapies to mitigate the neurological effects in vulnerable infants exposed to prolonged sedation. Our application aligns with the goals of the National Institute of Child Health and Human Development (NICHD) and National Institute of Drug Abuse (NIDA) to improve the lives of children throughout all stages of development. We established the feasibility serving critical data collection, and we assembled an interdisciplinary team of experts for its successful completion. This research is innovative in that it encompasses a selected group of term-born infants exposed to prolonged sedation associated with dependence to drugs of sedation; novel in that it will investigate underlying mechanisms and neurodevelopmental impact of complex thoracic non-cardiac critical care; and significant in that it is expected to evolve pediatric critical care by developing novel adjunct therapies for age-specific pain and sedation treatment in the United States and the world.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract T cells play a central role in immunity and are of great therapeutic importance. Studies on T cell development provide direct insights into the pathophysiology of a wide variety of diseases and can facilitate adoptive T cell immunotherapies. Though a large effort has been made to identify pathways that regulate T cell differentiation, these studies have mostly focused on secreted proteins, cell surface receptors, and transcription factors known to directly drive developmental processes. Recently, we discovered that manipulation of an epigenetic regulator of lymphoid potential promotes the generation of mature, functional T cells from human induced pluripotent stem cells (iPSCs), demonstrating an important role for epigenetic modulators in T cell development and function. To date, the epigenetic regulation of T cell development remains elusive. In this application, we propose to test the hypothesis that iPSCs provide a platform to identify novel mechanisms regulating T cell development, and new insights into epigenetic regulations of T cell differentiation can be used to facilitate the generation of more robust iPSC-T cells for adoptive T cell immunotherapy. Previously, we have established a stroma-free differentiation system that faithfully recapitulates T cell development in culture. In Aim 1, we will perform small molecule screens at different stages of iPSC- T cell differentiation to discover new epigenetic regulators that can affect lymphoid specification and T cell maturation. We have provided a proof-of-concept by conducting a pilot screen using a library of small molecules that modulate the activity of epigenetic modification enzymes. The screen identified several epigenetic modulators, such as histone methyltransferase G9a, that promote T cell fate commitment. In Aim 2, we will transition our study to mechanistically examine how epigenetic regulations govern lymphocyte fate decisions via modulating chromatin structure. The impact of epigenetic modulators, including G9a, on lymphopoiesis will be probed in both iPSC-derived blood cells and zebrafish models. Lastly, Aim 3 will focus on using small molecule-mediate epigenetic modulation to facilitate the production of mature iPSC-T cells with enhanced function. Success in the proposed study will not only improve our understanding of the molecular mechanisms underlying the formation of immune cells but also open new avenues for stem cell-based immunotherapies. The career development award will allow me to develop new skills necessary to fulfill the proposed goals and foster my development into an independent investigator.

NIH Research Projects · FY 2025 · 2023-09

Project Summary From a Drosophila mutant screen for defects in the formation of the embryonic neuromuscular junction (NMJ), we have learned that mutations in components of the kinetochore complex are needed for the transformation of a growth cone into a correctly shaped synaptic connection. Loss of these proteins also alters the structure of sensory dendrites. In cultured mammalian neurons, kinetochore proteins also appear to guide development: their knockdown by RNAi causes an excess of filipodia like protrusions to form on hippocampal dendrites. This is a completely novel function for the kinetochore, a protein complex previously known only to function at the centromere of chromosomes where it is required in dividing cells to “catch” and stabilize spindle microtubules and thereby enable the segregation of chromosomes to the daughter cells. The neuronal phenptypes cannot be explained by defects in chromosome mechanics and therefore we hypothesize a postmitotic function for a “neuro-kinetochore”, a function that is likely to involve the same core property of the centromeric kinetochore: the ability to bind to the plus-ends of microtubules and stabilize them. We propose to test the hypothesis that a complex very much akin to that found at centromeres will function locally in post-mitotic neurons to assist in the transformation of dynamic growth cone microtubules into stable bundles of synaptic microtubules and similarly to stabilize dendritic microtubules and thereby arrest dendrite growth. The proposal makes use of the advantages of both Drosophila and mammalian systems. Aim 1 of this proposal therefore seeks to characterize in greater depth the nature of the defects at the embryonic fly NMJ and in hippocampal dendrites. Aim 2 delves into the mechanism underlying the phenotype by asking whether structure function studies support the hypothesis of a kinetochore-like structure that must bind to microtubules. Aim 3 focuses on the microtubule cytoskeleton and asks whether there are defects in microtubule dynamics and stabilization in the mutants, and how this novel role for kinetochore proteins fits into our knowledge of the processes that transform growth cones into mature endings and determine the morphology of dendrites.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ ABSTRACT Polycystic ovary syndrome (PCOS) is a major health concern that affects up to 10% of all reproductive-aged women and is the leading cause of female infertility. This complex, heterogeneous condition is associated with ovulatory dysfunction, hyperandrogenism, and cardiometabolic dysfunction and incurs an estimated $8 billion in annual U.S. healthcare costs. PCOS is associated with alterations in both ovarian factors (folliculogenesis, gonadotropin secretion and action, ovarian androgen biosynthesis) and non-ovarian factors (insulin secretion and action, weight and energy regulation). These contributing factors are all interconnected, and the inciting causes of PCOS remains unknown. As a result, the clinical care of PCOS is currently confined to managing the manifestations of PCOS rather than treating the underlying causes. The overall hypothesis for this study is that PCOS arises from the perturbation of multiple metabolic and reproductive endocrine pathways, some of which are shared and others distinct between women, men, and children. To test this hypothesis, this project will take advantage of a recently completed genome wide-association study meta-analysis that has doubled the number of genetic loci that influence PCOS risk. Because these genetic variants are present in all individuals, this discovery enables examination of the phenotypic effects of genetic risk factors for PCOS in not just women but also men and children. This project leverages the power of the UK Biobank, a cohort of nearly 400,000 adults in the UK, and four pediatric cohorts, Avon Longitudinal Study of Parents and Children (N>6,000), Copenhagen Studies on Asthma in Childhood (N>500), Project Viva (N>500), and the HOLBAEK Study (N>4,000). In adults, genetic risk factors for PCOS will be assessed for sex-biased effects on cardiometabolic and other outcomes and clustered with PCOS-related traits, and gene-set enrichment analysis will be used to gain mechanistic insights into these groups of PCOS genetic risk factors. In children, genetic risk scores (i.e., estimated genetic susceptibility to PCOS) will be calculated and tested for associations with cardiometabolic and androgenic traits as well as diverse metabolites. This proposal promises to implicate causal biological mechanisms underlying the pathogenesis of PCOS and its associated features, which could allow for the deconstruction of the causes of PCOS into distinct subgroups, and thereby pave the way for a precision- medicine approach to the diagnosis, risk stratification, and treatment of PCOS in women and its associated conditions in adults and children. Through this proposal, Dr. Zhu will attain new skills in advanced computational genetic methods, experience in applying principles in computational biology and bioinformatics, and a deep understanding of the impact of reproductive endocrinology on a variety of health conditions. The K08 award will provide Dr. Zhu the critical training and mentorship to achieve her goal of becoming an independent physician-scientist applying innovative computational methods to understand complex reproductive endocrine disorders.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY Pediatric feeding disorders are extremely common, affecting 5-20% of children, and they cause significant patient morbidity, decreased quality of life for children and parents, and increased healthcare utilization and cost. Many of these patients require feeding tube placement, but even this does not ensure successful feeding and growth. Up to 50% of children with gastrostomy tubes suffer from feeding intolerance, defined as vomiting or inability to tolerate adequate feed volumes to sustain growth. Despite the high prevalence and large health impact of this problem, no prospective studies exist to help inform management. Furthermore, the gastrointestinal mechanisms underlying feeding intolerance remain largely unknown, hindering our ability to effectively target treatments toward patient pathophysiology. Two proposed mechanisms of feeding intolerance are (1) delayed gastric emptying and (2) impaired gastric accommodation. We have shown that 49% of children with feeding intolerance have delayed gastric emptying; however, no studies have correlated delayed gastric emptying with feeding intolerance symptoms or assessed the role of other abnormalities of gastric physiology such as impaired accommodation. In Aim 1 of this proposal, we will use a cross-sectional study design to fully characterize (using both ultrasonography and scintigraphy) gastric emptying and accommodation patterns in gastrostomy-fed patients with and without feeding intolerance. In Aim 2, we will use a prospective cohort design to assess changes in gastric physiology and feeding intolerance symptoms after initiating therapy with erythromycin (which improves gastric emptying) or cyproheptadine (which improves gastric accommodation). Dr. Suzanna Hirsch, MD is an Instructor at Harvard Medical School (HMS) and a subspecialist within the Aerodigestive Center at Boston Children’s Hospital (BCH). She has gained substantial clinical research experience during her medical training and has demonstrated commitment to an academic career in patient- oriented research. Her career goal is to fundamentally advance scientific understanding of gastric dysfunction in pediatric feeding disorders and to use this mechanistic knowledge to drive evidence-based care and fuel treatment innovation. The mentorship and training afforded by this career development award will be critical for Dr. Hirsch’s academic development. Her primary mentor, Dr. Rachel Rosen, is an expert in pediatric motility disorders, and her co-mentor, Dr. Odd Helge Gilja, is an expert in gastrointestinal ultrasonography – both are exceptional researchers with longstanding commitments to mentorship. Dr. Hirsch has carefully crafted a career development plan with opportunities to gain skills in gastric ultrasonography and clinical research methods. She will pursue a Master’s in Public Health including coursework in repeated measures, longitudinal data analyses, and clinical trial design. Her research activities will be conducted in the unparalleled academic environments of BCH and HMS, which are firmly committed to her successful transition to independence.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY The choroid plexus (ChP) comprises a network of cells that form a critical brain barrier that can mediate secondary damage in certain brain disorders and trauma. The Lehtinen lab has developed a suite of tools to study the ChP across development ex vivo and in vivo. This project applies imaging technology to study blood- CSF barrier permeability regulation at the cellular level. Our overarching hypothesis is that the intracellular messenger cAMP regulates endothelial tight junctions between ChP epithelial cells and thereby blood-CSF barrier integrity as it does in the nearby blood-brain barrier (BBB). The main effectors of cAMP, PKA and Epac, regulate endothelial tight junction redistribution and barrier permeability. Gi/o-linked G-Protein Coupled Receptors (GPCRs) are strong upstream regulators of cAMP. The Lehtinen laboratory's single nucleus sequencing data suggest that ChP epithelial cells selectively and developmentally express mGlur8, which via Gαi, activation in other brain tissues inhibits cAMP production. To elucidate mechanisms of neurotransmitter alteration of the blood-CSF barrier we will study the cellular mechanisms of neurotransmitter-activated GPCR- cAMP signaling in ChP epithelial cells. The Lehtinen lab, in collaboration with co-sponsor Mark Andermann's lab, recently established a protocol for ex vivo and in vivo imaging of ChP structure and function based on using fluorescent reporters of calcium activity. I will apply these techniques to reveal how GPCRs modulate cAMP levels using fluorescent cAMP indicators in ChP explants (Aim 1). In Aim 2 we will use an in vivo preparation to map the populations of receptors that are accessible to central vs peripheral ligands. Those located on the apical membrane are in contact with the CSF and those on the basal surface are exposed to the blood. With functional assays including peripheral delivery of low molecular weight fluorescent dyes, we will assess the effects of central (CSF) vs peripheral (intravenous) delivered ligands such as mGluR8 agonists on cAMP and blood-CSF barrier permeability. Together these studies will reveal mechanisms how neurotransmitters, specifically glutamate, may contribute to blood-CSF barrier integrity in health and disease. The research and training proposed will take place at Boston Children's Hospital, a world-renowned pediatric hospital that offers an exceptional research environment and countless opportunities to carry research from bench to bedside. Importantly, the research proposed will take place under the guidance of Dr. Maria Lehtinen, an expert in the field of choroid plexus and CSF biology. In addition, Dr. Mark Andermann (co-mentor) is a leader in in vivo optical imaging techniques. The results from this proposal will result in first-authored publications and a wealth of preliminary data for a competitive K99/R00 application. This fellowship will provide the candidate with the opportunity to begin training in choroid plexus biology and make significant scientific contributions while also helping launch an independent academic career.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Sickle cell disease (SCD) and transfusion-dependent b-thalassemia (TDT) are severe, prevalent blood disorders for which fetal hemoglobin (HbF) induction can bypass the fundamental hemoglobin defects and hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative potential. Allogeneic and autologous trans- plant approaches can succeed, nonetheless, the short- and long-term toxicities of genotoxic alkylating chemo- therapy-based conditioning regimens remain a substantial barrier to the widespread application of curative HSCT for SCD and TDT. Immunotherapies targeting HSPC antigens have been proposed as a safer conditioning strat- egy, however the pharmacokinetics of these agents currently hamper their clinical efficacy. The long-term goal of our proposal is to address this unmet medical need by developing an effective, novel strategy for the engraft- ment and progressive enrichment of autologous gene modified HSPCs in SCD and TDT by coupling non-geno- toxic immunotherapy-based myeloablation with epitope-engineering. Our central hypothesis is that precise multiplexed base editing of HbF determinants and targeted epitopes within HSPCs can endow hematopoietic lineages with both HbF induction capacity and selective resistance to monoclonal Abs or CAR-T cells without affecting protein function or regulation (so-called stealth status). We have identified defined minimal amino-acid changes within the extracellular domains (ECD) of the cytokine receptors KIT, FLT3 and IL3RA, each expressed in long-term repopulating HSCs, that abrogate recognition by therapeutic Abs while preserving physiologic responses to stimulation with their respective ligands. Here, we will capitalize on these results and further expand the reach of these innovative genetic engineering tools with the objectives to i) generate “stealth” g-globin derepressed HSPCs by multiplex CRISPR-Cas base-editing; ii) validate efficacy of this approach on suitable pre-clinical models of b-hemoglobinopathy, and iii) further optimize and scale the manufacturing process for production of efficiently and precisely engineered cellular products suitable to progress to the clinic. We aim: 1) to optimize multiplex base editing approaches to simultaneously derepress HbF and engineer stealth HSPC epitopes that will generate immunotherapy resistant hematopoietic stem cells capable of ameliorating sickling and globin chain imbalance in SCD and TDT patient erythroid cells; 2) to maximize the engraftment of edited HSCs by optimizing immunotherapy regimens to enrich multiplex edited HSPCs through modeling parameters for therapeutic selection of edited HSPCs, and thereby obtaining proof-of-concept chemotherapy-free engraft- ment and selection of edited patient HSPCs; and 3) to identify conditions that produce efficient on-target base edits without measurable off-targets at clinical scale. This project will provide fundamental advancement of a new chemotherapy-free gene therapy approach to HSCT for hemoglobinopathies that should additionally have broad applicability to other hematopoietic disorders.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Malaria is an important cause of illness and death worldwide, with most of these deaths resulting from Plasmodium falciparum infection. The signs and symptoms of human malaria results from the asexual replication of parasites in human red blood cells. During this clinically important blood stage, P. falciparum parasites divide by schizogony – a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The generation of the invasive daughter parasites, known as merozoites, occurs with high fidelity, ensuring that each daughter has a single nucleus and the required organelles. The molecular mechanism underlying this high fidelity of nuclear and organellar partitioning is incompletely understood in Plasmodium. Studies in the related Apicomplexan parasite Toxoplasma gondii identified a family of proteins, referred to as striated fiber assemblins (SFAs), that are critical for parasite cell division. The SFAs are hypothesized to form a connection between the centrosome of dividing nuclei and the newly forming apical ends of the two daughter parasites during T. gondii endodyogeny. The P. falciparum orthologs of the SFAs, PfSFA1 and PfSFA2, have been reported to be dispensable in a genome-wide transposon screen. In contrast to this data, we demonstrate that these two proteins are essential for asexual replication in P. falciparum. Based on our preliminary data, we hypothesize that PfSFA1 and PfSFA2 localize to a filament-like structure during parasite segmentation. In the current proposal, we will test this hypothesize in a series of microscopy assays. Furthermore, we will also evaluate the function of PfSFA1 and PfSFA2 by characterizing parasite arrest and morphologic defects following inducible knockout. To gain a more complete understanding of the molecular functions of PfSFA1 and PfSFA2, we will determine the direct protein interactions by co- immunoprecipitation and the indirect or transient interactions by proximity labeling. By determining a robust protein interaction network for the Plasmodium SFAs, we will establish the foundation to understand their molecular function. We hypothesize that the SFAs proteins are critical for organization of segmentation during the asexual replication of P. falciparum. Together, the proposed studies will interrogate the function of these two essential proteins and directly identify their role during P. falciparum segmentation.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Chronic pancreatitis (CP) results from progressive inflammation and fibrosis, most commonly from acute pancreatitis (AP) and recurrent episodes of AP (RAP), and is associated with long term complications of diabetes and exocrine pancreatic insufficiency. Pain is one of three diagnostic criteria for AP and multiple studies have now confirmed that pain is also the most significant symptom for patients with CP (77-93%). While there are a variety of interventions available to treat pain, many of which are not efficacious in CP patient: only ~25% chronic pancreatitis patients achieve meaningful pain relief with current standard of care for pain management. As of now, there are no chronic pancreatitis pain biomarkers to stratify pain based on severity and/or type, which would guide physician decision making or monitor therapeutic responses. This contributes to the immense burden of pain in this CP patient population. To address this gap in knowledge, we will leverage the PROspective Evaluation of Chronic Pancreatitis for EpidEmiologic and Translational StuDies (PROCEED) which has been realized under the auspice of the NIDDK-/ NCI-funded Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer (CPDPC). PROCEED, which will be a key resource for this proposal, is the first prospective longitudinal CP cohort in the U.S. It has enrolled 1638 (and counting) participants with CP, AP, and RAP, as well as healthy and non- pancreatitis symptomatic controls. All participants of this unique cohort undergo deep phenotyping, provide detailed information on clinical symptoms, and general QoL variables. Furthermore, 10 pain-associated variables are recorded providing detailed information about pain types, severities and interventions. Due to the size and multi-site nature of PROCEED, the cohort was split into independent discovery and validation cohorts to enable FDA-compliant biomarker validation. In Specific Aims 1 and 2 of this proposal, we will use plasma and urine samples from the PROCEED discovery cohort (n >500) to generate quantitative multi-omics datasets of the proteins, chemokines, cytokines and neuropeptides. Mass spectrometry-based discovery proteomics and multiplexed antibody-based assays will be used for the biomarker identification. Upon discovery of promising pain biomarkers, we will transition to the validation phase (Specific Aims 3 and 4). In this phase, we will use the urine and plasma samples from the independent PROCEED validation cohort (n >500) to validate the potential biomarkers identified in the Specific Aims 1 and 2. We will use targeted liquid chromatography/mass spectrometry methods as well as antibody-based method for the validation. The large number of PROCEED samples and their superb annotation will allow for an exquisitely granular pain-focused analysis of the resulting large scale proteomic and cytokine/chemokine assay data.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Research: Children exposed to indoor air pollution are known to more likely develop asthma, and in those with asthma, indoor air pollution can worsen asthma symptoms. Radon (Rn), a radioactive gas known for its lung cancer effects, decays into radiation-emitting progeny which has potential to induce cellular damage once inhaled. While residential Rn exposure is common, less is known about the non-cancer health effects of Rn decay products. Current health regulations and priorities are based solely on its carcinogenic effects in the lung. Indoor exposure to radon has been recently associated with COPD morbidity and mortality and an increased risk for hospital admissions. Given the association linking residential Rn exposure to COPD morbidity, in this exploratory application, we propose adverse health effects associated with Rn decay products via particle radioactivity in asthma morbidity, a common condition of obstructive lung disease in the pediatric population. Candidate: Dr. Banzon’s long-term goal is to become an independent NIH-funded investigator focused on the role of assessing the physical environment with a specific focus on air pollution and asthma morbidity in inner- city children. This proposal details a five-year project to provide Dr. Banzon the training and expertise necessary to study the effects of radon progeny and particle radioactivity in asthma by measuring its health effects in an established cohort of inner-city children with asthma enrolled in the School Inner-City Asthma Study (PI: Phipatanakul). Her aim is to identify a novel and modifiable environmental exposure that contributes to asthma morbidity in children. The findings have potential to aid in the design of public health intervention trials, directly inform activities regarding radon mitigation as an intervention to alleviate disability from asthma, and identify biomarkers associated with particle radioactivity which may help phenotype patients and help predict those who may have a more favorable response to personalized asthma treatment based on biomarkers. Environment: Dr. Banzon will be mentored by Dr. Phipatanakul, an expert in epidemiology, clinical trials, and clinical investigation in asthma and allergic diseases. She has assembled an extraordinary team of advisors, including Drs. Petros Koutrakis, Jonathan Gaffin, and Brett Coull, who have committed their time, resources, and expertise to facilitate Dr. Banzon’s career development and successful completion of the proposed project. During this award period, Dr. Banzon will complete complementary coursework through the Harvard School of Public Health and Harvard Catalyst Program, with a focus to hone on her skills in environmental epidemiology targeted toward air pollution and childhood asthma. The academic environment created by the mentor, institution, Harvard University, and its affiliates provides fertile ground for learning and collaborating specific to her research. Dr. Banzon will emerge an expert in the field of environmental epidemiology, with a unique understanding of the physical environmental that will shape her into a successful independent investigator.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Reactivation of fetal hemoglobin expression in adult erythroid cells is a validated approach to genetic therapy of both b-thalassemia and sickle cell disease (SCD). As currently practiced, however, genetic therapy (either lentiviral delivery or CRISPR/Cas9 editing) cannot meet the large disease burden due to its reliance on myeloablative preconditioning of patients for hematopoietic reconstitution and the medically intensive nature of the procedure. Drug (small molecule) therapeutics are needed to treat the many patients with these disorders. The vision of our research is to use small molecule therapeutics to reactivate y-globin gene expression, and do so both robustly and safely. The most potent, validated repressor of g-globin expression is BCL11A. In this project we will extend our recent studies in which we leveraged new chemical methods of targeted protein degradation (TPD) to deplete BCL11A and reactivate HbF production. The project consists of two broad aims. First, we will determine the dynamics of globin gene transcription using a platform in which erythroid cells at different phases of the cell cycle can be isolated for nascent transcription analyses. Using acute TPD of BCL11A, we will then ascertain at which stage(s) of the cell cycle g- (HBG) globin is reactivated upon loss of the repressor. These data will provide temporal resolution of globin gene transcription at unprecedented resolution. In a second aim, we will explore the use of erythroid-specific E3 ubiquitin ligases, TRIM10 and TRIM58, to target degradation of BCL11A. The TRIM proteins will be directed to BCL11A with BCL11A-specific nanobodies. We will initiate discovery of ligands for the TRIM proteins in order to develop tool compounds for cell-specific TPD of BCL11A as a new therapeutic strategy. Our work will lay the groundwork for novel small molecule approaches for robust and safe reactivation of HbF for the hemoglobin disorders.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Cardiomyopathy and heart failure are leading causes of morbidity and mortality world-wide. In addition to ventricular dysfunction, heart-failure associated ventricular arrhythmias cause sudden death with few disease-modifying therapies. Changes in myocardial conduction, increased fibrosis, alterations of ion channel characteristics and genetic susceptibilities have all been postulated to underlie the increased risk of arrhythmia in heart failure, but no unifying mechanism is known. Post- translational modifications (PTMs) of cardiac proteins have emerged as critical factors in mediating normal physiologic function or leading to heart disease when dysregulated. Recently mutations in the N-terminal acetyltransferase complex type A (NatA) have been identified in patients with congenital heart disease, cardiomyopathy, and arrhythmia. This protein complex acetylates the N-terminus of nascent proteins regulating stability, subcellular localization, and complex formation, with nearly 40% of the proteome as potential targets. We have recently identified a large family with a novel mutation in the catalytic subunit of NatA, NAA10. Male patients have severely prolonged QTs, recurrent arrhythmias, developmental delay, learning disabilities, and cardiomyopathy, with female patients more variably affected. We created models of NAA10 dysfunction using induced pluripotent stem cells (iPSCs) derived from several affected male patients. Electrophysiologic analysis of differentiated iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated action potential duration (APD) prolongation, abnormalities of sarcomeric structure, calcium handling and corresponding dysregulation of sodium and potassium currents. Establishing a network of collaborators, we investigated the mechanism of NAA10 dysfunction and developed an animal model for cardiac- specific ablation of NAA10. We propose to use our scalable model systems to investigate the currently unknown role of N-terminal acetylation within the heart as an entry point to understanding the mechanisms of arrhythmia risk in heart failure. In Aim 1, we will determine the mechanism of how N-terminal acetylation regulates sodium and potassium ion channels along with the discovery of other target proteins. In Aim 2, we will use recently developed murine models to selectively ablate Naa10 and the paralogue Naa12 within the heart to determine the causative mechanisms of N-terminal acetylation in heart failure and arrhythmogenesis. In Aim 3, we examine the contribution of N-terminal acetylation in acquired forms of heart disease including human heart failure. This transformative proposal will provide novel mechanistic insight into the poorly understood role of N-terminal acetylation in cardiovascular disease with potential for improved arrhythmia risk stratification and therapeutic development.

NIH Research Projects · FY 2025 · 2023-08

Concussion, also referred to as mild traumatic brain injury (mTBI), is a neurological disorder that causes disability in children and predisposes them to challenges later in life, including significant physical, cognitive, and psychological disability. The trigeminal nerve extends from the central nervous system to innervate the greater portion of face and eye, and may offer insight into head trauma pathophysiology. In particular, painful sensitivity to light (photophobia), corneal nerve pathology, and atypical functional activity within the brain have all been reported in preliminary work evaluating persons with mTBI and trigeminal nerve pathology. Our overall hypothesis is that the trigeminal nerve is sensitive to mTBI and accounts for pain-related symptomatology. To test this hypothesis, in aim 1, we will define trigeminal nerve pathology in persons with mTBI using quantitative sensory testing (QST), in vivo corneal nerve microscopy (IVCM), and diffusion tensor imaging (DTI). In aim 2, we will define central nervous system changes to light-induced pathways with mTBI-related photophobia using fMRI and DTI. This study is likely to yield (1) a diagnostic marker that is sensitive to mTBI, (2) evince a neurological source of mTBI-related pain symptoms. Data generated from this investigation can be used to improve the diagnosis, treatment and monitoring of patients suffering from head trauma and provide an objective marker to base clinical decision making.

NIH Research Projects · FY 2025 · 2023-08

(PLEASE KEEP IN WORD, DO NOT PDF) Title: The role of Septin6 Group in Murine and Human Hematopoiesis 1 R01 DK137172-01 Septins are highly conserved GTPase proteins which regulate a variety of cellular functions including cytokinesis, polarity, cell cycle, vesicle trafficking, exocytosis, and creation of diffusion barriers. We have recently identified a non-syndromic newborn with severe neutropenia (Renella, AJH 2022) with a novel X-linked germline mutation in the C-terminus of SEPTIN6 gene (SEPTIN6 c.1282T>C p.428Glnext*9) associated with dysmyelopoiesis. Editing the germline mutation into normal male CD34+ hematopoietic stem and progenitor cells (HSC/P) phenocopied key pathologic features of the clinical syndrome, including large, multinucleated nuclei and reduced myeloid progenitor growth in vitro (preliminary data). The C-terminus has been proposed to play a key role in filament stabilization, bundling and bending as well as interactions with other septins and in silico modeling suggest the addition of 9 amino acids (aa) associated with the SEPTIN6 c.1282T>C p.428Glnext*9 mutation would alter function by interfering with SEPTIN6 protein interactions. Based on our initial publication, a second patient with severe neutropenia has since been identified in Seattle with a distinct mutation in the same codon (SEPTIN6 c.1282T>A) (Mohamad, Allensbach, Williams, accepted for presentation, ASH 2022). While the aa substitution is different, modeling predicts that SEPTIN6 variants adopt the same structure. Both identified patients had a high degree of myeloid tetraploidy in the marrow and were refractory to G-CSF.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Maternal prenatal stress and nutrition influences fetal growth and long-term child outcomes. Maternal psychological stress and undernutrition frequently co-occur and affect overlapping biological stress axes involved in fetal growth and neuroendocrine development. Still, little is known about the interactive effects of maternal prenatal stress and nutrition on fetal and long-term child outcomes. The proposed K99/R00 research will leverage data from the ongoing “Enhancing Nutrition and Antenatal Infection Treatment for Maternal and Child Health” (ENAT) trial in rural Ethiopia to examine interactive effects of maternal prenatal stress and nutrition intervention on fetal and child outcomes. ENAT randomized 2390 pregnant women to receive an “enhanced nutrition” package or “standard care” and collected data on maternal prenatal stress (Cohen's Perceived Stress Scale) throughout pregnancy. Existing ENAT data will be used to determine independent and interactive effects of maternal prenatal stress and nutrition intervention on birthweight-for-age (Aim 1, K99). During the R00 I will conduct a new follow up study with ENAT mother-child dyads at 36 months postpartum to assess children's stress-sensitive outcomes (Aim 2, R00). Specifically, the developmental follow-up study will assess child outcomes related to attention, stress reactivity, memory, and socio-emotional functioning. I will also use biospecimens from ENAT to examine whether maternal and newborn telomere lengths can serve as biomarkers of in- utero programming of BWAZ and child outcomes in relation to maternal prenatal stress and nutrition (Aim 3, R00). I hypothesize that maternal prenatal stress and nutrition intervention will have main and interactive effects on newborn and childhood outcomes. I also hypothesize that maternal and newborn telomere length will serve as biomarkers for prenatal programing effects of maternal stress and nutrition. My long-term career goal is to lead independent multi-disciplinary research delineating the complex ways in which biological and psychosocial factors influence fetal and child development in low resource environments. This award would allow me to acquire basic science knowledge of maternal-fetal nutrition and telomere biology, technical skills in advanced neurodevelopmental assessments including eye-tracking and electrocardiography, and practical skills in primary data collection and research management in a low-income country. During the R00 I will independently lead an innovative child development research study in rural Ethiopia. The proposed research aligns with NICHD's goal to improve pregnancy outcomes to maximize the lifelong health of women and children and to help all children thrive. The proposed study will generate epidemiologic and biologic evidence linking maternal prenatal nutrition and stress with newborn and childhood outcomes that are sensitive to prenatal stress and predict long-term outcomes related to school achievement and mental health. I intend to use this knowledge to guide public health decisions regarding pre- and postnatal intervention for the millions of women living in poverty both domestically and globally and to develop future intervention to support child development in low resource settings.

NIH Research Projects · FY 2026 · 2023-08

SUMMARY In tissues, immune cells are well-organized spatially and perform specific functions to dictate tissue homeostasis and inflammation. Yet, it is rarely understood how the organization and functions of immune cells are regulated by their surrounding tissue stroma. The overall vision of the lab is to elucidate the impacts and mechanisms of the interactions between immune and non-immune cell types, in order to understand general principles of how immune functions are regulated within tissues. This may ultimately lead us to program immune responses to restore tissue homeostasis from disease. The lab has discovered that macrophages and fibroblasts, two cell types that are commonly present in mammalian tissues, self-assemble into a tissue-like system that maintains a stable and robust population composition. These homeostatic features rely on paracrine communication of growth factors and direct contact between these cells. This macrophage-fibroblast system provides a unique, accessible and modular platform to discover immune functions in the cellular context of stromal cells and to dissect the underlying mechanisms. Our published and preliminary data formulate the overall hypothesis of the lab: distinct cellular responses emerge from the interactions between macrophages and fibroblasts. The goal of the lab in the next five years is to identify the cellular and molecular bases of the interactions between macrophages and fibroblasts and to characterize how these interactions regulate immune responses during tissue homeostasis or inflammation. We aim to elucidate general principles that regulate functions and organization of immune cells in tissues, by developing new methods to study their interactions and interactive partners, discovering interaction receptors to perturb them, and defining cellular functions dependent on such interactions. To reach this goal, 3 directions will be pursued in my lab: 1) develop fucosyl-labeling, a new cell- based chemical genetic approach, to identify cell-cell interactions, 2) identify molecules responsible for the physical association between macrophages and fibroblasts, leveraging candidate-based genetic approaches, 3) determine how the interactions between macrophages and fibroblasts impact their cellular responses under inflammatory conditions using transcriptome profiling, imaging and immunological assays. Research proposed in this application is innovative because it presents a comprehensive strategy to directly tackle the mechanistic nature of immune-stromal interactions, combined with exploration of a new research paradigm and the development of cutting-edge technologies that will have a fundamental impact in basic cell biology and immunology. The proposed research is significant because it will identify the cellular mechanisms that regulate immune functions critical for maintaining tissue homeostasis and regulating inflammatory responses. My lab will advance our knowledge of inter-cellular processes that may lead to inflammatory disorders and define new possible therapeutic targets. The reagents, tools and experimental systems developed here will benefit the broad scientific community to explore multi-cellular interactions.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY The twin epidemics of obesity and type 2 diabetes mellitus (T2DM) continue to worsen, highlighting a growing need to understand the dysregulation of appetite and insulin secretion that contribute to these diseases. Enteroendocrine cells (EECs) are key regulators of both appetite and insulin secretion. Thus, the long-term goal of this project is to understand the transcriptional and epigenetic regulation of EEC differentiation and function, and how this is perturbed in disease states. The overall objective of this application is to identify and evaluate factors that are necessary for EE differentiation and hormone production/secretion. The central hypothesis is that EEC growth and function are controlled by intrinsic and extrinsic factors. Intrinsically, we have shown that FOXO1 inhibition (AS) and, separately, CB1/JNK inhibition (RSP) induce human EE differentiation, but the mechanisms driving this are not known. Extrinsically, nutrients are known to regulate EE hormone production, but it is unclear how other factors, including disease-related cytokines and hormones, alter this response. The rationale for this proposal is that through a better understanding of EEC differentiation and function, unique therapies can be developed to regulate appetite and insulin secretion via EE hormones. The central hypothesis will be evaluated in two specific aims: 1) to identify the role of HES6 and LMX1B in human EE differentiation and to identify the epigenetic and transcriptional changes driven by AS and RSP during early EE differentiation; and 2) to evaluate the impact of cytokines and hormones on nutrient-stimulated EEC function. In Aim 1, we will study gene expression changes (using CRISPR/Cas9, SHARE-seq), epigenetic changes (using SHARE-seq, bulk ATAC-seq), and protein interactions (using co-immunoprecipitation) in human duodenal organoids to investigate the roles of HES6 and LMX1B and to identify new factors involved in early stages of EE differentiation. In Aim 2, we will assess the ability of EECs (derived from duodenal and rectal organoids) to recapitulate the response of native EECs to nutrient stimulation. We will assess hormone production and secretion (using ELISA, qPCR), and how these responses are dysregulated following exposure to obesity/T2DM-associated cytokines and hormones. The candidate for this K08 proposal is Daniel Zeve, MD, PhD, a pediatric endocrinologist with expertise in developmental biology and metabolism. With his mentor, Dr. David Breault, Dr. Zeve has designed a career development plan to achieve scientific independence. This plan will be performed at Boston Children’s Hospital, allowing the applicant access to a multitude of resources throughout the Harvard Medical system. During the award period, the applicant will gain additional experience in SHARE-seq, epigenetics, CRISPR/Cas9, and bioinformatics through multiple avenues, including coursework, seminars, and high-level collaborations. This innovative project, which seeks to unravel the regulation of EEC differentiation and function, will highlight potential novel therapeutic targets for obesity and T2DM, and provide a strong foundation for Dr. Zeve’s scientific independence and future R01 submissions.

NIH Research Projects · FY 2026 · 2023-08

Wiskott-Aldrich Syndrome-Like, WASL, is essential for F-actin dynamics within cells. WASL also has fundamental, yet not well characterized, roles in transcription and epigenetic regulation in the nucleus that are actin-dependent and actin-independent. The balance between these multifaceted roles of WASL is key in understanding its regulatory function tying external environmental signals to cellular behavior and differentiation. As dysregulation of these cellular processes have been tied to increased cancer invasiveness and neoplastic cell transformation, it is essential to understand the dynamics of WASL regulation. We recently uncovered a surprising role for WASL in regulating developmental patterning: We find that WASL is necessary for the formation of skeletal pattern and increased WASL signaling leads to the formation of novel skeletal elements. Importantly, we find that this patterning role for WASL is conserved across vertebrates. A developmental function for WASL was previously unknown and unexpected given its core functionality in the cell. However, as misregulated WASL activity results in disruption of both cytoplasmic F-actin regulation as well as changes in transcription, it remains unclear how WASL orchestrates specific signals underlying these developmental events. Here, we capitalize on new paired gain- and loss-of-function models in the zebrafish and the mouse to address the central hypothesis that WASL regulates skeletal development through modulation of transcription and is independent of its role in cytoplasmic F-actin dynamics. We outline three independent approaches to directly address this hypothesis. In Aim 1, we will take advantage of the modular nature of WASL protein and remove specific regions of the protein required for establishing F-actin nucleation. These ∆VCA mutant alleles of WASL will be compared against the specific gain-of-function WASL allele we have identified, both separately as well as in cis, through analysis of WASL localization within the cell, cytoplasmic F-actin formation, Hox gene transcription, as well as skeletal patterning. Then in Aim 2, we will use our models of loss and gain of WASL activity to define the specific transcriptional and epigenetic changes associated with WASL regulation during limb and fin development. This allows us to identify definitive transcriptional signatures of WASL in development and their dependence on Wasl F-actin binding. We will further assess the dependence of F-actin formation in WASL regulation of chondrogenic differentiation of limb bud cells and how this affects transcriptional modulation during development. Lastly, in Aim 3, we capitalize on natural variation in WASL amino acid sequence to refine specific phosphorylated residues as potential key regulators of skeletal diversification. Using our new experimental tools, we will parse the regulation of WASL activity and function in skeletal growth and patterning during development. Through the completion of these approaches we will broaden our understanding of the intricate, and instructive roles of WASL in cell behavior and differentiation and how shifts in this integration can lead to generation of novel structures and variation in form.

NIH Research Projects · FY 2024 · 2023-07

ABSTRACT Both private entities and academic groups are pioneering B cell editing for therapeutic purposes, either to express therapeutic antibodies from their native Ig loci, or other transgenes from an ectopic locus. BCR editing is currently performed with CRISPR/Cas system and a homology- directed repair template. Non-BCR trasngenes are introduced by retroviral transduction or transposon-based random insertion. Both nuclease-based and insertion-based B cell engineering techniques carry risks associated with chromosomal deletion (CRISPR/Cas) or insertional mutagenesis (retroviruses/transposons). Furthermore, efficient editing is only possible by isolating and editing B cells ex vivo meaning that therapies based on these editing protocols will likely be very expensive. We have discovered a method of B cell editing that requires no exogenous nucleases and does not rely on random insertion. Our method relies on transducing class-switching B cells with a DNA template supplied by a recombinant adeno-associated virus (rAAV) vector. The inverted terminal repeat (ITR) sequences in the rAAV naturally integrate into double-strand breaks created by the B cell class-switch machinery. With the right expression cassette designs, we can replace the endogenous heavy chain variable (VH) segment or even express a non-antibody transgene from within the BCR locus. This nuclease-free technique has potential advantages in terms of safety and, because it requires only a single rAAV transduction event, it also promises a simple, cost effective means of editing B cells in vivo. Here we aim to develop our nuclease-free editing technique and provide proof-of-concept for therapeutic applications. In Aim 1, we will optimize the design of our rAAV-delivered repair template, and demonstrate the relative safety of our approach compared to CRISPR/Cas-based editing. In Aim 2, we test different expression cassette designs for antibody and non-antibody transgene expression and determine whether or not inclusion of cis-acting genetic elements that increase somatic hypermutation can enhance affinity maturation of our edited B cells. In Aim 3, we will address in vivo editing. We will determine whether or not in vivo nuclease-free editing efficiency can be enhanced by vaccinating mice prior to rAAV administration to drive B cell class switching and optimize our rAAV doses and timing relative to the pre-vaccination step. We will also demonstrate the ability of our edited B cell system to produce recombinant antibodies (BCR editing) and erythropoietin (non-antibody transgene expression) in mice.