Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2024-09

This proposal investigates mechanisms that combine T2D (type 2 diabetes) with NAFLD (non-alcoholic fatty liver disease), which progress during chronic hepatic IR (insulin resistance) to life-threatening NASH (non- alcoholic steatohepatitis). Several studies argue that selective hepatic IR is required to integrate T2D with NAFLD/NASH. Regardless, we find that complete hepatic IR exacerbates NAFLD/NASH and T2D in mice fed the western GAN diet—which models common sugar-sweetened food and beverages associated with T2D and NAFLD/NASH in people. We model complete hepatic IR with ‘LDKO’ mice that lack hepatic Irs1 (insulin receptor substrate 1) and Irs2, which activates FoxO1 mediated transcription to induce hepatic Fst (follistatin) expression and secretion. Circulating hepatic Fst causes WAT (white adipose tissue) IR and uncontrolled lipolysis in LDKO mice. Our human data show that FST is elevated in NAFLD patients and stimulates lipolysis in human adipocytes in vitro. Interestingly, phenotypic clustering identifies an LDKO-like subset of patients with higher liver fat and circulating FST, WAT IR, and reduced lipid storage in gluteal-femoral fat. Our proposal investigates whether hepatic FoxO1 and Fst accelerate progression of NAFLD to NASH during hepatic IR. We use mouse genetics to determine whether inactivation of FoxO1 or Fst in LDKO mice fed the GAN diet can attenuate both NAFLD/NASH trajectory and liver inflammatory gene expression to identify pathways causing NAFLD/NASH during hepatic IR. Re-esterification of circulating fatty acids with hepatic Gro3P (glycerol-3- phosphate)—a fructose metabolite—is a major source of hepatic triacylglycerol in NAFLD patients. We use a simpler high-fructose diet (HFruD) to investigate whether FoxO1 and/or Fst establish NAFLD from hepatic fructose metabolism during complete or selective hepatic IR. To understand whether fructose accelerates NAFLD/NASH during hepatic IR through re-esterification of circulating fatty acid, we investigate LDKO mice fed the HFruD after deletion of hepatic FoxO1 or Fst—or deletion of Khk (Ketohexokinase) to prevent hepatic fructose metabolism. We feed mice [13C]fructose and use LC-MS to determine the incorporation of fructose metabolites into the glycerol or fatty acid moieties of liver or circulating triacylglycerol. In humans and mice, NAFLD might arise from ‘selective insulin resistance’—owing to uncontrolled hepatic glucose output in conjunction with some insulin-stimulated lipogenesis; however, we posit that selective hepatic IR might have the opposite effect and attenuate NAFLD/NASH owing to some inhibition of FoxO1 and Fst. Since chronic nutrient excess suppresses hepatic Irs2, we investigate GAN diet-induced NAFLD/NASH and T2D in mice lacking hepatic Irs2 (LKO2 mice) or Irs1 (LKO1 mice), as well as our novel Irs2tg transgenic mice with durable Irs2 expression in hepatocytes of Cntr or LDKO mice. Completion of our proposal can impact human health by identifying systemic and hepatic metabolic regulatory mechanisms by which hepatic IR integrates T2D with NAFLD/NASH.

NIH Research Projects · FY 2025 · 2024-09

Neurodevelopmental disability can be caused by pathogenic variants in any one of numerous genes, many of which are still being identified. The identification of a variant of uncertain significance (VUS) in an NDD gene in a proband leads to families, clinicians, and scientists wondering if the VUS is causal or a “red herring.” Ideally, reliable, cell-based, protein-based, or biochemical assays would test functional consequences of such variants, helping add experimental evidence for variant reclassification under ACMG criteria. However, for many NDD genes, there are no in vitro or ex vivo assays, and the effect of a VUS must be tested in vivo. This exploratory grant proposes an efficient and innovative strategy to evaluate VUSs in genes that cause NDD when haploinsufficient in humans, that cause no phenotype or a subtle phenotype when haploinsufficient in mice, and that cause a clear fetal phenotype when knocked out (KO) in mice. This is the situation for many human disease genes, including the NDD gene ANKRD17. De novo heterozygous nonsense, frameshift, and canonical splice site variants in ANKRD17 found in multiple probands with NDD indicate the mechanism of mutational effect is haploinsufficiency. However, Ankrd17 haploinsufficient mice are viable and fertile, without a reported neurodevelopmental phenotype. In contrast, Ankrd17 KO mice exhibit a lethal cardiovascular phenotype at E10.5. ANKRD17 and Ankrd17 are 96% identical and 98% similar at the amino acid level. The advantage of our strategy is that is simpler to determine if a hemizygous or homozygous variant is a loss-of- function because it causes a fetal KO phenotype than it is to determine if that variant causes a subtle heterozygous phenotype postnatally. The former only requires efficient zygotic editing, and fetal phenotyping and genotyping. The latter requires newly generated mouse strains and postnatal neurobehavioral testing. As proof of concept, we will use i-GONAD to introduce Ankrd17 pathogenic and benign variants in E0.7 zygotes. i-GONAD is an efficient in vivo zygote gene-editing method for introducing hemizygous, heterozygous, homozygous, and compound heterozygous variants. We will phenotype fetuses at E10.5 and correlate phenotypes with genotypes. We expect fetuses that are hemizygous and homozygous for pathogenic variants to exhibit the same phenotype as KO fetuses, while fetuses hemizygous or homozygous for benign variants will appear normal. We will then test VUSs in ANKRD17 that were identified in patients with NDD and make the results of these experiments accessible via the NIH-supported ClinVar portal. Successful completion of these experiments will establish a new paradigm for testing VUSs in genes that cause highly penetrant haploinsufficiency phenotypes in humans, subtle or no haploinsufficiency phenotype in mice, and highly penetrant fetal phenotypes in knockout mice.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY / ABSTRACT The composition and organization of the cell surface are critically important for cell-to-cell communication and for the ability of cells to interact with their surrounding environment. The cell surface is decorated with myriad glycosylated transmembrane proteins for which there is a molecular understanding of their function and cell surface presentation. Surprisingly, RNA and RNA-binding proteins (RBPs) are also glycosylated and components of the cell surface. However, is unknown what glycosylated RNAs and RBPs do at the cell surface. This unexplored area of biology represents a major knowledge gap in our understanding of both the RNA and cell surface fields. Thus, the immediate goal of this proposal is to close this knowledge gap and to create the conceptual and technical foundations that will enhance our understanding and exploration, respectively, of the biology of RNA at the surface of the cell. To achieve this, we propose to explore three fundamental, but outstanding, hypotheses. Importantly, these hypotheses are established on sound logic of the known biology of RNA and RBPs and supported by the published literature and preliminary data. Hypothesis 1 explores the molecular, biochemical, and biophysical activities of the cell surface localized RBPs DDX21 and NCL in the extracellular space. Hypothesis 2 takes an unbiased genetic approach to determine how cell surface RBPs are trafficked to the cell surface, given that they lack a classical membrane-targeting signal. Hypothesis 3 explores the intersection between cell surface RBPs and RNA in disease pathogenesis by focusing on the functional characterization of cell surface RBPs that possess cell-penetrating activities. The expected outcomes of this research are to reveal that the biochemical activities of RBPs are not restricted to the intracellular space of the cell, but also to the cell surface. The significance of the proposed research lies in expanding the field of RNA to the surface of cells and providing proof of concept of the importance of cell surface RBPs in human disease. While high-risk, the proposed research addresses an important problem and has the potential to be transformative. Hence its suitability for the Transformative Research Award.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Cognitive dysfunction (CD) is a common neuropsychiatric manifestation of childhood-onset systemic lupus erythematosus (cSLE) that remains poorly diagnosed and undertreated. CD and other neuropsychiatric symptoms of cSLE can negatively impact school function, self-management, and psychosocial health, as well as lifelong health-related quality of life and occupational opportunity during adulthood. While prior studies in patients with cSLE point to the association of CD with volumetric reductions of gray and white matter, the functional neurobiological aberrancies underlying CD and their relationship to negative health-related behaviors remain largely understudied. Furthermore, in a heightened inflammatory state, the homeostasis of the CNS is likely compromised, implicating neuroimmune systems such as the choroid plexus, which serve as blood- cerebral spinal fluid or immunological barriers. Disruptions localized to the choroid plexus may initiate a range of outcomes, including neuroinflammation, alterations in gray and white matter structural properties, or neuropsychiatric manifestations including CD. The goal of this R21 is to characterize the biobehavioral basis of CD in cSLE by cross-sectional interrogation of (i) prefrontal and parietal cortices with fNIRS and fMRI, (ii) microstructural white matter properties using diffusion tensor imaging (DTI), (iii) volumetric properties of the choroid plexus using high-resolution structural MRI, (iv) neurometabolite (e.g., choline (Cho) and myo-inositol (mIns)) concentrations using single-voxel MRS, (iv) cognitive function using the Pediatric Automated Neuropsychological Assessment Metrics (PedANAM) and supplementary cognitive tests (e.g., Digit span task)40, 41, and (v) psychiatric symptom (brief psychiatric rating scale [BPRS]42) and disease (SLE Disease Activity Index- 2K [SLEDAI-2K]43) severities. We will prospectively enroll 20 cSLE patients (12-18 years old, male or female) and 20 age-sex matched healthy controls (HCs). To better understand the role of comorbid mood symptoms in cSLE, we will also enroll 20 children with depressive or anxiety disorders. We will leverage existing structural MRI datasets (30 cSLE patients + 30 matched HCs). Our hypothesis is that CD in cSLE is underpinned by a combination of dysfunction of the prefrontal cortex, altered white matter microstructure, and compromised choroid plexus barrier function marked by morphological change. Understanding the biological basis and behavioral outcomes of CD can improve recognition of neuropsychiatric cSLE and realign therapeutic strategies with neurobiological phenotypes. We propose the following Specific Aims: Aim 1: Investigate functional CNS properties associated with CD in cSLE. Aim 2: Determine the associations among white matter microstructure with severity of CD in cSLE. Aim 3: Quantify the choroid plexus volume in cSLE and its relationship with CD, glial activity, and disease activity. To carry out this work, we assembled a team with complementary experience in the treatment and investigation of cSLE, neuroscience, immunology, psychiatry, neuroradiology, and statistics.

NIH Research Projects · FY 2024 · 2024-09

Project Summary Impairments in language development are common in children with autism and can have a profound influence on their future developmental outcomes and quality of life. There is great variability in both language ability at the time of autism diagnosis and in the rate of language gains during preschool years. Importantly, language ability by early school age is one of the best predictors of future academic success, behavioral functioning, and independence. However, our understanding of factors, including underlying neurobiological mechanisms, impacting early language development in autism is limited, reducing our ability to develop effective interventions and improve outcomes. Through harmonization of longitudinal and cross-sectional data collected from 165 autistic children across four studies, the proposed secondary analysis aims to identify early EEG indices of delays in language acquisition in ASD, and identify neurobiological, behavioral, and environmental factors that prevent or promote further language gains. The goal of using this approach is to identify specific mechanistic factors that impede language early language acquisition in autism. To do this we will use neuroimaging data, language assessments, and natural language samples collected during early development to (1) Characterize resting state electrophysiological differences in autistic children with and without language impairment at 2–3 years of age; (2) Identify early neural markers that predict limited language gains in ASD; and (3) Identify and characterize neurobiological, behavioral, and environmental factors that predict greater language gains in 2-year-olds with ASD. In alignment with the goals of the NIH Tackling Acquisition of Language in Kids (TALK) initiative, this proposal leverages existing longitudinal and cross-sectional data sets to understand developmental trajectories of late talking children with autism. Research activities include harmonization of EEG processing and behavioral measures across multiple data sets, additional transcription and behavioral coding of parent-child interactions, and re-consenting participants to make data available to NIH data repositories. Results from this study will advance our understanding of neurodevelopmental pathways preventing or promoting language development in autism and inform new methods for early detection and therapeutic intervention.

NIH Research Projects · FY 2025 · 2024-09

Daily cannabis use among young adults aged 18-26 years is at historically high levels and >4.8 million young adults meet criteria for cannabis use disorder (CUD). Heavy cannabis exposure during brain development adversely affects cognition, emotions, risk for psychiatric disorders, and educational and other social outcomes. However, most young adults with CUD do not receive treatment. Brief motivational interventions may be effective for CUD in young adults, but effects are small and evidence is of low quality. Combining motivational interventions with other approaches may enhance effectiveness, and provision in primary care and use of telehealth may improve reach. We have developed a novel intervention, MOMENT-V, that combines telehealth motivational enhancement therapy (MET) with mobile health (mHealth) ecological momentary intervention (EMI) to provide a fully remote brief intervention for CUD in young adults seen in primary care, building on our prior research on cannabis use in daily life, a brief intervention with in-person MET plus EMI, adapting in-person counseling for the virtual environment, and brief interventions in primary care. In an open pilot, 100% of young adults were retained, and satisfaction and engagement with MOMENT-V was high. We now propose a pilot randomized controlled trial of MOMENT-V vs. enhanced usual care in 60 young adults aged 18-26 years with CUD recruited from primary care clinics, using mixed methods and with follow-up at 3 weeks, 3 months, and 6 months. The specific aims are to determine 1) intervention feasibility (primary outcomes: intervention completion, EMI engagement, acceptability), 2) trial feasibility (primary outcomes: screening, eligibility, enrollment, retention), and 3) preliminary efficacy (primary outcomes: cannabis use frequency, problems from/negative consequences of cannabis use). Primary outcomes will be evaluated against a priori benchmarks. Secondary outcomes will include MET adherence to motivational interviewing principles, therapeutic alliance, duration of study activities, barriers, facilitators, CUD symptoms, amount of delta-9-tetrahydrocannabinol (THC) used in standard units, motivation to change use, psychological distress, cognitive function, and quality of life. All intervention and trial procedures will be conducted remotely. Additionally, we will explore the feasibility of videorecorded and live video-observed oral fluid testing for THC and of using these results to assess cannabis reduction in a fully remote trial. This proposal is responsive to PAR-22-183 areas of special interest, including research that uses innovative technologies and Stage I treatment development research testing behavioral interventions within primary care. Results will be used to develop an R01 proposal for a fully powered randomized controlled trial to test the effectiveness of MOMENT-V. Findings from the proposed study will enhance efforts to provide high-quality brief treatment to young adults with CUD and inform research using telehealth, mHealth, remote trial implementation, and oral fluid testing to objectively assess cannabis use reduction.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Primary language impairment (PLI) affects 6-8% of children in the U.S. and the longer it goes undetected or untreated, the greater the negative cascading effect on children’s functioning. Late talking (LT) in toddlerhood (not meeting expressive language milestones between 18-24m) is an often-used marker of PLI risk (approximately 40% of LT have persistent delays and go on to receive a diagnosis of PLI). To date, research on LT has identified several factors that are associated with poorer language in the second year, including demographic, family history, and child neurocognitive variables. However, there is little extant research focusing on predictors of delayed expressive language in the first year, before the onset of major language milestones. For the proposed secondary data analysis, we will analyze data from three existing longitudinal studies conducted in our lab. These projects collectively provide a rich, densely sampled longitudinal dataset from 2-36m encompassing a diverse sample of children (n>300) with distinct factors (e.g., socioeconomic, familial history of language disorder or autism) that put them at elevated likelihood of developmental language delay. From these projects, we have access to a suite of demographic and environmental data, language measures, resting-state electroencephalogram (EEG) data, and videos of parent-child interactions (PCX). We plan to process EEG data, code PCX videos for language and parenting behaviors, and harmonize variables across studies to establish a unified dataset to retrospectively investigate the biopsychosocial factors that contribute to late talking and predict risk for PLI in early infancy. We have identified approximately n=64 late talkers in this sample and plan to select a group of matched peers who met typical expressive language milestones at 24m. The specific aims of the project are: 1. To trace longitudinal trajectories of EEG features from 2-24 months that are associated with late talking. 2. To identify features of the psychosocial environment with the greatest explanatory power for individual differences in EEG developmental trajectories and language outcomes at 24 and 36m. Our goal for this project is to establish a comprehensive foundation for identifying the neural mechanisms and environmental factors associated with language development that may, in combination, improve predictive models of delay and impairment. In addition, we plan to make this dataset available on NIH and public databases to facilitate future secondary analyses that may uncover nuanced patterns in the neurodevelopmental trajectories and psychosocial environments of late talkers.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Alzheimer’s disease (AD) and other neurodegenerative diseases are characterized by age-related onset and progressive neuronal loss. Neuroinflammation mediated by disease-associated microglia is increasingly implicated in AD pathogenesis. As the primary immune cells in the brain, abnormally reactive microglia have been recently found to create a neuroinflammatory environment that results in neuronal death, yet the mechanisms by which age and genetic factors interact remain largely unknown. Somatic mutations accumulate in various cell types during the development and aging process of the human body. Clonal expansion, driven by somatic mutations in genes regulating cell proliferation, is associated with an increased risk of cancer with age, but has only recently been linked to a growing list of non-cancer neurological diseases. Notably, somatic BRAF mutation in the microglial lineage has been implicated in histiocytosis-associated neurodegenerative conditions. Our preliminary results from three AD cohorts and with three sequencing technologies consistently show an excess of clonal somatic mutations in AD brains, particularly in proliferation- related genes of microglia. This new study aims to examine if the accumulation of somatic mutation contributes to an age-related increase in AD risk by driving clonal expansion of microglia, which subsequently induces neuroinflammation and neuronal loss in AD brains. The first Aim of the study is to identify somatic mutations by re-analysis of the existing bulk RNA-seq datasets from large AD cohorts, and compare the transcriptome-wide burden and distribution of somatic mutation between different brain regions of AD patients and matched controls. In the second Aim of the study, molecule-barcoded ultra-deep panel sequencing will be applied to screen for somatic mutations more sensitively among genes that regulate cell cycle and proliferation in AD and control brains; we will further use amplicon sequencing to quantify the mutant allele fraction across different cell types sorted from AD brains, to verify if these somatic mutations are specifically carried by microglia. The third Aim of the study will focus on the functional impact of proliferation-related somatic mutations in AD microglia, by using both an in silico method based on AD single-nucleus RNA-seq data and an in vitro method based on a human stem- cell differentiated microglia model. Our proposed study will shed new light on the contribution of somatic mutation to increased AD risk, and highlight the clonal expansion of microglia driven by somatic mutations in proliferation-related genes as a potential mechanism involved in AD pathogenesis.

NIH Research Projects · FY 2025 · 2024-08

Children’s Interstitial and Diffuse Lung Diseases (chILD) are disorders of the lung tissue that present in infancy and early childhood. ChILD pose major burdens on thousands of children across the U.S., on their families, and on the healthcare system more broadly. For most, the chILD-causing genes have not yet been identified and current treatments are non-specific and are all too often ineffective. The long-term goal of this project is to define the genetic landscape of chILD to inform the development of more specific and biologically rational therapies. Our approach is informed by our implementation of two complementary approaches: (i) whole-exome sequencing of patients seen in our chILD ambulatory clinics, which has to date established a genetic diagnosis in 20% of cases, including discovery of 6 novel candidate chILD genes; and (ii) in situ gene expression profiling of lung biopsy specimens, which we find differentiates samples with similar chILD histopathology based on their underlying genetic causes, suggesting that patients with similar profiles may have forms of chILD caused by shared molecular factors. In Aim 1, we will expand on these observations by profiling 300 available chILD lung biopsy samples to define patient groups (clusters) with similar molecular profiles. A chILD molecular classifier will be developed using 50 samples with definitive diagnoses and then validated in an independent set of 50 samples. The model will then be applied to 200 additional samples from patients lacking a molecular diagnosis and assess the biologic significance of each cluster using gene set enrichment and rare variant genetic analyses. We will assess clinical significance of cluster membership by testing for differences in clinical outcomes across clusters. In Aim 2, we will analyze whole exome sequence data from a cohort of 400 children with unexplained childhood parenchymal lung disease using family- and population-based methods to map new ChILD-causing genes. To improve power, we apply integrative genomic and network-based analytic approach that incorporates in situ transcriptomic data from Aim 1. We will replicate findings in the ChILD Research Network (ChILDRN) registry and will evaluate the functional impact of candidate variants on target protein expression using patient- derived biobanked samples. In Aim 3, to better understand how newly discovered ChILD genes contribute to disease pathogenesis, and to characterize the spectrum of their clinical presentations, we will identify patients harboring rare genetic variation in 6 novel chILD genes by screening the Genomic Information Commons (GIC) database – an NIH-supported, federated infrastructure facilitating cross-institutional query of genotype– phenotype databases at leading U.S. children’s hospitals. Children with these variants will be invited with their parents to undergo standardized phenotypic assessments (clinical evaluations, biochemical analyses, and molecular profiling) to characterize the spectrum of phenotypic consequences and inheritance patterns. Ultimately, the findings from these studies will provide novel insights into the pathobiology of chILD and inform the development of targeted therapies for these disorders.

NIH Research Projects · FY 2025 · 2024-08

This project develops artificial intelligence (AI)/machine learning (ML) methods for real time monitoring and updating of clinical decision support (CDS) systems to improve model reliability across patient populations. As AI/ML systems mature and their use in CDS expands, their potential to influence health outcomes grows. A key concern is the reliance on retrospective patient data, which often under-characterizes certain diseases and contains unwarranted differences in treatment and outcomes that naïve models may reproduce. This has motivated methods that embed performance constraints into models to mitigate undesirable behavior. Current approaches face persistent challenges including performance degradation with changes in clinical practice, maintaining consistent performance across patient populations without reducing accuracy, and limited ability to adapt to existing workflows. This project develops AI-based postprocessing methods to address these gaps, focusing on resource- and time-constrained settings where AI/ML tools help prioritize patient care. Our primary application is an ML system that predicts inpatient admissions in the emergency department (ED) to improve patient flow. We also evaluate generalization across endpoints, sites, and time. The central hypothesis is that continuous monitoring and updating of model performance can ensure reliable CDS operation. In the first two aims, we train models on retrospective data and assess their impact on ED attending decisions using a silent prospective study. In the third aim, we extend postprocessing methods for multicalibration to real-time operation, enabling adaptation to health data streams. The fourth aim implements a real-time AI auditing and calibration system for predicting patient disposition in the emergency department at Boston Children’s Hospital. This work supports the National Library of Medicine’s vision of sustainable computational infrastructure and seeks to reduce systematic performance degradation in CDS systems that may influence care decisions.

NIH Research Projects · FY 2024 · 2024-08

Transgender and gender-nonbinary (TNB) adolescents may receive gender-affirming hormone therapy (GAHT) with estradiol or testosterone to induce desired secondary sex characteristics, relieve gender dysphoria, and improve mental health. Because GAHT is given during a crucial phase of brain development, many parents and caregivers raise concerns about the potential effects of GAHT on brain development. Currently, the field lacks empirical data on the effects of GAHT on brain structure and function in TNB adolescents. To address this gap, we will conduct an observational, longitudinal study that will enroll 80 TNB adolescents (40 transfeminine and 40 transmasculine) who are starting GAHT as a part of their clinical care, and we will study participants before and after 12 months of GAHT using state-of-the-art neuroimaging techniques. We will specifically evaluate the structure and functional connectivity of the dorsolateral prefrontal cortex (DLPFC) region of the brain, which is involved in regulating core executive functions, which are key determinants of goal-directed behaviors, academic success, and personal autonomy. To account for normal age-related changes in these neuroimaging outcomes, we will compare changes in our cohort to developmental changes seen in cisgender adolescents from the general population using data from the Adolescent Brain and Cognitive Development (ABCD) study, a prospective investigation of 11,878 children recruited nationwide that has made data freely available to researchers. We will also investigate the association of neuroimaging changes in our cohort with changes in the three core executive functions: response inhibition, cognitive flexibility, and working memory. In addition, we will explore the correlations between changes in neuroimaging findings and changes in symptoms of anxiety and depression. To account for changes in these neurocognitive and psychological outcomes that occur normally with age, we have carefully selected measures that give scores that are standardized for age. Our study will a) evaluate the effects of GAHT (estradiol and testosterone) on DLPFC structure and function, and b) characterize the associations of GAHT-related changes with executive function tasks and with symptoms of anxiety and depression. Our investigation will provide pragmatic data about the effects of GAHT on brain changes in TNB adolescents and its relation to executive function to enable clinicians to counsel patients and families about these effects and to eventually develop approaches to optimize cognitive and educational outcomes.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Accumulating evidence suggests robustness of transcriptional regulation, yet human genetic studies indicate phenotypic sensitivity to transcription factor (TF) dosage in both complex traits and rare disorders, motivating quantitative studies of TF dosage effects. These features are exemplified by the development of the human face, which is frequently disrupted in a range of syndromes with typical features. The aim of this proposal is to develop and apply tools to precisely modulate dosage of craniofacial TFs in cranial neural crest cells, the progenitors of the face, and assess effects across multiple biological levels – molecular, cellular, and morphological. The proposal initially focuses on SOX9, using diverse approaches to understand the rules that determine which regulatory elements and genes are sensitive to SOX9 dosage (Aim 1), as well as their phenotypic consequences (Aim 2). Then, such rules will be generalized to five additional craniofacial TFs (Aim 3) and used to dissect mechanisms underlying genetic variants associated with craniofacial morphology or disease (Aim 4). These studies will reveal drivers of craniofacial morphological and disease variation, provide general insights into transcriptional regulation, and help transform our approach to studying TF function, from binary to quantitative in nature. The proposed research, along with a comprehensive plan for my career development, combines my scientific background and postdoctoral training in functional genomics, gene regulation, and human genetics with opportunities for additional training in machine learning, mouse genetics, and morphological analysis. I will be mentored by Drs. Joanna Wysocka and Jonathan Pritchard in a vibrant scientific environment (Stanford Chemical and Systems Biology) and receive additional training from my advisory committee of Drs. James Ferrell, Anshul Kundaje, and Benedikt Hallgrimsson. My transition to independence will be facilitated by participation in both scientific and non-scientific trainings and workshops, as well as experience in presenting my work at scientific conferences. I will also gain experience mentoring students and technicians, and will hone my grant management and writing skills. Collectively, my mentors have guided over 25 of their former postdocs to independent academic research positions, and they will provide practical advice and feedback during my faculty job search. My long-term career goal is to direct an independent research program aimed at understanding the molecular underpinnings of quantitative variation in craniofacial morphology and disease risk. So far I have achieved significant progress towards this goal through my research experience, publications, and engagement with the broader scientific community. I firmly believe, however, that a K99 mentored phase will maximize my chances for success by providing access to key resources and training that would be otherwise lacking from my postdoctoral experience.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY Childhood obesity and food insecurity are escalating and intersecting public health threats. Childhood obesity prevalence has been rising in the U.S. and tracks into adulthood, increasing the risks of chronic diseases such as type 2 diabetes. Food insecurity is associated with poorer dietary quality, including higher energy-dense food intake, and a higher prevalence of obesity and diabetes in adults; however, data are inconsistent and less known regarding longitudinal health effects in children. Further, there is evidence that the neighborhood food environment, including limited access to sources of healthy food, is associated with elevated body mass index in children. Because food insecurity and childhood obesity tend to co-occur in Black, Hispanic, and lower-income populations, there is an urgent need to examine the multilevel contributions of household food insecurity and neighborhood food access to rising childhood obesity prevalence. Dr. Allison Wu, a pediatric gastroenterology and nutrition specialist with advanced training in epidemiology and health services research, is well-suited to address this need. Dr. Wu seeks to expand on her research in the determinants, risks, and disparities in childhood obesity to develop and test interventions to support food and nutrition security in children and families. Specifically, her proposal aims to examine the extent to which household food insecurity and neighborhood food access contribute to increasing childhood obesity prevalence (Aim 1), test a caregiver education workshop in families with food insecurity (Aim 2), and explore the facilitators and barriers associated with the intervention’s implementation (Aim 3). These are the critical next steps toward achieving her long-term goal to develop evidence-based interventions and policies ensuring nutrition security for children and families. This career development award will provide Dr. Wu with the necessary mentored training and research opportunities to 1) develop expertise in multilevel and longitudinal analyses, 2) conduct a phase II clinical trial, and 3) obtain a foundation in implementation science and training in qualitative methods to translate interventions into routine practice. She is supported by outstanding mentorship provided by experts in obesity epidemiology, health services, and community health (Dr. Elsie Taveras), food insecurity and implementation science (Dr. Lauren Fiechtner), social and behavioral epidemiology (Drs. Henning Tiemeier and Izzuddin Aris), clinical trials in pediatric nutrition (Drs. Christopher Duggan and Enju Liu), and qualitative research in child health (Dr. Karen Kuhlthau). Her training and research activities will be conducted in the unparalleled academic environments at Boston Children’s Hospital and Harvard Medical School, which are firmly committed to Dr. Wu’s successful transition to independence as a physician-scientist.

NIH Research Projects · FY 2025 · 2024-08

Project Summary. Stress granules (SGs) – cytoplasmic aggregates formed from stalled translation initiation complexes upon cell stress – have emerged as critical regulators of oncogenesis. SGs can confer cancer cells the ability to withstand harsh biological conditions and can impart tumorigenic translational states through mRNA sequestration. However, a mechanistic understanding of how SGs endogenously regulate tumorigenesis in vivo remains elusive. My research proposal leverages a humanized zebrafish melanoma model where tumor development can be visualized and manipulated from the precursor stage to its single cell of origin and beyond. Using this system, I have found that SGs are formed in melanoma precursor cells and persist during tumor initiation and progression. Furthermore, my preliminary data shows that CRISPR/Cas9-mediated ablation of a key SG component, g3bp1, in melanoma initiating cells delays the establishment of the cancer precursor zone, an essential initiating cell phase in the emergence of melanoma. Taken together, the overarching hypothesis of my proposal is that SGs are formed in melanoma precursor cells to enhance viability upon tumorigenic stress and once formed, subsequently accelerate melanomagenesis by enabling an oncogenic translational profile. To test my hypothesis, I will first determine the impact of stress granule formation on melanomagenesis by perturbating key components using genetically engineered zebrafish and chemical biology. I will subsequently test whether modification of stress granule formation alters melanoma cell oncogenicity using flow cytometry and subcutaneous transplant assays. In addition, I will elucidate the contribution of RNA sequestration by stress granules toward melanoma development using proximity labeling and translatome profiling. I will then functionally resolve the contribution of SG-sequestered mRNA using zebrafish genetics. Lastly, I will perform immunostaining of human melanoma samples for proteins encoded by mRNAs with preferential translation in melanoma initiating cells, assessing the conservation of these molecules and their potential to serve as melanoma biomarkers. Ultimately, the results obtained herein will provide mechanistic insight toward the functional role of stress granules during cancer initiation. As stress granules are detected across many cancer types, this work will elucidate nodes of tumor biology that can be exploited to halt a broad spectrum of cancers before they arise. In this proposal, I establish training goals that will expand my conceptual and technical expertise in cancer biology and RNA processing. In addition, I present aims that will enable the acquisition of the professional skillsets necessary to achieve my long-term research goal, which is to run my own lab at an academic research institution studying the basic biology of RNA processing during cancer initiation.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Amblyopia is the leading cause of vision loss in children. Delayed diagnosis and/or inadequate treatment of amblyopia in early childhood may result in permanent vision loss. The underlying social determinants of health contributing to the outcomes of children with amblyopia are poorly understood. There is an urgent need to understand the factors contributing to diagnostic delay and treatment failure in amblyopia – an effort that may offer novel opportunities for vision screening policies and community-based interventions that improve vision care. This proposal aims to investigate the delivery of preventive vision care in the US and develop tools to identify children with preventable vision loss. This research will leverage an array of large data sources – including nationally representative surveys, insurance claims, and electronic health records – to identify modifiable factors that may improve the visual outcomes of children with amblyopia. The central hypothesis is that household- and neighborhood-level social determinants of health influence the receipt of vision screening (Aim 1), access to eye specialty care (Aim 2), and treatment success (Aim 3). The investigations will evaluate existing vision screening policies in the US, identify geospatial factors influencing health care access, and help develop a prediction model to identify children who may lose vision from amblyopia. The candidate, Dr. Isdin Oke, is a pediatric ophthalmologist, data scientist, and health services researcher whose long-term goal is to become an independent investigator with expertise in data-driven approaches to reduce preventable vision loss in children. The career development plan will provide expertise in study design, research methods, and advanced biostatistical approaches for conducting health services research. The mentorship team includes expertise in health services research, vision screening, social epidemiology, geospatial analyses, and predictive modeling. The training environment at Boston Children's Hospital provides access to rich data sources and computational infrastructure. The unique combination of the training plan, mentorship team, and institutional environment will accelerate the candidate's development into an NIH-funded independent investigator and leader in data science and health services research in ophthalmology.

NIH Research Projects · FY 2026 · 2024-08

ABSTRACT The genetic mechanisms contributing to neurodevelopmental disorders are highly variable across children and families. This is especially true for attention-deficit/hyperactivity disorder (ADHD), which affects approximately 7% of school aged children. In the last decade, databases containing genomic information about individuals and families with ADHD have been developed. However, the individuals within these databases are highly heterogeneous, due to varying recruitment methods, ADHD diagnosis, and variable inclusion/exclusion criteria. For example, some individuals with ADHD may have comorbid autism (ASD) or intellectual disability (ID), and many individuals self-report their ADHD status, without a standardized approach to confirming the diagnosis. As a result, much of the underlying genetic etiology of well-defined primary ADHD (i.e., ADHD without comorbid ID or ASD) remains unknown. Common variants account for a minority of variance in ADHD, and our pilot data suggests that up to 50% of individuals with primary ADHD have a rare pathogenic variant accounting for their diagnosis. We aim to improve the understanding of the genetic underpinning of primary ADHD through the following major goals: 1) Developing the largest family-based repository of children with primary ADHD and their family members using consistent, precise, and comprehensive diagnostic criteria, as well as standardized evaluations of parental and sibling ADHD status. 2) Identify novel candidate genes for ADHD and expand on previous studies that have focused on de novo (spontaneous) genetic mutations, by also examining co-segregation of ADHD and inherited genetic variants, thereby providing the most comprehensive analysis of the genomic architecture of ADHD to date. 3) Validate our gene discovery by assessing genome-wide significance in an existing sample of 5,595 cases and 62,739 healthy controls. 4) Develop a more robust clinical variant classification approach through integrated family histories and functional studies that will increase understanding of the impact of variants of unknown significance and will provide a valuable resource for future genomic and neurobiological functional studies. To do this, we will analyze genomic data from 1,400 children and their families who have been diagnosed with ADHD (without autism or intellectual disability) using comprehensive neuropsychological evaluation from two sources: 402 from our own Developmental Medicine Center (DMC) at Boston Children's Hospital, including 187 already recruited; and 998 from the IMAGE Study, for which data is available through the NIH RGR. We will validate our findings in a sample of 5,595 cases and 62,739 controls from the NIH All of Us Research Program. We will conduct whole exome sequencing (WES) on all participants, perform mRNA-sequencing from hiPSC- derived neural cells to clarify the impact of a subset of variants of unknown significance, and perform patient- and CRISPR-derived iPSC analyses on a subset of 5 damaging variants of interest.

NIH Research Projects · FY 2024 · 2024-08

Project Summary/Abstract: Continuous functional T and B lymphoid output is critical to overall health and life, as delayed and reduced lymphopoietic output has been associated with serious illness and even mortality. The lymphoid system is particularly sensitive to hematopoietic perturbation, both in the context of steady state and regeneration. Hematopoietic stem cell transplantation (HSCT) into irradiated recipients and stress from infection or other insults in native hematopoietic systems have been shown to negatively regulate lymphoid output. One of the challenges to developing clinical therapies to restore healthy immune function is that we don’t have enough information on lymphoid development in vivo. This is true for both the embryonic immune system which hPSC blood differentiation systems attempt to reproduce and the adult immune system. Understanding the mechanisms by which lymphoid output from HSPCs is controlled at clonal, transcriptional, and epigenetic levels is critical for revealing how lymphoid output is established and regulated in steady state, how it is suppressed with regeneration, and whether there is a specific signature associated with reduced lymphoid output that is conserved between native and non-native systems. It was recently shown that the HSC specific transcription factor Tcf15 is indispensable for long term self-renewal, following HSCT. Intriguingly, I have demonstrated that Tcf15 also negatively regulates both T and B lymphoid differentiation. This was true both in native hematopoiesis and regeneration. It is well established that HSCs display multilineage priming. This allows HSCs to readily regenerate the hematopoietic system, upon perturbation and to differentiate into lineage restricted progenitor cells to meet native hematopoietic demands. In addition, reduced lymphoid lineage priming has been shown to promote HSC expansion. For the mentored K99 phase of this proposal, I will transition from a focus on Tcf15 in native hematopoiesis, to a focus on Tcf15 in lymphopoiesis, a focus that will allow me to establish research independence from my mentor. With the evidence of multilineage HSC priming, the evidence of native antagonism between HSC self-renewal and lymphoid differentiation programs, and my preliminary data hypothesize that Tcf15 expression attenuates lymphoid lineage priming in HSCs and that this is conserved between steady state and regeneration. I propose to test this hypothesis in two aims: In Aim 1, I will apply scRNA- seq, scATAC-seq, and proteomic analyses to elucidate the transcriptional and epigenetic program by which Tcf15 regulates T and B lymphoid differentiation from HSPCs. In Aim 2, I will use Tcf15 reporter and CRISPR- based lineage tracing (CARLIN) mouse models generated in our lab to explore the effect of endogenous Tcf15 on T and B cell specification from HSCs. For the R00 component of this award, I will seek to continue my examination of regulation of lymphoid lineage priming, but downstream of HSCs, exploring whether I can apply lineage and transcriptional tracing methods to retroactively connect T and B cell differentiation outcomes to distinct lympho-myeloid primed progenitors (LMPP) identities in health and age, in an unbiased manner.

NIH Research Projects · FY 2025 · 2024-08

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. During fetal life or in adulthood, hematopoietic stem cells (HSCs) are positioned adjacent to venous sinusoids, specialized veins that provide support for stem cell homeostasis. Previously, we found that sinusoids interact directly with HSCs in a process we termed “cuddling,” and this supports HSC division. We investigated the transcriptional program of venous sinusoids and published a method to overexpress transcription factors (TFs) to reprogram ectopic regions of the zebrafish embryo to become venous sinusoids. These new vessels support local blood stem cell self-renewal. We now plan to examine vascular reprogramming in adults to provide an alternative location for hematopoietic stem cells to form blood during stress or infiltrative disorders of the marrow. Through comparing single cell RNA-seq analyses of adult liver sinusoids and marrow sinusoids from zebrafish, mice and humans, we found five new TFs specifically expressed in the marrow sinusoids. We plan to overexpress these TFs in the hope of transforming the adult liver, which typically does not support hematopoiesis, into a hematopoietic site. Preliminary data overexpressing TFEC and MAFB using a liver-sinusoid-specific enhancer demonstrates activation of much of the marrow sinusoid fate in vivo. Overexpression of these factors in human iPSC-derived venous sinusoids leads to enhanced proliferation of hematopoietic stem and progenitor cells (HSPCs) in co-culture. We plan to overexpress the human orthologs of other TFs found in zebrafish to examine the support of HSPCs in vitro. The reprogrammed venous sinusoids could be used to expand HSCs in vitro. In an effort to understand how niche endothelial cells (ECs) cuddle HSCs, we undertook a chemical genetic screen to find small molecules that regulate the process. We found that antagonists of the G-coupled receptor, cysteinyl leukotriene receptor 1 (CysLTR1), stimulate stem cell division specifically nearby cuddling ECs in vivo. The receptor is specifically expressed by the sinusoids in zebrafish and humans. Using lipidomics, we found that the CysLTR1 ligand, LTD4, is present in the zebrafish larval niche where HSCs divide. LTD4 suppresses the HSC division nearby cuddling ECs. Using genetics, chemical biology, and biochemistry, we plan to define the mechanism by which LTD4 regulates endothelial cells to support HSC self-renewal. Our work will provide a new understanding of the mechanism of endothelial cuddling and its role in HSC division, potentially leading to new chemical or cell therapy to induce HSC division and maintenance for the treatment of blood disorders.

NIH Research Projects · FY 2026 · 2024-08

Project Summary/Abstract Twin-to-Twin Transfusion Syndrome (TTTS) is a serious complication of twin pregnancies, affecting up to 15% of twins sharing the same placenta (monochorionic twins). TTTS occurs when monochorionic twins develop abnormal connections (anastomosis) in placental vessels resulting in imbalanced blood flow: one twin (the recipient) receives more blood at the expense of the other (donor) twin with both at increased risk for demise and compromised neurodevelopmental outcomes. The natural history of TTTS is dismal with perinatal losses up to 70-100%. Although fetoscopic laser photocoagulation of the placental anastomoses has improved outcomes, losses remain as high as 34% with cerebral palsy and neurodevelopmental impairment occurring in 10-18%. Thus, margins for improvement remain and controversy exists over optimal type of laser surgery, appropriate definition of TTTS and when to intervene. Surgical planning requires accurate localization of the umbilical cord insertions and placenta surface vessels, which remains challenging with 2D magnetic resonance (MR) or ultrasound (US) images. Diagnosis of TTTS and response to laser surgery is based on user dependent, indirect measures of placental function (e.g. umbilical arterial and venous Doppler assessment, maximum vertical pocket of amniotic fluid, fetal bladder size). In addition, fetal growth discordance, a critical reflection of TTTS severity, is also based on user dependent 2D US techniques. Improved preoperative placental vascular visualization, direct measures of placental function and user independent measures of fetal size are needed. We propose to build on our prior work developing CNN to perform rapid 3D uterine reconstructions with 2D HASTE images with flattening of placental images to improve visualization of the placental surface vessels. To directly assess placental function, we will build upon our prior work where we found oxygen transport correlated with discordance and predicted birth weight in monochorionic twins. Instead of monitoring relative T2* changes, we propose to quantify T2* at baseline and with maternal oxygen. We also use intravoxel incoherent motion (IVIM) with and without flow compensation (FC) to explore pulsatile flow in the fetal villous tree. Finally, we will extend our prior work that identified key points for fetal pose providing limb length and motion statistics in singletons, to twins and add estimation of fetal intracranial, bladder and body volumes. In summary, to address the unmet needs in monochorionic twins, we propose the following aims: Aim 1) Provide rapid visualization of the placental surface vessels and umbilical cord insertion in native and flattened space; Aim 2) Characterize local placental function pre and post laser ablation; and Aim 3) Calculate discordance of intracranial, bladder and body volumes, fetal limb biometrics and motion statistics pre and 4-6 weeks post treatment. If successful, we will be poised to perform a clinical study assessing the impact of these advancements on surgical planning and outcomes and determine application to earlier gestational ages.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT The genetic mechanisms contributing to neurodevelopmental disorders are highly variable across children and families. This is especially true for attention-deficit/hyperactivity disorder (ADHD), which affects approximately 7% of school aged children. In the last decade, databases containing genomic information about individuals and families with ADHD have been developed. However, the individuals within these databases are highly heterogeneous, due to varying recruitment methods, ADHD diagnosis, and variable inclusion/exclusion criteria. For example, some individuals with ADHD may have comorbid autism (ASD) or intellectual disability (ID), and many individuals self-report their ADHD status, without a standardized approach to confirming the diagnosis. As a result, much of the underlying genetic etiology of well-defined primary ADHD (i.e., ADHD without comorbid ID or ASD) remains unknown. Common variants account for a minority of variance in ADHD, and our pilot data suggests that up to 50% of individuals with primary ADHD have a rare pathogenic variant accounting for their diagnosis. We aim to improve the understanding of the genetic underpinning of primary ADHD through the following major goals: 1) Developing the largest family-based repository of children with primary ADHD and their family members using consistent, precise, and comprehensive diagnostic criteria, as well as standardized evaluations of parental and sibling ADHD status. 2) Identify novel candidate genes for ADHD and expand on previous studies that have focused on de novo (spontaneous) genetic mutations, by also examining co-segregation of ADHD and inherited genetic variants, thereby providing the most comprehensive analysis of the genomic architecture of ADHD to date. 3) Validate our gene discovery by assessing genome-wide significance in an existing sample of 5,595 cases and 62,739 healthy controls. 4) Develop a more robust clinical variant classification approach through integrated family histories and functional studies that will increase understanding of the impact of variants of unknown significance and will provide a valuable resource for future genomic and neurobiological functional studies. To do this, we will analyze genomic data from 1,400 children and their families who have been diagnosed with ADHD (without autism or intellectual disability) using comprehensive neuropsychological evaluation from two sources: 402 from our own Developmental Medicine Center (DMC) at Boston Children's Hospital, including 187 already recruited; and 998 from the IMAGE Study, for which data is available through the NIH RGR. We will validate our findings in a sample of 5,595 cases and 62,739 controls from the NIH All of Us Research Program. We will conduct whole exome sequencing (WES) on all participants, perform mRNA-sequencing from hiPSC- derived neural cells to clarify the impact of a subset of variants of unknown significance, and perform patient- and CRISPR-derived iPSC analyses on a subset of 5 damaging variants of interest.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Premature infants are vulnerable to long-term social, cognitive, and behavioral difficulties. However, not all premature infants go on to have developmental delays. Moderate to late preterm infants (born after 32 weeks) represent a missed clinical population as they do not automatically qualify for federally provided health screening and intervention programs. Therefore, the investigation into the neural mechanisms underlying heterogeneities in behavioral development is a promising approach to improve our ability to identify infants in need of additional services and inform treatment responses. This is also an important public health mission and a NICHD funding priority. The proposed K99 study will chart brain development using electroencephalography (EEG) during well-baby visits (4, 9, and 12 months) at a primary care clinic and a follow-up lab visit (24 months). Participants (N = 720 target recruitment) will be drawn from an ongoing R01 (PI Nelson: 5R01NS120986) study that examines if EEG is feasible in the context of a primary care facility and aids in the early identification of children who go on to receive an autism diagnosis. To this end, I will assess the longitudinal trajectories of brain development in preterm and full term infants across the first two years of life (aim 1). Next, I will determine the link between brain development and development delays (aim 2). Finally, in the R00 phase, I will recruit and longitudinally follow a new sample of preterm (1 month chronological, 1 month corrected, 4 months chronological, and 4 months corrected) and full term infants (1 month chronological and 4 months chronological). Using a multimethod neuroimaging approach (simultaneous functional Near- Infrared Spectroscopy and EEG) I will explore when disruptions in cerebral blood flow responses and brain activity first emerge and their associations with developmental delay. Overall, understanding neurodevelopmental processes before and when delays first emerge provides a tractable approach to improving psychological outcomes and well-being across the lifespan. The training and experience gained during the award period will support my transition to becoming an independent investigator and will contribute to my long-term research goals of investigating infant brain mechanisms underlying heterogeneities in social and cognitive development.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY What is the shortest path from the human antibody repertoire to sera that protects from an HIV-1 challenge? We propose here that this pathway starts from B-cell receptors (BCRs) with long, tyrosine-sulfated heavy- chain CDR3s (HCDR3s) encoded by D3-family of diversity (D) chain segments. This path ends with broadly neutralizing antibodies (bnAbs) targeting the V2-glycan or ‘apex’ epitope of the HIV-1 envelope glycoprotein (Env). That is, the most direct way to prevent many HIV-1 transmission events through conventional vaccination is to induce a defined subset of circulating HCDR3s to bind the Env apex. This hypothesis is supported by several observations: (1) Apex bnAbs do not require extensive or rare hypermutations. (2) Neutralization by key apex bnAbs, notably those of the PG9/PG16 and VRC26 families, is largely mediated by their HCDR3 regions. (3) These key bnAbs bind their distinct apex epitopes through HCDR3 encoded by long (>24 amino-acid), tyrosine-sulfated D3-family diversity (D) segments, specifically via ‘YYDF’ motifs encoded by the D3-3 segment. (4) BCR bearing HCDR3 with these properties are present in humans at a frequency of 1 in 2000, far more frequently than other proposed bnAb precursors. Further, (5) when we introduced with CRISPR/Cas12a only the HCDR3s of PG16 and VRC26.25 into a diverse population of murine BCRs, B cells encoding these chimeric BCRs affinity matured and generated potent neutralizing sera in recipient mice immunized with trimeric Env (SOSIP-TM) antigens. (6) When we similarly edited murine B cells to express the HCDR3 of the VRC26-family unmutated common ancestor (UCA), they similarly affinity matured and generated potent neutralizing sera in recipient mice. Finally, as we show here, (7) SOSIP-TM proteins can be modified to bind common D3-3-encoded HCDR3 from HIV-negative persons while continuing to engage mature apex bnAbs. In summary, potential apex-bnAb precursors with long D3-family HCDR3 are common, these precursors can bind SOSIP-TM variants with unmodified apex epitopes, and they can affinity mature in response to SOSIP-TM antigens in wild-type mice engrafted with human HCDR3-edited B cells. Our goals then are to refine our definition of accessible apex bnAb precursors found frequently in uninfected persons, and to identify sets of SOSIP-TM antigens that affinity mature these precursors so that they generate polyclonal sera and monoclonal bnAbs that protect from multiple HIV-1 isolates. To do so, we will use our useful variant of mammalian display technology (Yin et al., PNAS 2021) and our novel mouse models for vaccine evaluation (He, Ou et al., Immunity 2023; Yin et al., Nat Biomed Eng, 2024). These models rely on our ability to introduce human HCDR3, or whole human antibodies, directly into their appropriate VDJ (VJ)- recombined loci of murine B cells, and then affinity mature the resulting BCR chimeras in vivo. Collectively, these studies will determine the best antigens and vaccination strategies to generate apex bnAbs in uninfected persons. In doing so, they will complement parallel efforts to elicit other classes of bnAbs.

NIH Research Projects · FY 2026 · 2024-07

Summary A nanobody that recognizes immunoglobulin light chains, conjugated to a molecular entity that recognizes a virus-infected or a cancerous cell, is an effective therapeutic: A single injection of fusion constructs comprising an anti-kappa light chain nanobody (VHHkappa) and zanamivir, a small molecule that targets influenza neuraminidase, protects mice from a lethal challenge with both A- and B-strains of influenza. In the model that established protection by VHHkappa adducts against influenza, the underlying mechanism of action involves antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but the relative contribution of each is not known. We shall therefore use FcgR common g chain-deficient mice and C3- deficient mice to assess the relative contributions of ADCC and CDC. The generation of Fc constructs of different Ig isotypes and bearing FcR-engagement disabling mutations, similarly modified with zanamivir, will be used to complement this analysis. Having established proof-of-concept for influenza and optimized parameters for elimination of influenza virus- infected cells, we will explore nanobodies that recognize other pathogens (Ebola virus, SARS-CoV-2, HIV) in combination with VHHkappa in a series of collaborative experiments. The agents to be developed may inspire novel immunomodulatory therapeutics, to be used as a stand-alone approach, or in combination with approved drugs. The possibilities of post-exposure prophylaxis against viral infections (Ebola, SARS-CoV-2, HIV) in the absence of pre-existing immunity, deserve particular emphasis. We shall further enhance the activity of the proposed VHHkappa fusions through the generation of the corresponding drug adducts, using cytotoxic drugs such as maytansinoids as compounds that have shown clinical utility. Enveloped viruses (e.g., HIV, SARS-CoV-2) export viral proteins to the surface of the infected cell during budding. Infected cells can thus be distinguished from uninfected cells based on the surface display of viral proteins. We now extend these in vivo observations to fusions of VHHkappa with anti-checkpoint (PD-L1, CTLA-4) nanobodies. We generated maytansinoid-modified VHHkappa fusions with the anti-PD-L1 and anti-CTLA-4 VHHs. Our preliminary data show enhanced anti-tumor activity in the MC38 and B16.F10 mouse tumor models in comparison with commonly used monoclonal antibodies. However, not all such fusions (examples: fusions of VHHkappa with nanobodies that recognize Class II MHC or CD8) have shown the intended depletion efficacy in vivo. This proposal seeks to establish the parameters -including biodistribution and surface expression levels of the targeted molecules- that determine success or failure of VHHkappa fusions. The availability of VHHs that recognize human kappa light chains suggest the possibility of clinical translation of this approach.

NIH Research Projects · FY 2024 · 2024-07

Abstract: Nephrotic syndrome (NS) is the second most common cause of end-stage kidney disease (ESKD) before age twenty-five. The spectrum of disease is defined by response to steroids, divided into steroid resistant nephrotic syndrome (SRNS) and steroid sensitive nephrotic syndrome (SSNS). SRNS has limited therapeutic options and often progresses to chronic kidney disease (CKD) and ESKD, while SSNS carries significant morbidity due to long-term treatment with non-specific immunosuppression. We aim to improve our understanding of the genetic drivers of NS to more precisely guide clinical management and create targeted therapies. The past two decades have seen an increased understanding of the genetic basis of NS, particularly in the discovery of Mendelian causes of NS. Mendelian forms of NS are primarily recessive, with two pathogenic variants being essentially fully penetrant for the more severe form of NS, SRNS. The first recessive Mendelian NS gene discovered was nephrin (NPHS1). In keeping with this inheritance model, the heterozygous state has been thought to be clinically silent – a true “carrier” state. Recently, a published analysis of a Finnish population cohort (FinnGen) challenged that model by showing that carriers of “Fin-Major”, a specific NPHS1 loss-of- function Mendelian founder mutation in Finns, have increased odds of kidney disease. Additionally, genome wide association studies (GWAS) have implicated common variants in NPHS1 with increased risk of SSNS, reduced estimated glomerular filtration rate (eGFR), and lower serum albumin. Taken together, these discoveries suggest that forms of genetic variation beyond bi-allelic Mendelian variants, such as heterozygous rare pathogenic coding variants and common regulatory variants, may alter NPHS1 function and contribute to kidney dysfunction and disease. The overall goal of my project is to discover the association of coding and non-coding NPHS1 variants across the allele frequency spectrum with diverse kidney diseases and traits. More specifically, I hypothesize that in the general population, heterozygous, pathogenic Mendelian NPHS1 variants and common risk variants discovered by GWAS are associated with lower eGFR, higher urine protein levels, and increased odds of nephrotic syndrome. To test this hypothesis, I propose testing the following specific aims amongst 1.5 million people enrolled in multiple large population- and hospital system-based biorepositories: Aim 1: Discover the clinical consequences of Fin-Major carrier state in the FinnGen cohort. Aim 2: Discover the prevalence and clinical impact of rare and common NPHS1 variants in 5 hospital based and population cohorts collectively totaling approximately 1,500,000 individuals.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Despite fewer deaths as a result of the Safe Sleep campaign, the Sudden Infant Death Syndrome (SIDS) remains the leading cause of post-neonatal infant mortality in the United States (~0.39/1000 live births). Our research strongly implicates biological abnormalities within the brainstem that underlie an infant's vulnerability to SIDS, including abnormalities in the serotonin (5-HT) neurotransmitter systems. MicroRNA (miRNA) biology has emerged rapidly with clear evidence that miRNAs play a critical role in regulation of protein expression, including in the regulation of proteins involved in the development and function of the central nervous system (CNS) and in the regulation of the 5-HTergic system within the CNS. We recently performed an unbiased sequencing screen of microRNAs in a dissected area of the medulla concentrated with 5-HT neurons to identify differentially expressed miRNAs in SIDS compared to controls. In this proposal we focus on two of the miRNAs identified with altered expression – miR-34a-5p (upregulated in SIDS) and miR-122-5p (downregulated in SIDS) and two mRNAs that these miRNAs target, hyperpolarization-activated cyclic nucleotide gated 3 (HCN3) and cytoplasmic polyadenylation element binding protein 1 (CPEB1), respectively. These specific miRNAs were selected based on the reported roles that HCN channels and CPEB1 play in 5-HT function and inflammation, respectively, the latter of interest given evidence of an effect of inflammation on 5-HT metabolism. Here we test the following hypotheses, directly in SIDS tissue and serum that: 1) cell-specific dysregulation of miR-34a-5p and miR-122- 5p in the medulla of SIDS, results in aberrant expression of their respective mRNA targets, HCN3 and CPEB1 (Specific Aims 1 and 2) and, 2) that aberrant levels of medullary miR-34a-5p and miR-122-5p are reflected within the serum of SIDS infants and serve as potential biomarkers of SIDS pathogenesis (Specific Aim 3). We will use our unique database of SIDS and non-SIDS controls, multiplexed in-situ hybridization techniques, machine learning quantitative software, and qPCR directly in human SIDS and control tissue and serum. Data generated from this R21 will be utilized to further develop novel hypotheses and animal models aimed at this important regulatory system.