Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2025-08

Project Summary This K01 award provides Dr. Taehyeung Kim with the advanced research training, protected time, and mentoring needed to become an independent investigator specialized in functional genomics and committed to understanding the regulatory role of genetic variants conferring disease risk through Tregs. Candidate: The PI is an Instructor at Harvard Medical School in the laboratory of Dr. Peter Nigrovic in the Division of Immunology at the Boston Children’s Hospital. His previous training has provided him with extensive knowledge in the genetics of human diseases, as well as technical expertise in gene and genome editing, molecular biology, biochemistry, and manipulation of human primary immune cells. Research: Epigenetic analysis of RA GWAS variants implicates Tregs in disease risk, but how these variants affect Tregs remains largely unexplored. The PI found that the non-coding variant rs3087243 affects the immune control gene CTLA4, likely mediated by the transcription factor EGR2. Remarkably, healthy individuals carrying this risk allele have lower CTLA4 expression and fewer Tregs, potentially due to CTLA4 signaling inhibiting mTORC1. Further, we experimentally identified 197 SNPs as potential functional variants linked to RA/JIA risk via Tregs, based on allele-specific binding to Treg nuclear proteins. In Aim 1, we will investigate how rs3087243 controls Treg abundance. In Aim 2, we will uncover novel SNP-Gene-Phenotype pathways along with their regulatory mechanisms in Treg immunity, opening up the possibility of therapeutic targeting. Mentoring/Training: The PI's goal is to become an independent investigator and a tenured faculty member at a premier academic institution. The proposed research and training plan is crafted to position the PI to obtain independent R01 funding and to become an internationally recognized authority in the field of functional genomics of immune cells in autoimmune diseases. By leveraging a network of experts in his field of study, he aims to gain a deep understanding of how the human genome orchestrates cell phenotypes and the implications of dysregulation in autoimmune conditions. His research training will encompass CRISPR-based genome editing, computational biology to analyze immune cell functions through single-cell RNA-seq, and advanced techniques in immune cell phenotyping and manipulation. Additionally, he will acquire essential laboratory leadership skills to smooth the transition to his independent laboratory. This endeavor will unfold in an exceptional institutional setting, under the guidance of Dr. Nigrovic, an authority in immunology of autoimmune arthritis, Dr. Maria Gutierrez-Arcelus, an expert in functional genomics and systems biology, and an Advisory Committee comprised of Drs. Talal Chatila, a Treg biology specialist; Dr. Nir Hacohen, a luminary in molecular genomics; and Dr. Matthew Weirauch, an expert in transcription factor biology.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Neuroendocrine signaling is central to many aspects of life, but the computational mechanisms underlying the many facets of neuroendocrine biology receive relatively little attention. The cell biology of neuroendocrine signaling differs from that of neuron-to-neuron signaling, and the timescales of regulation are many orders of magnitude longer—implying different computational mechanisms. In responding to stress, behavioral changes that result from neuroendocrine signaling often require sustained or repeated triggers. Once initiated, stress- induced changes in behavior can outlast the triggering stimuli. This implies evidence accumulation and state- persistence. Understanding the computational mechanisms behind these effects would be extremely valuable, as they may explain inter-individual differences in susceptibilities to stressors and disease states in humans. I present a new system for assessing and understanding neuroendocrine computations that determine the entry into, and exit from, a state of stress-induced suppression of ovulation in Drosophila. Our preliminary data implicate conserved hormones under the control of a multi-organ neuroendocrine signaling pathway. Precise genetic access to three anatomical nodes, together with the unparalleled genetic toolbox of Drosophila melanogaster, will allow a detailed investigation of the molecular and electrical mechanisms that measure the amount of stress being experienced and determine the extent and duration of the response. Flies produce insulin-like peptides in a variety of tissues, including 14 brain neurons. We find that stress causes a lasting increase in the activity of the insulinergic neurons, and that the resulting increase in insulin triggers a lasting suppression of ovulation. The brain-derived signal is received through the insulin receptor on an endocrine gland called the corpora cardiaca. We have designed a novel 2-photon fluorescence lifetime imaging paradigm to monitor the activity dynamics of insulin producing cells in response to time spent in stressful situations, and lingering effects in the following days. We will explore the computations underlying the accumulation and dissipation of stress-derived information over time within the insulin producing neurons. A genetic screen targeted to the insulin-receiving cells in the corpora cardiaca indicated cAMP as a critical mediator of resiliency to stress. We will explore the hypothesis that cAMP levels set the threshold that stress levels must cross to trigger entry into the state of suppressed ovulation. Our preliminary data implicate the GnRH homolog Adipokinetic Hormone (AKH), which is produced by the corpora cardiaca, as the readout of this thresholding mechanism. We will work to understand how the insulin signal is translated into an AKH signal, with specific focus on long timescale mechanisms. We will work to understand the signal transduction mechanism downstream of GnRH signaling, which we localize to the adipose tissue of the fly. The result will be an anatomical, electrical, and molecular description of neuroendocrine computations with profound effects on behavior and reproduction.

NIH Research Projects · FY 2026 · 2025-08

Project Summary Strategies focused on reducing pathological burden in Alzheimer’s Disease (AD) have had limited effect in reversing cognitive decline. One novel approach to treating AD may be targeting resilience-promoting factors that are recruited in the 10-15% of individuals who have normal cognition but have high levels of AD pathology upon post-mortem analysis. This resilience to AD pathology has been well-characterized clinically, but the molecular mechanisms that enable resilience are largely unknown. By using a mouse model of environmental enrichment (EE), which is known to be a potent stimulator of resilience, we recently demonstrated that EE increases activity of the Mef2 transcription factors (Mef2a, Mef2c) in cortical neurons. Overexpression of Mef2 in the absence of EE was sufficient to induce cognitive resilience in a mouse model of neurodegeneration. Motivated by this discovery, this proposal aims to (1) elucidate whether Mef2 mediates survival of specific neuronal subtypes vulnerable to AD pathology, (2) assess whether Mef2 activation may be protective, and (3) leverage human single-nucleus transcriptomic data to study molecular mechanisms of resilience in humans. AD is characterized by massive neuronal loss, and Mef2 is known to promote the survival of both excitatory and inhibitory neurons. Therefore, in Aim 1, we will test whether Mef2-mediated survival of a particular neuronal subtype is critical for inducing cognitive resilience to AD pathology. After engineering custom-tailored viral vectors to express Mef2a or Mef2c in either excitatory or inhibitory neurons, we will introduce these constructs into multiple mouse models of neurodegeneration. This will allow us to evaluate both whether a particular Mef2 paralog is critical, and whether Mef2 expression in either excitatory or inhibitory neurons is the prime determinant of cognitive resilience. The specific constructs that induce a state of cognitive resilience will be further analyzed to determine the neural activity and molecular signatures of resilience. In Aim 2, we will test whether a small molecule that activates Mef2, AR-42, is able to induce cognitive resilience in multiple mouse models of AD. As AR-42 has already successfully completed Phase I clinical trials for the treatment of hematologic malignancies and was found to be safe without any significant toxicities or adverse events, our preclinical testing could directly lead to the initiation of clinical trials to assess if Mef2 activation via AR-42 could be used in the treatment of AD. Finally, in Aim 3, we will take advantage of large existing datasets of single- nuclear RNA-sequencing (snRNA-seq) data from the prefrontal cortex of hundreds of humans to identify the gene programs and cell-types that are differentially active in cognitively resilient individuals. This proposal will advance my career by providing training in the fields of aging and AD research, while enabling the development of further skills in systems neuroscience, neuroengineering, and computational biology. It will also enable me to launch an independent research program aimed at dissecting the mechanisms underlying how the environment mediates risk and resilience to the development of age-related disorders.

NIH Research Projects · FY 2026 · 2025-08

ABSTRACT Hematopoietic stem cell transplantation (HCT) is an essential treatment for high-risk non-malignant and malignant hematologic diseases. However, outcomes remain inadequate, with multiple immune-mediated complications contributing to mortality. With 5-yr survival often <60%, there is an urgent need to improve results. We will address this by deciphering the mechanisms driving HCT outcomes by studying the biology of HCT patients at a depth and scale not previously achieved. To accomplish this, we have partnered with the Blood and Marrow Transplant Clinical Trials Network (BMT CTN), the NMDP, and the Pediatric Transplant Consortium (PTCTC) to perform detailed biologic studies on patients with both non-malignant and malignant hematologic diseases enrolled on multicenter HCT trials. Our goal is to leverage the power of systems immunology, genomics, and state-of-the-art microbiome analysis to determine, from patient samples, the underlying mechanisms that drive clinical transplant outcomes. We will do so through the following Aims: Aim 1: The Tradeoff Between T Cell Reconstitution and GVHD: Optimizing Immunomodulation to Optimize Transplant Outcomes. In this Aim, we will test the hypothesis that a GVHD prophylaxis strategy can be identified that improves immune reconstitution compared to PT-Cy (50mg/kg), while still controlling GVHD. Aim 2: The Host:Microbe Dyad and Its Influence on Transplant Outcomes. In this Aim, we will test the hypothesis that microbiome dynamics at the taxonomic and subtaxonomic (strain) level, driven by conditioning regimen and GVHD prophylaxis choice, are predictive of (a) pathogen domination and infectious complications of HCT, (b) gut GVHD, and (c) immune reconstitution. Aim 3: Cellular and Molecular Analysis To Predict Relapse and Graft Failure: In this Aim, we will examine the interface of immune reconstitution and both relapse and graft failure. This proposal seeks to create a new paradigm for transplant trials: an integrated, embedded scientific hub that can efficiently collaborate across multicenter clinical trial organizations to address the most pressing biologic questions linked to patient outcomes. If successful, it will lead to the discovery of biologic mechanisms driving HCT results, such that fully safe and effective transplant approaches can be developed.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract Late language emergence occurs in approximately 10-20% of children. There is significant variability in language outcomes among late-talking children, further complicated by the heterogeneity of this population, including children with neurodevelopmental disorders such as Autism Spectrum Disorder (ASD). Our group recently found that ASD diagnoses do not persist at school age for over one-third of children diagnosed as toddlers (termed “persistent ASD” and “non-persistent ASD”). Gaps remain in our knowledge about language outcomes and their predictors in children diagnosed with ASD at a young age, particularly those who no longer fulfill ASD criteria. Identifying predictors for late-talking children most at risk for persistent language impairment would strengthen initial counseling recommendations regarding intervention and prognosis. Understanding domains of language impacted in children with persistent and non-persistent ASD could support clinicians in monitoring specific language domains, such as language syntax and morphology, during developmental follow- up visits for children initially diagnosed with ASD. Thus, we will leverage a unique extant database to characterize language profiles and outcomes in 213 children diagnosed with ASD as toddlers (<36 months) and re-evaluated at school-age (5-7 years). Our objectives are to: 1) Examine language outcomes in school-age children diagnosed with ASD as toddlers and identify predictors of language outcomes in these children, and 2) Compare aspects of language as a function of ASD status (persistent vs. non-persistent) in 5-7 year old children diagnosed with ASD as toddlers. Our methods will include a latent class analysis based on available data in the extant BOAT database to identify groups of children based on language function. Latent classes will be formed by variables including functional communication (Vineland-3), expressive and receptive language (PLS-5), verbal IQ (DAS), and speech (parent report of speech disorder). We will also analyze predictors of overall class membership, including baseline cognition, language, and adaptive skills abstracted from an initial clinical evaluation and persistence of ASD. Finally, we will complete subdomain-level analyses of PLS-5 protocols and language sampling analysis using Systematic Analysis of Language Transcripts (SALT) to characterize individual aspects of language/ communication in children who continue to meet diagnostic criteria for ASD vs. those who do not. This project will address an unmet clinical need to better identify predictors of language outcomes in late- talking children diagnosed with ASD, characterize language profiles with a focus on functional impairment, and assess differences in language in children who continue to meet ASD criteria vs. those who do not.

NIH Research Projects · FY 2025 · 2025-08

Abstract This research proposal aims to elucidate the mechanisms underlying the activation and regulation of the NLRP3 inflammasome, focusing on conformational changes, post-translational modifications (PTMs) such as ubiquitination and lipidation, and novel regulatory factors. These regulatory mechanisms ensure precise activation and prevent excessive inflammation that could lead to tissue damage or chronic inflammatory diseases. By understanding these regulatory processes, we can gain valuable insights into NLRP3 biology and its role in various diseases, ultimately aiding in the development of targeted therapies for inflammatory and autoimmune conditions. Significant progress has already been made towards these aims, including high-resolution cryo-EM map and identification of novel regulators through mass spectrometry. My preliminary data have already revealed new conformational states of NLRP3, and identified specific ubiquitination and lipidation sites on NLRP3 that may be crucial for its activation. The K99 phase will focus on improving the resolution of my cryo-EM maps, verifying PTM sites, and determining the functional roles of these modifications. In the R00 phase, I plan to explore the roles of novel regulatory proteins in NLRP3 activation. Using mass spectrometry and protein interaction studies, I will identify and characterize new regulators that interact with NLRP3 in a temporal and spatial manner. TurboID technology will be employed to map the interactome of NLRP3 during different stages of activation, providing a comprehensive understanding of the dynamic changes in protein interactions. I will determine the cryo-EM structures of NLRP3 in complex with these identified regulators to reveal their mechanisms of action. My long-term interest lies in understanding immune responses following pathogen infection, both in health and disease. With the experience gained from a K99/R00 Award, I aim to establish an independent laboratory dedicated to basic and translational research on inflammasome activation, control, degradation, and its interaction with pathogens. This research will provide a solid foundation for developing novel therapeutic strategies to modulate inflammasome activity, offering new hope for patients suffering from chronic inflammatory and autoimmune diseases.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The proposed research and training program aims to equip the PI with the skills to become an independent investigator and to advance the understanding of neurodevelopmental disorders (NDDs) while developing innovative therapies. Building on previous training in epigenomics, the PI will use emerging single-cell epigenomic technologies to link pathogenic variants in chromatin regulators to specific neuronal phenotypes. NDDs affect more than 3 million people in the U.S., impairing language, learning, motor skills, and neurological function. Despite their prevalence, the molecular mechanisms underlying these disorders are not well understood, hindering the development of effective treatments. Mutations in chromatin regulators, such as KMT2D, are major contributors to NDDs, including intellectual disability, autism spectrum disorder and schizophrenia. KMT2D is essential for differentiation and development by modulating chromatin accessibility and 3D chromatin structure. Loss-of-function mutations in KMT2D are associated with brain malformations, memory defects and neuronal differentiation problems, with Kabuki syndrome being a notable example. However, the precise molecular mechanisms by which KMT2D haploinsufficiency leads to brain abnormalities and cognitive impairment are not well understood. This project will use mouse and human models of KMT2D haploinsufficiency to profile single cell chromatin accessibility and 3D chromatin structure, and examine the impact on regulatory programs in different cell types. The research and training will be guided by a distinguished mentoring team of experts in several relevant fields: Drs. Walsh (neurobiology), Bodamer (NDDs), Lee (single cell genomics), and Gussoni (stem cells). The results of the project will advance our understanding of chromatin regulators in NDDs and provide broadly applicable methods and tools for other NDDs.

NIH Research Projects · FY 2025 · 2025-08

We all age and grow old. Age is the primary cause of disease and heath burden in today’s society. Thus understanding how to mitigate the shared mechanisms of the manifestation of age-related diseases will have significant impacts on healthcare and, more generally, quality of life. To understand how aging is mitigated with time, we can leverage cases in nature where organisms have evolved mechanisms that permit them to live exceptionally long lives – the inference being that aging also must be inhibited to support such extended lifespans. These natural experiments provide unique solutions to shared physiological constraints as occurring in humans. Here we detail the genome of the longest-living animal, the Ocean Quahog, Arctica islandica, and ask what genes and novel functions allow for the attainment of exceptional lifespan to over 500 years. We also capitalize on the differential selection on lifespan across different populations of the Quahog across an ecological cline, ranging from 35 to 507 years. This dynamic evolutionary selection on lifespan in this species permits both macroevolutionary comparisons of lifespan, but also fine detail population genetics of what local selective forces and genes may underlie this shift. Our central hypothesis is that factors associating with exceptional longevity- -in even distantly related animals such as the Ocean quahog--will have core physiological similarities that will inform of mechanisms that can be altered to extend lifespan and healthspan. In Aim 1, we first propose to look at patterns of selection across the genome in comparison with lifespan variation among bivalve species as well as within different populations of A. islandica. As the distance between bivalves and more commonly used model animals is quite large, the orthology of genes is often hard to pin down, in Aim 2 we will assess conservation and homology of genes across the A. islandica genome though structural mapping. This will provide the codex of gene conservation and potential novelty within this large family of animals. To provide context for changes we observe associating with lifespan, we will use the evolutionarily defined genesets from A. islandica to refine human GWAS data for humans that live at the fringes of expected limits. Lastly in Aim 3, we look to functional changes in proteostatic mechanisms functioning within A. islandica. We will assess variation and function of small chaperone proteins in Arctica and ask if particular variants are sufficient to impart proteostatic resiliency in experimental models in Drosophila and zebrafish of aging and lifespan regulation. Lastly, we have evidence of specific variants of metabolic regulator, gapdh, shared across animals with exceptional longevity, including Arctica islandica. We will ask if these variants are sufficient to render proteostatic resiliency, as well as to extend healthspan and lifespan of Drosophila and zebrafish strains. Together, these aims will provide essential window into understanding the core principles and factors that underlie capacity for exceptional long life, and the heath implication that follow.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Malaria remains an important global cause of morbidity and mortality. The majority of the burden of this disease is due to infection with the Plasmodium falciparum parasites and affects children and pregnant women. Asexual replication of the parasite, during the human blood stage, causes the signs and symptoms of the disease when repeated rounds of asexual replication generate a dramatic increase in the number of parasites. P. falciparum asexual replication occurs via schizogony, wherein the nuclei and organelles for 20-36 daughter cells are produced within a common cytoplasm without cytokinesis. Then, in a complex and highly organized process known as segmentation, the parasite partitions the nuclei and organelles into each daughter parasite with remarkable fidelity. There is a major knowledge gap surrounding this divergent method of cell division. We aim to use cryo-electron tomography to learn new molecular insights into this process. Segmentation is largely driven by two parasite-specific structures, the inner membrane complex (IMC) and the basal complex. The IMC consists of two lipid bilayers and multiple associated proteins that form adjacent to an underlying cytoskeletal network of intermediate-like filaments. Together, the IMC and associated cytoskeleton are critical for the parasite morphology and rigidity. The basal complex is a multi-protein molecular machine that forms at the posterior/basal end of the IMC. It is highly essential for parasite cell division and is hypothesized to be the contractile ring that mediates abscission during cytokinesis. Importantly, the basal complex is unlike the contractile rings of model organisms and lacks dependence on actin-myosin interactions. Thus, the mechanism of action for this presumed contractile ring is fully unknown. Several basic questions are ready for answers – does the basal complex form a ring or a coil, are concentric structures present or is it a single “ring”, is the complex symmetrical or are there distinctly different regions with it. In the current proposal, we will utilize cryo-electron tomography (cryo-ET) to gain a molecular understanding of basal complex function. We have generated a parasite strain where an essential component of the basal complex, PfCINCH, has been tagged with 3xFLAG and mNeonGreen. This allows visualization of the basal complex in intact parasites and purification of the basal complex from lysed parasites. In the first aim, we will determine the native structure of the basal complex using in-situ cryo-electron tomography. In the second aim, we will use affinity-purification to obtain basal complexes from lysed parasites. Cryo-ET will be applied to these isolated basal complexes to obtain a high-resolution structure via sub-tomographic averaging. Together, the proposed experiments will provide the first structural information about this essential multi-protein machine. The resulting structural insights will begin to reveal the molecular mechanism of the basal complex – and potentially lead to possible interventions to block its essential activity.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract With age, hematopoietic stem cells (HSCs) accrue mutations, some of which provide a competitive advantage and lead to the expansion of clones bearing the mutations, a phenomenon called clonal hematopoiesis (CH). CH is associated with inflammation, susceptibility to infections, high risk of cardiovascular disease, and development of myeloproliferative disorders and cancer. Together, these conditions represent the leading causes of death in older adults. Recent studies indicate that ~95% of healthy middle-aged adults bear CH-associated mutations. Although adults with these mutations are at much higher risk of hematologic disorders, progression from CH to disease states still only occurs in a small subset of these individuals. Many genes are associated with CH, but mutations in DMNT3a represent a majority of CH-associated mutations. We have shown that, within DNMT3a mutant HSCs, expansion is driven by a small set of “super-competitor” clones. Given that higher variant allele frequencies (VAF) of DNMT3a mutations indicate higher risk for hematologic disease, identifying factors which drive expansion in these clones is of high clinical relevance. We conducted a CRISPR- based ex vivo expansion screen of genes that are upregulated in DNMT3a mutant cells. Results of the screen highlighted the gene SLC3a2, whose knockout inhibits rapid expansion and induces synthetic lethality in DNMT3a mutant cells ex vivo and in transplantation settings. This proposal seeks to investigate the mechanisms of clonal competition in CH via a three-pronged approach. To validate SLC3a2’s effects in vivo, we will assay the expansion of DNMT3a mutant, SLC3a2 knockout HSCs and their gene expression profiles in unperturbed hematopoiesis. SLC3a2 is a component of the CD98 complex, which has dual roles in amino acid transport and integrin-mediated adhesion. To dissect the functional mechanism of SLC3a2’s effects in clonal competition, we will conduct pharmacological and genetic disruptions to components of the complex required solely for amino acid transport, and perform metabolic assays aimed at either phenocopying or rescuing the effect of SLC3a2 KO. Lastly, we will assess SLC3a2’s role in human clonal hematopoiesis using human cord blood CD34+ cells genetically altered to model DNMT3a-mediated clonal hematopoiesis. As a postdoctoral fellow, Dr. Quach will conduct his research in the laboratory of Dr. Fernando Camargo at Boston Children’s Hospital. Dr. Quach will build on his solid background in hematology and translational research, developing new skills in single-cell transcriptomics and stem cell biology to generate insights into the mechanisms of clonal hematopoiesis. His mentor, Dr. Camargo, is an expert in hematopoiesis and has developed innovative technologies for cellular barcoding and clonal analysis. Dr. Camargo’s mentorship and expertise, a strong scientific advisory committee, and a rigorous research and training plan provide all the necessary elements for success in the proposed project and Dr. Quach’s transition to independence.

NIH Research Projects · FY 2025 · 2025-08

Abstract Medical progress in many fields like oncology, transplantation, surgery, neonatology and rheumatology is hurt by the rising incidence of invasive fungal infections. Developing novel antifungal therapeutics is challenging because fungal and human cells share many homologous essential proteins. A novel approach to antifungal development could be to target non-conserved domains within conserved proteins, following detailed functional and structural characterization. On the other side, the cell wall has proven to be an excellent target: it commands a significant portion of signaling- and metabolic resources of the fungal cell, and the entire organelle is absent in humans. Identifying non-conserved regulatory elements in cell wall stress endurance, and testing non-conserved protein regions to identify targets of domain-based drug discovery, could expand the spectrum of targets for novel compounds that synergize with cell wall-active agents and protect them from resistance development. Mutants in Candida albicans Target of Rapamycin kinase 1 (Tor1) truncated for its least conserved region, N-terminal HEAT repeats, are hypersensitive to cell wall stress. Among transcripts aberrantly regulated in Tor1 N-terminal truncation mutants are a cell wall stress transcription factor and its putative regulator, both not present in humans. This transcription factor mediates C. albicans responses to echinocandins, antifungal agents that inhibit biosynthesis of the major structural polysaccharide in the fungal cell wall. We propose to identify targets in C. albicans cell wall stress endurance, whose inhibition potentiates echinocandins and protects them from resistance development, or has a cidal effect on its own. To this end, we will characterize a key cell wall stress transcription factor and its regulator, and two non-conserved regions of Kog1, the essential regulator in Target of Rapamycin Complex 1. Human oncologic target investigations and mechanistic analysis of neurodegenerative diseases combined, provide important background understanding of domain-based drug discovery and inhibitors of prion-like motifs. We intend to leverage this understanding towards a proof of the principle, that the paradigm for antifungal drug discovery can expand beyond the biosynthetic enzymes that currently are major targets, to include non-conserved regulatory elements which the fungus needs to protect its cell wall.

NIH Research Projects · FY 2025 · 2025-08

Project Summary / Abstract Severe chronic neutropenia (SCN) is an immunodeficiency that leads to severe, often life-threatening, infections in patients with both congenital and acquired forms of neutropenia. Despite granulocyte colony- stimulating factor therapy, patients remain at long term, often life-long, risk of serious infections and additional comorbidities. The Severe Chronic Neutropenia International Registry (SCNIR) was established in 1994 to study the natural history and clinical consequences of SCN and its treatment. Based on the research resources of the Registry, SCNIR investigators have published 255 journal articles, and 41 reviews and book chapters related to SCN. The SCNIR has provided evidence-based clinical guidance, advanced our understanding of neutrophil biology, and broadly informed the care of neutropenia and immunodeficiencies. Scientific technology developments have opened new opportunities to investigate SCN biology. Ongoing evolution of clinical medicine and diagnostics requires continued reassessment of natural history, treatments, and outcomes. The objective of the SCNIR is to provide a platform to leverage innovations in database science, molecular and computational biology, patient-reported outcomes, and other advances in basic and clinical research to enable discovery of new treatments and inform medical management. This project builds on the history of productive collaborations within and outside the SCNIR to expand available NIAID resources and provide a model for the study of other immunodeficiencies and rare diseases. Our SPECIFIC AIMS are: Aim 1: Transition the SCNIR database to a hybrid model at Boston Children’s Hospital We will transition SCNIR operations to Boston, with a hybrid data collection system engaging both physicians and patients; collect and analyze patient-reported outcomes for SCN; and develop a pilot project in natural language processing for data extraction and entry. Aim 2: Generate clinically annotated genomic datasets for translational research in SCN We will characterize the germline genomic features of chronic neutropenia for the study of genetic interactions, and novel genetic causes of SCN by the research community; characterize the somatic mutational landscape of SCN; and provide resources for innovative translational research and clinical trials. Aim 3: Provide resources and outreach for education, recruitment, and retention. The SCNIR will engage patients, families, and medical providers to provide education and promote retention and will share expertise and foster collaborations with related patient advocacy and research groups. Continued operation of this longstanding, productive registry will permit otherwise unfeasible long-term assessments of SCN natural history, generate resources for both clinical and scientific research investigators, and provide up-to-date educational resources and outreach to medical and patient communities.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Melanoma is a devastating type of cancer, and despite recent advances in targeted or immunologic therapies, many patients relapse. We study melanoma using a zebrafish model with melanocyte-specific expression of human BRAFV600E or other oncogenes in the background of a variety of tumor suppressor mutations. With zebrafish genetic and chemical biology tools, we hope to reveal critical pathways that participate in the initiation and formation of melanoma. We have observed the onset of melanoma using a neural crest specific transgenic reporter for crestin, a neural crest progenitor gene that is expressed early in neural crest development and shuts off by day 5 of zebrafish development. When a melanoma arises, crestin expression reactivates in single cells transforming into melanoma. We recently found that the initiating cell is derived from a zone of hundreds of cells that express higher levels of the master melanocyte regulator MITF and display aberrant morphology. We called this region a cancer precursor zone (CPZ) and found analogous regions that express MITF and another conserved gene, ID1, in early human melanocyte lesions and melanoma in situ. Using novel cellular barcoding technologies such as CRISPR-based GESTALT, we have shown the CPZ is oligoclonal from which a single malignant clone expands to form melanoma. We also showed that BMP, which induces ID1 clonally expands CPZs. Using the color-based Palmbow, we will study gene expression in clones of CPZ and early tumors and use imaging to study the clones. Clonal mapping using mitochondrial sequencing of human melanomas will confirm our observations in zebrafish. We plan to use proteomics and zebrafish genetics to examine the pathways involved in neural crest reactivation from the CPZ. Using proteomics, we have found growth factors and receptors that are expressed in crestin+ patches and not CPZs, such as midkine b that is known to regulate neural crest development. Overexpression and knockout experiments will be used to test the sufficiency and requirement of these proteins for causing tumor initiation. The CPZ is also associated with increased oxidative stress, and we found the antioxidant gene ApoD is expressed at higher levels in early crestin+ patches and drives the transition to overt tumors. One function of ApoD is to convey resistance to ferroptotic death and to enable tumor initiating cells to handle more reactive oxygen species (ROS). Overexpression of ApoD leads to an increase in both tumor incidence and abundance in zebrafish. We plan to use genetics and cell biology, including leveraging MAZERATI, a tissue-specific CRISPR technology, to understand the mechanism by which ApoD controls ferroptosis and how the protection from ROS and ferroptosis plays a role in melanoma initiation. We also plan to use our color based and CRISPR cellular barcoding technologies to investigate if ROS resistance and suppression of ferroptosis by ApoD leads to clonal selection and tumor initiation. Our studies will improve the understanding of cancer initiation and progression. This may lead to the identification of new biomarkers or drug targets, yielding early diagnosis and new preventative therapies for melanoma.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The circadian clock is an endogenous timekeeping mechanism that drives daily rhythms in gene expression, physiology, and behavior. The key components of the circadian clock in mammals are transcription factors and regulators that drive rhythmic transcription. Recently, RNA has emerged as a biophysical modulator of transcription factors and other chromatin-associated factors via direct interactions. However, how these RNA- protein interactions might shape circadian rhythms remains unexplored. My preliminary data suggests that a master circadian transcription factor BMAL1 directly interacts with RNA. This led to the hypothesis that BMAL1- RNA interactions modulate circadian rhythms. To test this hypothesis, I propose the following aims: (1) Identify and characterize BMAL1-binding RNAs, (2) Define structural determinants of BMAL1-RNA interaction and determine their role in regulating circadian transcription and translation. I will carry out this study using a variety of molecular biology and genomic techniques, including a biochemical assay to capture RNA-protein interactions, RNA sequencing, and genetic manipulation. This work will uncover a novel modulatory mechanism of circadian rhythms and provide a foundation for studying the mechanistic link between the circadian clock and a wide range of RNA-mediated cellular functions. This fellowship will provide an excellent opportunity to gain expertise in molecular biology and RNA-protein interaction. This will prepare me to become an independent academic investigator studying circadian rhythms at multiple levels.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Ischemic heart disease is the leading cause of death worldwide, in part because human heart muscle fails to regenerate after ischemic injury. A primary barrier to human heart regeneration is the near-complete absence of cardiomyocyte cell division following a myocardial infarction (MI). Fortuitously, injured hearts of adult zebrafish and neonatal mice are capable of robust cardiac regeneration achieved through cardiomyocyte proliferation. However, neonatal mice lose their innate cardiac regenerative capacity between P1 and P7 due to cardiomyocyte cell cycle exit, the regulatory details of which remain incompletely understood. Deciphering the pro- and anti-proliferative pathways regulating cardiomyocyte proliferation and cell cycle exit in these highly tractable model organisms will identify druggable pathways for stimulating heart regeneration as a permanent remedy for myocardial infarction. Herein, I propose to expand our fundamental knowledge of the gene regulatory networks controlling cardiomyocyte proliferation through mechanistic studies on the AAA+ ATPase Pontin and high-quality candidate downstream targets. We present preliminary data identifying Pontin as a novel regulator of cardiomyocyte proliferation in zebrafish and mice. Zebrafish pontin mutants are devoid of cardiomyocyte proliferation, and cardiomyocyte-specific overexpression of Pontin (pontinOECM) hyperactivates cardiomyocyte proliferation during cardiac development, growth, and regeneration. Remarkably, Pontin overexpression also improves the efficiency of heart regeneration, as evidenced by reduced scar size. We also demonstrate that myocardial-specific deletion of Pontin in embryonic mice suppresses cardiomyocyte proliferation, leading to embryonic lethality. Interestingly, Pontin levels decrease naturally in cardiomyocytes soon after birth, consistent with the possibility that Pontin downregulation creates a barrier to regeneration by facilitating cardiomyocyte cell cycle exit. Accordingly, cardiomyocyte-specific overexpression of Pontin delays cell cycle exit and, potentially, expands the regenerative window. Accordingly, myocardial infarction of PontinOECM hearts at P8 resulted in improved ejection fraction at P29, consistent with a regenerative response. Here, I propose to test the central hypothesis that Pontin regulates cardiomyocyte proliferation by controlling the chromatin accessibility of high-quality candidate genes identified through ‘omics efforts detailed in preliminary data. In Aim I, we will corroborate the candidate Pontin targets regulating CM proliferation during zebrafish heart development. In Aim II, we will corroborate the direct Pontin targets regulating CM proliferation during zebrafish heart regeneration. In Aim III, we will determine if Pontin is a pro-regenerative factor in mice and whether the zebrafish targets are conserved in mammals. Overall, the proposed studies will produce novel biological insights and positively impact ongoing efforts to stimulate cardiomyocyte proliferation in the context of human heart disease.

NIH Research Projects · FY 2026 · 2025-08

Motion and distortion-robust quantitative MRI of early brain developments The objective of this research is to significantly enhance quantitative imaging technology for in-vivo analysis of neurovascular structures and the early developing brain's response to perinatal stroke. Quantitative MRI (qMRI) is becoming essential in neuroimaging, offering superior capabilities over qualitative MRI. Specifically, quantitative susceptibility mapping (QSM) and R2* mapping provide early insights into developmental abnormalities such as brain hemosiderin deposits, intracranial hemorrhage, and blood oxygenation levels. However, quantitative perinatal MRI faces significant challenges: 1) fetal motion restricts imaging to 2D and often introduces artifacts, and 2) geometric and field distortions are amplified by fetal and maternal organ movements. Currently, our understanding of early brain development and common neurodevelopmental abnormalities is primarily derived from postmortem (in-vitro) studies due to the lack of advanced quantitative imaging technology. This project aims to bridge these gaps by developing an innovative, motion- and distortion-robust quantitative MRI technique. This involves creating and evaluating a novel approach based on 3D motion-robust radial multi-echo MRI combined with advanced image processing and reconstruction techniques. These methods will correct for motion and reconstruct high-resolution quantitative data directly from the data space, allowing detailed visualization of the neurovascular structure in small fetal and neonatal brains. The project has three specific aims: 1) improving neonatal quantitative MRI for moving subjects, and 2) achieving high-resolution susceptibility-weighted, quantitative susceptibility and R2* maps in the developing brain despite subject movements 3) quantitative evaluations of the proposed approach against Echo planar imaging-based approaches on healthy and patients with perinatal stroke. This research is significant because it enables high-resolution in-vivo mapping of the neurovascular structure in the fetal brain despite motion, simplifies MRI research for neonates and preterm infants with motion-robust acquisition and reconstruction techniques that compensate for small head movements, reduces the need for sedation and anesthesia in clinical MRI of neonates and uncooperative patients, corrects motion while increasing the spatial resolution of quantitative MRI, thereby dramatically improving the analysis of neural structure and connectivity in early brain development.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This project aims to develop a solution for high-throughput brain connectomics by integrating GridTape-based automated section collection and handling with advanced serial electron tomography (ET). ET is an indispensable tool for ultra-high resolution imaging, typically used to study very small regions in detail by computationally reconstructing 3D volumes from 2D projections. Using intermediate voltage transmission electron microscopy (IVEM), ET generates volumetric information through thick sections (up to 2 μm). By combining ET of thick sections with automated section collection and handling, we aim to significantly advance whole mouse brain connectomic imaging in terms of robustness and practicality, improving upon capabilities for high- and multi-resolution imaging for mammalian brains, and more efficiently elucidating the organizational principles of brain circuits. Our approach will further facilitate sample-preserving imaging, allowing for multi-scale interrogations without sample destruction, thereby significantly reducing the risks associated with data acquisition and amplifying the value of prepared samples. The efficacy of this solution will be validated using the mouse cerebellum, providing insights into cell-to-cell communication within this brain region crucial for motor control. The project will be driven by three complementary aims structured to: 1) Evaluate ET scalability by assessing imaging conditions, lossless sectioning, and GridTape characteristics, 2) Develop tape-enabled serial ET through the design and testing of a reel-to-reel tape system, a tape tilting system, and related software, and 3) Augment tape-enabled ET with advanced machine learning algorithms, energy-filtered TEM strategies (e.g., automated most-probable loss tomography (MPL)), continuous imaging, and modifications for large section tomography. Deliverables from this work will include the development, testing, and troubleshooting of prototype FAST-ET platform, critical reference datasets for the neuroscience community, and scalable metrics for mammalian whole-brain connectomics. Our goal is to significantly advance understanding of brain function and dysfunction with broad implications for neuroscience, engineering, and artificial intelligence.

NIH Research Projects · FY 2025 · 2025-08

Project Summary To survive in the harsh environment of the human host, pathogens such as the gram-negative bacterium Pseudomonas aeruginosa (Pa) must utilize a broad array of virulence factors. Aberrant firing of these often metabolically costly pathways can result in immune detection or out competition, thus their production is tightly regulated. Recent techniques have allowed for the expanded study of small RNA (sRNA) mediated regulation in bacteria and it is increasingly clear that these ubiquitous molecules exert important regulatory effects, even on systems whose regulation was previously thought to be well-understood. Recently, work from the Dove lab utilized RNA interaction by ligation and sequencing (RIL-seq) to assess the global RNA-RNA interactions mediated by the RNA chaperone Hfq in Pa. Among this data I found an abundant uncharacterized sRNA, that we refer to as BecO, which appears to be highly conserved in Pa. To begin understanding the pathways impacted by this sRNA, I performed RNA-seq. Excitingly, my preliminary data shows that BecO significantly regulates the expression of hundreds of genes. Among these genes, those encoding for the Type III secretion system (T3SS) and pyochelin synthesis appeared to be strongly regulated. These pathways are both critical for the virulence of Pa, suggesting an important role for BecO during infection. Here I propose to investigate the outsized regulatory role of this sRNA and build a skillset that can be broadly applied to investigating other sRNA regulatory systems. This will be aided by my sponsor, Dr. Simon Dove’s expertise on sRNA regulation in Pa. In Aim 1 I will determine how BecO controls T3SS gene expression. I will investigate whether BecO exerts its regulatory effects by base- pairing with the mRNA transcripts of one or more of the master transcription regulators that govern T3SS gene expression. I will also determine which of the three putative seed regions that I have identified in BecO are involved in mediating T3SS control. In Aim 2 I propose to determine how BecO controls expression of the pyochelin biosynthesis genes. To do this I propose to develop a more sensitive assay based on RIL-seq to identify additional RNA interaction partners for BecO. This technique would have broad utility in the identification of regulatory targets for sRNAs in Pa and other bacteria. Preliminary experiments have shown the growth of the human pathogen Acinetobacter baumannii is altered when co-cultured with Pa in a manner that is influenced by BecO. In Aim 2, I also plan to determine if this altered growth is due to any positive regulatory effects BecO might have on pyochelin production. Together the work proposed will further our understanding of the complex regulation imposed on P. aeruginosa virulence factors and generate new insights into how sRNAs fit into these regulatory circuits. By performing this work in the highly collaborative and intellectually stimulating environment of the Boston Children’s Hospital and Harvard Medical School communities I will have an excellent opportunity to develop the skills needed to become an independent investigator.

NIH Research Projects · FY 2025 · 2025-08

Tuberculosis (TB) is one of the largest causes of global infectious disease death. Current treatments of TB infection involve complex drug regimens that require months of treatment and are plagued by drug toxicities and drug resistance. Furthermore, TB has been the cause of death in a third to a half of the approximately 40 million AIDS deaths and remains a major cause of death in those HIV-infected individuals especially those who are immunosuppressed. Therefore, host-directed therapeutics (HDT) that increase innate immune responses to TB when combined with current antimicrobial therapies, could potentially enhance TB cure and clinical outcomes. However, the critical knowledge about the human innate immune response to MTb required to develop such HDTs is lacking as well as physiological in vitro lab models to study and validate such early innate host responses to TB infection. Here, we propose to overcome these barriers by establishing a new physiological in vitro lung alveolar epithelial cell organoid model from human bronchoalveolar lavage fluid (BALF) (Aim 1). Specifically, building on our studies with co-investigator Carla Kim, where we developed a new model for Mycobacterium tuberculosis (MTb) infection using pulmonary alvelolar epithelial type II (AT2) organoids derived from human lung tissue, in Aim 1, we propose to develop an AT2 organoid MTb infection model from easily obtainable human bronchalveolar lavage fluid (BALF), which contains primary AT2 cells and other cells including macrophages from the same individual. These BALF-AT2 organoids will allow us to use advanced imaging microscopy in collaboration with Dr. Tom Kirchausen to study MTb infection of AT2 and macrophage cells and their interaction, as well as the effect of nitazoxanide (NTZ), an experimental MTb HDT, which inhibits MTb growth in concert with broadly amplifying type I and III IFN signaling. Using AT2 tissue organoids and NTZ as a probe, in Aim 2 we will perform bulk and single-cell (sc)-seq analyses to identify factors that are associated with NTZ inhibition of early MTb infection and growth in collaboration with Dr. Brad Rosenberg. Critical pathways and novel factors in innate immune restriction of early MTb infection will then be functionally validated using CRISPR approaches. We anticipate will provide new model physiological platforms for study of TB infection and that these studies will elucidate critical innate immune mechanisms that may serve as HDT targets for further study and inhibition of MTb infection.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Heart malformations are the most common form of serious birth defect, with more than 1% having congenital heart disease (CHD)1, and about 1 in 4 of these requiring an interventional procedure in the first year of life2. As a result, extensive efforts have been made to understand normal heart development and how it is perturbed by genetic or environmental insults3. A small number of cardiac progenitor cells, marked by the expression of MESP1, differentiate into the many cell types of the heart. Clonal analyses have outlined relationships between these progenitor populations, as well as their ultimate contribution to the major differentiated cell types of the heart10,11. Due to technical limitations, these lineage maps are incomplete, lacking clear connections between major progenitor populations as well as information about intermediate steps of lineage diversification of cardiac cells. Building on revolutionary lineage tracing, spatial, and transcriptomic technologies with single cell resolution, we will create a high-resolution spatiotemporal lineage map of cardiac development. Furthermore, we will deploy this approach to investigate how gene mutations that cause cardiac malformations disrupt cardiac lineage specification and result in congenital heart disease. We will pursue the following Specific Aims: Aim 1. To spatiotemporally map early cardiac progenitor lineages and their clonal relationships. Aim 2. To spatiotemporally map the phylogeny of the major myocardial cell types. Aim 3. To define the effect of Tbx5 knockout on cardiac lineage diversification. This proposal will produce a high-resolution spatiotemporal lineage map of cardiac development. Our studies of Tbx5 inactivation within this framework will illustrate its utility for studying the mechanisms by which genetic or environmental insults cause heart malformations. The methods and analytical tools developed in this project will have widespread applications in other fields of developmental biology as well as heart regeneration and acquired heart disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY: Congenital heart disease (CHD) is the most common congenital anomaly and affects approximately 1% of people. Neurodevelopmental delay and disability (NDD) are the most common extracardiac complications among people with CHD, but clinical and surgical factors currently only explain approximately one-third of NDD risk. Without knowledge regarding the molecular mechanisms that underly shared risk for altered heart and brain development, people with congenital heart disease cannot benefit from early diagnosis, accurate prognosis, and targeted treatments. Many genetic syndromes affect heart and brain development; we anticipate that these seemingly divergent organs will share dysregulated gene expression during development. Therefore, we propose to identify shared nodal biology disrupted in cardiac and neuronal progenitors with haploinsufficiency of CHD7 or KMT2D, causes of genetic syndromes characterized by CHD/NDD. In support of this goal, we have already generated human induced pluripotent stem cells (iPSCs) that are haploinsufficient for CHD7 or KMT2D. With these reagents in hand, we will combine our expertise in iPSC methods and computational biology to determine sources of shared genetic risk for heart and brain development, as well as advance our approaches for functional assessment of variants. The Overall Aim of this proposal is advance our understanding of the connection between CHD and NDD by (1) identifying mechanisms by which syndromic CHD genes lead to alterations in both heart and brain development, and (2) comparing the yield of transcriptomics, epigenetic and high-throughput high-dimensional cell imaging to assess the functional impact of variants of uncertain significance from people with CHD in iPSC models of heart and brain development. First, we will compare single nucleus and bulk transcriptomics, chromatin accessibility, and cell morphology features of iPSCs that are haploinsufficient for CHD7 and KMT2D as they differentiate to cardiac and neuronal progenitors. This combination of techniques will identify shared and divergent direct targets of CHD7 and KMT2D in each cell lineage. Next, we will compare the ability of transcriptomic and cell morphology approaches to determine whether missense variants of uncertain significance from people with CHD are likely pathogenic, as cell morphology assessment can be higher throughput and significantly lower in cost than transcriptomics. Together this proposal will employ multivariate analysis of biological data in a way that builds upon the ongoing work supported by my K08 award and generates new results that will support a future independent R01 grant application in 2027. These results will contribute towards the long-term objective of understanding the molecular basis of heart development and human disease to improve diagnosis, better define risks, and inspire novel treatments for patients.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This conference and associated consensus process will create a bundle of NIH-endorsable consensus Common Data Elements (CDE) specifically targeted to advance research efforts according to FAIR principles in Juvenile Idiopathic Arthritis (JIA), the most prevalent pediatric rheumatic disease (PRD), for which no current effort to create a CDE exist or is in-process. Creation of consensus-driven CDEs for JIA will facilitate study of this and other PRDs diseases across research projects in pediatric rheumatic diseases and other autoimmune diseases, furthering the scope and generalizability of research in rheumatic diseases across the lifetime. The JIA disease domain appears readily amenable to the processes needed for consensus CDEs to be formally defined, mapped to existing data elements from major existing efforts, and submitted in a format that will enable endorsement by the PRD community and NIH. We therefore propose a consensus driven process to enable a final product of an NIH-endorsable CDE for JIA, with the aims of (1) expert identification and collation of existing data elements, including from major existing research efforts; (2) a virtual conference for JIA CDE consensus development, conducted using modified Delphi methodology that has been successfully utilized for generation of multiple consensus efforts in the past; and (3) creation of NIH-endorsable bundles of consensus- based CDEs for JIA. As a final product, we will collate and compile responses from the respective consensus conference meetings into a JIA CDE bundle. This will be prepared in both text-based, human readable format and machine-interpretable formats (e.g. data dictionaries, code lists), with inclusion of metadata and conversion to formats suitable for inclusion in the NIH CDE repository.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY This R01 proposal outlines a collaborative research initiative aimed at elucidating how mobilization of transposable elements (TEs) and alterations in mRNA translation disrupt the function of neural progenitor cells (NPCs) in Down Syndrome (DS). DS, or Trisomy 21, is the most common chromosomal cause of intellectual disability and is associated with impaired neurogenesis, leading to cortical maldevelopment in early life. Our data from the human prenatal brain has identified dramatic de-repression of TEs in DS cell subpopulations, which we have linked to activation of the antiviral innate immune system. Further, we have identified profound disruption in the mRNA translational machinery, including in many components of RNA granules. Based on this data, we first seek to map the TE landscape and cellular response in human induced pluripotent stem cell- derived NPCs, and test interventions to attenuate TE activation. Next, we will investigate changes to the translatome and proteome associated with DS, identifying novel TE-associated translation events. Finally, we hypothesize that DS NPCs exhibit significant alterations in the composition of subcellular cytoplasmic biomolecular condensates – RNA granules – that are critical mediators of mRNA metabolism and translation. We will map the protein and RNA composition of translation-associated condensates in DS NPCs and investigate how cytoplasmic phase separation dynamics are altered in DS. The knowledge derived from this research plan will not only enhance our understanding of the mechanisms through which DS impacts NPC function, but will also identify targetable pathways for reversing abnormal corticogenesis, offering potential avenues for intervention and prevention.

NIH Research Projects · FY 2025 · 2025-07

Project Summary DADA2 is an inborn error of immunity characterized by systemic inflammation with vascular involvement and bone marrow failure. Biallelic mutations in ADA2 are responsible for this recessive condition, though symptomatic carriers with a single deleterious variant are increasingly recognized. How pathogenic ADA2 variants translate to the heterogeneous disease manifestations remains unclear. Underscoring the importance to better understand DADA2, our work demonstrated the potential for ~35,000 patients worldwide. We found that missense ADA2 variants with residual enzymatic activity are associated with the inflammatory phenotype while loss of function (LOF) variants are linked to bone marrow failure in DADA2. We now provide evidence that missense ADA2 variants cause protein misfolding and retention in the endoplasmic reticulum, leading to activation of the unfolded protein response (UPR). We hypothesize that activation of UPR in monocytes by misfolded ADA2 protein induces tumor necrosis factor production to drive inflammation in DADA2, while the absence of ADA2 protein impairs hematopoiesis. This mechanism also potentially explains features of DADA2 in some heterozygous carriers of missense ADA2 variants. We will pursue this hypothesis through 2 complementary but independent aims. Aim I will examine the activation of three main UPR pathways and cytokine production in monocyte-derived macrophages from healthy controls, patients with DADA2, and heterozygous carriers of pathogenic ADA2 variants. We will validate the results in gene-edited cell lines with disease-associated variants. We will evaluate UPR activation in inflamed tissues from patients, and compare the transcriptomic landscape, including signatures of TNF and UPR activation, between disease phenotypes. We will explore how carriage of misfolded ADA2 may impair the trafficking and function of wildtype ADA2, leading to inflammation. Aim II will evaluate the therapeutic effects of TNF inhibitors, UPR inhibitors, gene therapy and enzyme replacement therapy on cytokine production and immune activation by monocyte-derived macrophages from patients with DADA2. Extending our analysis to the BMF phenotype, we will study the impact of these therapies on in vitro differentiation of ADA2-deficient hematopoietic stem and progenitor cells. In an exploratory sub-aim, we will seek to establish an in vivo model of DADA2 by engrafting gene-edited hematopoietic stem cells into humanized mice. The proposed studies will inform the development of personalized medicine for DADA2 and more broadly illuminate how defects in one gene can lead to variable disease phenotypes.

NIH Research Projects · FY 2025 · 2025-07

Project summary β2 integrins are leukocyte-specific heterodimeric adhesion molecules consisting of α- and β- subunits. β2 integrins consist of αLβ2, αMβ2, αXβ2 and αDβ2 with αDβ2 being cloned most recently. αDβ2 is highly homologous to αMβ2 and αXβ2, both of which are also known as complement receptors (CR3 and CR4, respectively). Although the biological role of αDβ2 still needs to be studied in depth, in vivo studies have shown that this may be a promising target for acute lung injury in sepsis. Our preliminary data and our published study showed that αDβ2 deficiency was associated with less bacterial loads and less development of acute lung injury in septic mice. αDβ2 was expressed on human and mouse injured lung in sepsis. Acute lung injury is among the leading causes of morbidity and mortality in sepsis (~40% mortality), and is managed only supportively including the use of mechanical ventilation, which by itself can cause lung injury. Thus, delineating its fundamental, biological role is of great importance to understand the mechanism of how αDβ2 contributes to the disease pathophysiology. Considering its high homology to αMβ2 and αXβ2, we hypothesize that αDβ2 would be a novel complement receptor. Our preliminary data showed that sheep erythrocytes opsonized by iC3b formed rosetting on HEK cells stably expressing αDβ2, supporting our hypothesis. In Aim 1, we will characterize the binding profile of αDβ2 to different complement fragments and delineate the contribution of αDβ2 to the binding of complement fragments by different leukocytes in vitro. We will rank order the binding of αMβ2, αXβ2 and αDβ2 to iC3b. In our preliminary data, we found that lung αDβ2 knockout neutrophils had more phagocytosis than WT counterparts from septic mice. We will delineate the underlying mechanism of αDβ2-mediated phagocytosis regulation in myeloid cell population such as neutrophils and macrophages under the hypothesis that αDβ2 serves as a negative regulator of αMβ2 and αXβ2. In Aim 2, we will determine the interaction between αDβ2 and complements in vivo using two disease models mimicking sepsis. One will be polymicrobial abdominal sepsis model, one of the most commonly used sepsis models. In this model, we will examine the role of αDβ2 in lung injury caused by extrapulmonary sepsis. Another model will be Pseudomonas aeruginosa pneumonia model, given it is associated with a high mortality. In this model, we will examine the role of the role of αDβ2 in direct lung injury. We will also examine the expression pattern of αDβ2 and complement fragments in the existing discarded lung specimens biopsied from patients with the diagnosis of acute hypoxemic lung injury and sepsis. Upon the completion of the proposed study, we will solidify that αDβ2 is a novel complement receptor in vitro and in vivo. Our future plan is to screen αDβ2 antagonists and test them in vivo to further delineate if αDβ2 would serve as a potential target for acute lung injury. The ultimate goal of the project is to intervene αDβ2 in patients with acute lung injury in sepsis.