Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2025-07

Most patients with Atopic dermatitis (AD) have a skin barrier defect. This allows cutaneous sensitization to antigens that leads to allergic skin inflammation (ASI). In a minority of patients, AD arises from an immune deficiency. Patients with DOCK8 deficiency and WASP deficiency have a normal skin barrier, but impaired Treg cell function. Dock8-/- mice developed exaggerated ASI following application of ovalbumin (OVA) to tape stripped skin. Adoptive transfer experiments demonstrate that defective Tregs underlie their exaggerated ASI. We propose to use the DOCK8 deficiency model to study of the pathways that drive AD independently of skin barrier defects and determine the mechanisms by which Tregs restrain ASI. DOCK8 activates the GTPase CDC42. CDC42 activates the Ser/Thr kinase PAK2 as well as WASP, which drives actin filaments branching. Preliminary data show that AD is more severe in DOCK8- deficient than WASP-deficient patients. Similarly, ASI is more severe in Dock8-/- than in Wasp-/- mice. Tregs in OVA sensitized skin and their FOXP3 expression are decreased in Dock8-/-, but not Wasp-/-, mice. DOCK8 deficient induced Tregs (iTregs) have reduced stability in vitro particularly in the presence of IL-4. Tregs from Dock8-/, but not Wasp-/-, mice, as well as WT Tregs treated with PAK inhibitors, have decreased levels of pY-STAT5 following IL-2 stimulation due to accelerated dephosphorylation. This suggests that the DOCK8-PAK2 axis, but not the DOCK8-WASP axis, is essential to maintain pY-STAT5 levels in Tregs AlphaFold Multimer modeling predicts a direct interaction between STAT5B and the tyrosine phosphatase SHP2, abolished by mimicking PAK2-mediated serine phosphorylation of STAT5B. We will test the overall hypothesis that in DOCK8 deficiency, loss of WASP activation, which disrupts the cortical branched actin cytoskeleton and Treg cell interaction with target cells, and loss of PAK2 activation, which reduces the levels of IL-2 driven pY-STAT5 and Treg stability, synergize to promote ASI. We will test the following hypotheses 1) Reduced IL-2 driven STAT5 phosphorylation in DOCK8 deficient Tregs impairs their stability in a Th2 milieu and ability to restrain ASI. 2) Defective WASP activation and defective PAK activation independently impair Treg ability to suppress ASI in DOCK8 deficiency. 3) Serine phosphorylation of pY-STAT5 by IL-2 driven DOCK8 mediated activation of PAK2 promotes Treg stability by inhibiting the docking of SHP2 to STAT5. Many patients with AD remain resistant to current therapies. Understanding the roles of DOCK8 and associated proteins as well as the regulation of the STAT5 signaling axis in Treg function and the molecular pathogenesis of AD could inform novel therapies in AD, particularly those designed to boost Treg cells.

NIH Research Projects · FY 2025 · 2025-07

Project Summary The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The nucleocapsid (N) protein of SARS-CoV-2 plays several functionally critical roles in the life cycle of the virus, similar to those in other coronaviruses. It assembles with the genomic RNA into an RNA- protein complex, which is packaged into virions. The protein also forms a replication-transcriptional complex with the RNA synthesis machinery to create replicating organelles for efficient transcription and replication. It protects viral double-stranded RNA (dsRNA), which would otherwise be degraded by the RNA interference (RNAi)-based antiviral immune defense mechanism in the host cells. N protein adopts a modular architecture with two well- folded domains, including N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three intrinsically disordered regions. Crystal structures of the NTD and CTD have been determined, but high- resolution information of the full-length N protein, in particular, in complex with RNA is still lacking. We hypothesize that intrinsically disordered regions of the N protein from SARS-CoV-2 adopt defined structures when bound to RNA that are critical for viral assembly and replication. In this project, we plan to determine the high-resolution structure of the full-length N protein in complex with the viral RNA by cryogenic electron microscopy (cryo-EM). The goal is to visualize detailed structural features of the full-length N protein in the context of RNA to advance our understanding of its function and to inform development of intervention strategies. Our specific aim is to obtain a high-resolution structure of intact SARS-CoV-2 N protein in complex of RNA.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT Although a cause-and-effect relationship between the microbiome and obesity—and its related metabolic diseases—has been identified for almost 20 years, there are still no obesity therapies targeting or utilizing the microbiome. This is due, in part, to a lack of mechanistic understanding. The long-term goal is to delineate the mechanisms commensal organisms use to benefit host metabolic regulation and to develop new therapeutics targeting those pathways. We and other groups have identified Blautia species as candidate commensals that supports metabolic and mucosal health. Our prior studies of human subjects demonstrated an association between low Blautia levels and obesity, loss of control eating, and greater fat ingestion in a buffet-meal setting. The overall objective of this proposal is to determine how Blautia, and its metabolites activate signaling events within the gut epithelium. Our central hypothesis, based on our prior studies, is that acyl amines synthesized by Blautia species activate enteroendocrine cell (EEC) production of GLP-1 and PYY, which benefit host metabolism, and GLP-2, which benefits the structure and function of the gut mucosa. The rationale for this project is that a fundamental understanding of how Blautia benefits host metabolism and epithelial health will allow us to harness Blautia, its metabolites and other beneficial commensals to treat obesity and related disorders. The central hypothesis will be tested via two specific aims: 1.) Identify the synthetic processes and microbial products that augment EEC activation by Blautia; and 2.) Delineate the mucosal and metabolic effects of Blautia acyl amine production in vivo. Under the first aim, we will use a CRISPRi system to perform genome editing in Blautia wexlerae—suppressing the expression of native genes in this key commensal organism has not yet been done. To define the bacterium’s genetic requirements for synthesizing EEC-activating acyl amines—as measured by LC-MS—we will knock down candidate acyl transferase genes. We will also screen a library of microbial metabolites for molecules that synergize with acyl amines to boost EEC activation. This will be done high- throughput in a cell culture model of EECs and validated in EEC-containing human organoids. The second aim will delineate the physiologic consequences of B. wexlerae acyl amine production in mice. Here, we will utilize our newly established model of B. wexlerae colonization in germ-free mice, which we have shown increases acyl amine production in the gut by 5-10-fold. We will determine the effects of B. wexlerae on glycemic control, insulin sensitivity, food intake, weight and epithelial proliferation. The work is technically innovative in that it will uniquely combine new bacteriological methods, microbial metabolite analyses, and high-throughput screening of host phenotypes to maximize the likelihood of discovering new host-microbe interactions. The proposed studies are scientifically innovative because they will test a series of mechanism-based hypotheses that focus on advancing our understanding of how Blautia influences the host. Ultimately, this more precise understanding will open new horizons for utilizing Blautia and other microbes for clinical applications.

NIH Research Projects · FY 2025 · 2025-07

Abstract Development of effective adaptive immunity following infection and vaccination requires recognition of antigens and secretion of antibodies by B cells. T and B cells are the two major types of lymphocytes in the immune system. T cells can sense MHC-bound antigens by the T cell receptors (TCRs) and B cells can recognize free, unprocessed antigens by the B cell receptors (BCRs). Despite the critical importance of BCRs, how BCRs are assembled and regulated remain unclear. In this application, we propose to overcome certain technical obstacles in our pursuit of BCR assembly and regulation by engineering appropriate cell lines and using the native membrane environment for complex formation.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY Tracheobronchomalacia is a rare disease involving excessive airway collapse due to weakening of the airway walls. It is diagnosed in 13% of adults and 30% of children who undergo bronchoscopy for respiratory distress. The disease is characterized by the percentage collapse and collapse-morphology in the weakened airway segment. The collapse-morphology can vary depending on the relative anatomical region(s) of tissue weakening. Mild to moderate Tracheobronchomalacia can be treated non-invasively via positive pressure ventilation. Positive pressure ventilation, while noninvasive, requires the patient to be attached continuously to a pressure generating device for effective treatment. Severe Tracheobronchomalacia usually requires surgical intervention that involves attaching supportive materials outside the trachea that thwarts regional collapse. Surgical interventions are complex and require long post-operative recovery. Airway stents provide a simple, economical and minimally invasive treatment mode. Unfortunately, available stents are avoided or only implanted for monitored short periods due to stent-associated complications of excessive granulation tissue formation and mucus plugging resulting from the foreign body reaction against the implanted stent. Stent physical characteristics play a significant role in inducing excessive granulation tissue formation and mucostasis. The stent physical characteristics which include the stent material, geometry and size, need to be optimized at the design and development stage to minimize the physical impact during implantation. Existing benchtop and animal models can neither reliably generate the grades and morphologies of Tracheobronchomalacia, nor do they possess the granularity needed to perform design optimization. In this proposal we will establish new research tools and protocols that will substantially advance the stent design process and propel the development of viable airway stents. We present the first benchtop platform for rapid stent testing capable of generating specific grades and morphologies of Tracheobronchomalacia and accurately simulate dynamic airway collapse. Our system evaluates stent effectiveness in the clinically relevant parameter of airway lumen cross-sectional area. Furthermore, the platform can map the device-to-airway contact force distribution during dynamic airway collapse. In aim 1, we will first establish the protocols for reliably generating the clinical grades of Tracheobronchomalacia. Next, we will evaluate the performance of helical and axial stent designs of varying geometric feature values. This will determine the geometric parameter for each stent design for treating Tracheobronchomalacia using minimum material and contact forces. In aim 2, we will study the effect of stent geometry and oversizing on the spatial distribution and severity of granulation tissue formation and mucostasis. At the conclusion of experiments from both aims, we will attempt to establish a relationship between the spatial characteristics of granulation tissue recorded from the animal study and the benchtop contact forces. This will enable assessing stent designs prone to granulation tissue complications before testing stents in animals.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Immunotherapy has revolutionized cancer therapy, but most solid cancers do not respond. Thus, immunotherapy needs to be improved. Developing cancers undergo tumor editing to evade immune surveillance. Because most editing occurs before cancers are detected, surprisingly little is known about what genes are edited. Studies of immune editing have examined candidate genes in antigen processing and presentation but have largely ignored genes active in innate immunity, which plays a critical role in self:non-self or normal cell:tumor discrimination and in inducing and amplifying adaptive immunity. Genome-wide studies of tumor immune editing have not previously been done. Our first goal is to identify genome-wide changes in gene expression early in tumorigenesis in a variety of cancers by taking advantage of genetically engineered mouse models (GEMM) of cancer to capture and compare early vs late spontaneously arising tumors, using scRNA-seq, spatial transcriptomics and multiplexed flow cytometry and immunofluorescence microscopy to identify the genes/pathways whose expression is suppressed during early tumorigenesis. We will also identify changes in gene expression in the tumor and metastatic niche in the transition from dormancy at tissue sites of colonization to macrometastases. Comparing both tumor and infiltrating immune cell gene expression, cell subset abundance, cell-cell communications and spatial organization will provide a detailed picture of how tumor editing leads to immune exhaustion and evasion of immune surveillance and identify the genes, pathways, and processes that are suppressed. Preliminary data suggest that early tumor editing is highly focused on genes involved in both adaptive and innate immunity, including interferon, inflammasome/pyroptosis, necroptosis, and inflammatory cytokine pathways, which are epigenetically repressed. The dominant genes/pathways edited in GEMM will also be examined in human cancers, comparing gene expression and immune cell infiltration in carcinoma in situ with advanced carcinomas. Reversing immune editing could potentially convert immunologically cold tumors into immune responsive tumors. Epigenetic repression of gene expression by DNA hypermethylation of promoters is a major mechanism of gene repression that is prominent during tumor editing. Our next goal is to determine how much DNA hypermethylation and other epigenetic changes contributes to tumor editing and whether inhibiting DNA methylation or other epigenetic modifications that maintain heterochromatin can reignite protective immunity or induce responsiveness to checkpoint inhibition. Another goal is to investigate in more detail the role of suppression of specific innate immune pathways in the tumor and in specific immune cell subtypes in tumor resistance to immune control. We will determine whether activating specific innate immune pathways, repressed during tumor editing, using small molecule activators or inhibitors or tumor-targeted RNA-based gene knockdown, knockout or mRNA expression, can induce immune responsiveness in immunologically cold tumors. These studies will identify novel drug targets that could potentially reverse editing and restore tumor immunity.

NIH Research Projects · FY 2025 · 2025-07

Project Summary/Abstract Renal transplantation is widely recognized as the treatment of choice for children with end stage renal disease (ESRD). The life expectancy benefit is significant and a functioning renal transplant enables children to grow well, develop almost normally and improve their school educational performance levels. However, current data indicate that virtually all grafts in pediatric recipients will eventually fail due to chronic allograft dysfunction, and as such, the goal of preserving long-term allograft function is the key area for future progress. Furthermore, registry data indicate that opportunistic infections are now the most common cause for hospitalization and death in the pediatric population. Little is known about pathogen-specific protective immunity in pediatric recipients who are exposed to multiple novel infectious agents throughout the post-transplant period in the absence of Tmemory. Our approach in this trial is based on the concept that successful preservation of long-term allograft function requires an immunosuppressive regimen that targets donor specific alloantibody (DSA) production while preserving pathogen-specific immunity. We also propose that pediatric recipients require precision tools to monitor, identify and prevent silent subclinical intragraft inflammation/rejection, which is common at early times in the post transplant period. Based on a recent pilot study using de novo Belatacept therapy in combination with an mTOR inhibitor (mTORi) in pediatric recipients, we will test the hypothesis that early introduction of a Belatacept/sirolimus maintenance immunosuppressive regimen is safe and efficacious in children to augment immunoregulation, prevent DSA production and enhance long-term allograft function. EBV seropositive primary renal transplant recipients, aged between 6 and 21 yrs, from eleven experienced pediatric clinical centers will be randomized to receive induction therapy with anti-thymocyte globulin and either Belatacept therapy in combination with sirolimus or remain on standard immunosuppression therapy using tacrolimus and mycophenolate mofetil. Primary endpoint analysis includes de novo DSA development and assessment of allograft function after 36 months of follow up. Associated studies include surveillance monitoring using a novel automated point-of-care urine biomarker assay, and in-depth mechanistic studies on the cellular basis for pathogen-specific immunity and evaluation of functional antibody responses to vaccine. Extensive mechanistic studies will also be performed to assess the impact of Belatacept/mTORi on cellular and humoral alloimmunity and the further development of urinary biomarkers to differentiate subclinical rejection from infection. There are significant unmet clinical needs in pediatric recipients who have unique pathogen-specific and alloimmune responses following transplantation. Overall, the relevance of this proposal is that it builds upon previous trials to test if a novel agent (Belatacept) targets allograft dysfunction; in-depth mechanistic monitoring will allow for the prediction of patient course, and our findings will be applicable to recipients of other solid organ transplants.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY This R21 proposal outlines a collaborative research initiative aimed at elucidating how human microglia-like cells (iMGLs) leverage mRNA translational control mechanisms to respond to environmental cues. Human microglia must leverage significant protein diversity to mount immune responses and shape behavior in response to external stimuli, but the role of mRNA translation in microglial function is poorly understood. Further, mRNA translation is far more complex than previously appreciated, as translation occurs within regions of the genome annotated as non-coding – so-called non-canonical open reading frames (ncORFs). ncORFs include small open reading frames (sORFs) less than 300 nucleotides that encode microproteins less than 100 amino acids. Microproteins have been implicated as critical biological effectors of the immune response, but the role of such microproteins in human microglia is unknown. We hypothesize that microproteins may be an important and indispensable component of human microglia function and the response to environmental stimuli. As such, we will define the translational landscape – the “translatome” – of iMGLs using ribosome profiling, which allows us to map translation at single nucleotide resolution. We will define open reading frames in iMGLs, and identify both novel sORFs and putative microproteins. We will similarly use ribosome profiling to identify translational responses to external stimuli, such as myelin debris and amyloid. We will confirm our findings using size-selected proteomics to validate stimuli-dependent human microglial microproteins. The knowledge derived from this research plan will not only enhance our understanding of the mechanisms through which human microglia respond to the local microenvironment, but will also identify candidate microproteins that can be functionally interrogated as critical effectors of human microglia function.

NIH Research Projects · FY 2025 · 2025-07

Abstract Genome-wide association studies have unveiled millions of single nucleotide polymorphisms (SNPs) associated with common diseases. Yet, the functional mechanism of most SNPs in these diseases still needs to be discovered. My recent studies suggested that hundreds of thousands of SNPs strongly associate with ligand gene expression in the GTEx dataset, suggesting the impacts of these SNPs on many cell types in a tissue niche via ligand-receptor interactions. However, the lack of robust technology that adequately considers the context of tissue niches constrains the systematic characterization of SNP functions in health and diseases. This project, empowered by functional genomics approaches, aims to address this challenge. I will develop robust computational technologies for single-cell functional genomics, allowing comprehensive identification of SNPs regulating tissue niches and elucidating their regulatory roles in health and diseases. In Aim 1 (K99 phase), I will develop computational frameworks with deep learning models to decode the epigenetics response of a cell to intercellular communications with other cells. The performance of my algorithm will be systematically validated by RNA-seq and ATAC-seq profiles under control and ligand treatment conditions. In Aim 2 (K99 and R00 phase), I will develop robust statistical methods to identify the impact of SNPs on the tissue niches via ligand-receptor communications. The effects of SNPs on ligand expression will be elucidated by integrating the SNPs with genome-wide transcription factor binding sites and chromatin status. In Aim 3 (R00 phase), I will employ robust computational technologies to identify clinically relevant SNPs that regulate tissue niches via intercellular ligand-receptor communications. I will also validate the SNP functions by prime editing, focusing on endothelial cells and adipocytes in adipose tissue, which are tightly associated with many diseases, including obesity, diabetes and complications, and cardiovascular diseases. This study will lay a solid foundation for future research as an independent scientist focusing on genetic diseases. My extensive experience in epigenetics, intercellular communication modeling, and single- cell data analysis positions me uniquely to execute this proposal. The project will be supervised by an interdisciplinary team, including Dr. Kaifu Chen for bioinformatics, Dr. Yu-Hua Tseng for metabolic diseases, and five collaborators with expertise in deep learning, big data integration, statistical genetics, and prime editing. The training will also include professional skills such as scientific writing, communication, and mentoring. The outstanding research environment at Boston Children’s Hospital and Harvard Medical School will further enhance the execution of the proposed study. Completing this training and proposal during the K99 phase will well prepare me for a future role as an independent researcher in translational genetics and genomics research, ensuring a seamless transition into the R00 phase.

NIH Research Projects · FY 2025 · 2025-07

Cells respond to nutrient availability and adjust their functions and performance to ensure survival. This adaptation requires rapid, nutrient-responsive decision-making and prioritization of essential cellular processes. For example, we recently showed that depletion of the essential vitamin folate in erythroid cells results in premature differentiation that improves cell survival on the expense of proliferation rate. Our work, that showed that erythroid cell lines as well as primary murine erythroid progenitors prematurely differentiate under folate deficiency, revealed a potential etiology for folate deficiency-induced anemia. To further reveal the cellular response to folate deprivation and other perturbations of the one-carbon (1C) metabolism pathway in erythroid cells, we performed a genome wide CRISPR screen and RNAseq in folate deprived erythroid cells. Our CRISPR screen revealed all five genes of the post translational modification (PTM) UFMylation pathway as top hits, and the RNAseq data showed differential gene expression induction following folate deprivation through key signaling pathways including mTOR. Although recent literature defines the crucial role of UFMylation in mediating ER stress and homeostasis of translation, the specific triggers and consequences of UFMylation remain largely unknown, warranting further exploration of conditions which trigger this PTM. We hypothesize that regulation of UFMylation maintains cellular fitness during folate depletion. To address this hypothesis, we will validate our CRISPR screen results, characterize the role of UFMylation in cellular adaptation to 1C disruption, identify UFMylation targets, and explore the role of UFMylation in nutritional status-dependent ribosomal regulation. We further hypothesize that 1C perturbation induces a signaling cascade to promote cellular fitness. To address this hypothesis, we will pharmacologically and genetically target pathways known to be differentially activated during nutrient sensing and probe the role of these pathways in cell function regulation during 1C metabolism disruption. Completing these aims will mark a new role of UFMylation in a signaling pathway activated in response to metabolic perturbation and define a new cellular adaptation mechanism in response to 1C metabolism disruption. As a Ph.D. student in the Kanarek lab I have the commitment and support of my sponsors Dr. Kanarek and Dr. Toker, as well as access to the expertise of leaders in the metabolism and cell signaling fields, and to state-of-the-art facilities and equipment. Leveraging these resources, I plan to achieve the following over the next three years: (1) Gain proficiency in molecular and cellular biology techniques for rigorous scientific research in metabolism; (2) Deepen my knowledge of nutrient sensing and erythroid biology, contributing to basic biomedical research; (3) Enhance communication skills through writing, presenting, and networking to build a mentorship and peer network to support my future in biomedical research; (4) Develop leadership skills through mentoring students at various academic stages, drawing on my own experiences.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Animal sense light for perception as well as the regulation of physiology and behavior. While cells in the retina that transmit photic information to the brain to support these latter ‘non image’ functions have been identified (the intrinsically photosensitive retinal ganglion cells: ipRGCs), it remains unclear what information they convey to the brain and how it is processed to support particular tasks. I propose to relate molecular and cellular mechanisms of ipRGCs to their signals within the brain, their influence on postsynaptic cells, and their drive of behavior. To enable these investigations, I established a paradigm for imaging visually-evoked Ca2+ dynamics and neurotransmitter release in the Olivary Pretectal Nucleus (OPN), the retinorecipient region that drives the pupillary light reflex (PLR), while simultaneously monitoring pupillary constriction and other mouse behaviors. Using this paradigm, I will investigate how melanopsin’s presence within the distal axon and the release of specific neurotransmitters from ipRGC axon terminals shapes the properties of the PLR (Aim 1). Adjustments in pupil size are likely to diverge from ipRGC signals in several important respects; for instance, due to arousal. To test this hypothesis, I will measure photic signals in postsynaptic cells of the OPN and assess their regulation by internal states (Aim 2). Together, these experiments will define how mechanisms in ipRGCs determine the photic information available in the brain and how downstream cells transform this information to meet the requirements of a visual reflex.

NIH Research Projects · FY 2025 · 2025-06

ABSTRACT RUNX1 Familial Platelet Disorder with Propensity to Develop Myeloid Malignancy (RUNX1-FPD/MM) is a rare genetic disorder due to germline heterozygous loss-of-function mutations in the gene encoding the key hematopoietic transcription factor RUNX1. Affected individuals have thrombocytopenia, platelet dysfunction, autoinflammatory symptoms, early clonal hematopoiesis (CH), and a high risk of developing myelodysplastic syndrome (MDS) and leukemia (~35-45% lifetime risk with median onset age of 33 years). Unfortunately, the mechanisms that predispose to early CH and hematologic malignancy remain poorly understood. Moreover, no interventions have been identified to reduce the risk of leukemia development. A major obstacle in the field has been the lack of practical and faithful experimental models to study RUNX1-FPD/MM and do high-throughput drug screens. Mice are not as sensitive to RUNX1 haploinsufficiency as humans, and mouse models do not develop leukemia. Non-human primate models show greater phenotypic similarity with human disease but are costly and not amenable to high throughput analysis. CRISPR-gene editing of human primary CD34+ cell has been attempted to knock-in heterozygous patient mutations, but challenges remain in avoiding alteration of the wild type allele. shRNA models have been generated to knock-down RUNX1 to ~50% levels. However, this represents the cell population average and not necessarily levels in individual cells. Availability of primary patient samples is limited by the rarity of the disease, and xenotransplantation of these samples to make long-lived models has been challenging. Human induced pluripotent stem cell (hIPSC) lines have been valuable but have only been examined in 2D cultures whose conditions are tailored to specific lineages. 3D organoid culture systems have been developed for many solid organs and have served as valuable experimental models since they recapitulate the complex microenvironment including stromal elements, contain more physiologic cytokine/chemokine levels, and are amenable to drug screening/testing. Very recently, 3D organoid culture systems have been established for the human bone marrow (BM). The objective of this 1-year pilot proposal is to develop BM organoid system to study RUNX1-FPD/MM. This will involve generating BM organoids from two hIPSC lines derived from patients with RUNX1-FPD (a splice-site acceptor mutation and gene deletion) along with isogenic gene corrected controls. Comparative analysis will be performed to determine the extent to which the BM organoids recapitulate RUNX1-FPD/MM phenotype. Proof-of-principle experiments will be performed to assess their utility in drug testing. Lastly, chimeric organoids containing small clones with an additional somatic mutation in BCOR, a common occurrence during CH in RUNX1-FPD/MM patients, will be generated and used to determine if the organoids can be used to study clonal dynamics in this disorder. The successful outcome of this study will be the development of a new tractable experimental system to study human RUNX1-FPD/MM in a more physiologic setting and one that is amenable to future drug development.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract The blood system deteriorates with age with the function of hematopoietic stem and progenitor cells (HSPCs), specifically, declining. Frequently with age, driver mutations cause a smaller number of individual HSPCs, or clones, to contribute disproportionally to differentiated blood progeny leading to hematopoietic clonal dominance and increased rates of hematological malignancy and cardiovascular disease. This accumulation of somatic mutations is common among many aged tissues. Alteration in the metabolic state of aged HSPCs leading to impaired function is well documented however, metabolic heterogeneity among aged HSPCs has only recently been appreciated. Whether the metabolic heterogeneity of aged HSPCs is clone specific is unknown. The overarching hypothesis of this proposal is that hematopoietic clonal dominance in aging is caused by a divergent metabolic state and correction of this divergent metabolic state would restore hematopoietic function and decrease disease acquisition in older individuals. I have demonstrated that HSPC clonal imbalance occurs in zebrafish in the absence of known driver mutations, similar to humans, using a genetic barcoding strategy. Aim 1 of this proposal pairs single-cell metabolomics and genetic cellular barcoding to identify metabolic alterations in dominant HSPCs in the absence of known driver mutations during the K99 phase of this award. The second half of Aim 1, to be accomplished during the R00 phase is to interrogate the metabolic pathways that differentiate dominant from non-dominant HSPCs to identify therapeutic targets to prevent hematopoietic clonal imbalance with aging. Using a method of combining mosaic editing of genes frequently edited in HSPCs of older humans and colorimetric barcoding I have demonstrated that the choline pathway is perturbed in dominant HSPCs. Pharmacological inhibition of choline lipid species production was sufficient to reduce mutant HSPC clonal expansion. During the K99 phase of this award, I will determine the mechanism of this inhibitor to reduce clonal expansion by performing intracellular methylome profiling. In the R00 phase I will test the relevance of this pharmacological intervention in human HSPCs in a xenotransplantation setting. By understanding the metabolic state of dominant HSPC clones during onset and progression in non-mutant and mutant contexts, interventions can be designed and applied to prevent age-related declines of the hematopoietic system. I have developed training goals that will center on 1) expanding my single-cell analysis skillset, 2) developing my intracellular methylation experiment capabilities, 3) mastering human HSPC culture, 4) enhancing my scientific communication, 5) growing my biology of aging footprint, and 6) growing my mentorship and leadership skills. The research and training plan designed for this K99/R00 award will support my long-term career goal to become an independent investigator at a research-intensive institution studying how metabolism underlies the hematopoietic dysfunction seen in individuals as they age.

NIH Research Projects · FY 2026 · 2025-06

Lymphatics are vital for fluid recycling, immune surveillance, and lipid uptake. Abnormal lymphatics lead to lymphedema, metabolic changes, inflammation, and infections and contribute to the pathophysiology of many chronic illnesses. In response to the NIH mission (NOT-HL-23-099) promoting research on the biology of the lymphatic system to advance discovery of novel drugs for lymphatic diseases, we have identified Neuropilin-2 (NRP2) as a pivotal manipulator of lymphatic function. NRP2 is a unique transmembrane receptor in lymphatic endothelial cells (LEC) capable of signaling through disparate and opposing ligands. Our central hypothesis is that NRP2 acts as a dynamic rheostat by toggling between its ligands and downstream signaling pathways to either promote or restrain fluid and lipid uptake into lymphatic vessels. Using innovative biochemical, genomic, and pharmaceutical strategies in new animal models, we will test our hypothesis by defining, modulating, and controlling NRP2 ligand-dependent signaling in lymphatics. NRP2 binds VEGFC in complex with VEGFR3 to increase lymphatic function. Our compelling new data reveals that NRP2 is necessary for VEGFC-induced activation of VEGFR3, adult lymphangiogenesis, and proper lymphatic drainage functions in vivo. On the other hand, the inimitable NRP2 receptor can also bind SEMA3F in complex with PlexinA1 to inhibit lymphatic functions such as absorption and immune cell trafficking. In Aim 1, we will alter Nrp2 expression and ligand-dependent functions in the skin to sustain proper fluid drainage and ameliorate lymphedema. In preliminary studies, systemic neutralization of the endogenous Sema3F ligand in adult wildtype mice increases VEGFC binding to NRP2 and accelerates lymphangiogenesis following injury to restore lymphatic drainage and alleviate secondary lymphedema. However, increasing lymphatic drainage is not always advantageous. In the GI tract, specific lymphatic capillaries called lacteals facilitate lipid absorption. In Aim 2, we will temper Nrp2 expression and ligand-dependent functions in the intestine to block lipid influx and prevent diet-induced obesity. To date, the role of NRP2 in lacteal function, lipid absorption, or lipid transport has not been investigated, especially in relation to adult weight gain or digestive inflammatory disease. In accordance, global or conditional Nrp2 loss in lacteals correlates with lower basal body weight and reduced weight gain with high fat diet compared to controls. Specifically, we will establish the therapeutic potential of inhibiting differential ligand-binding domains in Nrp2 using systemic antibodies or exogenous competitive proteins in preclinical models to either promote VEGFC/NRP2 signaling to alleviate lymphedema or to limit VEGFC/NRP2 signaling in lacteals to hinder lipid absorption to prevent obesity or metabolic disease. Overall, our proposal is significant and warrants careful consideration as it will reveal distinct lymphatic regulatory mechanisms and use inventive approaches to curtail lymphedema or obesity and their comorbidities. Since humanized versions of these drugs already exist, our results may quickly translate into viable and effective treatments to improve lymphatic diseases.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY/ABSTRACT Younger adults aged 18-30 years are using cannabis frequently and experiencing the dysfunction and distress of cannabis use disorder (CUD) at unprecedented rates. Facing numerous barriers to treatment and lack of diverse representation in research, younger adults with CUD need expanded opportunities to access interventions and to participate in clinical trials. Remote implementation offers a promising means to addressing these critical gaps in care and research. However, valid remote procedures for biologic assessment of a key outcome, reduction in cannabis use days, have not been established. We propose an approach to assessing cannabis use and non-use remotely via oral fluid testing, which can be directly observed, is easily and quickly accomplished with minimal training, has a short detection window (≤24-72 hours after cannabis use) and is less prone to alteration or falsification vs. urine and other biologic specimens. Using our experience with studying cannabis use in younger adults, telehealth preferences, fully remote interventions, and collecting and testing biologic specimens, we propose to evaluate a community sample of 200 individuals aged 18-30 years with past- 30-day cannabis use ≥1x/week. Participants will videorecord of daily oral fluid delta-9-tetrahydrocannabinol (delta-9-THC) testing on 6 consecutive days and live observed testing on the 7th day. We will assess completion of daily oral fluid testing and its performance in identifying cannabis use/non-use intervals, vs. self-report on timeline follow-back calendar interview. To do so, we will analyze performance of oral fluid test results over each period of 3 consecutive days, corresponding to a cannabis use/non-use interval of 4 days, which, based on our research, is a meaningful period of non-use following treatment in individuals who use cannabis frequently (5 possible 3-day testing intervals/participant, resulting in ≤1,000 intervals). We will also contrast oral fluid results with urine results to demonstrate the relative utility of oral fluid testing for establishing use/non-use over short intervals. The specific aims are 1) to establish the feasibility of remote testing for oral fluid delta-9-THC using video recordings on a personal device and live observation via videoconference call. We will used a mixed methods approach, including staff observation, participant survey, and participant interview to evaluate implementation, completion, acceptability, burden, and technical aspects of the oral fluid testing procedures; 2A) to determine agreement between oral fluid test results over 3 consecutive days (any positive = use, all negative = non-use) and self-reported cannabis use/non-use over the corresponding days on TLFB (4 days, 3 plus 1 day earlier); and 2B) to demonstrate agreement between results on a single oral fluid test and a single urine test obtained contemporaneously in individuals who have used cannabis ≥1x/week in the past 30 days. Identifying an approach to biologic assessment of cannabis non-use days that can be accomplished under video observation will substantially add to the rigor of remote clinical trials for CUD. Younger adults will particularly benefit from the enhanced evidence on remote interventions for CUD.

NIH Research Projects · FY 2026 · 2025-05

Project Summary/Abstract Cardiac arrest affects ~700,000 patients annually in the US, with a survival rate between 10% and 30%, and survivors often suffering from neurologic injury. Despite advances in resuscitation, maintaining adequate oxygenation during early resuscitation remains a significant challenge, irrespective of the cause of cardiac arrest. In asphyxia cardiac arrest (ACA), severe hypoxemia is often refractory to conventional inventions such as mechanical ventilation. In out-of-hospital cardiac arrest (OHCA), where none-asphyxia cardiac cause is common, existing ventilation methods such as bag-valve-mask and advanced airway placement are often ineffective to insufflate the lung during early resuscitation. To address this problem, we have developed a new gas carrier that allows safe intravenous injection of oxygen (IVO2), based on pH-responsive polymeric microbubbles (PMBs). PMBs feature a thin polymer shell encasing an oxygen gas core (~5 µm), providing high oxygen content (50% vol/vol) and storage stability. Designed to dissolve immediately in physiological media via a pH trigger, PMBs deliver a high volume of oxygen without requiring a diffusion sink (e.g., hyperoxic blood), thereby minimizing risks of embolism or vascular obstruction. The dissolved PMB shells revert to soluble, excretable components, reducing long-term side effects. In a realistic swine model of ACA, we demonstrated that administering very small doses of oxygen via PMBs effectively alleviates severe hypoxemia and significantly improves neurologically intact survival. We propose IVO2 may be developed as a new oxygen therapy for early resuscitation of cardiac arrest. The project has 3 specific aims. In Aim-1, we will optimize the molecular structures of PMBs shell polymer to accelerate their clearance. Tissue deposition presents a main risk for material toxicity, this study will allow minimize long-term adverse effects of IVO2 gas carriers. In Aim-2, building on our preliminary data, we will further interrogate how dose and treatment timing may influence physiologic response and mitochondrial health in rodent ACA models. This knowledge is essential to understanding the therapeutic efficacy and limits of IVO2 as a transitional treatment for ACA. In Aim-3, we will investigate whether IVO2 can replace and outperform existing ventilation methods in the early resuscitation of a ventricular fibrillation OHCA swine model to improve neurologically intact survival. By replacing early ventilation efforts, IVO2 may rapidly restore normoxia in a more reliable way and offer various physiologic benefits e.g., reducing pulmonary vascular resistance, allowing care provides to focus on chemist compressions and medication provision. All these factors contribute to earlier ROCS and neurologically intact survival. If success, the proposed therapy may transform the current paradigm for the early cardiac arrest resuscitation.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Therapeutic genome editing holds immense promise for treating genetic diseases, but off-target effects remain a critical safety concern, particularly when considering human genetic diversity. Most existing methods for identifying off-target sites do not account for genetic variants, potentially missing important risks for individual patients. We have developed CRISPRme, a computational tool that nominates off-target sites based on genetic variants, and ABSOLVE-seq, an experimental method to verify these sites in relevant cellular contexts. This proposal aims to optimize and integrate these approaches into a comprehensive pipeline for assessing variant- associated off-target risks in genome editing therapeutics to support IND applications. Aim 1 focuses on optimizing CRISPRme by incorporating larger variant datasets, integrating advanced prediction tools, and developing standardized protocols for off-target nomination. We will extend the search space to include genetic variants from the All of Us Research Program and implement a risk assessment framework that considers functional annotations and cancer-related somatic mutations. Aim 2 will optimize the ABSOLVE-seq experimental methodology and analysis pipeline. We will improve vector design, plasmid fidelity, and transduction protocols to enhance sensitivity and scalability. The analysis pipeline will be refined to provide robust statistical modeling of editing outcomes across various editing modalities, including nucleases, base editors, and prime editors. Throughout the project, we will collaborate with SCGE Consortium members to generate benchmark datasets for lead gRNAs being explored for clinical development. We will work to extend the methods to diverse cell types and tissues relevant to in vivo gene therapy of the liver, eye, and central nervous system. The deliverables will include standardized software modules, SOPs, and comprehensive data packages suitable for regulatory submissions. Our user-friendly software tools and interactive reports will facilitate the translation of genome editing therapeutics, meeting the highest standards required for supporting IND-submissions. This work will significantly advance our ability to assess off-target risks in genome editing therapies, accounting for genetic diversity and potentially accelerating the clinical translation of these promising treatments. By contributing valuable tools, datasets, and expertise to the SCGE Consortium and the broader gene editing community, we aim to foster collaborative progress in developing safe and effective genome editing therapeutics suitable for diverse patient populations.

NIH Research Projects · FY 2026 · 2025-05

Project Summary RNA-guided DNA sequence recognition by CRISPR/Cas9 has been exploited to develop an array of genome and epigenome editors, including base editors, primer editors, CRISPR-activators (CRISPR-a) and CRISPR-interference (CRISPR-i). These editors consist of either nuclease dead (dCas9) or DNA-nickase (nCas9) variants of Cas9 fused at N and C termini to “accessory protein” which contain the editor-specific activity. The enzymatic activities of these accessory proteins are being evolved very rapidly to increase the accuracy, efficiency, and types of editing possible. Mice harboring these editors as transgenes are invaluable research tools. However, generating mice containing transgenes that express the latest suite of Cas9 editors is challenging, given their rapid rate of change and ever-increasing number. These editors can be delivered using adeno-associated virus (AAV) vectors, but due to packaging limits of AAV most require a dual AAV strategy involving intein-mediated protein splicing, which hampers editing efficiency and homogeneity. Here we propose to generate mouse lines that express the core dCas9 or nCas9 RNA-guided DNA sequence recognition platforms, functionalized at N- and C-termini with protein ligation tags. The accessory proteins and guide RNAs will be introduced using single AAV vectors. This combination will achieve higher level and more homogeneous editing than possible with AAV-only strategies, while retaining flexibility and the ability to exploit the latest advances in editor enzyme engineering. In Aim 1, we will create Cre-triggered mouse/AAV systems for CRISPR-i/a. We will create mice with Cre-activated expression of dCas9 with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered N- or C-terminal epigenetic editors to implement CRISPR-i/a. In Aim 2, we will create Cre-triggered mouse/AAV systems for base and prime editing. We will create mice with Cre-activated expression of nCas9T (nicks the target strand) with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered N- or C-terminal base editors. We will further create mice with Cre- activated expression of nCas9NT (nicks the non-target strand) with N- and C-terminal protein ligation tags, which will efficiently join to AAV-delivered reverse transcriptase. These mouse/AAV systems will be invaluable reagents to probe disease biology, perform proof-of-concept therapeutic gene editing, and enable efficient in vivo CRISPR screens using genome or epigenome editing. This proposal is responsive to PAR-21-167. Development of Animal Models and Related Biological Materials for Research.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY/ ABSTRACT Polycystic ovary syndrome (PCOS) is a major health concern that affects up to 10% of reproductive-aged women and incurs an estimated economic burden of $8 billion in annual healthcare costs in the U.S. This complex, heterogenous condition is characterized by ovulatory dysfunction and hyperandrogenism and is associated with increased risk for metabolic dysregulation. Not included in cost estimates are the impact on offsprings of mothers with PCOS, who are at increased risk for adverse birth outcomes as well as the metabolic and androgenic features of PCOS. Evidence from clinical studies suggest that maternal PCOS could have causal biological effects in the intrauterine period that lead to adverse child outcomes; however, the role of PCOS genetic factors in the development of these adverse child outcomes is not known. Distinguishing between the contributing roles of the intrauterine environment and transmitted PCOS genetic factors to these adverse offspring outcomes would directly impact the timing (peri- vs. postnatal) and population (mothers vs. offspring) of targeted interventions to prevent PCOS and its associated comorbidities in children. The central hypothesis for this proposal is that maternal PCOS genetics, by acting through pre- and perinatal factors, play an integral role in the development of adverse birth outcomes and childhood metabolic and androgenic phenotypes in offspring. To test this hypothesis, this project will take advantage of a recently completed genome wide-association study meta-analysis that has doubled the number of genetic loci that influence PCOS risk and the increasing availability of mother-offspring data in biobanks. This project leverages the power of four pediatric cohorts, Avon Longitudinal Study of Parents and Children (N>6,000), Copenhagen Studies on Asthma in Childhood (N>500), Project Viva (N>500), and the Growing Up Today Study (N>1,000). In mothers and their offspring, PCOS genetic risk scores (i.e., estimated genetic susceptibility to PCOS) will be calculated. The effect of maternal PCOS genetic risk on the relationship between perinatal factors and adverse birth outcomes will be assessed, and the pre- and perinatal factors associated with maternal PCOS genetic risk that influence metabolic and androgenic phenotypes in offspring will be defined. This proposal promises to dissect the roles of transmitted PCOS genetic factors and the intrauterine environment in the development of adverse birth and childhood outcomes, and thereby pave the way for a precision-medicine approach to care that breaks the transgenerational cycle of PCOS and its related comorbidities.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY/ABSTRACT Adolescents and young adults (AYA) account for nearly 20% of new HIV diagnoses in the United States. However, very few sexually active adolescents have used pre-exposure prophylaxis (PrEP), despite it being safe, highly effective at preventing HIV transmission, and FDA-approved for youth. Pediatric health care providers could have a major impact on PrEP use for youth, as they are trusted sources of health information for AYA, and every individual who uses PrEP must obtain it from a clinician. Although CDC recommends that providers speak with all sexually active youth about PrEP, pediatricians infrequently discuss or prescribe this medication, in large part due to lack of knowledge and training in PrEP. Without strategies to train and engage pediatricians in effective implementation of PrEP, including discussing, prescribing, and supporting persistence, its impact for youth will remain unrealized. Academic detailing, an evidence-based, 1-on-1 educational outreach technique for clinicians, could improve PrEP prescribing and persistence for this population. Academic detailing has been associated with substantial increases in PrEP prescribing by clinicians in adult settings and has the potential to improve pediatric PrEP. Dr. Carly Guss’ long-term goal is to improve PrEP use and impact for youth. The objective of this proposal, the first step towards her goal, is to develop an implementation strategy for PrEP based on academic detailing that can improve prescribing to AYA. The research setting is a large children’s hospital and affiliated community health center in an HIV priority jurisdiction (Boston, MA) with a major unmet need for PrEP. The specific aims are to: 1) Explore barriers and facilitators to PrEP discussions, prescribing, and persistence among AYA and identify educational needs of pediatric health care providers. Using; 2) Develop a novel implementation strategy for PrEP in pediatric settings by locally adapting an existing PrEP academic detailing campaign; and 3) Conduct a pilot study of our PrEP implementation strategy for AYA. This proposal is significant in addressing the National HIV/AIDS Strategy goals to prevent new HIV infections among youth. The research is innovative in its application of rigorous implementation science methods and academic detailing in a novel context in urban pediatric settings. The research strategy directly aligns with Dr. Guss’ career development goals in behavior-change interventions for providers, qualitative and quantitative methods, and implementation science for PrEP. This K23 award will allow Dr. Guss to receive necessary training and experience to develop skills and knowledge in HIV prevention and implementation science. Dr. Guss is supported by an experienced and multidisciplinary mentorship team to help her achieve her research and training goals. Dr. Guss has assembled a scientific advisory board of national experts in HIV prevention for youth to provide additional guidance and feedback to ensure that this project and my scientific training are successful. Dr. Guss’ career goal is to become an independent clinician-investigator, and she will use the preliminary data obtained during this study to apply for NIH R01 funding in the final years of this award in pursuit of her career goal.

NIH Research Projects · FY 2026 · 2025-04

Understanding the mechanism of pre- to naïve- to formative-pluripotency transitions Abstract Pregnancy loss affects about 15% of couples globally. Of which, over 50% are due to developmental abnormalities at peri-implantation. Peri-implantation represents a specific developmental window during which the inner cell mass (ICM) goes through a number of differentiations and pluripotent transitions to prepare for gastrulation. Understanding the molecular, cellular, and epigenetic events that occur during peri-implantation is critical for determining how this process goes awry. However, the limited number of mammalian peri-implantation embryos, as well as the technical complexity in handling these cells, has been a major constraint in progressing research. Central to peri-implantation development is the pluripotency transitions of epiblast (Epi) cells from pre- pluripotency (E3.5 ICM cells that co-express Oct4, Nanog, and Gata6) to naïve, formative and primed pluripotent states with distinct developmental potentials. To overcome the technical difficulties in working with peri- implantation embryos, in vitro ESC culture systems have been developed to mimic the various in vivo pluripotent states, which have largely contributed to our current understanding of the pluripotency transition. However, several factors shown to be important for pluripotency transition in vitro failed to generate the expected phenotype, or even no phenotype in mouse knockouts, indicating that studies using peri-implantation embryos are urgently needed. Thus, understanding how the various pluripotency transitions take place in vivo is critical not only for understanding peri-implantation development, but also for understanding peri-implantation-related diseases and infertility. To be able to work with limited peri-implantation embryos, we have established innovative techniques to overcome the technical hurdles. With our state-of-art tools and unique mouse models, we have established the following three aims: 1) Identifying key TFs and dissecting the mechanisms of ICM to epiblast transition 2) Identifying key TFs and dissecting the mechanism of naïve to formative transition 3) Understanding how DNA methylation regulates naïve to formative transition Completion of the proposed studies will not only reveal the mechanisms of pluripotent transitions, but may also reveal targets for therapeutic intervention of the peri-implantation-related diseases.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Self-antigen-specific T cells prevalent within the adaptive immune system pose an ever-present threat to health. Accordingly, they are heavily regulated at steady-state by mechanisms of peripheral tolerance. Infections and inflammation, however, offer an opportunity for these cells to become activated and initiate autoimmunity. While characterizing the response of self-antigen-specific T cells during inflammation with the use of peptide:MHC class II tetramers in an experimental mouse model of lung injury, we recently provided direct evidence that the immune system generates a polarized response towards tolerance during inflammation through the selective expansion of endogenous self-antigen-specific Foxp3+ Tregs. Moreover, we have preliminary evidence that these self-antigen-specific Tregs remain enriched within the T cell repertoire for months after the injury. We hypothesize that the “experienced” self-antigen-specific Tregs expanded during acute lung damage develop into a long-lived tissue-resident population that limits recurrent injury. Characterizing the development of lung- resident self-antigen-specific Tregs following lung injury addresses the broader question of how the adaptive immune response reinforces tolerance towards self-antigens presented during inflammation. To investigate our hypothesis, we have improved upon our experimental mouse model to harbor greater frequencies of polyclonal lung self-antigen-specific Tregs that respond to tissue injury, generated new “barcoded” tetramers for large scale transcriptomic studies, and implemented a parabiosis approach to monitor trafficking of these Tregs. In Aim 1, we will study the population dynamics of self-antigen-specific Tregs and determine whether they establish tissue- residence in the lungs. In Aim 2, we will characterize the phenotype and the function of self-antigen-specific Tregs expanded in the context of prior lung injury promote tolerance in future lung disease. Understanding how “experienced” self-antigen-specific Tregs guard against future lung injury will provide new insights into the ability of self-antigen-specific T cell repertoire to prevent of autoimmunity. Dr. Daniel Shin will perform the work for this K08 proposal, sponsored by Boston Children’s Hospital, at the Center for Immunology and Inflammatory Diseases, a state-of-the-art multidisciplinary research center, at Massachusetts General Hospital under the mentorship of Drs. James Moon and Andrew Luster. With the guidance of his co-mentors, Dr. Shin has set forth a comprehensive career development plan that will provide new technical skills in mouse immunology, develop greater mastery of RNA transcriptomics, and hone academic and professional skills training. To accomplish these goals, he has also assembled an exceptional Training Advisory Committee composed of Drs. Medoff, Villani, Chatila, and Oettgen who will provide invaluable expertise and assistance in the fields of lung inflammation, systems biology, Treg biology, and career development. The goal of this K08 award is to equip Dr. Shin with the intellectual and technical foundation to become a successful independent, NIH-funded investigator with expertise in peripheral tolerance, tissue-resident Tregs, and the prevention of organ-specific autoimmunity.

NIH Research Projects · FY 2026 · 2025-02

Abstract Pseudomonas aeruginosa is an important opportunistic pathogen of humans. It is the principal cause of morbidity and mortality in people with cystic fibrosis, is a major cause of hospital-acquired pneumonia and is particularly problematic in chronic wound infections. Post-transcriptional regulators are known to play important roles in controlling the virulence of P. aeruginosa and the best characterized of these are the RNA-binding proteins Hfq and RsmA. However, apart from these two regulators, little is known about the roles played by other RNA-binding proteins in this gram-negative bacterium. Here we propose to study a putative RNA-binding protein in P. aeruginosa called PhaF that we have recently found to be a global post-transcriptional regulator. In Aim 1 we propose to identify the direct regulatory targets of PhaF in P. aeruginosa strain PAO1, a commonly used laboratory strain, at different phases of growth and investigate the activity of PhaF in clinical isolates of P. aeruginosa. We will also identify PhaF target transcripts in cells grown under conditions that more closely mimic infection conditions, as well as in an animal model of chronic infection. In addition, we propose to test whether PhaF influences the virulence of P. aeruginosa through its effects on several of the targets we have already identified that are linked to biofilm formation and virulence. These experiments will help better define the PhaF regulon under standard laboratory growth conditions, as well as conditions that are relevant to infections. Furthermore, they will provide the first assessment of the extent to which the PhaF regulon is conserved between laboratory strains of P. aeruginosa and clinical isolates. The putative RNA-binding domain present in the C- terminal domain (CTD) of PhaF is remarkable in that it contains 20 or more repeats of the motif KPAA found in histone H1 from eukaryotes. In Aim 2 we will determine whether the CTD of PhaF constitutes a novel RNA- binding determinant, investigate the mechanism by which PhaF positively regulates the translation of target transcripts, and determine what governs the apparent switch in PhaF target specificity that occurs in response to varying growth conditions. Our studies are expected to illuminate the regulatory roles and mechanism of action of a newly identified global post-transcriptional regulator that controls the expression of important determinants of biofilm formation and virulence in P. aeruginosa. The experiments we describe are relevant for understanding post-transcriptional control exerted by RNA-binding proteins in clinical isolates of this bacterium.

NIH Research Projects · FY 2026 · 2025-02

Project Summary / Abstract Somatic cells undergo continuous evolution with heritable modifications through genetic and epigenetic changes, influencing cellular competition and selection. Depicted as a detailed branching lineage map, a cellular genealogy provides an essential scaffold to understand the causes and consequences of the somatic evolutionary dynamics. However, the impact of various somatic modifications on genealogical dynamics and cell states remains unclear, due in part to our limited ability to track somatic modifications within genealogical contexts. To address this limitation, I propose to develop a somatic mutation-resolved lineage tracing system that reconstructs a single-cell genealogical tree coupled with joint detection of somatic mutations and epigenetic alterations (Aim1). To demonstrate the biological significance of this system and to advance our understanding of hematopoietic stem cell (HSC) somatic evolution, I will trace the acquisition histories of somatic mutations on HSC genealogical architecture and chart the evolutionary trajectory of normal aging hematopoiesis and the leukemic transformation (Aim2). To tackle the causality in somatic evolution, I will develop a genealogy-resolved massively parallel single-cell CRISPR screen to survey the causative effects of somatic modifications on the genealogical dynamics and cellular functions at a large scale (Aim3). This project will advance the frontier of studying genomic evolution in somatic cells, by revealing how genomic modifications impact cellular dynamics. It paves the way to decipher the driving forces that shape somatic evolution in broad areas essential for human health, including aging hematopoiesis, cancer progression, etc. This project is a critical step for my long-term goal to advance our ability to observe, understand, and control cellular behaviors and cell fate choices. Through the K99 phase of this proposed career development plan, I will further strengthen my expertise and knowledge in mouse modeling, functional genomics, and leukemia biology, as well as professional skills such as leadership, and lab management which are essential for my successful independent career. These training goals will be achieved through joint mentorship between Dr. Jonathan Weissman and Dr. Vijay Sankaran labs together with my advisory committee in a highly enriched cross-institutional environment (Boston Children’s Hospital, Whitehead Institute, Broad Institute). When combining these new skills with my rigorous training in single-cell genomics, stem cell, and computational biology, I will be better prepared to transition into a successful independent research career.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY/ABSTRACT Interpretation of fetal brain MRI continues to be a challenge due to fetal motion even though single-shot techniques such as T2 weighted Half Fourier Single-shot Turbo spin-Echo (HASTE) acquisitions are used. Unpredictable fetal movement causes artifacts and images acquired in oblique orientations. As a result, the radiologist must interpret multiple stacks of imperfect HASTE images in varying oblique orientations, visually interrogating up to 1,000 independent 2D images. Additionally, multiple linear measurements are used to assess brain development. This approach is time-consuming and mentally taxing. Ironically, methods to create coherent fetal brain volumes have existed for over a decade in the research world but none have transitioned into clinical workflows due to long reconstruction times, the need for specialized hardware, and no infrastructure to deposit the reconstructed volumes/biometrics into the picture archiving and communication system (PACS) for a radiologist’s review. We will leverage the latest deep-learning strategies to perform rapid, robust, and accurate fetal brain reconstructions and automatically generate fetal brain biometrics. We will build on the unique open- source Children’s Hospital Research Integration System (ChRIS) to integrate results into the clinical workflow. In Aim 1 we will develop fast methods for reconstructing coherent 3D fetal brain volumes from a collection of clinically acquired 2D HASTE slice stacks, providing accurate automated metrics to support clinical interpretation. Our deliverable is an algorithm that i) successfully performs fetal brain reconstructions in >95% of cases in under 2 min and ii) provides important brain biometrics within 1 standard deviation of expert human measures. In Aim 2 we will integrate fetal brain reconstructions and derived metrics into the clinical workflow using ChRIS, within 5 min of study completion. In Aim 3 we will assess the impact of coherent fetal brain volumes and automated metrics on radiological interpretation. We hypothesize that expert fetal brain interpretations will be faster, more sensitive, specific, and concordant in the detection of abnormalities when viewing reconstructed volumes and using automated metrics compared to viewing 2D HASTE stacks as acquired. Ground truth will be provided by neonatal brain MRIs and the cohort will be enriched with cases where neonatal MRIs discovered findings missed on fetal MRIs. The deliverable is a rigorous quantitative evaluation of the impact of coherent volume reconstruction and automatic brain biometrics on the efficacy of radiological interpretation of fetal MRI. If successful, this project will enable radiologists to interpret fetal brain MRIs as one volume instead of numerous stacks of images in varying obliquities, saving time, and increasing accuracy. The automated metrics will further enrich the information available to radiologists without requiring additional physician time. The ChRIS infrastructure is freely available on GitHub and will support the next phase of dissemination of this and other innovations to interested institutions for integration into their clinical workflows.