Boston Children'S Hospital

NIH Research Projects · FY 2025 · 2023-07

Summary Structural insights from our recent work provide a strong scientific premise for exploring the membrane-related components of HIV-1 envelope glycoprotein (Env), including the membrane proximal external region (MPER), the transmembrane domain (TMD) and the cytoplasmic tail (CT), for vaccine development. Our data indicate that all these regions influence the stability and antigenicity of the Env ectodomain. Major broadly neutralizing antibody (bnAb) epitopes on HIV-1 Env include CD4 binding site, V1V2-glycan, V3-glycan, the fusion peptide/gp120-gp41 interface and the MPER. The optimal presentation of these epitopes, critical for their antigenicity and immunogenicity, depends on the Env trimer organization and conformation. Recently, the fusion peptide-based immunogens have induced robust cross-clade neutralizing responses in animal models, suggesting that bnAbs may be elicited by vaccination. In this research project, our central hypothesis is that rationally designed HIV-1 Env immunogens in different conformations with various degrees of bnAb epitope exposure induce different B cell responses, which may lead to production of diverse bnAbs in animal models and to new strategies for HIV-1 vaccine immunogen design. The research team is formed by a group of outstanding investigators with diverse yet complementary expertise to carry out the proposed studies. This group has extensive experience in protein engineering, production and characterization, in B cell biology and vaccinology in animal models, and in detailed analysis of vaccine-elicited antibody responses. The group members have an extensive history of working together on HIV-1 and SARS-CoV-2 related projects. The team will capitalize on the newly determined structures of the membrane-related components of HIV-1 Env to develop two innovative immunogen-design strategies: (1) soluble Env trimer immunogens and (2) membrane-bound intact Env trimer immunogens. We propose two Specific Aims to test the hypothesis: Aim 1. We will design, characterize and produce Env-based immunogens in both the protein and mRNA forms and perform structural studies of Env-based immunogens and their complexes with antibodies. Aim 2. We will evaluate immunogenicity of novel Env-based protein immunogens and mRNA vaccines in VelocImmune human antibody mice.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY Calcitonin gene-related peptide (CGRP) is secreted by neurons and is important for vasodilation, nociception, and immune responses. Drugs targeting CGRP have been a breakthrough in the treatment of migraine headaches. Increasing clinical evidence suggests that CGRP signaling may also be involved in gastrointestinal (GI) health and disease. For example, constipation is one of the most common side effects of the new anti-CGRP therapies, hinting that this signaling pathway is important for normal gut motility. Converging with the real-world experience of migraine patients, three genome-wide association studies recently identified the CALCB locus as strongly linked to stool frequency and diverticular disease. CALCB encodes the β-isoform of CGRP, which is highly conserved between humans and mice. The functional significance of CALCB in the bowel, however, has not been identified. The overarching goal of this proposal is to define the enteric neurons that secrete CALCB and determine which aspects of GI motility that CALCB signaling is necessary for in vivo. CALCB and CALCA, the α-isoform of CGRP, differ by only 3 amino acids making it challenging to distinguish them at the protein level by immunohistochemistry or ELISA. The two isoforms are encoded by distinct loci, however, enabling their expression to be readily distinguished at the transcript level. Previous gene expression studies showed that while CALCA is the major CGRP isoform in most of the body, CALCB dominates in the intestine. Despite this dominance, virtually all of the studies that have probed the roles of CGRP in gut immunity and visceral pain have identified CALCA originating from gut-extrinsic afferent neurons as the key isoform and found CALCB to be dispensable, leaving its essential functions unclear. Utilizing the anti-CGRP agents in clinical use as well as new genetic tools that we have generated to enable selective labeling and manipulation of CALCB neurons in the enteric nervous system (ENS), we will accomplish three objectives. One, determine which segments of the GI tract require CALCB, the major ENS-derived CGRP, for normal motility in mice. Our preliminary data show that these requirements are sex-specific, suggesting that the cellular-molecular wiring of the CGRP pathway in the intestine may be different in males and females. Two, using genetically encoded reporters, calcium indicators and chemogenetic proteins, we will define the neurons in the male and female ENS that release CALCB and how gut motility is affected by altering their activity. Three, given the accumulating evidence for CGRP involvement in GI homeostasis, we will determine the mechanisms that regulate the intestinal levels of both isoforms. In addition to advancing the fundamental understanding of neuromuscular function in the bowel, the impact of this work will be to explain how a widely used class of drugs causes adverse GI effects and, conversely, if isoform- or ENS-specific targeting of CGRP signaling may be beneficial for treating GI dysmotility.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ ABSTRACT The cell surface is a platform for physical and regulatory control over cell biology, positioning it to be a key interface for diagnostic targeting and therapeutic intervention. While RNA is a central polymer in biology most thought and experimental effort devoted to RNA biology has been confined to intracellular spaces and excluded from participating in cell surface biology. On the cell surface, carbohydrate polymers (glycans) are of critical importance due to biophysical and signaling activities. Interestingly, despite both polymers playing central roles in biology, RNA and glycans have largely existed in entirely non-overlapping fields of study. However, my work has provided evidence of a hybrid molecule, an RNA-glycan conjugate (glycoRNA); this new class of biomolecule represents a direct link between RNA and glycobiology. Critically, glycoRNAs are localized to the external surface of living cells and can engage with immunomodulatory Siglec receptors. Thus, glycoRNAs are positioned on a surface of critical regulatory importance, with access to cell-cell interactions, pathogens, and signaling receptors on the cell surface. However, we currently lack facile tools to study this new cell surface molecule, we do not understand the molecular or atomic composition of glycoRNAs, and we have a poor understanding of how many species biosynthesize glycoRNAs. This MIRA proposal is focused on developing and implementing methods to uncover functional roles of RNA glycosylation and we will approach the complex biology of glycoRNAs in a systematic fashion. Initially we will develop novel chemical approaches to label glycans in the context of RNA. My proposed strategy of selective carboxylic acid labeling represents an innovative new approach to detecting glycoRNA, without the need for synthetic metabolic reporters. These tools will be easily implemented across cell types and species enabling others in the scientific community. We will apply these tools and other molecular assays to expand our understanding of the composition of the cell surface in the context of glycoRNA. Biochemical, biophysical, and imaging-based strategies will be used to define the molecular neighborhoods of glycoRNAs as well as the chemical nature of the RNA-glycan linkage; all together providing a more complete picture of the mammalian cell surface. Finally, we will develop the first evidence of glycoRNAs in non-mammalian organisms. First focusing on two major strains of yeast (S. cerevisiae and S. pombe) with robust culturing, functional, and genetic tools that will allow for rapid dissection of the biogenesis pathway for eventual engineering purposes. Expanding to other organisms including prokaryotes (pathogenic and not) as well as other multicellular eukaryotes like C. elegans will better define the scope of glycoRNA biosynthesis and more robustly equip us to generate synthetic glycoRNAs. More broadly, we intend to advance the general model of how cells interact with each other, pathogens, and exogenous molecules, as without cell surface glycoRNAs, they are likely incomplete. This proposal develops innovative methods to establish new conceptual and physical layers of regulation between a cell and its environment.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Despite recent progress, clinical heterogeneity has likely hindered efforts to clearly delineate the genetic architecture of psychotic disorders like schizophrenia and bipolar disorder. However, this heterogeneity also presents an opportunity for studying individuals with extreme phenotypes, virulent forms of the illness with putatively more homogeneous etiologies. Early onset psychosis (EOP, onset prior to 18 years) represents such an extreme phenotype, with dramatically higher rates of rare deleterious mutations in EOP than adult-onset psychosis. Consequently, studying EOP cohorts provides a unique opportunity to discover rare genetic loci influencing illness risk. We will deep phenotype and sequence 1900 EOP probands and 1900 non-psychotic, demographically matched youth. For 400 probands, both parents and a non-psychotic sibling will be recruited to facilitate the search for inherited and de novo mutations associated with EOP (n=1200 family members). Children and adolescents and their families will be recruited from a single, large public pediatric psychiatric hospital in Mexico City. To date, most psychiatric genetic studies focus on European-ancestry (EA) cohorts, while excluding of other ancestry groups. Yet, no single population is sufficient to fully illuminate the genetic architecture of complex traits like psychosis, and the EA focus could exacerbate health care disparities. Latinos make up ~8% of the world population (~18% of the US population) but appear in less than 1% of published genome-wide studies. Complicating matters, Latinos are genetically heterogeneous, with substantial differences between Central and South American and Caribbean populations, reflecting continental-level ancestral group admixture and the substructure of local Indigenous American populations. As 62% of the Latinos in the US are of Mexican origin findings from the Mexican population are directly relevant for most individuals in the nation’s largest racial/ethnic minority. During our initial 1-year project, we recruited 1000 participants from the same psychiatric hospital and using identical procedures, thus demonstrating the feasibility of the current study. Combining these 1000 individuals with the additional 5000 participants we now propose to acquire, we aim to: 1) characterize EOP probands and siblings in terms of cognitive and psychosocial functioning, frequency of adverse life events, social determinants, and cannabis use; 2) document the prevalence of rare loss of function mutations and CNVs previously associated with schizophrenia or autism spectrum disorder in EOP participants relative to their unaffected family members and demographic and population controls; and 3) utilize ancestry analysis to identify chromosomal regions and runs of homozygosity shared in common by multiple unrelated EOP cases but not by unaffected individuals. David Glahn (BCH), Laura Almasy (CHOP), Humberto Nicolini (Instituto Nacional de Medicina Genómica) and Carlos Bustamante (Stanford) lead this project.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Our laboratories have developed novel techniques for the editing B-cell receptors of human primary B cells. Using newly identified CRISPR/Cas proteins and innovative homology-directed repair templates, we can efficiently overwrite the endogenous variable heavy (VH) and variable light (VL) segments of a mature VDJ- recombined BCR with the variable genes of broadly neutralizing HIV antibodies (bNabs). Importantly, these variable genes are placed in their respective natural loci, and – excepting the new VH and VL segments – these edited B cells are indistinguishable from unmodified mature, naïve B cells. We describe these edited B cells as “chimeric antigen receptor B cells”, or CAR B cells, evoking the more familiar CAR T cells. These CAR B cells replicate, differentiate, affinity mature, and secrete antibodies in vivo providing an efficient delivery vehicle for bNabs. CAR B cells represent a key advance over passive infusion or gene therapy delivery of bNabs, because they do not raise anti-drug antibodies against their novel BCR, and because the can affinity mature in response to an antigen, including HIV-1 emerging from a reactivated reservoir. They can thus adapt in real time to the specific viral variants in the reservoir. To date, however, we have only transformed CAR B cells ex vivo by isolating primary B cells from a particular host, transforming them by electroporation of CRISPR/Cas RNPs and DNA repair templates, and re-infusing the CAR B cells into the host. Although ex vivo CAR B transformation could be clinically viable, it would likely be a prohibitively expensive procedure for most HIV-positive persons. Here we propose to perform the CAR B transformation procedure in vivo by developing a B cell-tropic adeno-associated virus (AAV)-based gene therapy vector and CRISPS/Cas editing cassette that could be administered intravenously; a comparatively fast and inexpensive procedure. This proposal is divided into three aims. In Aim 1, we draw upon our extensive experience modifying AAV capsids to target specific cell types to create a B cell-tropic capsid. In Aim 2, we develop and evaluate a range of AAV-delivered CRISPR/Cas editing cassette designs for their ability to efficiently transform CAR B cells. Lastly, in Aim 3, we optimize an immunogen and immunization strategy to drive proliferation and affinity maturation of newly transformed CAR B cells. These studies will make clinically viable a promising approach for suppressing an established HIV-1 infection or preventing a new one in high-risk persons.

NIH Research Projects · FY 2026 · 2023-07

Project Summary Sudden infant death syndrome (SIDS) remains the leading cause of post-neonatal mortality in the U.S.– an unchanging and devastating fact despite implementation of safe sleep practices (extrinsic risk reduction). Addressing this 21st century health crisis now requires discovery of intrinsic biological vulnerabilities and plausible molecular pathways that might lead to biomarkers and preventative interventions. In multiple independent SIDS tissue datasets, serotonergic (5-HTergic) abnormalities in the brainstem were consistently identified; in animal models of reduced brainstem 5-HTergic activity, compromised autoresuscitation (AR) was observed – the ability of mouse pups to recover from cycles of asphyxial apneas and bradycardia (resembling the cycles of apnea and bradycardia observed in some SIDS cases) was significantly diminished. Such 5- HTergic system dysfunction, as an intrinsic vulnerability, may be caused or exacerbated by extrinsic stressors such as pre- and/or postnatal hypoxia (e.g., placental insufficiency, parental smoking) and/or antemortem infections. Hypoxia and infection are each risk factors for SIDS and can increase neuroinflammation, which can impair AR. Notable new findings in some SIDS cases as compared to controls are elevations of the neuroinflammatory markers IL-1β, IL-2, IL-4, IL-17, and GM-CSF and/or in neopterin (a marker of Th1 (proinflammatory) cellular activation) in the cerebrospinal fluid. We postulate that neuroinflammation, triggered by hypoxia and/or antemortem infections (bacterial or viral), interact to create a vulnerable 5- HTergic system, reduce AR effectiveness, and increase the risk for sudden death, and may underlie some SIDS cases. We propose: 1) To quantitate inflammatory mediators within SIDS brains and determine whether a profile of mediators associates with 5-HTergic brainstem abnormalities. We will test the hypothesis that specific inflammatory profiles associate with low 5-HT1A and 5-HT2A receptor binding and low 5-HT levels. 2) To map at single-cell resolution, differences in gene expression profiles and overall cell-type composition/states of brainstem tissue across SIDS cases (the SIDS subsets identified through Aim 1) and controls. We hypothesize that SIDS subsets will be distinguished by specific inflammatory profiles in glia, neurons, and/or endothelial cells, and gene expression differences will identify novel, previously unrecognized SIDS-related pathways for mechanistic testing in cell and animal models. 3) Assess the interaction between chronic intermittent hypoxia (gestational to P8) and postnatal antemortem infection on molecular, cellular, inflammatory, and physiological readouts, including the autoresuscitation response (AR). We will test the hypothesis that the combined effects of antemortem hypoxia and infection interact to create greater neuroinflammation, more severe 5-HTergic deficits, and increased likelihood of AR failure, compared to either hypoxia or infection alone. SIDS research must address the missing mechanistic links between risk factors and postmortem pathology to develop life- saving interventions.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT Use of continuous glucose monitors (CGM) has been shown to significantly improve glycemic control and decrease the risk of complications for pediatric patients with type 1 diabetes (T1D). However, despite the rapid advancements in technological capabilities over the past twenty years, access to and meaningful use of CGM are not distributed equitably across the population. The continued advancement in sophistication and potential for improved outcomes coupled with population-level increases in CGM use contributes to widening disparities in care and makes essential an improved understanding of optimizing use of personal medical devices for T1D. I am a public health trained pediatric endocrinologist; through this proposed K23 Award I aim to characterize disparities based on race/ethnicity and socioeconomic status (SES) in CGM use for children with T1D utilizing statistical analysis in disparate care settings and nationwide claims data. These analyses of the sources of disparities will inform my pilot clinic-based intervention to narrow gaps in CGM use. This project builds upon my research describing the experience of Hispanic caregivers of children with T1D, my analysis of demographic predictors of metabolic control in the T1D program at Boston Children’s Hospital (BCH), and my training in health services research and public health. This grant will enable my acquisition of new skills in time- to-event analysis, claims-based data use, and intervention design, implementation, and evaluation. Adding depth to my health services research methods and establishing a foundation in clinical trials, this grant, along with the unique environment and diverse mentorship at BCH, Department of Population Medicine (DPM), and Boston Medical Center (BMC) will position me well to begin an independent research program. In Aim 1, I will utilize a national health insurance plan claims database to analyze personal medical device use and acute care utilization among children with T1D with a focus on the impact of race/ethnicity and SES on CGM prescription and adherence. In my second aim I will employ a mixed methods approach to quantify CGM use among children with T1D cared for at a quaternary referral center (BCH) and a safety net hospital (BMC) and conduct a qualitative assessment of barriers to consistent CGM use in Black, Hispanic, and low SES patients at both sites to inform the development of a clinic-based intervention to improve CGM uptake and adherence in diverse, marginalized, and low resource patient populations. In Aim 3, I will develop and pilot the intervention informed by Aim 2 in a 6-month RCT at BCH and BMC to assess the impact of culturally effective education and patient navigation for CGM use on disparities in its uptake and associated clinical outcomes. I aim to establish a career as an independent physician-scientist with a background in the drivers of pediatric T1D care and outcomes, with the long-term goal of providing equitable access to rapidly improving technological innovations to better the lives of children with T1D. The mentorship, formal training, and research experience that this K23 will provide will position me well to begin an impactful independent research career.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY The mechanisms by which the hematopoietic stem and progenitor (HSPC) niche is affected by clonal hematological disorders such as myelodysplastic syndrome (MDS) remain poorly understood. Furthermore, the heterogeneity and clonal response of endothelial and stromal cells (the main components of the HSPC niche) in MDS in vivo remain unexplored. To tackle these aspects, I developed a new zebrafish model of MDS by driving the protooncogene CMYC overexpression specifically in blood cells. Additionally, I crossed a genetic lineage tracing zebrafish line called GESTALT to a double transgenic zebrafish line carrying two fluorescent reporters allowing to purify specifically niche endothelial and stromal cells. This way, I created a new GESTALT line that permits CRISPR-CAS9 based barcoding during zebrafish embryonic development, purification of adult marrow niche cells and recovery of niche DNA barcodes by sequencing. Combining these novel tools, I induced MDS in barcoded zebrafish and read out the clonality and the transcriptome of endothelial and stromal cells. I discovered that clones of stromal cells selectively expand, and endothelial cells are transcriptionally remodeled in MDS. Given these data, I hypothesize that MDS remodels the clonality and transcriptional profile of the HSPC niche and that mechanisms involved in HSPC-niche interactions promote disease progression. Under the mentorship of Dr. Leonard Zon, I will investigate the mechanisms by which MDS remodels the niche using a combination of in silico computational approaches, genetic (GESTALT) and color based (Zebrabow) lineage tracing, confocal microscopy and in vivo mosaic mutagenesis. Once I establish my laboratory, I will build a multidisciplinary team to deepen my computational analyses and broaden my in vivo genetic and biochemical perturbations of the clonal mechanisms of niche involvement in MDS. My overarching goal is to identify novel targetable mechanisms specific to the HSPC niche that would prevent and/or halt MDS progression. This K99/R00 award will enable me to develop new technical skills, participate in courses that will improve my ability to manage a laboratory, and attend conferences that will broaden my network and my knowledge of hematological disease modeling, Zebrabow lineage tracing paired with confocal microscopy and zebrafish mutagenesis. The scientific advisory committee I have put together includes experts in the fields of hematopoiesis, lineage tracing, and stem cell biology and, along with Dr. Zon, will give me feedback on my research and career progress. These proposed research and career development activities will pave the way for me to become an independent investigator discovering and studying new mechanisms responsible for hematopoietic disorders progression mediated by the blood stem cell niche.

NIH Research Projects · FY 2026 · 2023-07

Abstract This application is entitled ‘Anatomical regulation of glucose and lipid metabolism by insulin signaling in hepatocytes’. The liver is a multitasking organ, performing diverse functions that are critical for maintaining glucose and lipid homeostasis. Previous studies of hepatic insulin signaling have been done with the assumption that all hepatocytes are equivalent. Recently single-cell transcriptomics has revealed that around half of hepatocyte genes are expressed in a zoned manner, in which periportal hepatocytes might coordinate fasting metabolism, whereas pericentral hepatocytes might manage postprandial metabolism. A clear understanding of how insulin signaling coordinates energy homeostasis at spatial levels is necessary. This proposal brings our focus to the important problem of how anatomical segregation of insulin signaling in the liver regulates glucose and lipid metabolism in physiological and pathological conditions. By using promoter knock-in mouse models, we will perform the functional study in vivo by using Gls2CreER mouse line to target periportal hepatocytes and Cyp1a2CreER mouse line to target pericentral hepatocytes. This real-time molecular strategy is innovative as previous work that has relied upon static approaches. By intercrossing with floxed mice targeting insulin signaling components, this project has the potential to reveal important insight into the anatomical segregation of insulin signaling in the liver to control energy homeostasis. First, impaired insulin signal transduction in periportal hepatocytes is expected to promote hepatic glucose production but might retain insulin sensitivity in pericentral hepatocytes for lipid metabolism, producing the pathological combination of hyperglycemia and hyperlipidemia. This strategy might provide an innovative model to investigate the metabolic features of insulin resistance in humans. Second, our preliminary data shows that total hepatic insulin signaling deficiency impairs hepatic de novo lipogenesis and prevents diet-induced fatty liver in mice, which contradicts the excess lipogenesis in insulin-resistant humans. Direct investigation of insulin signaling in pericentral hepatocytes can reveal the relationship between insulin resistance and NAFLD. Third, selective insulin resistance has implications for therapy; however, how to precisely target this paradox is still unresolved. It would be desirable to employ drug targets that could alleviate both T2D and NAFLD. Thus, we propose to identify novel candidate genes that contribute to HFD-induced hyperglycemia and hepatic steatosis. Together, the proposed experiments can discern the function of insulin signaling in the regulation of glucose and lipid metabolism in the periportal and pericentral hepatocytes, which would reveal foundational mechanisms coordinated by hepatic insulin action that moderate glucose and lipid metabolism under physiological and pathological conditions.

NIH Research Projects · FY 2026 · 2023-06

Inflammation evolved to lead to recovery from sterile or microbial injuries. The induction of the inflammatory process not only activates the immune cells, but also alters their metabolism and thus forge the immune response. Accumulating evidence shows that a proper inflammatory process requires the coincident recognition by pattern recognition receptors (PRRs) of exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). We recently demonstrated that the coincident recognition of lipopolysaccharide (LPS), the major component of Gram-negative bacteria, and host-derived oxidized phospholipids known as oxPAPC (a class of DAMPs) leads to the formation of phagocytes characterized by a unique metabolic profile that increases the production of interleukin (IL)-1β, a potent pro-inflammatory cytokine. Whether, and how, the simultaneous encounter of LPS and oxPAPC alters other inflammatory activities of phagocytes remains largely unknown. Based on new compelling data, here we hypothesize that the coincident recognition of LPS and oxPAPC alters key metabolic checkpoints to drive hyper-inflammation. Also, that these changes can be harnessed against septic shock. Sepsis is a complex inflammatory syndrome characterized by a hyper-inflammatory phase called septic shock. Although it was previously proposed that oxPAPC protects against the hyperinflammatory phase of sepsis by inhibiting the capacity of LPS to signal, our new unpublished data show instead that oxPAPC production follows LPS or bacterial encounter in vivo and that oxPAPC increases inflammation and lethality in mouse models of sepsis. Notably, we found that, to exert its functions, oxPAPC directly interacts with, and inhibits, AKT. AKT is a central metabolic checkpoint that regulates the metabolism of phagocytes and their inflammatory activity. AKT inhibition by oxPAPC prevents the production of IL-10. IL-10 is a pluripotent immunoregulatory cytokine indispensable for maintaining immune homeostasis and restricting inflammation during sepsis. Mechanistically, oxPAPC-dependent inhibition of AKT potentiates the methionine cycle and favors the trimethylation of the histone H3, thus switching off IL-10 transcription. Supported by our new solid data, we will employ biochemistry, transcriptional and epigenetic analyses, as well as metabolomics in vitro to further dissect the signaling cascade initiated by oxPAPC during LPS encounter. By using new transgenic or conditional knock-out mice, as well as commercially available drugs, we will test in vivo the possibility to target the newly identified metabolic pathways regulated by oxPAPC to protect against sepsis. Altogether we will characterize the molecular components that mediate host-derived inflammatory ligand-dependent immunometabolic functions. Our study will offer potential therapeutic targets for modulating immune system activation and sepsis, a devastating inflammatory syndrome that is widespread in western countries.

NIH Research Projects · FY 2024 · 2023-06

PROJECT SUMMARY Combined anti-retroviral therapy (cART) and pre-exposure prophylaxis (PrEP) represent major milestones in the effort to eliminate AIDS and prevent new HIV-1 infections. They nonetheless have limitations. For example, a life-time use of two or three compounds delivered to most every cell and tissue in the body will likely come with undesirable, difficult-to-anticipate side effects. Access and compliance also remain concerns, especially among infected persons who have not yet been reached by our healthcare infrastructures. Similarly, PrEP requires both access and a conscious effort before a potential transmission event, something that is not always realistic for the hardest-to-reach demographics here and abroad. Here we will develop an approach that provides robust prophylaxis and perhaps effective viral suppression for six months or more after a single injection. Specifically we will optimize eCD4-Ig, a very broad and potent antibody-like molecule, for its delivery in an injectable hydrogel, and we will optimize this hydrogel for delivery of eCD4-Ig. eCD4-Ig provides highly effective protection in rhesus macaques from high-dose challenges with both SHIV-AD8 and SIVmac239. It also has the breadth and potency to suppress an established SHIV-AD8 infection. This breadth appears sufficient to suppress the wide diversity of viruses in an individual and in a population. As importantly, HIV-1 has not developed easily accessible pathways of escape from eCD4-Ig as it has from neutralizing antibodies. It is therefore an ideal payload for a safe, effective, and sustained hydrogel delivery system. As we show, these hydrogels are well-tolerated, non-immunogenic, easily manufactured, and deliverable with a high-gauge need. Importantly, they and their payloads can be immediately withdrawn in case of an adverse event. Modeling suggest that they can sustain eCD4-Ig concentrations that could provide effective prophylaxis for well over six months. We will test this possibility in human FcRn- transgenic mice and in rhesus macaques, and confirm that our best eCD4-Ig/hydrogel formulations could replace PrEP and/or cART.

NIH Research Projects · FY 2024 · 2023-06

PROJECT SUMMARY/ABSTRACT One of the leading causes of visual dysfunction in developed countries is cortical visual impairment (CVI). CVI is very commonly a comorbidity with neurological and neurodevelopmental disorders, and significantly contributes to altered development. CVI occurs when deficits in the eyes alone cannot explain the defects in perception, indicating that visual processing in the cortex is responsible for altered visual function. No treatments or effective therapies are currently available. Elucidating the circuitry underlying CVI in neurodevelopmental disorders will guide in designing targeted treatments not only for visual impairment, but also to improve other core features of neurological functioning. One neurodevelopmental disorder with high rates of CVI is CDKL5 deficiency disorder (CDD). CDD is an epileptic encephalopathy characterized by seizures beginning in the first months of life, severe developmental delay, often including lack of speech and independent walking. About 75% of individuals with CDD experience CVI and this impairment is also reflected in mouse models of CDD which have been shown to have reduced visual evoked response and reduced visual acuity. Although CVI is a prominent feature of CDD, we do not understand how CVI arises and the underlying circuits. Recently, our laboratory discovered that CDD mouse models exhibit an increased functional callosal connectivity across cortical hemispheres. Callosal interhemispheric connectivity is key for higher order processing. In neurotypical development, callosal projection neurons (CPNs) prune their axons from layer 4 pyramidal neurons and refine selective synapses in superficial and deeper cortical layers allowing the acquisition of adult visual function. Our hypothesis is that in the absence of CDKL5, callosal projections fail to refine and to acquire proper mature function giving rise to CVI. By combining a multi-level approach, I will test this working hypothesis in two aims. In aim one I will analyze anatomically the number, cell type, and distribution of CPNs and their synaptic partners in Cdkl5 knockout mice. Training for this aim will be provided by imaging core facilities and Dr. Michela Fagiolini who is an expert in visual cortical structure and development. In aim two I will examine physiologically the neuronal activity and dynamics of visual cortical circuits with and without modulation of CPNs in the visual cortex of freely behaving Cdkl5 knockout and littermate WT mice. Training for this aim will be overseen by Dr. Michela Fagiolini, as well as the animal behavior and physiology core. Additional mentorship will be provided by Dr. Heather Olson as the head of CDKl5 clinic at Boston Children’s Hospital and by Dr. Bo Zhang on statistical technique and rigor. Together these aims will provide critical insight into the role of interhemispheric connectivity in cortical visual impairment in CDD opening the door to innovations in therapeutics.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Diffusion-weighted magnetic resonance imaging (dMRI) is the most promising tool for studying brain microstructure. However, the application of dMRI to the assessment of fetal brain in-utero is challenged by unpredictable motion, low signal-to-noise ratio, low spatial resolution, and imaging artifacts. While much effort has been spent on improving image acquisition and motion compensation techniques, data processing and analysis methods have remained largely unchanged. Existing biomarker estimation methods in fetal dMRI suffer from low accuracy and low reproducibility. Moreover, cross-subject and population studies require delineation of white matter (WM) tracts, which currently can only be performed via highly subjective and time-consuming manual segmentation. These shortcomings have significantly limited our ability to study the brain at this critical stage and to detect subtle changes in brain microstructure due to disorders. This proposed project will develop and validate a new generation of methods for analysis of fetal dMRI data. Unlike existing methods, which are based on biophysical models of the diffusion signal and mathematical model fitting, the new methods will rely on data-driven and machine learning techniques. Building on our pioneering works that have shown the potential of these methods, we will develop deep learning techniques for estimating microstructural biomarkers such as fractional anisotropy, neurite orientation dispersion, and fiber orientation distribution. The new methods will be based on two-stage transformer networks, which will be trained using dMRI data from preterm infants and fetuses. Moreover, we will develop methods that work with undersampled scans and provide a calibrated measure of estimation uncertainty. We will develop convolutional neural networks to segment WM tracts in the fetal brain based on the local fiber orientations. To address the noise in the input and target labels, we will build on our prior works on segmentation with noisy data and labels, shape-aware segmentation, and use of uncertainty to improve segmentation accuracy. The new technique will generate tracts automatically, with tracts that are indistinguishable from those created by the best human experts. We will evaluate the new methods using test-retest and bootstrapping methods and via assessment by experts in fetal brain microstructure and with histological knowledge of transient fetal fiber pathways. The new methods will enable assessment of fetal brain microstructure and the impact of neurodevelopmental disorders on tract-specific microstructure with a level of accuracy, detail, and reproducibility that is currently beyond reach. To definitively demonstrate the value and significance of the new methods, we will use them to assess the alterations in WM micro-structure due to congenital heart disease (CHD), which is the most common birth defect. In the process, we will produce the most comprehensive and detailed picture of the impact of CHD on the fetal brain microstructure ever attempted.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY Combined antiretroviral therapy (ART) has revolutionized the treatment of HIV but ART regimens are not without drawbacks. Cost, the need for daily administration, side effects, and social stigma all contribute to reduced patient compliance. Moreover, despite treatment, some 15-55% of people living with HIV will develop some form of HIV-associated neurocognitive disorder (HAND). Because of these problems with ART regimens, we and many other investigators have been studying the use of recombinant adeno-associated virus (rAAV) gene therapy vectors to deliver antibodies and other HIV therapeutics to people living with HIV. Because expression from these vectors is essentially permanent; patients could be protected for life from HIV infections with only a single AAV treatment (i.e. a ‘functional’ cure). Although numerous nonhuman primate experiments and two human clinical trials have been conducted to study the use of AAV for functional cure, however, we are unaware of any efforts to determine whether or not rAAV-expressed biologics can prevent or treat HAND. We have developed an anti-HIV biologic called eCD4, a fusion of CD4-Ig with a carboxy-terminal co-receptor (CCR5/CXCR4) mimetic peptide. We hypothesize that eCD4 is uniquely suited to preventing replication of the neurotropic strains of HIV that preferentially infect the brain (macrophage-tropic isolates) because these viruses necessarily evolve high affinity for CD4 to compensate for the low abundance of CD4 on macrophages and microglia. The organizing hypothesis of this project, then, is to determine if rAAV-delivered eCD4, either expressed from the periphery or within the central nervous system, can prevent or treat HAND. To test this hypothesis, we will use a pigtail macaque model of SIV-induced central nervous system disease, developed in our laboratories, in which co-infection with an immunosuppressive swarm (SIV/DeltaB670) and neurotropic clone (SIV/17E-Fr) establishes a highly reproducible CNS infection. Animals will be treated with ART until aviremic and rAAV will be used to deliver a pigtail macaque version of eCD4 to skeletal muscle and/or brain tissue. ART will be withdrawn to determine whether or not rAAV/eCD4 can prevent the re-emergence of CNS viremia. If successful, these studies may open new avenues to the functional cure of HIV and treatment of HAND.

NIH Research Projects · FY 2026 · 2023-05

Innate immunity is an absolutely essential host defense mechanism that, if perturbed, can itself cause a large number of human diseases. Among innate immune defense mechanisms, inflammasomes are cytosolic supramolecular complexes that recruit and activate inflammatory caspases, in particular caspase-1, to mediate proteolytic maturation of proinflammatory cytokines in the IL-1 family, and induce the rapid inflammatory form of cell death known as pyroptosis. Cytokine release and pyroptosis both signal danger to the rest of the immune system and pyroptosis kills infected or damaged cells to curtail the spread of the disease. NLRP3 is the inflammasome sensor that has caught the attention of the field and is emerging to be a general sensor of membrane damage and cellular stress, induced by pathogens and endogenous danger signals such as bacterial toxin nigericin, extracellular ATP, uric acid crystals, cholesterol crystals, hyaluronan and amyloid-β fibrils. Uric acid crystal-induced inflammasome activation is causal to severe joint inflammation in gout, and other stimuli could contribute to cardiovascular and neurodegenerative diseases. NLRP3 has a tripartite organization with an N- terminal effector domain known as PYD, a central nucleotide-binding ATPase domain (NBD, also known as NACHT) and a C-terminal LRR domain. Upon activation, NLRP3 recruits the adaptor ASC through PYD-PYD interactions and ASC further recruits caspase-1 through CARD-CARD interactions to induce proximity-promoted caspase dimerization and activation. Despite the great academic and clinical interest on NLRP3, the molecular pathway and mechanism for NLRP3 activation remain unclear, likely due to the complicated conformational transitions and intracellular trafficking that are just beginning to be elucidated. In this application, we propose to elucidate the functional mechanism of NLRP3 inflammasome activation by investigating the conformational transitions and intracellular trafficking.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Good hearing and cognitive skills are critical to maintaining later-life quality yet decline with advancing age. Hearing thresholds and cognitive abilities are correlated, and a diagnosis of age-related sensorineural hearing loss increases risk for Alzheimer’s disease and related dementias. These findings suggest that shared genetic and environmental factors influence presbycusis, cognitive decline, and dementia risk. We call this the “com- mon pathway” hypothesis. In our previous study of individuals from randomly selected Mexican American pedi- grees, we found genetic correlations between hearing and cognitive abilities, providing support for the common pathway hypothesis. However, the specific common genetic and environmental pathways have yet to be identi- fied. Here, we propose to measure hearing abilities, cognitive abilities, and putative dementia biomarkers (t- tau, p-tau, Aβ42/40, and NF-L) in an additional 600 Mexican American participants, increasing our total sample size to 1,300 and thereby providing sufficient power for further analyses, including identification of specific ge- netic/environmental risk factors. Our specific aims are (1) to quantify age-related and shared genetic influences on hearing, cognition, and dementia biomarkers; (2) to interrogate whole-genome sequence data, medical in- formation, and geospatial data in order to identify specific genetic and environmental influences on these traits; (3) to provide direct evidence of pleiotropy between hearing loss and dementia risk per se; and (4) to validate measures of cochlear synaptopathy, a physiological early warning sign of hearing loss that is undetectable via traditional hearing tests, as phenotypes for future aging studies. Measuring hearing abilities, cognitive abilities, and dementia biomarkers together in the same individuals may dramatically improve the odds of delineating specific factors influencing one or more of these crucial aspects of aging. Such discoveries may in turn provide insights and strategies for increasing the numbers of Americans who successfully age. Drs. Samuel Mathias and David Glahn at Boston Children’s Hospital and Dr. John Blangero at University of Texas Rio Grande Val- ley are co-principal investigators on this application. Dr. Amy Garrett at University of Texas Health Science Center San Antonio will lead a subcontract. Given the wealth of phenotypic, environmental and genetic data already available in this cohort, the proposed study represents a readily available, cost-effective, and powerful resource for elucidating the mechanisms of aging.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Transcriptional cis-regulatory elements (CREs), such as enhancers and promoters, play an essential role in all biological processes by controlling the expression of their target genes. Sequence variants in these CREs can perturb their target gene expression by altering the binding of transcription factors (TF). It is now clear that the substantial risk is encoded within these noncoding regulatory variants in most human disorders. However, systematic identification of regulatory variants and their causative transcriptional machinery for human diseases remains challenging. Over the past decade, we have pioneered to solve these important problems and have made significant progress in developing machine-learning-based methods to predict CREs (gkm-SVM) and regulatory variants (deltaSVM) from DNA sequence. We recently demonstrated that these regulatory variants predicted by deltaSVM significantly contribute to the heritability of human traits and diseases in a tissue- and cell-specific way. Here, we will extend these methodologies to further improve the discovery of regulatory variants in the human genome and explore their contribution to human diseases and traits. Toward this end, we will employ a two-step training approach. We will first build multiple sequence-based models to predict regulatory variants trained on a compendium of genomic data. We will then train ensemble models to find optimal combinations of these models to predict experimentally identified regulatory variants that exhibit allelic imbalance in chromatin accessibility. Uniquely, we will build this model in a cell-type resolved manner using human kidney single-cell chromatin accessibility data. Next, we will systematically assess these models using a broad range of human traits and diseases from well-powered genome-wide association studies (GWAS). We will then computationally identify targeted genes of these predicted regulatory variants and prioritize genes based on their contribution to traits and diseases relevant to tissues and cells using co-localization analyses. Lastly, we will experimentally validate these putative regulatory variants with massively parallel reporter assays and their predicted target genes with multiple CRE deletion experiments using CRISPR-cas9. As an exemplar, we will focus on kidney traits and use kidney relevant cell lines for these validation experiments. Our framework will enable us to further improve regulatory variation discovery and ultimately help us better understand how gene regulatory mechanisms are perturbed in human diseases and trait variation.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract: Investigating the origin of cell types is key to understanding basic processes in developmental biology and to enable in vitro production of cells and tissues for therapeutic benefit. While major advances in our understanding the ontogeny of hematopoietic cells have been made, significant technical limitations have precluded us from having a full picture of the lineage relationships of blood cells during development. For instance, the prevailing view in the field is that hematopoietic stem cells (HSCs) that emerge in the embryo are the cells responsible for lifelong blood production in the mammal. However, limited data exist that analyse the long-term fate of the earliest hematopoietic progenitors entirely in situ. Furthermore, the field still lacks a conclusive understanding of the true anatomical site of origin of the cells that will become the lifelong HSCs/progenitors in the adult. The Camargo and Hormoz laboratories have pioneered the use of in situ mammalian barcoding strategies to perform lineage tracing at the single cell level. Our proposal here aims to utilize these systems investigate the developmental origins of hematopoiesis. Our application is based on our identification of a population of non-transplantable embryonic multipotent progenitors (eMPPs) that contribute long-term to post-natal hematopoiesis. These findings imply that progenitor populations in addition to traditional HSCs have an active and substantial contribution to adult multilineage blood production. Our first goal in this application is to further characterize eMPPs molecularly and functionally. We will also explore the idea that functional differences in hematopoietic lineages can arise based on their eMPP or HSC ontogeny. Finally, we will extend the use of our barcoding approaches to broadly and unbiasedly characterize developmental waves of blood production and their exact site of origin. Our work has the potential to uncover basic mechanisms of blood development that could be useful for therapeutic manipulation.

NIH Research Projects · FY 2026 · 2023-05

Project Summary Epilepsy affects about 1% of people, and one-third of cases do not respond effectively to drug treatment. Patients with drug-resistant epilepsy are candidates for surgical resection of the epileptogenic zone, a potentially curative treatment. Clinical functional MRI plays a critical role in planning for neurosurgery in epilepsy. FMRI provides data to localize eloquent cortex, to assess the risks and benefits of a planned surgical resection, and to allow a resection to be tailored to the individual patient. The primary challenge to acquiring high quality functional MRI is motion of the participant. Motion reduces the temporal signal-to-noise ratio (tSNR) by misaligning the BOLD signal, motion creates spin history artifact, and motion can move parts of the brain out of the imaging field of view. These artifacts in turn lead to both false positive and false negative detections of functional activity, which compromise the fidelity of functional localization. This is usually detected and corrected to the extent possible, by discarding motion corrupted data, and using only motion-free segments. Since sufficient data must be acquired for such an analysis, fMRI acquisitions are designed to acquire redundant data to allow for loss to motion. At our institution, and others, this additional imaging time alone has been estimated to more than double the cost of fMRI imaging studies. The loss of fidelity and increased cost due to motion compromises the utility of the fMRI in planning for surgery. This is especially critical in patients who have difficulty following instructions, such as elderly, ill, or pediatric subjects. There is an unmet need for improved motion monitoring, prospective and retrospective correction for motion for fMRI. To improve the utility and decrease the cost of fMRI, we propose to develop, apply and evaluate novel technology to enable real-time self-navigated motion monitoring and improved correction for fMRI, through the following four specific aims: Aim 1: Develop and evaluate reduction of motion enabled by real-time slice-by-slice motion monitoring during fMRI; Aim 2: Develop and evaluate the reduction of motion artifact from slice by slice retrospective motion correction; Aim 3: Develop and evaluate the reduction of motion artifact from real-time slice by slice prospective motion correction (PMC); Aim 4: Assess the utility of motion monitoring, retrospective motion correction and prospective motion correction for improving functional MRI for planning for epilepsy surgery.

NIH Research Projects · FY 2026 · 2023-05

Project Summary/Abstract Rheumatoid arthritis (RA) is a prevalent autoimmune disorder characterized by chronic inflammatory processes that lead to joint destruction. Immunologically, autoreactive CD4+ T cells play a central role in disease pathology and activate B cells leading to pathologic lymphoid aggregates within the synovium and autoantibody production. Although identifying the molecular targets recognized by pathogenic CD4+ T cells is a critical first step in understanding the molecular basis of RA, we still do not know the antigenic targets for the vast majority of synovial CD4+ T cells and how such reactivities relate to autoantibody responses. We have developed a pipeline for CD4+ T cell antigen discovery in RA that relies on a new, cell-based genetic-screening technology that enables mapping of TCR specificities at genome scale. Based on our preliminary single-cell transcriptomic data, we have identified several interesting CD4+ T cell populations in synovial fluid that are clonally expanded and have begun to discover their TCR targets. This proposal is a five-year research and training plan with a scientific focus on identifying the antigenic targets of clonally expanded CD4+ T cells from RA synovium and understanding how such antigens relate to autoantibody responses. We propose in Aim 1 to map the antigenic epitopes and assess the corresponding HLA-restriction of clonally expanded synovial CD4+ TCRs by performing peptidome-wide antigen discovery screens. Aim 2 dissects T cell-B cell collaboration in the arthritic joint by interrogating antibody repertoire binding specificities and performing CD4+ T cell-B cell co-culture assays. Finally in Aim 3, we will engineer an antigen discovery platform to enable our ability to uncover synovial TCR reactivities against citrullinated-peptide antigens, a prominent post-translational modification observed in RA. This study combines cutting-edge genetic and transcriptomic technologies with mechanistic work to critically evaluate the antigen-specific landscape of RA. It will provide the candidate new training in several scientific areas to pursue translational immunology research. The candidate’s immediate career development goals are to gain experience with bioinformatic analysis, antibody profiling technologies, and human immunology assays. A specific career development plan is described by both the candidate and the mentors, Dr. Stephen Elledge, an expert in functional genomics and technology development, and Dr. Michael Brenner MD, an expert in lymphocyte biology and RA, taking advantage of the powerful resources available at Brigham and Women’s Hospital and Harvard Medical School. The candidate’s long-term career goal is to attain a tenure-track faculty position leading a diverse group of collaborative scientists dedicated to studying antigen specific immune responses in rheumatic and autoimmune diseases and their potential applications for therapy.

NIH Research Projects · FY 2025 · 2023-05

SUMMARY Type one diabetes mellitus (T1D) is a debilitating disease with no cure. After an initial partial remission with improved residual β-cell function, the “honeymoon period”, less than 17% of children achieve the glycemic targets recommended by the American Diabetes Association (ADA), placing millions at risk for complications and early mortality. Conversely, tight glycemic control - as achieved with greater insulin doses to match carbohydrates consumed - has also been linked to complications, namely weight-gain, insulin resistance, and metabolic syndrome. Preliminary evidence suggests that a very low-carbohydrate, high-fat, (i.e., ketogenic diet, KD) in T1D may (1) improve glycemic control by mitigating postprandial glycemic excursions and (2) reduce insulin exposure and associated adverse effects on peripheral tissues. Children with incident T1D may experience additional benefits, as a KD may also (3) prolong the honeymoon period - as seen in case reports - via immune and/or metabolic effects on β-cell function. Specifically, improved glycemia and insulinemia may promote β-cell rest, and the physiologically elevated β-hydroxybutyrate (βOHB) levels on a KD have been linked to decreased inflammation and gut microbiome changes that may reduce ß-cell autoimmunity. We propose to test the hypothesis that a KD vs. standard diet (SD) will prolong diabetes remission and improve diabetes control in children with incident T1D. In a 9-months parallel randomized controlled trial (RCT), fifty-two children (26 per arm) aged 5-12 years will receive a family-based intervention with food deliveries and intensive nutrition and diabetes education to promote a KD vs SD. Continuous glucose monitoring (CGM) and Bluetooth enabled insulin pens will be used for cloud-based data collection. Anthropometrics, fasting biomarkers and stimulated C-peptide area under the curve following a mixed meal tolerance test will will be assessed at baseline, 1, 5 and 9 months. The primary endpoint will be percent change in stimulated C-peptide between 1 and 9 months. Secondary endpoints will include percent children with clinical diabetes remission (insulin dose adjusted HbA1c [IADD1c] <9) at 9 months, indices of glycemic control from continuous glucose monitoring, and markers of metabolic health (BMI, indices of insulin resistance, and the ratio of triglycerides to HDL cholesterol). To explore pathways related to improved ß-cell function, we will also evaluate gut microbiome, metabolome, and immunologic biomarker responses to a KD vs SD and test interactions of targeted biomarker groups with changes in β-cell function and glycemia. Compared with technological and pharmacological treatments, dietary intervention is inexpensive, relatively free of major side-effects and directly translatable. A KD may have benefits on ß-cell function, glycemia and insulinemia, and would thereby provide a major therapeutic advance for children living with T1D. Regardless of outcome, our research will close an important knowledge gap on the safety and efficacy of a KD for children with T1D, an approach with increasing patient popularity despite lack of high-quality research.

NIH Research Projects · FY 2026 · 2023-04

Project Summary The goal of this project is to enhance the capabilities of diffusion-weighted magnetic resonance imaging (dMRI)for neonatal and pediatric subjects. Currently, dMRI is the only viable non-invasive method for probing brain microstructure. The past two decades have witnessed development of more powerful and more complex modelsof brain microstructure based on dMRI signal. Unfortunately, accurate and reliable estimation of these models require large numbers of high-quality measurements, which may be difficult or impossible to obtain in neonatal and pediatric subjects. Therefore, there is an urgent need for methods that can accurately and robustly estimatethe micro-structural biomarkers from reduced and low-quality measurements. To address this need, this researchwill develop and validate data-driven and machine learning (ML) techniques methods for estimating dMRI biomarkers for neonatal and pediatric subjects. The potential of these methods has greatly increased by the availability of large high-quality dMRI datasets such as the Human Connectome Project (HCP) data. Recent works, including our own studies, have demonstrated that ML techniques have a great potential to overcome limitations of the existing analysis tools and to achieve superior estimation accuracy. This research will substantially extend our preliminary work and generate important new capabilities that currently do not exist. Specifically, we will develop and validate novel methods for estimating important micro- structural models and biomarkers, ranging from diffusion tensor to advanced multi-compartment models, with far fewer measurements.In this regard, the two main novel aspects of our work will include 1) the use of spatio- temporal atlases to improvethe accuracy of subject-level analysis and 2) development of new deep neural network architectures based on self-attention. Furthermore, we will develop new techniques for enhancing the reliability, robustness, and explainability of ML methods for dMRI analysis. This will include techniques for computing well-calibrated uncertainty estimations, techniques that can detect corrupt, noisy, and out-of- distribution measurements, and techniques that enable interpretation and explanation of the predictions of these ML methods. We will evaluate the new methods using test-retest and bootstrapping methods and via assessment by experts in brain anatomyand micro-structure. The methods developed in this research will enable quantitative assessment of neonatal and pediatric brain micro-structure and the impact of developmental factors and neurological disorders at thesecritical stages in brain development with accuracy, detail, and reproducibility that is currently beyond reach.

NIH Research Projects · FY 2026 · 2023-04

Project summary Somatic mutations accumulate in normal tissues and are increasingly recognized as a crucial determinant of disease risk, especially in age-related conditions and cancer. Somatic mutations show enrichment in portions of the noncoding genome that show “open” chromatin structure, such as active promoter and enhancer elements, because open chromatin is more vulnerable to mutagens. Furthermore, transcription factors binding appears to obstruct DNA repair, increasing the likelihood of forming fixed, double- stranded mutations. The channeling effects of these mechanisms result in a concentration of somatic mutations in restricted, yet critical, regions of the genome. Somatic mutations with an increased likelihood of causing diseases frequently arise at recurrent genomic sites, and often even recurrent mutations at specific bases, allowing for the development of targeted methods with greater sensitivity, lower cost, and higher throughput to identify somatic mutations than traditional sequencing techniques. Present methods for identifying somatic mutations generally utilize deep (≥250X) whole genome sequencing (WGS) and tend to be expensive, create large datasets that are computationally challenging to analyze, and have limited ability to detect somatic variants with very low allele fractions. We propose a two phase approach to developing a new tool to address these shortcomings. In the first phase we will develop a method of detecting somatic mutations using ATAC-seq. ATAC-seq targets the open chromatin regions of the genome so is focused on regions with increased somatic mutations that have an increased likelihood of being biologically meaningful, only incorporates a fraction of the genome creating a more manageable dataset, and allows for deeper sequencing to increase the sensitivity of somatic mutation detection. This phase of the proposal includes three aims: modification of the ATAC-seq protocol to allow for detection of somatic mutations; development of analysis software to analyze the data; and testing of the protocol. In the second phase of the protocol, data obtained from phase one will be used to develop a panel sequencing protocol to further narrow the genomic regions looked at, reduce the cost of the analysis, and allow for extracted DNA to be directly analyzed (rather than the intact chromatin needed for ATAC-seq). This phase will also involve three aims: expansion of ATAC-seq analysis to determine the best regions to include on the sequencing panel; development of the sequencing panel; and testing of the panel on a range of individuals and tissue types. This project will provide rapid and inexpensive methods for the detection of potentially critical somatic mutations in any tissue type. At a research level, it will allow for the analysis of a large number of samples to provide critical information on biologically important somatic mutations and thus be an important tool that will help illuminate the spectrum of somatic mutation in the noncoding genome.

NIH Research Projects · FY 2026 · 2023-04

Our decade of experience in developing experimental and computational tools to study somatic mutations has revealed temporal, cell-type specific, and mechanistic patterns of somatic mutations across multiple tissues and across the human lifespan. Yet our view of somatic mutations has been limited due to cost. The field of somatic mutations requires a technique with the flexibility of bulk tissue with the allele- and molecule-specific power of single-cell techniques. To build the unified somatic variants catalog of human genome the proposed research aims to develop a Tn5 based duplex sequencing (Tn5-duplex-seq) method, a scalable single-well workflow to capture the landscape of somatic mutations at the single-molecule level from a range of cell or DNA input using Tn5 transposase, which resolves complementary strands by strand orientation from simple PCR-based capture. Duplex sequencing, the capture of complementary strand information in single molecules, offers unprecedented accuracy for detection of somatic mutations, but can be error-prone or laborious depending on the method of capturing complementary strands. The core concept of Tn5-duplex-seq has been successfully applied to single- cells with high accuracy (META-CS) but requires optimization. In the proposed study we aim to 1) determine parameters for pooled cell and bulk DNA sequencing to retain single-molecule information and cost- effectiveness, and 2) harness the power of single-molecule duplex detection which provides the basis for a suite of analytical tools to evaluate different types of somatic mutation including single nucleotide variants, indels, microsatellites and copy number variants with high accuracy. Tn5-duplex-seq can transform our understanding of somatic mutations through the innate comprehensiveness of this technique: capturing both clonal as well as the rarest somatic mutations and providing the basis of accuracy for analytical approaches to determine both somatic single base and structural variation. Ultimately, our tool will be accessible, cost-effective, and scalable to be used by the larger scientific community and easily integrated with pipelines of the Somatic Mosaicism across Human Tissues (SMaHT) Network. Tn5- duplex-seq with comprehensive profiling of genetic mutations has the potential to answer fundamental questions about our genomes in human biology and medicine. This study will reveal the landscape of somatic mutations and their accumulation in human tissue at single-molecule resolution, enabling detection of both clonal and cell-specific somatic mutations using a flexible-input workflow. The proposed research is significant for the comprehensive, results-based development of strategies for discovering the mutational burden and landscape in human development and aging. Together with the planned characterization of mutational signatures, the anticipated results should provide knowledge that may help to develop new strategies for preventing aging and disease progression.

NIH Research Projects · FY 2026 · 2023-04

Pediatric neurodevelopmental disorders (NDD), including pediatric epilepsy, autism spectrum disorder, and intellectual disability, represent a major source of morbidity, yet our therapeutic options remain limited. Development of novel therapies for NDD will require a deeper mechanistic understanding of normal and abnormal brain development. This proposal focuses on genetic programs that are activated in response to neuronal activity and that are fundamental to normal neurodevelopment and on-going neuronal plasticity throughout life. Fos is a major activity-dependent transcription factor that binds to distal enhancer elements and regulates downstream activity-dependent genetic programs in a cell-type-specific manner to promote key processes, including synaptic pruning and the recruitment of inhibition in the developing brain. Despite its role in key developmental processes, we do not understand how Fos is differentially targeted to cell-type-specific binding sites, nor how genetic variation at these sites impacts neurodevelopment and neuronal function. Interestingly, Fos has been shown to physically interact with the BAF chromatin remodeling complex, and it is possible that this interaction is critical to Fos function. Many BAF subunits, most frequently ARID1B, are implicated in human NDD, but whether the BAF complex regulates neuronal activity-dependent genetic programs, and how this underlies aspects of BAF complex-related NDD, is previously unexplored. The research in this proposal will address these gaps in knowledge by: (a) profiling Fos and BAF complex neuronal binding sites across the human genome; (b) assessing human genetic variants at these sites in individuals with NDD vs controls; and (c) determining the effects of BAF complex perturbation on neuronal activity-dependent genetic programs in vitro and in vivo. Overall, this work will lead to greater insight into how activity-dependent genetic programs contribute to NDD pathogenesis. Additionally, by identifying specific activity-regulated genes and pathways that are mis-regulated downstream of Fos and the BAF complex, these experiments could highlight novel therapeutic targets for NDD. This research is the basis for a five-year career development program designed to build on Dr. Trowbridge’s background in molecular neuroscience, pediatric neurology/epilepsy, and neurogenetics, by providing her with additional training in analysis of human sequencing data, use of in vitro and in vivo models of NDD, and next- generation sequencing technologies. Her primary mentor, Dr. Mike Greenberg, and her scientific advisory committee, Drs. Annapurna Poduri and Chris Walsh, will provide guidance in these areas, as well as mentorship in the rigorous and ethical conduct of translational neuroscience research. Thus the proposed training plan will position Dr. Trowbridge to launch her independent career as a clinician-scientist focused on understanding the role of activity-dependent genetic programs in NDD.