Univ Of North Carolina Chapel Hill

NIH Research Projects · FY 2025 · 2024-09

Modified Project Summary/Abstract Section More than 10 million youth in the United States live in poverty and lack access to neighborhood and material resources. Experiences associated with poverty and neighborhood resource scarcity are well known to confer risk for adverse mental health outcomes in part by shaping neural structure and function. At the same time, brain development is highly plastic and shifts to meet the needs of the environment. Despite emerging research finding behavioral and neural differences between adversity and non-adversity exposed youth – differences that reflect adaptive developmental processes – few research studies have explored the processes that develop to support youth in navigating environments marked by resource scarcity. For individuals who lack material resources, cognitive processes are likely to be shifted towards meeting the environmental challenge of obtaining those material resources needed for day-to-day survival. One way in which individuals impacted by resource scarcity achieve this is through forming, utilizing, and maintaining social relationships. Social relationships provide a range of resources, including social capital and social support. Underlying the maintenance of supportive social relationships is mentalizing– the ability to understand and take the perspectives of others’ thoughts and emotions. Based on available evidence, we hypothesize that increased engagement in mentalizing serves as a protective factor against psychopathology for adolescents who lack access to neighborhood and material resources. The proposed project will leverage a multi-modal longitudinal study to examine the impacts of neighborhood resource availability on mentalizing, cognitive empathy, and associated neural correlates that may support the acquisition of social support and promote positive mental health outcomes. Three aims guide this research: (1) exploring how availability of resources impacts mentalizing, associated neural networks, and cognitive empathy, (2) examining how mentalizing supports the maintenance of social support, and (3) evaluating mentalizing and cognitive empathy as an underlying protective mechanism against psychopathology among youth who lack access to neighborhood and material resources. These aims will be addressed by leveraging a large study of adolescent youth (expected n = 275) who will participate in a passive neuroimaging task and complete behavioral and self-report measures. Resources that will support this research include access to this longitudinal sample, multiple research facilities, and a strong mentorship team with expertise in developmental neuroscience and resilience. The proposed research will enrich theories regarding how neurodevelopmental processes are shaped by the environmental experience and identify protective factors that can be leveraged to inform mental health treatments.

NIH Research Projects · FY 2024 · 2024-09

Abstract: Unraveling the molecular mechanisms that link SNPs and genes identified in GWAS studies to the disease is a challenge that must be overcome to translate these genetic discoveries into actionable health insights. We would like to build a machine learning tool on the basis of visible neural networks (vNN) that recently showed success to provide predictive and explanatory power on cellular responses to gene regulations or disease treatment. Key to the vNN approach is an understandable network such as Knowledge Graph that contains rich annotations of relevant entities and relationships among entities organized by integrative data sources. Our hypothesis is that organizing and integrating data from the Common Fund Data Ecosystem (CFDE) can enhance the explanatory power of vNN to illuminate GWAS results. We propose combining the ROBOKOP Knowledge Graph with diverse biological data from the CFDE, and vNN as a knowledge-based architecture to provide high interpretability in supervised learning. ROBOKOP Knowledge Graph will serve as an organizational hub for integrating CFDE data with existing knowledge. This query-able resource for CFDE data will be our first deliverable. We will extract network-based relationships from this data and train vNN using genotypes and phenotypes from T2D-focused GWAS, providing our second deliverable. The trained vNN, our third deliverable, will enable the prediction of T2D phenotypes from genotype data. Lastly, we'll provide the code base as a platform to expand this KG and vNN approach to other GWAS studies and potentially be generalized for genome wide ‘omic analyses with large data sets.

NIH Research Projects · FY 2025 · 2024-09

NOT APPLICABLE no Abstract

NIH Research Projects · FY 2024 · 2024-09

Abstract In utero exposures to insults such as pregestational diabetes can have significant repercussions for proper growth and development. Exposure to the diabetic milieu can lead to diabetic embryopathy, a disruption in normal cellular programs during critical stages of development which leads to congenital anomalies affecting multiple organs. Kidney anomalies resulting from genetic and in utero perturbations are the leading cause of chronic kidney disease in children, markedly affecting quality of life. However, there is a significant gap in our understanding of how such insults affect these early developmental programs, precluding the generation of interventional strategies. The limitations of mouse models and differences between human and mouse kidney programs necessitate the development of alternate systems in which to study in utero exposures. Kidney organoids represent an attractive model for their recapitulation of developmental programs in an easily manipulatable environment. Yet, studies with kidney organoids thus far have almost exclusively focused on genetic diseases and injury affecting cells of the mature nephron. To this end, we will exploit iPSC-derived kidney organoids for their ability to recapitulate human developmental processes and test how in utero exposures to the pregestational diabetic milieu affect kidney development programs. We will focus on programs of nephron progenitors, the precursor of all the cells in the nephron, and podocytes, a nephron progenitor descendant critical to establishing and maintaining proper blood filtration. We will mimic the pregestational diabetic environment in culture and interrogate the effects on nephron progenitor programs by single cell (sc)RNA-seq and the assay for transposase-accessible chromatin with sequencing (ATAC-seq) as the organoids progress through this stage of differentiation (Aim 1). We will also assess the effects on maturing podocytes utilizing similar methodology (Aim 2). We expect to identify alterations to the normal differentiation programs of these cells and uncover critical pathways affected by the diabetic milieu. These findings will serve as the foundations for future studies and aid the development of novel interventional strategies then help mitigate the effects of in utero insults. Our studies will enable new directions for kidney organoid research, laying the groundwork for future studies into in utero insults and congenital anomalies in this ex vivo, manipulatable system.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Therapeutic Potential of Macromolecules: The human interactome, potentially comprising over 650,000 protein-protein interactions (PPIs), remains an underexplored frontier for therapeutics discovery. As just one example, the interaction between the melanoma antigen, MAGE-A4, and an E3 ligase, RAD18, is hypothesized to increase DNA damage tolerance, leading to increased resistance of cancer cells to chemotherapies. Disrupting this interaction could increase the efficacy of chemotherapies. Macromolecule therapeutics (e.g., peptides and peptidomimetics) are well-suited to disrupt disease-causing PPIs. However, their design is non-intuitive with challenges including entropic costs associated with protein binding and limited cell permeability. While existing strategies to decrease the entropic cost, like peptide stapling and cyclization, have yielded potent therapeutics, they often result in molecules locked in a rigid conformation that struggle to access their targets within cells due to poor permeability. Interestingly, some bioactive peptides and peptidomimetics have been identified benefit from some lack of rigidity for cell permeability. Identifying the role of macromolecule conformation, i.e., degree of disorder, in desirable properties including protein binding and cell permeability will facilitate the development of a new class of therapeutics. We propose that compact, yet non-rigid, macromolecules offer valuable, yet under exploited scaffolds for bioactive macromolecules. This proposal aims to address key biological questions: How does macromolecule conformation impact binding to “undruggable” protein surfaces and other essential characteristics, such as cell permeability? Accelerating Innovation in Macromolecule Therapeutic Design: Our vision is to accelerate the design of macromolecule-based therapeutics by developing rapid characterization strategies that guide predictive algorithms. These methods will provide unprecedented comparisons of macromolecule conformation, guiding the design of macromolecular therapeutics. Three key challenges are addressed in this proposal: 1. Conformational Characterization: We will establish colorimetric and fluorimetric assays to assess average conformation and dynamics of disordered peptides and peptidomimetics. 2. Predictive Algorithm Development: Develop algorithms capable of identifying sequence spaces that favor conformations with a desired degree of disorder. 3. Protein-Protein Interaction Disruption: By leveraging favorable sequences with desired conformations, we will identify effective, cell permeable, disruptors of protein-protein interactions using the MAGE-A4 and E3 ligase interaction as a model system. Innovation and Impact: We aim to provide powerful predictive capabilities that leverage rapid conformational characterization to guide the design of potent macromolecule therapeutics, specifically for PPI disruption.

NIH Research Projects · FY 2025 · 2024-09

Project Abstract Suicide is the second leading cause of death for youth aged 10-12. Recent data from the CDC show that girls have greater rates of STBs than boys—1 in 3 adolescent girls consider attempting suicide. However, we know relatively little about what factors contribute to risk for suicide in preteen girls aside from exposure to childhood adversities such as experiencing threat (e.g., interpersonal violence). Our team’s research program investigates biological responses to interpersonal stressors as a mechanism linking childhood threat exposure and suicidal thoughts and behaviors (STBs) in adolescent girls. However, to improve early identification and intervention efforts, there is a critical need to identify STB risk factors that precede this biological sensitivity to interpersonal stressors that occurs later in pubertal development. Social motivation (SM) toward peers and peripubertal hormones typically increase during preteen years prior to adolescent biological sensitization to interpersonal stressors. The purpose of the current study is to investigate SM as a novel risk factor linking childhood threat exposure with STB risk in preteen girls. Aim 1 will refine existing assessments of SM and STBs for use in a diverse set of preteen girls. Aim 2 will test our theory that maladaptive SM (e.g., high desire to avoid social failure and low effort to engage with others) contributes to preteen emergence of STBs, particularly among girls with advanced pubertal timing. Aim 3 will evaluate whether childhood threat exposure predicts both STBs and maladaptive SM in the preteen years. Aim 3 will also test our preteen STB risk pathway, which posits that childhood threat exposure increases risk for STBs via maladaptive SM. Finally, exploratory Aim 4 will examine whether this novel risk pathway is exacerbated in girls with advanced pubertal timing. We will enroll 200 preteens (ages 8-11) assigned female at birth, premenarchal at baseline, and enriched for childhood threat exposure. Assessments will occur at baseline, 6- and 12-months. All visits will have questionnaires/interviews assessing STBs, psychopathology, childhood threat exposure, and SM, along with SM behavioral assessments. Visits will include 4-week ecological momentary assessments (2x daily) and saliva acquisition (x6) for hormone assay. Two Advisory Boards will collaborate with us on measure refinement, data collection, and result dissemination: (1) adult community leaders and caregivers and (2) preteen/teen girls with lived experience relevant to this investigation. Understanding the proposed associations and mechanisms is a critical first step to identifying specific and focused targets for preventive interventions. Thus, the current research proposal will have a high impact on the field by contributing important knowledge to suicide prevention efforts in preteen girls.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The F99 portion of this proposal investigates the role of the brain enriched E3 ubiquitin ligase TRIM9 in melanoma progression. The K00 portion of this proposal investigates the interface of tumors and neurons, and how this interface influences cancer progression. Tumors are composed of multiple cell types and heterogenous cancer cells. Cellular heterogeneity may be attributed to the dedifferentiation of cancerous cells, where characteristics of their differentiated states are lost, and new ones acquired. Melanoma, a cancer of melanocytes, cells derived from the neural crest, has been suggested to have undergone dedifferentiation. Interestingly, the brain-enriched E3 ubiquitin ligase TRIM9 is highly expressed in aggressive melanoma patient samples and multiple melanoma cell lines. Intriguingly, neurons, which also express TRIM9, are also derived from the neural crest. The Gupton lab identified TRIM9 as a potent regulator of exocytosis, actin dynamics, and directed axon navigation in developing neurons. The lab found that TRIM9 negatively regulates the actin polymerase VASP via non-degradative ubiquitination and negatively regulates exocytosis by preventing SNARE complex formation. My preliminary data suggest that TRIM9 modulates focal adhesion morphology and number, cell size, random migration, directed migration in response to extracellular stiffness, proliferation, and protease secretion in melanoma. In addition, preliminary TRIM9 immunoprecipitation mass spectrometry experiments identified several potential protein interactors that regulate focal adhesion dynamics, cell cycle progression, and trafficking in melanoma. The goal of this predoctoral training proposal is to define the role of TRIM9 in focal adhesion dynamics, migration, and exocytosis in vitro and tumor growth and metastasis in vivo in melanoma. I will use a combination of cell and molecular biology techniques, biochemistry, microscopy, and a mouse model of melanoma. The interaction between cancerous cells and other cells in the tumor microenvironment has been suggested to influence tumor progression. In patients, innervated tumors are correlated with morbidity. In addition, neuronal activity in small cell lung cancer tumors increases proliferation and metastasis in mice. Although progress has been made in delineating the mechanisms that underly cancer- nerve interactions resulting in increased tumorigenesis and poor prognosis, there is much yet to understand. The postdoctoral training proposal aims to identify mechanisms by which the reciprocal interactions between neurons and cancer cells increase tumor progression. To identify mechanisms, I will use cellular and molecular biology techniques, biochemistry, and MS assays, gained in the F99 phase, in combination with neuroscience techniques, which I will gain in the K00 phase. Together, the predoctoral and postdoctoral work aim to answer how cellular phenotypes and cancer progression are affected by either gaining the expression of a brain- enriched E3 ubiquitin ligase or interactions between neurons and cancerous cells in the tumor microenvironment.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT In low- and middle-income countries, young people with HIV (YPWH) experience the compounded effects of HIV and poverty stigmas that jointly contribute to suboptimal mental health and HIV care continuum outcomes. Our experience conducting research with YPWH in Zambia - which ranks among the ten countries with the highest HIV prevalence worldwide - has revealed linkages between intersectional HIV and poverty stigmas and mental health problems, which contribute onward to impact antiretroviral therapy (ART) initiation, retention in care, and medication adherence problems among YPWH. In turn, attrition along the HIV care continuum drives high mortality among YPWH. Our formative work establishes the promise for microeconomic interventions with YPWH as a foundation to improve well-being in this population of young adults experiencing the co-occurring challenges of HIV and poverty. However, our formative work also highlights the need to address intersectional stigmas related to HIV and poverty among young adults, in addition to providing livelihood intervention activities, as crucial components to jointly improve the continuum of HIV care and mental health outcomes. In response to PAR-23-190, we propose a combined intervention addressing intersectional HIV and poverty stigmas and strengthening economic capabilities to improve HIV care continuum and mental health outcomes in YPWH in Zambia. This intervention (tentatively named Kupambana or “to overcome”) includes an 8-week program involving two experimental components: (i) stigma reduction support group, consisting of eight weekly group-based educational sessions addressing self-care, health self-efficacy, resilience and empowerment, and coping strategies to minimize internalized stigmas and (ii) vocational and entrepreneurship training (VET) voucher, worth 2,500 Zambian kwacha [ZMW] (or ~150 USD), to pay for training fees and allow YPWH to obtain employable skills; and one usual care component: (iii) a one-time financial literacy group-based educational session. This application has two phases: adaptation and pilot test. Phase 1 will include interviews with ~25 YPLH and 15-20 key stakeholders and formation of a youth advisory board to inform the adaptation and implementation process. Phase 2 will include evaluation of the feasibility, acceptability, implementation costs, and preliminary effects of the Kupambana program on HIV care continuum, mental health outcomes, and mechanisms of change such as stigma. We will randomly assign 100 YPWH to either experimental (Kupambana) or control (usual care) group. We will conduct baseline, end-of-program, and two follow-up assessments at 3- and 6- months after end- of-program. We will also conduct exit interviews to assess fidelity and implementation procedures. If this research shows promise, we will use the findings to support a sufficiently powered R01 application to implement a type 1 hybrid effectiveness-implementation.

NIH Research Projects · FY 2025 · 2024-09

Abstract Viruses are the etiological agents of a number of different human cancers. Epstein-Barr virus (EBV) is a ubiquitous pathogen infecting over 95% of the human population worldwide. EBV is the etiological agent of several malignancies including Hodgkin lymphoma (HL), and multiple types of non-Hodgkin lymphoma (NHL) including post-transplant lymphoproliferative disease (PTLD), diffuse large B cell lymphomas (DLBCL), T cell lymphomas, NK/T cell lymphoma (NKTL), and Burkitt's lymphoma (BL). In addition, EBV is also associated with nasopharyngeal carcinoma (NPC) and gastric cancer. There is an increased incidence of EBV-associated NHL in HIV-infected patients. HIV-positive individuals have a 10–20% lifetime risk of developing Burkitt lymphoma and EBV-positive NKTL is seen with increased frequency in HIV infection as well as transplant recipients. HIV-infected EBV-positive NKTL patients are also at a higher risk for death when compared to NKTL patients without HIV. We propose to understand how EBV is associated with multiple types of NHL in the HIV-infected population. EBV is present in each and every lymphoma cell, which implies that EBV contributes to the initiation, development, and maintenance of the tumor. Several of these EBV-associated lymphomas e.g. NKTL and BL are highly aggressive and NKTL show resistance to chemotherapeutics. Hence, new therapies are needed to target these NHL. We have found that EBV-associated NHL display high expression of a cell cycle whose expression is upregulated following EBV infection of naïve B cells. We hypothesize that EBV activates this kinase to drive tumor growth and survival. In this proposal, we aim to identify how EBV upregulates this kinase and the viral genes that may be responsible for this phenomenon. We will investigate the mechanisms by which EBV, a causal agent of human cancer, modulates cellular processes that contribute to the early events in the carcinogenesis of EBV-associated lymphomas. We will also determine whether inhibiting this kinase can hinder EBV-driven lymphomagenesis in vitro and in vivo, and we will investigate whether inhibitors of this kinase can function as a novel therapeutic target to hinder the development and pathology of EBV-driven lymphomas. The identification of novel therapeutic targets will help overcome the morbidity and mortality of EBV-associated lymphomas around the globe and may also be relevant to non-viral cancers.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Dialysis vascular access (arteriovenous fistulae [AVF] and arteriovenous grafts [AVG]) is the Lifeline for the 500,000+ patients on hemodialysis in the United States (3million+ worldwide). Unfortunately, both AVFs and AVGs have high failure rates due to an aggressive venous segment stenosis which results in multiple interventional procedures, hospitalizations, and increased catheter (CVC) use. This results in vascular access also being the Achilles Heel of hemodialysis and an important unmet clinical need which results in a very significant morbidity, mortality, and economic cost (over 5B USD per annum). In this proposal we will combine expertise and experience from both industry and academia to create an innovative vascular conduit (the Functional INtegrated Electrospun [FINE] device) that comprises an inner Cu-PAS core surrounded by an outer P4HB sheath that will have (a) a precisely controlled microstructure to promote host cell integration (b) anti-bacterial and immunomodulatory properties (c) vein-artery-matched geometry to minimize upstream injury pathways and (d) an intrinsic self-sealing ability that allows for early cannulation. At an operational level we plan to (a) selectively design, refine and prototype FINE devices (b) document the safety and efficacy of selected FINE devices in a pig arteriovenous access stenosis model and (c) develop clinical scale production capabilities for the FINE device. In summary, this proposal brings together a trio of unlikely partners (industry x 1 and academia x 2) that between them have the science, the infrastructure, the experience, and the people to bring disruptive change to dialysis vascular access. While none of us on our own could have done what is described in this proposal, we would also venture to say that the sum of the individual parts will in this case be exponentially larger than the whole.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Alcohol-related blackout has traditionally been considered a warning sign of the development of an Alcohol Use Disorder (AUD). However, not all individuals who binge drink experience blackouts, and blackouts are not uncommon among individuals considered social drinkers. While pharmacological and behavioral factors contribute to blackout risk, they are not fully predictive. Recent work in the field has begun to consider individual variability in blackout susceptibility, and the literature indicates that some individuals have a genetic predisposition to experiencing blackouts. Preliminary work in our lab has indicated that having a history of certain non-REM parasomnias classified as Disorders of Arousal (DoA) may increase one’s risk of experiencing blackouts. Disorders of arousal, a diagnostic category that includes sleepwalking, are defined by inappropriate motor arousal during slow-wave sleep. These arousal events are preceded by decreases in functional connectivity between the motor cortex and prefrontal and subcortical targets, which allow for wake- like activity in the motor cortex to occur simultaneously with slow-wave sleep elsewhere. Importantly, atypical connectivity patterns persist into waking life in these individuals. As preliminary EEG work in our lab has demonstrated abnormal patterns of resting-state EEG activity in individuals with a history of blackout, we hypothesize that susceptibility to blackout and susceptibility to disordered arousal during sleep may be mediated by similar baseline patterns of neural connectivity. Given the role of GABAergic interneurons in modulating cortical functional connectivity, we hypothesize that individual variability in the efficiency of GABAergic inhibition of the motor cortex directly contributes to blackout susceptibility. Preliminary work from our lab has also indicated that self-reported feelings of stimulation in response to alcohol and self-reported resilience to the motor effects of drinking (e.g., stumbling) are correlated with number of blackouts experienced. We propose a model in which some individuals express a neurobiological phenotype defined by reduced efficiency of GABAergic inhibition of the motor cortex, which results in unstable functional connectivity at sober baseline and a relative resilience of the motor cortex to GABA-A mediated suppression of activity by alcohol. When paired with steep elevations in blood alcohol concentration, we hypothesize that this phenotype contributes to functional dissociation of the motor cortex and allows for motor activity past the point at which long-term memory storage is impaired and behavior is disinhibited (i.e., the blackout state). The purpose of this project is to investigate the role of GABAergic modulation of the motor cortex in blackout susceptibility using non-invasive neurophysiological metrics to examine circuitry in individuals with a history of blackout and binge- drinking controls. Identifying the neurobiological phenotypes associated with vulnerability to blackout could pave the way for targeted interventions to reduce the significant physical and psychological risks associated with alcohol-related blackout.

NIH Research Projects · FY 2025 · 2024-09

Project Summary School readiness skills prior to entering elementary school are crucial for later academic success and, importantly, these skills may act as a protective factor for low-income students as they transition into Kindergarten and continue through elementary school. However, extant studies of school readiness before elementary school have largely defined and measured this construct in narrow and potentially inaccurate ways. For the most part, studies have focused on cognitive assessments, ignoring important social-emotional characteristics that are required to perform well in the classroom. For studies that have taken these factors into account, this is often assessed by parent or teacher report or in the home or lab settings, which ignore the way in which the demands of the classroom environment may affect functioning. In addition, these assessments are primarily behavioral; very few studies have attempted to understand the interplay of children’s psychophysiological functioning and their classroom experiences. Indeed, there is a substantial gap in our knowledge regarding how psychophysiological functioning may impact students’ school readiness and early school success; this is particularly salient if we seek to improve academic outcomes for low-income children through early education. To address this gap, a diverse sample of families will be recruited for the proposed short-term longitudinal study. We will enroll 270 children from approximately 30 classrooms prior to their final year of preschool (preK). Across preK, children will participate in several data collection visits including fall and spring school readiness assessments. In addition, on two occasions across the school year, we will observe children in the classroom while also collecting cardiac data. Finally, at the end of Kindergarten, several assessments of school success will be conducted. These data will be used to examine how teacher behavior and children’s respiratory sinus arrhythmia (RSA), a key indicator of psychophysiological regulation and function, work together to predict school readiness and early school success. This study will be the first to examine school readiness in a sample of low-income children that includes conducting naturalistic observations (and cardiac monitoring) of children and their teachers in the preK classroom at two timepoints, with a focus on the parasympathetic nervous system (PNS) as a mechanism that may play a critical role the way in which children respond differently to teacher sensitivity and behavior, and subsequently predict school readiness and school success in Kindergarten.

NIH Research Projects · FY 2025 · 2024-09

The proposed project aims to address those gaps and to support and enhance high-quality STEM learning opportunities for all young children. By creating partnerships between researchers and practitioners, the project has the potential to impact many young children and their caregivers’ opportunities to engage in and benefit from STEM learning.    The proposed science education partnership (SEP) consists of researchers and practitioners in an informal learning space collaborating together in the co-development and implementation of STEM playgroups for children with and without disabilities and their caregivers. Researchers will support the practitioner team to collect and analyze preliminary efficacy data relative to child-caregiver engagement, child STEM play skills, and caregiver feelings of self-efficacy, specifically related to embedding foundational computational thinking skills into their daily routines and activities. The project will last for two years, using a waitlist control design comparing groups receiving the intervention to those who are on the waitlist. Both quantitative data and qualitative data will be collected and analyzed in order to assess the playgroup’s preliminary efficacy and preliminary changes in caregiver perceptions following playgroup participation. Qualitative data will additionally be used for ongoing modifications and adaptations of the playgroup model and materials to ensure that they are useful and relevant to caregivers and children who are participating in them.  The findings and products from the proposed project could inform future STEM playgroups both in informal learning spaces and beyond, and will be freely available on the partners’ websites. Overall, the project has the potential to increase STEM learning opportunities for many children and caregivers.

NIH Research Projects · FY 2024 · 2024-09

ABSTRACT Biomedical researchers need to identify novel disease genes and understand disease mechanisms; clinicians need to diagnose diseases and optimize treatments. An improved understanding of the genetic basis of disease helps achieve both goals. The Monarch Initiative makes this possible by integrating the fragmented data landscape into the most comprehensive open collection of genotype-phenotype data in the world. Our Knowledge Graph (KG) links together clinical, biomedical, and basic science research data spanning multiple organisms, and supports reasoning across a wide range of organisms, body systems, and diseases. Monarch has achieved demonstrable clinical and translational success using model organism data to perform rare disease diagnosis and gene-to-disease discovery, and our resources have become global standards. In Phase II, we integrated the Human Phenotype Ontology (HPO) into the UMLS; enhanced our variant prioritization algorithms and Exomiser tool, which was applied to 30,000 patients in the National Health Service (UK); developed the Biolink Model/API; released new ontologies (Mondo unified disease ontology, Unified Phenotype Ontology (uPheno), and the Environmental Conditions and Treatments Ontology (ECTO)), raised the number of harmonized data sources in our KG to 34; and overhauled our web Portal. We will leverage this foundation to make Monarch more intuitive for a diversity of users and contexts in phase III as follows: Augment the Monarch Portal with new visualizations and tools. Guided by user requirements, iterative user testing, and feedback, we propose to enhance the user experience and Portal functionality, focusing our work on improvements to Navigation, Visualization, and Query. Evaluate, optimize, and enhance algorithms for disease diagnostics, cross-species inference, and gene-disease discovery. We will develop a comprehensive, modular evaluation framework, ‘PhEval,’ that will allow us to monitor the diagnostic yield and performance of cross-species inference as our ontologies and data graphs evolve. This will assist basic science researchers and clinicians to reveal cross-species mechanistic evidence and evaluate potential precision disease modeling strategies. Disseminate computational tools, data, services, and tutorials to a broad translational community. We will expand access to our KG to enable users to process the KG for different domains and use cases, through simplified downloads, APIs, software libraries, R packages, Jupyter notebooks, and Dockerized resources, along with training materials. This will better support bioinformaticians and other researchers in leveraging our KG and phenotype data in their analyses. The Monarch Initiative aims to significantly improve the utilization, accessibility, and value of animal models for disease diagnosis and discovery.

NIH Research Projects · FY 2025 · 2024-09

PROJECT ABSTRACT U.S. physicians are at risk for poor mental health and work-related wellbeing. Obstetrician- gynecologists (OB-GYNs) are at particularly high risk due to recent changes and uncertainty in state-level reproductive health policies. Associated changes in the work environment may lead to burnout, poor mental health, and job turnover, which could have major health consequences for OB-GYNs and their patients. Our scientific premise is that the key to improving OB- GYNs' mental health and work-related wellbeing in a shifting policy climate is to focus on the role of organizational factors in buffering the impact of policy stressors. Accordingly, there is an urgent need to (1) better understand the relationships between state policy environment, work-related stressors, and mental health and work-related wellbeing, and (2) identify organizational-level factors that support OB-GYNs' health and wellbeing. Leveraging our interdisciplinary team's expertise in workplace health and wellbeing, experience with survey methods and policy analysis, and our preliminary data, we propose a convergent mixed- methods study of OB-GYNs from across the U.S. We will administer a quantitative survey to 800-900 OB-GYNs to (1) test whether OB-GYNs' mental health, work-related wellbeing, and turnover intention vary by policy context, and (2) evaluate the nature of the relationship between policy context, work experiences, and mental health and work-related wellbeing (Aim 1). We will conduct qualitative semi-structured interviews with 80 OB-GYNs from a range of state policy climates. We will then use mixed methods to synthesize survey and interview findings about the contributions of specific organizational policies and practices to OB-GYN mental health and work-related wellbeing, within and across policy contexts (Aim 2). Finally, we will draw on Aim 1- 2 findings and solicit input from key informant interviews (n=10) to develop and disseminate organizational-level resources to support OB-GYNs' mental health and work-related wellbeing (Aim 3, r2p). We will engage an interdisciplinary expert advisory board to inform the design, analysis, and dissemination. Our long-term goal is to protect and promote the work-related wellbeing of the healthcare workforce. By generating novel data from a national sample of OB-GYNs, this study will inform evidence-based recommendations and interventions to support OB-GYN mental health and work-related wellbeing. Its impacts support NIOSH's Total Worker Health® and Healthcare Worker Mental Health Initiatives, the NORA Healthcare and Social Assistance sector, and NORA's cross-sector program in Healthy Work Design and Wellbeing.

NIH Research Projects · FY 2026 · 2024-09

PROJECT SUMMARY/ABSTRACT Glioblastoma represents one of the most lethal types of cancers. Despite extensive molecular characterization, precision medicine efforts have largely failed for glioblastoma therapy, suggesting that these complex tumors are resilient ecosystems that overcome singular therapeutic approaches. Aging is strongly associated with increased incidence and mortality of most cancers, including glioblastoma. However, the cellular and molecular mechanisms by which aging promotes tumor initiation and progression remain poorly understood. Tissue-specific stem cells contribute to development, renewal, and regeneration of most organs. Neural stem cells (NSCs) undergo little to no cell division during normal homeostasis but become activated by tissue injury with wound responses. NSCs reside in specialized niches that provide maintenance cues and regulate the balance between quiescence and proliferation. Inflammation and nutrient constraints in NSC niches in the aging brain promotes quiescence and decreased regenerative potential. Like the normal brain, glioblastomas contain cellular hierarchies with stem-like tumor cells at the apex. While controversial, these cancer stem cells contribute to therapeutic resistance, invasion into normal brain, generation of new vasculature, and evasion of anti-tumor immunity. Cancer stem cells display remarkable plasticity, rendering therapeutic targeting strategies challenging. In the proposed studies, the Rich laboratory will leverage insights from aging in the nervous system and the molecular circuitry of cancer stem cells to understand how aging promotes tumor growth through cell autonomous mechanisms and interactions with the microenvironment. We will leverage preliminary observations that cancer stem cells reprogram essential metabolic pathways through integration of extrinsic signals from tumor-infiltrating immune cells and vasculature to promote epigenetic reprogramming, empowering sustained proliferation, self-renewal, and immune evasion. Based on this background, we hypothesize that aging promotes reprogramming of the tumor immune microenvironment and cell autonomous nuclear structural regulation that will inform the development of novel brain tumor treatments. Using spatial transcriptomics and metabolomics of patient tumors and organoids, we will investigate the metabolic alterations within the aging tumor microenvironment to derive multi-modal therapies that disrupt the dynamic growth and survival mechanisms within tumors.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Clostridioides difficile is a gram-positive, spore-forming, obligate anaerobic bacterium and opportunistic pathogen. C. difficile infection (CDI) is associated with hospital antibiotic use and can cause severe diarrhea, pseudomembranous colitis, and death. The primary causative agents of CDI symptoms are the secreted toxins TcdA and TcdB, which are regulated by the flgB operon through sigma factor sigma-D. The bacterial transcription termination factor Rho regulates the flgB operon by repressing transcription only when an invertible genetic “switch” mapping to the flgB transcript 5’ UTR is in the “off” orientation. Genetic deletion of Rho functionality restores flgB expression when the switch is oriented off. Loss of Rho functionality in C. difficile results in growth, sporulation, and virulence defects independent of loss of regulation at the flgB operon, indicating additional Rho-dependent regulation of C. difficile virulence factors. Efforts to identify a Rho recognition sequence in C. difficile, including at the flagellar switch, based on a multi-organism consensus have been unsuccessful, possibly due to the unusually GC-poor C. difficile genome (~29%). Rho-dependent regulation is also likely to be highly influenced by interactions with termination/antitermination factors NusA and NusG. Nus factors are essential for C. difficile growth but their influence on Rho-dependent termination and effect on the virulence factors expression is unknown. I hypothesize that Rho is a key factor in C. difficile pathogenesis through direct regulation of critical virulence genes in addition to the flagellar operon, and that this regulation is dependent on, or attenuated by, Nus factors on a gene-by-gene basis. The objective of this proposal is to identify and annotate Rho, NusA, and NusG dependent terminators in C. difficile to identify key Rho and Nus factor regulated growth, sporulation, and virulence genes for further study, and determine a predictive rut site sequence in C. difficile. I will compare RNA-seq and Term-seq data between the Rho-null and control C. difficile strains to identify Rho dependent terminators and verify these sites in vitro. I will use CRISPRi to generate NusA and NusG dependent transcript-depleted strains for Term-seq analysis. Completion of this study will improve our understanding of C. difficile virulence regulation, provide further annotation of the C. difficile transcriptome, and may reveal new potential targets for developing therapeutics to combat CDI. Dr. Tamayo is a leader in the field of C. difficile biology and her expertise in genetic approaches complements my graduate training in biochemical techniques. The University of North Carolina at Chapel Hill and the Department of Microbiology and Immunology both contribute to a productive, collegial research environment with numerous opportunities for career development.

NIH Research Projects · FY 2025 · 2024-09

This is an application for a five-year mentored patient-oriented research career development award (K23). The overall goals are to 1) adapt and pilot test a family-centered intervention designed to improve long-term outcomes for survivors of acute respiratory failure, and 2) educate and mentor the candidate clinician investigator while she transitions to independence. The candidate is an assistant professor in the Division of Pulmonary Diseases and Critical Care Medicine at the University of North Carolina Chapel Hill. She recently completed a three-year award period on UNC’s KL2 during which she generated key preliminary data to inform this proposal. The members of her mentoring team have an established record of mentoring junior faculty, high research productivity, and substantial peer-reviewed support. Her mentoring and advisory team includes experts in ICU patient and family caregiver outcomes (Carson), palliative care (Hanson), communication and decision-making support in the ICU and after ICU discharge (Cox), support of patients and caregivers during care transitions (Toles), and psychiatry (Gaines), clinical psychology (Trivedi) as well as a dedicated biostatistician (Lin). The research and training environment at the University of North Carolina Chapel Hill is strong and well established. UNC is a national leader in research, with over $1 billion in extramural support in fiscal year 2020. UNC-Chapel Hill faculty researchers receive more than $528 million in NIH research funding annually, and UNC is first in the nation for federally funded social and behavioral science research and development. The candidate’s career development objectives are as follows: 1) To gain skills and experience in the development and adaptation of behavioral health interventions; 2) To obtain training and experience in conducting randomized clinical trials; and 3) To gain proficiency in best practices for virtual participant engagement and intervention delivery. To achieve these goals, the candidate will: 1) participate in advanced, graduate level coursework; 2) gain experiential learning as a co-investigator on a multi-site randomized clinical trial of family support intervention; 3) attend The North Carolina Translational & Clinical Sciences Principal Investigator Development Series and join the R-Writing Group; and 4) conduct mentor guided research. The specific aims of the research project are: 1) To adapt the Self-Management Using Collaborative Coping Enhancement in Diseases (SUCCEED) intervention, a family-centered coping skills intervention for seriously ill patients and their family caregivers, for use in acute respiratory failure (end product: SUCCEED-ICU); and 2) To test the feasibility, acceptability, and preliminary efficacy of SUCCEED-ICU in a pilot randomized trial. The support and mentorship provided by a K23 is critical to achieve the candidate’s goal of transitioning to an independent clinician investigator in the field of improving long-term outcomes for patients who have experienced acute respiratory failure.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Cystic fibrosis (CF) is an inherited, multisystem disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes the CFTR epithelial anion channel. CFTR works in balance with the epithelial sodium channel (ENaC) to maintain hydration of the airway surface. Perturbations of this system in CF result in dehydrated airway mucus, defective mucociliary clearance, and lung infection that accounts for the majority of CF morbidity and mortality. Recently approved CFTR-modulator drugs have revolutionized the treatment of people with CF who have the most common pathogenic CFTR variants. However, approximately 10% of people with CF have class I CFTR variants, including variants caused by premature termination codons (PTCs), which result in no CFTR mRNA or protein. These people with CF are ineligible for CFTR modulators. There is an unmet need for innovative therapies to treat these individuals. The recently developed thalidomide derivative CC-90009 is a small-molecule drug currently in Phase 1 clinical trials for acute myeloid leukemia. Studies in our lab demonstrated that CC-90009 both rescues class I CFTR PTC variants and unexpectedly also reduces ENaC activity in primary human airway epithelial cells. These promising dual effects are predicted to help restore mucus hydration and improve mucociliary clearance in CF. However, much remains unknown about CC-90009, including whether it restores pathologically decreased PTC- CFTR airway surface layer height and mucus hydration in CF, the mechanism by which it reduces ENaC activity, and its effects in animal models of CF. Given these knowledge gaps, the specific aims for this proposal are to: 1) Interrogate the functional consequences and mechanism of CC-90009’s impact on ENaC activity 2) Evaluate CC-90009’s effects in animal models of CF to gauge its potential translational utility Completing these aims with guidance from my scientific and medical mentors in UNC’s Marsico Lung Institute will provide a rich opportunity to master a suite of innovative basic and translational biomedical research techniques. Collectively, this work will position me for a career as a physician-scientist invested in better understanding respiratory cell biology and disease. The results will also further our understanding of the therapeutic potential of thalidomide-derivative drugs and therapies directed at novel molecular pathways to treat class I CFTR variants, and all people with CF.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY There are hundreds of studies of Alzheimer’s disease, Alzheimer’s disease-related dementias, and other neurodegenerative diseases comparing post-mortem (PM) human brain tissues obtained from human brain repositories. Current approaches that compare multiple, separately measured, -omic profiles studies introduce variability from using multiple samples. Thus, a multi-omics approach that can utilize a single biospecimen is needed. We have developed a penta-omic extraction method for frozen tissue and propose a proof-of-concept study. This effort would create the first penta-omic database, utilizing normal PM human brain tissue from the NIH NeuroBioBank. A new penta-omic simultaneous metabolomic, proteomic, lipidomic, DNA, RNA extraction method called SiMPL-DREx will be applied to a single tissue sample which will minimize the heterogeneity associated with testing multiple samples. SiMPL-DREx has added value because it more efficiently uses small volume, highly requested brain tissue. PM tissue selection is generally based entirely on a single quality control measure, RNA integrity number (RIN), obtained from a single tissue sample, usually the occipital pole (OP). Of the four -omic macromolecules extracted with SiMPL-DREx, RNA is the most labile primarily due to the universal distribution of RNase in the body. Tissue with RIN >7 is highly desirable for genomic and transcriptomic studies, yet how the overall quality of the metabolome, proteome, and lipidome (MPL) varies as a function of RIN has never been fully investigated. Tissues with RIN<7.0 are rejected for most -omic studies but may have sufficient quality for MPL studies. The primary post-mortem factor affecting -omic quality is autolysis which is dependent on body temperature. The time to be placed in cold storage is often not easily determined so RIN serves as a surrogate. This study will assess the association of RIN and measures of -omic quality in OP from unaffected donors with three RIN values (9-10, 6-7, 3-5) having an immediate death (agonal duration <1hr). The overarching goal of this study is to validate the penta-omic extraction method and to identify markers of tissue quality for different -omics outcomes that are independent of RIN. To this end, we will pursue two specific aims. In aim 1, SiMPL-DREx will be validated against existing single-omic extraction methods. In aim 2A, we will examine association of RIN category with DNA integrity number (DIN), protein integrity number (PIN) and pH to identify potential additional measures of tissue quality. In aim 2B a new measure, lipid integrity number (LIN) based on lipase activity is defined and will be tested for independence from RIN. The metabolome is unique by contributions from macro- and micro-molecular degradation, and therefore individual metabolites will be examined for association with RIN as potential markers of quality. If successful, this study will establish a more efficient method for multi-omic tissue extraction and identify additional measures of tissue quality for -omic studies beyond RIN.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Craniofacial malformations represent the largest category of birth defects and a major expense to the healthcare system and affected families. As craniofacial morphogenesis is complex and requires the coordinated interplay of several cellular pathways, mutations in many genes in these pathways can give rise to craniofacial defects. Coiled coil domain containing protein 32 (CCDC32) is an understudied gene recently found to underlie a craniofacial syndrome known as Cardiofacioneurodevelopmental Syndrome (CFNDS). Patients with defective CCDC32 exhibit a number of morphological abnormalities including bilateral cleft lip/palate, hypo- or hypertelorism, and microcephaly among others. Preliminary cellular data have shown CCDC32’s involvement in endocytosis and ciliogenesis, although molecular data to explain these functions is lacking. I have found that CCDC32 interacts with the adaptor protein complex 2 (AP2), a key factor in regulating endocytosis, via two well characterized motifs and exhibits the novel function of disassembling this complex. Moreover, I discovered CCDC32 binds the related AP3 complex, which is involved in trafficking throughout the Golgi and endolysosomal compartments. My preliminary data shows that knocking down CCDC32 in cells results in defects in intracellular trafficking including abnormal and reversible accumulation of ciliary components, and previously unobserved ciliary assembly dynamics. I hypothesize that CCDC32 promotes ciliary maintenance via AP3, and also regulates ciliary function via AP2. My approach will use cellular, biochemical, and structural methods to address the molecular mechanism of CCDC32’s role in endocytosis and ciliary assembly. In Aim 1, I will characterize the membrane trafficking interactions of CCDC32 that regulate ciliary maintenance. To determine a role for AP3 in intracellular ciliary trafficking, I will knock down AP3 in cells and observe cilia dynamics. To determine the molecular mechanism of CCDC32 interaction, I will determine a high-resolution CryoEM structure of the AP3- CCDC32 complex. To confirm the cellular importance of this interaction, I will use targeted mutagenesis to disrupt complex assembly in vivo. In Aim 2, I will determine how AP2-mediated endocytosis supports CCDC32’s role in ciliary function. CCDC32 and AP2 both promote endocytosis and bind one another, although the necessity and nature of this interaction has not been established. To assess an AP2-dependent function for CCDC32 in endocytosis, I will use targeted mutagenesis in vivo. I have also shown that CCDC32 disassembles AP2. To elucidate this novel mechanism, I will obtain CryoEM structures of CCDC32 with AP2 alpha/sigma. I will validate that AP2 disassembly occurs at membranes using biochemical reconstitution. To understand the effect of CCDC32 on endocytosis at the cilium, I will use TIRF endocytosis assays. Via these experiments, I will build the first molecular model of CCDC32 in cilia biology. This study will impact basic research on ciliary maintenance and membrane trafficking, and on health and disease research in craniofacial disorders like CFNDS.

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY/ABSTRACT Experiences of childhood adversity are common in the United States and account for a substantial proportion of pediatric-onset psychopathology. Understanding mechanistic pathways that contribute to this heightened psychopathology risk is, therefore, of critical consequence to public health. The Dimensional Model of Adversity and Psychopathology (DMAP) proposes that adverse experiences characterized by deprivation (e.g., neglect) may have distinct neurodevelopmental consequences in the frontoparietal network that confer risk for pediatric psychopathology. While existing theory emphasizes the importance of early childhood experiences of deprivation in shaping development in the frontoparietal network, existing neurodevelopmental research on childhood adversity is primarily cross-sectional and has focused almost exclusively on neural and mental health outcomes in adolescent samples. In two highly related pediatric neuroimaging samples with rich assessment of early childhood adversity exposure and psychopathology (Sample 1: R01MH115004; Sample 2: R01MH120314), we seek to address these gaps in the literature and Goal 2 of the NIMH strategic plan by identifying neurodevelopmental pathways linking deprivation with transdiagnostic psychopathology risk first in early childhood and then longitudinally from early childhood through adolescence. The proposed study will first examine concurrent associations of deprivation with altered frontoparietal network activation during cognitive control (Aim 1). We hypothesize that deprivation will be associated with worse cognitive control performance and reduced activation in the frontoparietal network (Sample 1). Next, we will characterize early childhood deprivation-related alterations to trajectories of cortical structure in the frontoparietal control network (Aim 2). We hypothesize that deprivation will be associated with blunted intra-individual longitudinal trajectories of cortical thinning in the frontoparietal network (Sample 2). Finally, we will examine deprivation-related alterations to brain function and structural development as potential mechanisms contributing to childhood and adolescent psychopathology (Aim 3). We hypothesize that decreased activation in the frontoparietal network during cognitive control will mediate the association of early childhood deprivation with concurrent psychopathology (Sample 1). Furthermore, we predict that altered longitudinal trajectories of cortical structure in the frontoparietal control network will mediate the association of early childhood deprivation with adolescent psychopathology. Training aims include advanced training in functional magnetic resonance imaging (fMRI) analysis, longitudinal modeling of neuroimaging data, and latent modeling of pediatric psychopathology. The applicant’s training will be supported by her sponsor’s and consultants’ expertise in these areas and extensive institutional resources. Findings will have implications for population health by identifying pathways of transdiagnostic psychopathology risk that can inform future early intervention efforts for childhood adversity involving deprivation.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Ozone is a common ambient air pollutant that presents a significant public health hazard as a trigger for lung function decrements, airway inflammation, and exacerbations of asthma and COPD symptoms. Controlled human exposure studies have found that the severity of these responses varies widely across individuals while the severity of a given individual’s reaction is reproducible across multiple independent exposures, indicating a potential genetic contribution to one’s risk for a severe response. Results from animal models also indicate that response to ozone is a complex trait. The genetic variants that underly this variation are currently unknown and this data gap needs to be addressed to identify high-risk individuals. Mapping expression quantitative trait loci (eQTL), which are statistical associations between variants and gene expression, is one approach used by human geneticists to study the genetic basis of complex traits. However, most eQTL mapping studies have used unperturbed/unexposed samples which may miss eQTL that only manifest after exposure or have an altered magnitude of effect with exposure, termed “response eQTL.” These response eQTL are thus gene-by- environment interactions. Studying these interactions in humans is often difficult, making in vitro models an attractive approach. Primary human bronchial epithelial cells (HBECs) isolated from the airway are a highly relevant in vitro model. HBECs can be readily cultured into a well-differentiated pseudo-stratified epithelium similar to that found in vivo and are cultured at an air-liquid interface, enabling in vitro exposures which mimic in vivo exposures. Additionally, using primary cells from different donors provides the genetic variation necessary for mapping eQTL. The goal of my project is to use this in vitro model to identify ozone response eQTL, genome- wide, and identify overlapping signals with asthma and COPD GWAS loci. I hypothesize that mapping ozone response eQTL in a physiologically relevant cell culture model will identify variants associated with response variability. Further, I hypothesize that some of these response eQTL will colocalize with GWAS signals for asthma and COPD, which have both been shown to be affected by air pollution exposure. In Aim 1, I will characterize the transcriptomic response to ozone in HBECs from 202 donors with diversity in age, sex, genetic ancestry, and smoking history. In Aim 2, I will map baseline and ozone response eQTL, using the difference in gene expression between filtered air and ozone exposed culture pairs from each donor to map response eQTL. In Aim 3 I will perform colocalization between ozone response eQTL and GWAS for asthma and COPD, followed by Mendelian randomization analysis to evaluate a signal’s effect on disease via gene expression. In total, I will identify genetic drivers of increased risk for severe response to ozone using a response eQTL strategy to find context-dependent eQTL, which will improve identification of susceptible individuals and inform policy regarding safe levels of exposure.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY Glioblastoma multiforme (GBM) is a fatal and difficult to treat brain tumor with a dismal median survival of less than 2 years. Standard therapy consists of surgical tumor resection, radiotherapy, and temozolomide, which only delay tumor recurrence. Recent success of CAR T cell therapy against Non-Hodgkin’s Lymphomas have gener- ated significant excitement for the application of CAR T cells in GBM and several clinical trials have demonstrated efficacy of CAR T cells in patients with GBM. However, both immunosuppression and the blood brain barrier act as major impediments limiting CAR T cell efficacy in glioblastoma. Preclinical trials with localized administration for CAR T cells via intratumoral or intraventricular routes enhance CAR T cell infiltration to brain tumor and outperforms i.v. infusions. With locoregional control, CAR T cells are infused into the resected tumor cavity, followed by repeated infusions into the ventricular system. Multiple administrations are necessary to maintain a larger dose of CAR T cells without causing toxicity and to enhance persistence of functional CAR T cells over a longer time. However, this repetitive dosing is a major obstacle to clinical translation of CAR T cells against GBM. CAR T cell manufacturing takes weeks and carries high costs - ~$500,000 per dose. The long manufacturing time creates delays of weeks to months to infuse CAR T cells to patients with rapidly progressing disease. Additionally, lengthy ex vivo manipulations create CAR T cells with heterogeneous composition and terminal differentiation, limiting their engraftment and persistence. Taken together, the many shortfalls of current CAR T cell manufacturing urgently demand development of innovative tools to reduce manufacturing time and provide optimal CAR T cell phenotype and distribution. In this proposal, we describe the application of Multifunctional Alginate Scaffold for T cell Engineering and Release (MASTER) for use in GBM. MASTER will be implanted in the surgical cavity of GBM to generate and release CAR T cells in vivo with improved efficacy and persistence. Based on significant published and preliminary data, we show that MASTER provides bio-instructive ques to activate, transduce, expand, and release fully functional CAR T cells in vivo. The scaffold includes anchored activating antibodies and interleukins to guarantee T cell activation and proliferation. Scaffold macroporosity facilitates homogeneous distribution of T cells, creates an interface for interaction between viruses and T cells, and enables in vivo release of fully functional CAR T cells. MASTER reduces CAR T manufacturing times from weeks to a single day, substantially reducing costs. We demonstrate in preliminary data and propose further that MASTER seeded with naïve PBMCs and anti-B7H3 CAR-encoding retrovirus will be implanted in the resection cavity of a brain tumor. B7H3 is overexpressed in brain tumors and serves as a promising therapeutic target for CAR T cell therapy. This approach could have enormous clinical impact by significantly reducing therapy costs and dramatically expanding the patient population benefiting from CAR T cell therapy. These studies will provide a foundational technology platform for CAR T cell manufacturing and promote widespread patient access.

NIH Research Projects · FY 2026 · 2024-08

Abstract The gene Icam1 codes for intercellular adhesion molecule-1 (ICAM-1, CD54), a member of the Ig-like superfamily of adhesion proteins and a type 1 membrane-bound glycoprotein. It is expressed constitutively on most lung parenchymal cells, functioning as a receptor for interactions between these parenchymal cells and leukocytes. Its major ligands are the four members of the β2 leukocyte integrin family. Our laboratory has contributed to understanding both the adhesive and the signaling functions of ICAM-1. We identified the differential expression of ICAM-1 on AT1 compared to AT2 cells and its upregulation by lipopolysaccharide and S. pneumoniae on these cells and capillary endothelial cells. During inflammatory responses, cytokines such as IL-1, TNF and IFNγ regulate its expression differentially, depending on both the cell type and the inflammatory stimulus. Our studies of its cell-cell adhesive function document its critical roles in pneumonia induced by LPS and P. aeruginosa. Ligation of ICAM-1 by β2 integrins also induces intracellular signaling within both the parenchymal cell and the leukocyte. Furthermore, ICAM-1 is proteolytically shed from the surface of parenchymal cells, and this soluble ICAM1 (sICAM-1) has highly debated functions as an inhibitor of leukocyte adhesion or an activator of leukocytes. Thus, ICAM-1 plays very dynamic roles during inflammatory and immune responses. Due to recently identified issues with the Icam1 null mice generated by homologous recombination and the lack of cell-specific knockouts, we are at a standstill in understanding ICAM-1. We are generating both Icam1 null mice using CRISPR/Cas9 and mice with floxed Icam1 alleles. We will test the hypothesis that membrane-bound and sICAM- 1 mediate a range of cell-cell interactions that depend upon the particular ICAM-1-expressing cell type during both homeostasis and innate immune responses. Aim 1 determines the functions of sICAM-1 in the alveolar space and its therapeutic potential. The role of sICAM-1 in the trafficking of alveolar macrophages along the alveolar surface and the functions of sICAM-1 in recruitment and function of neutrophils and inflammatory macrophages during pneumonia will be identified. Aim 2 determines the functions of ICAM-1 on the alveolar surface during homeostasis and innate immune responses. Mice deficient in ICAM-1 in alveolar epithelial cells will be studied to determine its role in trafficking, motility and function of alveolar macrophages in healthy mice. The effect of alveolar epithelial ICAM-1 in recruitment and function of neutrophils, macrophage subpopulations and NK cells during pneumonia will also be determined. Outside-in signaling initiated by ICAM-1 ligation will also be assessed. Aim 3 determines the functions of endothelial ICAM-1 during innate immune responses. Mice deficient in ICAM-1 expression by endothelial cells will be studied to determine if the leukocytosis observed in Icam1 null mice is due to the absence of endothelial ICAM-1, as well as the functions of endothelial ICAM-1 in neutrophil, macrophage and NK cell trafficking during pneumonia. Signaling pathways will be assessed. These studies will expand our knowledge of ICAM-1 and its therapeutic potential.