Duke University

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT Identifying accessible tumor antigens that are not expressed in vital organs would allow genetic engineering of CAR T cells to avoid dysfunction at tumor beds. We have identified an olfactory receptor (OR) expressed in a variety of human tumors, ranging from ~70% of intrahepatic cholangiocarcinomas and 39% of prostate cancers, to ~10% of NSCLCs, and generated CAR T cells that specifically target its extracellular domain. The long-term goal of these studies is to translate these CAR T cells. Here, we will advance the preclinical work needed to apply for IND approval for a first-in-human clinical trial conducted at Moffitt, using OR2H1 CAR T cells generated in our Cell Therapy Facility under GMP conditions. Our central hypothesis is that genetically engineered OR2H1 CAR T cells can effectively control the progression of established tumors of different histologies, without the unacceptable on-target, off-tumor effects of other targets expressed in vital tissues. In Specific Aim 1, we will demonstrate the effectiveness and specificity of targeting OR2H1+ PDX with CAR T cells in vivo. These studies will support a rationale for subsequent IND approval for OR2H1 CAR T cell administration in patients with tumors expressing OR2H1 at variable levels. In Specific Aim 2, we will define the superiority of XBP1-ablated OR2H1 CAR T cells. These studies will support a rationale for genetic engineering of OR2H1 CAR T cells, to empower them to resist metabolic restrictions and inhibitory signals at tumor beds. In Specific Aim 3, we will optimize a method to identify eligible patients for OR2H1 CAR T cell targeting. Our work could exert a profound effect in the field by supporting a first-in-human clinical trial using OR2H1- targeted CAR T cells against a variety of solid human tumors, which could have significant benefits in a broad range of cancer patients, with limited or negligible toxicity.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Male germ cells (MGCs) are precursors to spermatogonial stem cells (SSCs), a stem cell population that both self-renews and differentiates, supplying spermatozoa for the entirety of a male’s reproductive lifespan. Disruptions in development of MGCs during fetal life leads to infertility in humans and mice. Just prior to birth, MGCs undergo a relatively long period of cellular quiescence. This G0 arrest phase is conserved between mice and humans and is considered an MGC reprogramming stage. During this time, MGCs lose their early GC fate and acquire the molecular profile of SSCs by undergoing vast epigenetic modifications. Although increases in mRNA levels of various epigenetic factors occur during G0, it is unclear how the transcripts of epigenetic factors are regulated post-transcriptionally throughout G0. DND1 is one of many RNA-binding proteins (RBPs) required for MGC development, and it is essential for both G0 arrest and for RNA expression of epigenetic regulators during G0. Transcript targets of DND1 during G0 include vital epigenetic regulators. Functionally, DND1 can promote transcript degradation, protect transcripts from degradation, or promote their translation. Because DND1 has multiple regulatory roles, it is unknown how DND1 post-transcriptionally regulates its target transcripts during G0 to ensure MGC-to-SSC development. In this proposal, how transcripts of epigenetic factors are post- transcriptionally regulated G0 in MGCs will be determined by focusing on the translational role of DND1, as supported by preliminary data. The central hypothesis is that an essential role of DND1 is to control when and where its target epigenetic regulators are translated during G0 to direct the reprogramming of MGCs into SSCs. To test this hypothesis, in Aim 1, DND1-bound transcripts that encode for epigenetic regulators and that are translated during G0 will be defined. The proteomes will be determined through LC/MS-TOF in two stages of G0 and will be cross-referenced to previously obtained DND1-target transcripts at their respective stages, creating DND1-directed translatomes. Epigenetic regulators in these translatomes will be assessed for nascent translation throughout G0 to determine their temporal translational regulation. In Aim 2, it will be determined where and how DND1 promotes the translation of its target transcripts. DND1-interacting proteins will be unbiasedly identified and using a variety of high-end microscopy tools, the localization of DND1, its target transcripts, and translational machinery will be determined. The insight provided by these aims will provide a novel paradigm for how an RBP regulates the epigenetic reprogramming of MGCS to ensure the development of SSCs. In addition to advancing scientific knowledge, this fellowship proposal also exhibits high training potential. The training plan describes a two-year blueprint designed to strengthen technical, professional, and conceptual skills, which will be implemented at Duke University in the laboratory of Dr. Blanche Capel. Overall, the enclosed research strategy and training plan can develop a trainee into an independent researcher while significantly advancing knowledge for improving reproductive health.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Falls result in substantial morbidity, mortality, and disability among older adults. Recently, hearing loss and hearing handicap have been identified as independent risk factors for falls. It is unclear the factors that mediate the association between hearing loss and falls. The long-term goal is to to develop novel interventions that will modify falls risk in the hearing impaired patient population. The current objective is to identify and understand the auditory and vestibular related factors that explain the association between falls and hearing loss and to characterize performance on these candidate factors. To that end, our proposal aims to examine the vestibular- related factors, centrally mediated auditory factors (spatial hearing and listening effort), cognitive, and psychosocial factors in an older adult patient population of fallers and non-fallers. The current proposal is innovative as the work will characterize the extent to which key theoretical factors explain the link between falls and hearing loss and specifically the extent to which unrecognized vestibular dysfunction may explain the association. These contributions will be significant, as they will inform strategies to implement targeted rehabilitation programs to reduce falls and falls-risk in this patient population.

NIH Research Projects · FY 2025 · 2023-08

The skin forms the first barrier against physical, biological and chemical insults and is essential for prevention of dehydration. To maintain this function epidermal keratinocytes undergo differentiation. During differentiation, the gene expression programs in keratinocytes switch from maintenance of cell proliferation to terminal differentiation. Calcium gradient formed between the basal and upper epidermal layers is a major factor underlying induction of terminal differentiation in keratinocytes. In addition, skin homeostasis relies on well- orchestrated intercellular communications mediated by cytokines that are produced by keratinocytes and other skin resident cells. We have identified that transcription factor FOXQ1 (a member of the Forkhead Box family of proteins) utilizes a novel, rheostat-like mechanism of transcriptional regulation of keratinocyte differentiation. Thus, in presence of low extracellular calcium, FOXQ1 repressed genes associated with epidermal differentiation in normal human keratinocytes (NHKs) and immortalized human keratinocytes (HaCaT). On the contrary, in calcium-treated (i.e. differentiation-induced) NHK and HaCaT cells, FOXQ1 activated the same set of genes. Therefore, in Specific Aim 1, we will identify the mechanisms underlying transcriptional regulation by FOXQ1 of keratinocyte differentiation. In addition, we demonstrated that under normal conditions, depletion of FOXQ1 in cultured keratinocytes decreased whereas its overexpression increased cell proliferation. Moreover, Foxq1-/- mice demonstrated decreased epidermal hyperplasia in response to treatment with imiquimod which induces psoriasis-like phenotypes in mouse epidermis. Therefore, in Specific Aim 2, we will generate keratinocyte-specific Foxq1 knock-out mice and identify the mechanisms of FOXQ1 regulation by pro-inflammatory cytokines and the role of Foxq1 in regulation of keratinocyte hyper-proliferation.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The ocular lens plays a crucial role in focusing incident light on to the retina, with aberrations in lens transparency and mechanical properties leading to impairment of vision. Despite continuing effort, our understanding of the various molecular pathways governing lens cytoarchitecture, homeostasis, deformability, clarity, and the etiological mechanisms of cataract remains incomplete. We recently identified that several members of the S100 family of calcium binding proteins are abundantly expressed and exhibit distinct distribution profiles in the lens. While expression of S100A6 and S100A10 distributed to both the lens epithelium and fibers, S100A4 distributes discretely to lens fiber cells. Absence of S100A4 but not S100A6 or S100A10 leads to opalescent late-onset cataract formation in a mouse model, together with the robust upregulation of S100A5, the expression of which was otherwise undetectable in the wild type lens. Other than these new data on S100A4, we currently have no knowledge of the role played by the S100 family of proteins in lens function. S100A4 is expressed in a cell and tissue specific manner and is involved in the pathophysiology of various diseases. The physiological function of S100A4 however, is poorly understood. A well-understood molecular function of S100A4 is its interaction with non-muscle myosin II (NM II), especially NM IIA, and regulation of NM II assembly, actin polymerization, contractile characteristics, cell migration, and plasticity. NM II plays a crucial role in lens cytoarchitecture, mechanics, and function. Though NM IIA mutations are associated with cataract development in humans, little is known about regulation of NM II activity in the context of lens function. While lenses derived from mature S100A4 null mouse lenses (2 to 6 month old) remain clear and maintain normal growth and size, those from six month-old mice exhibit impairments in assembly and phosphorylation of NM IIA, polymerization of actin, and alterations in the levels of aquaporin-5, CLIC5, connexin-50, cell adhesive proteins (integrin-β1, NrCAM, ZO-1) and α-crystallin, culminating in opalescent cataract formation in eight month-old mice. Based on these preliminary and novel findings, we hypothesize that S100A4 plays a crucial role in regulating NM II assembly in the lens, and that the absence of S100A4 disrupts NM II assembly and phosphorylation, actin polymerization, mechanical properties, and osmotic homeostasis in the lens, leading to late onset cataract formation. To test this hypothesis, in three interrelated specific aims, we will investigate: 1) the mechanistic role of S100A4 in regulation of lens NM II assembly and phosphorylation, actin polymerization, cell adhesion, and lens tensile properties, 2) the disruption of osmoregulation and solute carrier transport mechanisms underlying cataractogenesis in S100A4 null lenses, and 3) the effects of rescuing expression of S100A4 in S100A4 null mouse lens fibers (using a transgenic approach) on cytoskeletal integrity and lens phenotype. These novel and interrelated studies are expected to uncover the significance of S100A4 and other members of the S100 family proteins in lens function, which are known to participate in the pathophysiology of several diseases.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Epstein-Barr virus (EBV) is an extremely pervasive human herpesvirus, infecting approximately 95% of the global population by adulthood. EBV is transmitted through saliva and establishes infection in the oral cavity where it then establishes a latent infection for life in memory B cells. In most individuals this infection will remain benign, but EBV-associated diseases include infectious mononucleosis and cancers, more commonly in immune- compromised individuals. The balance between latent and lytic infection is under tight control and understanding the regulation of this process has broad implications for processes ranging from viral persistence in the oral mucosa to strategies to eliminate latently infected tumor cells. EBV reactivates in response to a diverse range of stressors including DNA damage, hypoxia, histone deacetylase inhibitors and activation of the B-cell receptor. A pervasive phenomenon, observed both in vitro and in vivo, is that cells have a heterogenous response to lytic induction stimuli. In a fraction of cells, the virus fully reactivates, while others remain completely refractory or only partially progress through the lytic cycle leading to an abortive infection. To better understand these cell fates after EBV lytic reactivation, my lab recently completed a single-cell RNA seq experiment of resting and reactivated EBV+ B lymphoma cells. We observed differential host gene expression patterns between refractory, abortive, and productive lytic cells. This included high expression of the known EBV restriction factors MYC and STAT3 in the refractory cells, but previously unknown markers of abortive cell populations: one characterized by elevated IL-6 receptor and the other defined by pro-survival signaling through the NFB pathway. Based on our single-cell data and prior studies, I hypothesize that an EBV induced DNA damage response leads to IL-6 production, which in turn promotes an abortive, antiviral state through the IL-6 receptor and ultimately pro- survival NFB signaling. In addition to defining mechanisms of host defense from EBV reactivation, this work also has important clinical ramifications as lytic induction therapies are currently in trials for EBV-associated malignancies. Understanding host factors that restrict successful lytic reactivation could lead to more effective therapeutic strategies in the future. Furthermore, these findings could have broad implications for how other herpesviruses reactivate and how latently infected cells communicate to regulate this process.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract: 30 lines Although Plasmodium vivax causes more than 7 million malaria cases each year, it has typically been excluded from malaria control programming in sub-Saharan Africa (SSA) due to the absence of reported cases and the assumption that the predominantly Duffy-negative population is invulnerable to P. vivax infection. However, there is growing evidence that P. vivax is indeed present in SSA and that Duffy-negative individuals can be infected, albeit at lower rates than their Duffy-positive counterparts. In addition, the recent documentation of Anopheles stephensi, a highly competent vector for both P. vivax and P. falciparum, in the Horn of Africa raises the possibility that P. vivax transmission may be enhanced by this emerging vector as it spreads southward into SSA. As Kenya approaches pre-elimination phase in its fight against malaria, it is facing the dual threat of the invasive An. stephensi vector and an unknown burden of the largely neglected P. vivax species. While models have shed some light on the potential spread of An. stephensi into SSA, these predictions and their potential impact on P. vivax transmission remain to be confirmed or quantified. Here, we focus on Turkana, a semi-arid region of northern Kenya where we recently documented low levels of year-round P. vivax for the first time. Turkana county borders Ethiopia, where P. vivax is endemic and An. stephensi presence has recently been confirmed. Across the border in Kenya, there is little to no information available on P. vivax prevalence, clinical burden, or its relationship with Duffy blood groups. Furthermore, An. stephensi surveillance has not been mounted in Turkana, despite the fact that it is predicted to have the highest risk of An. stephensi invasion. First, we propose to measure the clinical burden of P. vivax and its relationship with Duffy blood groups through passive case detection. By working with select health facilities across the county to screen and test patients seeking malaria treatment, we can measure the prevalence of P. vivax in suspected malaria cases and compare the rate of infections in different Duffy blood groups. Second, by conducting follow-ups with treated patients, we will quantify the rate at which P. vivax infections relapse due to dormant hypnozoite presence following the clearance of P. falciparum parasites, a phenomenon that has been well documented in many areas where P. falciparum and P. vivax are co-endemic. This will allow us to estimate the underlying silent reservoir of liver-stage P. vivax infection. Third, we will identify vectors likely involved in P. vivax transmission by collecting and classifying the species of mosquitoes and/or larvae from the homes of P. vivax cases, with particular emphasis on detecting An. stephensi. Evidence from this study will provide the foundation for understanding the conditions in which P. vivax could potentially spread from Turkana across Kenya and would have broad application, informing malaria surveillance and control strategies in Kenya and other areas across SSA where P. vivax and An. stephensi may have an increasing impact.

NIH Research Projects · FY 2026 · 2023-08

Adverse effects of environmental contaminants on human health are a major public health concern. We are all exposed to a complex mixture of different chemical contaminants through the air we breathe, the water we drink, the food we eat, and the products we use. As new industrial products are produced, leading to new direct and indirect exposures, there is a pressing need for new tools for assessing the adverse health effects in humans associated with exposure to chemical mixtures. Challenges include huge numbers of different possible mixtures, the curse of dimensionality in multivariate nonparametric regression and moderate to high correlation in different exposures. Building on compelling preliminary results from a highly successful NIEHS PRIME program R01, we develop a transformative statistical toolbox for inferences on health effects of chemical exposures, both in the high throughput screening context and for better disentangling health effects of chemical mixtures in epidemiology studies. The research proceeds through the following Aims. Aim 1 develops methods for inferring synergistic and antagonistic interactions from epidemiologic data, including for data collected longitudinally motivated by studies of exposure effects on childhood neurodevelopment. We improve substantially over current nonparametric regression approaches in interpretability and power to detect interactions; synergistic interactions in which chemicals amplify each other’s effects are particularly important. Aim 2 develops clustering methods to improve understanding of variation in exposure in relation to health. These methods will have broad impact in dramatically improving practical performance over current model- based clustering approaches. In addition, easily interpretable results are provided, adding additional insights over state-of-the-art regression-based methods. Aim 3 develops new methods for inferring relationships between chemical molecular structure and biologic activity. Given the sheer number of chemicals lacking any direct in vivo or in vitro data, it becomes crucial to use molecular structure to predict biologic activity. Leveraging on ToxCast/Tox21 and other data sources, we develop improved statistical models for relating chemical structure to activity, for inferring low-dimensional summaries of chemical activity based on molecular structure, and for optimally choosing the next chemicals to be tested. These methods can be used to predict effects of chemicals lacking any direct in vivo or in vitro data through targeted borrowing of information across related chemicals in the database. Aim 4 develops user-friendly and reproducible software, while using the methods to thoroughly analyze data from the motivating epidemiology studies, with a particular focus on the Mount Sinai Children’s Environmental Health Study and the UNC Early Life Factors Study, which both focus on assessing exposure effects on neurodevelopment in early childhood. We expect our methods to lead to important new findings.

NIH Research Projects · FY 2025 · 2023-07

Priming of antitumor immunity relies on a specialized subset of conventional Dendritic Cells (cDC1s), CD103+ DCs, that have unique capabilities of trafficking tumor antigens to lymph nodes for cross-presentation. The critical role of cDC1s in cancer immune surveillance make them highly coveted targets for immunotherapy; however, their scarcity, the difficulty of specifically engaging DCs, and the suppressive tumor microenvironment (TME) pose severe obstacles. We are investigating immunotherapy with attenuated, recombinant poliovirus (PV), PVSRIPO, because of the naturally evolved tropism of PV for DCs. In humans or chimpanzees, oral PV infection leads to remarkably effective targeting of CD11c+DCs in peripheral (gastrointestinal) and lymphoid tissues. PVSRIPO, engineered with an IRES from human rhinovirus type 2, lacks cytopathogenicity in normal cells, eg. DCs. Rather, DC infection yields ‘sustained’ type-I interferon (IFN) responses elicited by specific viral dsRNA patterns that engage the PV pattern recognition receptor (PRR) MDA5. Mechanistic investigations of PVSRIPO treatment in ex vivo human tumor slices revealed that: (i) the main outcome is sustained type-I IFN release from myeloid cell subsets in the non-malignant TME; (ii) PVSRIPO’s unique innate imprint provides optimal DC activation stimuli for CD4+T cell TH1-polarization and CD8+T cell priming; (iii) PVSRIPO’s ‘PRR-contextualized’ IFN response induces polyfunctional GzmB+, IFNg+, T-bet+ antitumor CD8+T cells that control tumor growth after adoptive transfer. We reported very promising Ph1 clinical trial results with PVSRIPO in challenging indications: recurrent glioblastoma and recurrent, nonresectable melanoma. Our premise is that PVSRIPO therapy recruits cDC1s, leads to local infection of these cells, induces migration to lymph nodes/spleen, and stimulates tumor antigen cross-presentation via the intrinsic innate inflammatory signature it elicits. We propose mechanistic and translational studies to gain insight into targeting of DCs by PVSRIPO, the way sublethal infection of distinct myeloid subsets activates tumor antigen cross-presentation, and rational means of enhancing DC engagement by intranodal virus administration. We propose the following Specific Aims: (1) Unravel mechanisms of cDC1 recruitment, activation, and lymph node migration upon PVSRIPO infection of the TME. (2) Determine the mononuclear phagocyte compartment(s) that mediate PVSRIPO antitumor effects; test the roles of T cell-intrinsic IFN signaling and chemotaxis. (3) Reinforce cDC1 engagement by perinodal delivery or upon infection of the PVSRIPO-reactive glioma myeloid infiltrate. Significance: these studies explore a unique opportunity for effectively engaging CD103+DCs in tumor antigen cross-presentation and CD8+T cell priming, based on the natural tropism for DCs and unique innate inflammatory imprint of PVSRIPO. The long-term objective of this research is to provide means for re-instating effective cancer immune surveillance in patients by overcoming tumor-associated deficits in DC function and CD8+T cell priming.

NIH Research Projects · FY 2024 · 2023-07

Peripheral artery disease (PAD) affects roughly 12 million Americans and accounts for over $21 billion in combined annual healthcare costs. Patients with PAD are at an increased risk of future cardiovascular events, including amputation, myocardial infarction, and death. Patients with PAD who undergo a peripheral vascular intervention (PVI) for claudication symptoms or for lower extremity ulcers or gangrene have a guideline-based indication to be on multiple evidence-based medications. These medications include high intensity statins, aspirin, a P2Y12 inhibitor (clopidogrel), and an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB). Adherence to these medications after PVI can significantly reduce the future risk of amputation, myocardial infarction, and/or death. However, observational data of patients with PAD after PVI has demonstrated poor medication adherence to these evidence-based medications. Additionally, there has been no prior examination of the barriers and facilitators of medication adherence in this population of patients. Moreover, there is little evidence regarding strategies to improve medication adherence in this vulnerable population of patients. This proposal seeks to identify the barriers and facilitators of medication adherence in a population of patients with PAD who have undergone PVI, and to develop a medication adherence tool to promote discussion of and mitigation of barriers to adherence in the outpatient clinic environment. In Aim 1, we will conduct semi-structured interviews of 40 patients with PAD who have undergone PVI to examine barriers and facilitators of medication adherence. In Aim 2, we will then use evidence-based quality improvement methods and a diverse research engagement panel, consisting of providers, patients, nurses, and experts in information technology, to develop a medication adherence tool for use in the outpatient clinic. This tool will be integrated into the electronic health record and will facilitate discussion of barriers to adherence and potential strategies for improving adherence. In Aim 3, we will perform a pilot feasibility randomized controlled trial of 200 patients in two sites within the Duke Health System to examine the feasibility and acceptability of the adherence tool, as well as the effect of the intervention on rates of medication adherence. This pilot randomized controlled trial will establish the foundation for a future, multi-center randomized controlled trial examining the use of the tool to improve rates of adherence and clinical outcomes. This research will occur in the setting of a comprehensive career development program designed to provide Dr. Rymer, an interventional cardiologist and a vascular proceduralist, with the training, experience, and leadership development needed to become an independent clinical research investigator. During the award period, Dr. Rymer’s team of exceptional mentors will guide her to develop expertise in qualitative research, implementation science, and clinical trial design. Dr. Rymer’s long-term goal is to improve the health of patients with PAD who have undergone an intervention by achieving optimal adherence to guideline-directed medical therapies.

NIH Research Projects · FY 2025 · 2023-07

This submission comprises two applications (Clinical and Statistical Data Coordinating Centers). We propose to conduct a large (N=1,100), simple, pragmatic, superiority trial in the US - IMPROVE-AD – comparing a strategy of medical therapy (MT) plus upfront thoracic endovascular aortic repair (TEVAR) to MT plus surveillance for deterioration in patients with uncomplicated type B aortic dissection (uTBAD). Surveillance will include clinically indicated TEVAR and/or open repair performed for deterioration during index hospitalization or follow up. This is the first trial of this size designed to establish guidance on uTBAD management. The trial leadership is comprised of an experienced group of investigators in a Clinical Coordination Center (CCC; Duke Clinical Research Institute, Durham, NC, Baylor College of Medicine, Houston, TX, and University of Washington, Seattle, WA) a Statistical and Data Coordination Center (SDCC; Duke Clinical Research Institute, Durham, NC) and a diverse, Executive and Steering Committee of experts in the field representing clinicians, trialists, and patient advocates. Aortic dissection (AD) is the most common fatal event involving the aorta occurring in 5 to 30 cases per million of population resulting in 12,000 deaths in the US annually. Type B aortic dissections involve the entire descending aorta. Based on evidence from the 1960s, the main strategy for uTBAD is medical therapy with lifelong surveillance. This strategy has been shown to have poor long-term outcome in 25-50% of patients (aortic related events). The emergence of TEVAR as a less invasive alternative to open repair, however, has resulted in debate over the use of upfront TEVAR to treat uTBAD. A pilot European trial (INSTEAD) compared the outcomes of upfront TEVAR to optimal medical therapy in 140 patients with uTBAD. Despite being significantly underpowered for all-cause mortality, the findings, along with observational data suggest that medical therapy plus upfront TEVAR may be associated with decreased all- cause and aortic-related mortality. We have also demonstrated from our completed surveys that there is equipoise among practitioners with respect to the most appropriate treatment strategy in uTBAD. We propose a pragmatic trial with centralized, telephone follow-up, remote blood pressure monitoring, a clinically relevant hierarchical primary endpoint (mortality / aortic-related hospitalization), and multi-disciplinary teams of investigators and patient advocates. The trial duration is 84 months with 5-month start-up. Average follow-up is 4 year with a minimum of 2.5 years and maximum of 6 years for individuals enrolled early. IMPROVE-AD will have 88% power to detect a 25% relative reduction in the incidence of the primary endpoint for patients randomized to upfront MT plus TEVAR compared to MT plus surveillance for deterioration, assuming a 5 year cumulative incidence of 20% death and 20% aortic-related hospitalization in the MT plus surveillance for deterioration arm.

NIH Research Projects · FY 2025 · 2023-07

This submission comprises two applications (Clinical and Statistical Data Coordinating Centers). We propose to conduct a large (N=1,100), simple, pragmatic, superiority trial in the US - IMPROVE-AD – comparing a strategy of medical therapy (MT) plus upfront thoracic endovascular aortic repair (TEVAR) to MT plus surveillance for deterioration in patients with uncomplicated type B aortic dissection (uTBAD). Surveillance will include clinically indicated TEVAR and/or open repair performed for deterioration during index hospitalization or follow up. This is the first trial of this size designed to establish guidance on uTBAD management. The trial leadership is comprised of an experienced group of investigators in a Clinical Coordination Center (CCC; Duke Clinical Research Institute, Durham, NC, Baylor College of Medicine, Houston, TX, and University of Washington, Seattle, WA) a Statistical and Data Coordination Center (SDCC; Duke Clinical Research Institute, Durham, NC) and a diverse, Executive and Steering Committee of experts in the field representing clinicians, trialists, and patient advocates. Aortic dissection (AD) is the most common fatal event involving the aorta occurring in 5 to 30 cases per million of population resulting in 12,000 deaths in the US annually. Type B aortic dissections involve the entire descending aorta. Based on evidence from the 1960s, the main strategy for uTBAD is medical therapy with lifelong surveillance. This strategy has been shown to have poor long-term outcome in 25-50% of patients (aortic related events). The emergence of TEVAR as a less invasive alternative to open repair, however, has resulted in debate over the use of upfront TEVAR to treat uTBAD. A pilot European trial (INSTEAD) compared the outcomes of upfront TEVAR to optimal medical therapy in 140 patients with uTBAD. Despite being significantly underpowered for all-cause mortality, the findings, along with observational data suggest that medical therapy plus upfront TEVAR may be associated with decreased all- cause and aortic-related mortality. We have also demonstrated from our completed surveys that there is equipoise among practitioners with respect to the most appropriate treatment strategy in uTBAD. We propose a pragmatic trial with centralized, telephone follow-up, remote blood pressure monitoring, a clinically relevant hierarchical primary endpoint (mortality / aortic-related hospitalization), and multi-disciplinary teams of investigators and patient advocates. The trial duration is 84 months with 5-month start-up. Average follow-up is 4 year with a minimum of 2.5 years and maximum of 6 years for individuals enrolled early. IMPROVE-AD will have 88% power to detect a 25% relative reduction in the incidence of the primary endpoint for patients randomized to upfront MT plus TEVAR compared to MT plus surveillance for deterioration, assuming a 5 year cumulative incidence of 20% death and 20% aortic-related hospitalization in the MT plus surveillance for deterioration arm.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Altered lubrication of articular cartilage following joint injury increases friction between the sliding cartilage surfaces, leads to deterioration of cartilage, and hastens the development of osteoarthritis (OA). OA due to joint injury, termed as post-traumatic osteoarthritis (PTOA), is estimated to account for at least 12% of all OA cases in the United States and approximately half of the individuals with an anterior cruciate ligament (ACL) injury develop PTOA regardless of the ACL reconstruction. Replenishing the synovial fluid and thereby restoring the articular cartilage lubrication has been demonstrated to benefit the joint. The overarching goal of the proposed study is to investigate the potential of cartilage-adhering, self-healing hyaluronic acid molecules to prevent or delay the degeneration of articular cartilage, and thereby osteoarthritis, following injury or trauma. The molecularly engineered HA-based lubricants are designed to integrate the function of both hyaluronic acid and lubricin, two key components of the synovial fluid, while exhibiting self-repairing ability. The self-healing properties are incorporated to ensure both long-term retention and adaptability of the lubricant within the mechanically active joint, while the cartilage adhering properties are expected to improve boundary lubrication. We hypothesize that the dynamic cartilage-adhering molecules that integrate the molecular features of HA and lubricin and simultaneously exhibit self-healing properties will protect the articular cartilage and prevent or slow down the progression of PTOA. Towards this, we will: (i) develop cartilage-adhering, self-healing HA molecules and determine the effect of molecular architecture-lubrication function relationship, (ii) determine the effect of the molecular architecture of the proposed novel HA-based lubricants on chondroprotection by using a joint-on-chip platform, and (iii) determine the effect of these lubricants on mitigating the development of post-traumatic osteoarthrits. The proposed studies will enable a new paradigm in which intra-articular injection of self-healing HA-bottle brush molecules could be an important treatment following acute injury to ameliorate or delay PTOA, or they could be used as an adjunct after surgical repair to improve the outcome.

NIH Research Projects · FY 2024 · 2023-07

1 PROJECT SUMMARY/ABSTRACT 2 Despite improvements in median life expectancy for sickle cell disease (SCD) from 14 years in 3 1973 to 61 years in recent cohorts from academic centers, adults with SCD experience 4 functional impairments. They develop deficits in physical, social, and cognitive function due to 5 both age-related changes and SCD complications, such as avascular necrosis, strokes, 6 cardiopulmonary disease, and sensory deficits. There are no validated assessment tools or 7 interventions to improve physical function in older adults with SCD. Failure to recognize and 8 manage vulnerabilities and functional decline can lead to disability and premature death. In a 9 pilot study we evaluated the feasibility if the Sickle Cell Disease Functional Assessment (SCD- 10 FA) and demonstrated that it was feasible, acceptable, and safe. In this study, we will evaluate 11 the validity and reliability of the SCD-FA in a prospective cohort of older adults with SCD (age ≥ 12 40 years) (Aim 1). We will evaluate the utility of the SCD-FA to predict number of days 13 hospitalized per year as the primary outcome, and Sickle Cell Day Hospital visits, emergency 14 department visits, dependence in Activities of Daily Living, and death as secondary outcomes. 15 We will also determine risk factors for impaired physical performance (Aim 2). The SCD-FA will 16 provide a method for: 1) assessing vulnerabilities in multiple health domains, 2) identifying 17 targets for interventions, and 3) measuring change in function. To address functional 18 impairments, we will adapt and pilot a personalized exercise training program in older adults 19 (age ≥ 40 years) with SCD (Aim 3). The intervention will be a virtual program adapted from 20 Gerofit, which is a supervised exercise and health promotion program for older adults that 21 includes exercises customized to each person’s health status and underlying functional deficits. 22 The expected outcome is to have pilot data to design a randomized control trial of a 23 personalized exercise intervention for older adults with SCD. 24 Dr. Oyedeji is an Assistant Professor of Medicine in the Division of Hematology at Duke 25 University. Her career development plan includes developing skills in health assessment tool 26 development and developing and implementing interventions with guidance from experts in 27 psychometrics, intervention development, implementation, geriatrics, and sickle cell disease. 28 This research coupled with coursework, professional development activities, and her mentorship 29 team will provide the necessary experience to support her success as she works towards her 30 goal of becoming an independent investigator and leader in SCD and aging.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract: INTRODUCTION: RNA viruses such as SARS-CoV-2 represent an ongoing public health threat. To replicate properly, RNA viruses must perform multiple functions in the same cytoplasm (i.e., genome replication, circularization, generation of sub-genomic RNA, packaging etc.) and for betacoronaviruses like SARS-CoV-2 the structural nucleocapsid protein (N) is required for all of these processes. How are RNA viruses able to perform multiple functions in the same cytoplasm with their limited proteome (for example ~24 proteins for SARS-CoV-2)? I hypothesize that one way RNA viruses achieve biochemical complexity is by condensation of RNA and protein components. Biochemical complexity can emerge by virtue of the co-condensing RNA as different viral RNA sequences confer different condensate properties. In this way, a single protein, N, can perform multiple functions in the same cytoplasm. I uncovered the mechanism by which SARS-CoV-2 N recognizes RNA to undergo condensation. Now, I am interested in understanding how the interaction of N with viral RNA controls condensation and impacts important viral functions. RESEARCH: In my K99/R00 research; (Aim 1) I will unbiasedly probe RNA-RNA interactions to understand how RNA is arranged and how interactions are modulated by condensation for genome circularization. (Aim 2) I will develop BSL2 assays to determine which viral processes require N condensation. (Aim 3) I will design synthetic RNA-binding proteins and small molecules to disrupt N condensation. TRAINING: I will complete my training period in Dr. Amy Gladfelter’s lab. During the training period, I will further develop my skills in RNA structure probing SHAPE (RNA structure), RNP-map (RNA/protein crosslinking), and SHAPE-Jump (RNA-RNA crosslinking) in collaboration with Weeks lab. I will learn how to design synthetic IDPs for the creation of bespoke RNA-binding proteins (Chilkoti). I will screen an existing small molecule library to identify inhibitors of N condensation (Fiorti). I will learn how to culture and genetically manipulate a BSL2 model betacoronavirus, MHV (Sheahan and Cameron). ENVIRONMENT: Dr. Amy Gladfelter is one of the world’s leading experts on the role of RNA in condensation. Additionally, UNC has some of the world’s leading experts in coronavirus biology including labs with BSL3 facilities offering the possibility to test newly developed N condensation inhibitors with live virus. I plan to further my growth through participation in RNA and condensation conferences, and attendance of courses in virology. I also plan on using my K99/R00 to ease my transition into running my own lab as an independent investigator. I am particularly interested in joining a university with strong biochemistry, molecular biology, virology, and biomedical engineering departments who are looking for an RNA structure enthusiast to join their ranks! IMPACT ON PUBLIC HEALTH: This work will advance our understanding of how betacoronavirus N recognize viral RNA sequences and may one day lead to novel therapeutics.

NIH Research Projects · FY 2024 · 2023-07

The outer membrane of Gram-negative bacteria forms a permeability barrier blocking antimicrobials from efficiently reaching their molecular targets residing within the bacterial cell wall or inside the bacterial cytosol. This barrier function is dependent on one of the outer membrane’s central building blocks, lipopolysaccharide (LPS). The LPS molecule is anchored in the outer bacterial membrane by its lipid A moiety. Lipid A, an acylated disaccharide, is sensed by the pattern recognition receptor TLR4 of the human immune system. To avoid TLR4 sensing, bacteria evolved mechanisms to modify their lipid A structure, for example by changing the number or lengths of its fatty acid side chains, or adding or removing terminal phosphate moieties. However, not all LPS modifications are important for TLR4 avoidance and the biological function of many LPS modifications is only poorly characterized. This proposal will test the novel hypothesis that specific lipid A modifications enable bacteria to escape from host immunity exerted by human guanylate binding protein 1 (GBP1). We recently showed that GBP1 is an additional bona fide LPS-binding protein. Intracellular GBP1 executes at least two functions: i) it accelerates the kinetics of LPS-mediated inflammasome activation and ii) it binds directly to the surface of cytosolic Gram- negative bacteria, where it acts as a surfactant operating synergistically with antimicrobials that need to penetrate the bacterial outer membrane. In Aim1 we will identify specific lipid A modifications that block the binding of GBP1 to the surface of two important human pathogens: the intracellular enteric pathogen Salmonella enterica Typhimurium and the extracellular pathogen Pseudomonas aeruginosa. GBP1 resides in the host cell cytosol but GBP1 is also secreted into the extracellular milieu. Secreted GBP1 can be found at high concentrations in plasma and cerebrospinal fluids of bacterial meningitis patients. However, the biological function of secreted GBP1 is unknown. Because we found that GBP1 binds to the extracellular bacterial pathogen Pseudomonas aeruginosa, we will test whether and how secreted GBP1 can exert host defense to extracellular bacteria in Aim2. Specifically, we will test the hypothesis that secreted GBP1 works synergistically with extracellular antimicrobial peptides. Conceptually related, Aim2 will also identify extant antibiotics that operate synergistically with GBP1. Together, Aims 1 and 2 provide a roadmap towards novel strategies for the treatment of many Gram-negative infections: targeting LPS-modifying enzymes involved in GBP1 evasion combined with the use of antibiotics that operate synergistically with GBP1.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Innovative interventions that support young cisgender women’s (YCW) use of PrEP are critically needed. Adolescent girls and young women (AGYW) in sub-Saharan Africa continue to have disproportionately high HIV incidence, particularly AGYW in Siaya County, Kenya. Existing intrapersonal-, interpersonal-, and clinic- level PrEP interventions will likely be more successful when embedded within supportive environments where prevailing social norms encourage YCW’s use of PrEP and normalize, rather than stigmatize, its use. Findings from our recent R21 highlight the negative impact on YCW’s PrEP use due to PrEP-related stigma by communities members, suggesting a need for broader-level interventions. The purpose of this R01 is to develop, pre-test, and pilot a community-level, ethnodrama intervention designed to transform community member beliefs about and foster support of YCW’s PrEP use, thereby reducing enacted PrEP-related stigma toward YCW and anticipated and internalized stigma among YCW. In Aim 1, we will develop the ethnodrama intervention. The Eldoret Film Festival, a Kenya-based drama group, together with Theater Delta, a U.S.-based organization focused on fostering social change through interactive theater, will use our R21 findings to create a series of ethnodrama performances designed to change the narrative on YCW’s PrEP use. In Aim 2, we will pre-test the ethnodrama intervention with YCW who have taken/are taking PrEP, female peers of YCW, male partners of YCW, healthcare providers, informal community leaders, and family members of YCW (n=60). During two rounds of pre-testing, we will assess the acceptability of the ethnodrama’s storylines and interactive components through small group discussions and explore the intervention’s effect on transforming participants’ PrEP-related perceptions using a validated Narrative Transportation Scale (NTS) and other assessments. In Aim 3, we will publicly pilot the ethnodrama intervention in its entirety in multiple areas in one sub-county of Siaya, focusing on assessing the feasibility of implementing the ethnodrama intervention and exploring the intervention’s perceived effectiveness on transforming beliefs, creating support of YCW’s PrEP use, and reducing stigma. Among a study cohort of YCW who have taken/are taking PrEP (n=20), we will administer a validated PrEP-related stigma scale pre-pilot, immediately post-pilot, and 3 months post-pilot and conduct in- depth interviews (IDIs) to explore responses to the PrEP stigma scale and perceptions of the intervention’s potential impact on reducing PrEP-related stigma. We will also administer the NTS and other assessments with randomly-selected audience members (n=20) for each performance, and administer the NTS and other assessments, including IDIs on the intervention’s potential impact on reducing PrEP-related stigma, with a community member study cohort (n=20), immediately post-pilot. By achieving our study aims, we will have the experience to rapidly initiate and implement the ethnodrama intervention on a larger scale in a subsequent effectiveness R01 that will measure its effect on PrEP adherence and persistence among YCW.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY The purpose of the Impact Neuroscience Program is to enhance rigorous scientific training in neuroscience at Duke University and modernize graduate education to meet the demands of a rapidly changing scientific and professional landscape. Traditional mentoring models are no longer sufficient to prepare students for the evolving workforce, where advances in artificial intelligence, data science, and translational applications are reshaping opportunities. This program equips graduate students, together with their peers and mentors, to succeed in both academic research and emerging careers in industry and entrepreneurial ventures. The program includes targeted neuroscience training and professional development activities such as advanced methods training within faculty labs, specialized instruction in data science and open science, and structured participation in a professional seminar series that provides resources for career development. In parallel, the program emphasizes community-wide practices and workshops that strengthen mentoring, streamline career planning, and improve the overall training infrastructure at Duke. In addition to activities with strong evidentiary support, the program will test and evaluate new interventions informed by the neuroscience of motivated learning. We predict improved retention, training efficiency, and long-term performance for the graduate student fellows, as well as broader benefits for the neuroscience community, including enhanced productivity and research outcomes across host labs.

NIH Research Projects · FY 2024 · 2023-07

Abstract In sub-Saharan Africa (SSA), people living with HIV (PLWH) face a new epidemic of uncontrolled hypertension, the leading risk factor for death worldwide. Current care models in SSA are inadequate to address the growing burden of hypertension in PLWH, yet few interventions targeting this population exist. We previously piloted Community Health Worker (CHW)-delivered Hypertension Management Pilot (CHAMP), a clinic-based hypertension educational intervention delivered by a CHW situated within a Tanzanian HIV clinic, and found it to be highly feasible and acceptable. However, the efficacy and sustainability of CHAMP remains unproven. In contrast, Control of Blood Pressure and Risk Attenuation (COBRA), a multi-component intervention consisting of community-based CHW-delivered education and blood pressure monitoring and protocolized referrals to physicians, demonstrated efficacy in improving hypertension control and reducing all- cause mortality in a cluster-randomized trial across 3 countries in Asia. Evidence-based interventions like COBRA hold considerable promise in achieving hypertension control among PLWH in SSA, but have not been studied in this population. In this 2-year study, we will integrate an evidence-based, multi-component, CHW-delivered hypertension intervention (COBRA) with our early experience deriving CHAMP to improve hypertension care and reduce blood pressure among PLWH in Tanzania. Using implementation science methods, we will adapt COBRA for delivery within the Tanzanian HIV clinic, examine implementation outcomes, and estimate effect size in preparation for a subsequent large-scale, hybrid effectiveness-implementation trial across Tanzania. In Aim 1, we will assemble a Design Consultation Team of key stakeholders to assist in intervention adaption. Adaptation and integration of COBRA and CHAMP will occur iteratively over 8 months, guided by the ADAPT- ITT model, and informed by our previously collected qualitative data describing barriers and facilitators to hypertension care for PLWH. In Aim 2, we will pilot the adapted intervention with a single arm pre-post feasibility trial in two HIV clinics and assess implementation (reach, adoptability, implementation, maintenance) and preliminary effectiveness outcomes (hypertension control, systolic and diastolic blood pressure, antihypertensive adherence, cardiovascular disease risk score, and hypertension knowledge). Our research team, comprised of experts in hypertension, HIV, and implementation science and our robust relationships with community agencies and health systems make us well-suited to conduct this work. The proposed program will lay the groundwork for an R01 application to evaluate implementation, effectiveness, and scalability of the adapted intervention in a fully powered trial across Tanzania and help stem the rising tide of cardiovascular comorbidities among PLWH in SSA.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT This Mentored Career Development (K01) Award proposal will provide an outstanding educational environment, mentoring, and training for Dr. Jillian Hurst, a pediatric infectious diseases researcher whose overarching career goal is to integrate clinical and biological data to further our understanding of the role of the microbiome and related host factors in modifying susceptibility to common childhood infections Dr. Hurst originally trained in biochemistry and molecular biology and has worked in translational pediatric research for the past five years. This career development award will allow her to receive training in omics and advanced statistical analyses using large datasets, permitting her to lead multidisciplinary teams that use large clinical and omics datasets to evaluate factors underlying common pediatric infections. The proposed research studies center on the role of the upper respiratory microbiome in the development and recurrence of acute otitis media (AOM; middle ear infection), the most common infectious disease of childhood. Dr. Hurst will test the hypothesis that early life antibiotic exposures disrupt the development of the URT microbiome, predisposing children to AOM and other common respiratory infections. She will use over eight years of electronic health records data, banked longitudinal upper respiratory specimens collected from children from birth to age 2, and samples and data from a prospective cohort of children with recurrent AOM to accomplish the following specific aims: 1) Assess the impact of early life antibiotic exposures on recurrent AOM development.; 2) Delineate the effects of early life antibiotic exposures on URT microbiome development; and 3) Evaluate associations between antibiotic exposures and the URT microbiome in children with recurrent AOM. Findings from these studies will guide the development of new strategies to prevent and treat AOM and associated infections, including guidance for antibiotic use and identification of therapeutic targets within the URT microbiome that could be leveraged to restore URT microbiome function and improve respiratory health among young children. Mentorship will be provided by a group of three established investigators with complementary expertise: Dr. Christopher Woods, an infectious diseases physician and clinical epidemiologist with expertise in the evaluation of host responses to infectious agents; Dr. Matthew Kelly, a pediatric infectious diseases physician with expertise in the human microbiome and clinical omics analyses; and Dr. Benjamin Goldstein, a biostatistician with expertise in the use of electronic health records data for population health analyses. Additionally, Dr. Hurst will be advised by internal and external advisors with expertise in antimicrobial stewardship, pediatric otolaryngology, health informatics and omics analyses, and the treatment and pathophysiology of AOM. Completion of the proposed training and research activities will establish Dr. Hurst as an independent investigator who will lead an NIH-funded research program investigating host factors and exposures underlying common infections in children.

NIH Research Projects · FY 2026 · 2023-07

Project Summary/Abstract Stroke is a leading cause of death and serious long-term disability and particularly devastating to rural communities. In the United States, rural areas have an estimated 30% greater stroke mortality compared to urban areas. Limited access to time-sensitive acute stroke therapies is an important cause of rural disparities in stroke morbidity and mortality. Regional stroke systems of care that coordinate emergency medical services (EMS), emergency departments, inter-facility transfer agencies, and hospitals can improve access to acute stroke care for underserved populations. However, evidence on effective and efficient stroke system designs and strategies is lacking. Prehospital stroke screening by EMS and routing directly to a stroke center specialized in providing advanced care can significantly reduce time to treatment and improve patient outcomes. However, optimal EMS stroke triage and transport strategies that maximize benefit to stroke patients and efficiently use emergency medical resources are largely unknown and depend on regional and local characteristics. Therefore, our overall objective is to develop a decision-analytic systems model that allows stroke system planners to compare potential effects of regionalized EMS triage and transport strategies on rural stroke outcomes and health care resource utilization. Decision-analytic modeling is a rigorous and flexible approach for integrating information sources to conduct in silico testing of system-level strategies under varying contexts. This comparative evidence provides valuable and timely information to decision makers to tailor interventions for real-world implementation and evaluation. Using existing real-world data sources, published evidence, and key stakeholder input, we will build a computer simulation model of regionalized stroke care for 70 rural counties in North Carolina. Our specific aims are to: (1) determine EMS stroke triage and transport strategies that optimize functional outcomes in rural stroke patients; (2) estimate the potential system-wide effects of rural EMS triage and transport on health care resource utilization; and (3) understand the influence of rural population and health care system characteristics on optimizing EMS stroke triage and transport strategies. In response to NOT-MD-20-025, this project aims to understand and address stroke disparities by improving access to timely acute care for underserved rural populations. Upon successful completion, we will have contributed a novel decision support framework to understand and improve regional stroke systems of care across various settings. Our future research will implement refined EMS stroke triage and transport strategies into rural systems and prospectively evaluate long-term patient outcomes and healthcare costs. This line of research has high potential to address significant morbidity and mortality and health disparities caused by acute stroke and other time- and resource-dependent medical emergencies (e.g., myocardial infarction, sepsis, and trauma).

NIH Research Projects · FY 2026 · 2023-07

Abstract Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a subgroup of prostate tumors with poor patient outcomes. Beyond systemic chemotherapy and palliative care, no targeted therapy has been successful reflecting our limited knowledge of how NEPC is developed. We have reported a t-NEPC-specific RNA splicing program driven by the neural splicing factor SRRM4, which promotes t-NEPC development. This splicing program has genome-wide impacts on cancer cells as it reprograms the functions of epigenetic modulators, transcriptional factors, and cancer stem cell regulators, resulting in prostate adenocarcinoma cells acquiring neuroendocrine lineage and becoming independent of AR signalling for survival. The FAK (focal adhesion kinase) gene splicing is a part of the t-NEPC splicing program, whereby neural FAK splice variants are highly expressed in t-NEPC. In contrast to the constitutive FAK (FAK-c) splice variant, neural FAKs promote neuroendocrine differentiation and AR-independent tumor growth. Further RNA-seq analysis indicated that these neural FAKs act mainly through a hypoxic transcriptome via HIF-1. Importantly, we found that when tumor cells gain the neural FAK signaling, they become vulnerable to FAK kinase inhibitors. These findings suggest that aberrant RNA splicing of the FAK gene promotes t-NEPC progression and that targeting the FAK signal may provide a new therapeutic option for t-NEPC patients. We have developed four specific aims to test this hypothesis. In Aim 1, we will establish the clinical relevance of neural FAK splicing in association with neuroendocrine carcinoma histology, castrate-resistant prostate cancer progression, tumor metastasis, and patient clinical outcomes. In Aim 2, we will decipher the intrinsic mechanism by which SRRM4 regulates neural FAK splicing, and determine the extrinsic therapy-induced stress conditions that regulate RNA splicing of neural FAKs. In Aim 3, we will characterize several FAK-associated kinase signaling that were known to enhance HIF-1 protein expression through either protein synthesis or protein degradation pathways. We will also employ global proteomic analyses to profile neural FAK interactomes and their activated phosphor- proteomics that promote t-NEPC progression. In Aim 4, we will study whether FAK inhibitors can block neural FAK actions in NEPC cells and test whether FAK inhibitors can be used as a combination therapy to block t- NEPC xenograft growth. In summary, these studies will gain new mechanistic insight into how prostate cancer cells develop therapy- resistance and nominate FAK inhibitors to be potential therapeutics for therapy-resistant prostate tumors.

NIH Research Projects · FY 2024 · 2023-07

Summary/Abstract Systemic lupus erythematosus (SLE or lupus) is a prototypic autoimmune disease that causes severe systemic manifestations, of which nephritis is the major cause of morbidity and mortality. SLE primarily affects young women, with African American patients showing much earlier and more severe disease than European Americans. An important feature of SLE is the expression of antibodies to nuclear molecules (anti-nuclear antibodies (ANAs)). ANAs provide serological markers for diagnosis, classification, and disease activity. While there has been extensive study of the pathogenesis of SLE, much remains unknown about the underlying mechanisms that promote inflammation and renal injury. As a result, SLE treatment is empiric and frequently ineffective. Current treatments can cause permanent organ damage and severe side effects, providing a strong rationale for the mechanistic studies necessary for more effective and less toxic therapies. In patients as well as animal models of SLE, the formation of immune complexes by ANAs is a key step in inflammation and injury. Of ANAs that can form immune complexes, antibodies to DNA (anti-DNA) have a prominent and well-validated role in nephritis, as shown by the isolation of anti-DNA from affected kidneys of patients, as well as the induction of nephritis in animal models by administration of monoclonal anti-DNA antibodies. While most models for immune complexes are based on antibody interaction with soluble protein or nucleic acid antigens of relatively low molecular weight, recent research suggests that immune complexes in SLE form on cell-generated particles known as extracellular vesicles. We propose that DNA can adsorb onto the surface of these particles to form a “corona” that provides a target for anti-DNA antibodies. The significance of this research is the development of well-controlled synthetic DNA-particles to address fundamental questions about the immunological properties of particles that are not addressable with naturally occurring particles. The outcome of this research will be the understanding of the mechanisms by which DNA binds to particles to form an antigenic substrate; the formation of immune complexes by particles; the effects of surface DNA on the interaction of particles with immune cells; and the role of surface DNA in immune stimulation. Aim 1 will determine the antigenicity of DNA adsorbed on particles as a function of particle diameter, DNA length, and a protein corona using a previously developed antibody binding assay as a readout. Aim 2 will use the antigenic particles identified in Aim 1 to elucidate the immunostimulatory activity of DNA-particles in vitro, measuring the immune response and cellular internalization of the DNA-particles. The development of a well-controlled synthetic particle system provides an innovative approach to the study of SLE. Beyond SLE, the role of naturally occurring, cell-generated particles as elements in disease pathogenesis has important implications for other autoimmune and inflammatory diseases. 0

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The average American adult consumes over 40 pounds of sugar per year. While sugar intake is necessary for energy metabolism and survival, this overconsumption has led to rampant obesity and diabetes. Therefore, it is critical to determine the gut-brain circuit that drives sugar overconsumption. Recently, specialized sensory cells in the intestinal epithelium, known as neuropod cells, were found to sense intestinal sugars and drive sugar appetite. Neuropod cells sense sugars using sodium-glucose transporters (SGLTs). Most studies on intestinal sugar sensing have focused on glucose transport ability itself, but little is known about sensing in the absence of transport. Here, we will use an anti-diabetic molecule specific to human SGLTs to probe whether it is glucose transport or sensing that is necessary to activate the neuropod cell sugar sensing circuit. My hypothesis is that sugar sensing, in the absence of transport, will activate neuropod cells, causing glutamate release and vagus nerve activity. Therefore, I am pursuing the following aims: 1) to determine whether specific SGLT activation leads to neuropod cell glutamate release and 2) to determine whether an anti-diabetic molecule leads to rapid, neuropod cell dependent vagal activity. My approach includes neurogenetic manipulations of intestinal organoids and in vitro and in vivo electrophysiology. These studies may uncover a pharmacological target for modulating rapid gut-brain control of food choice without perturbing life-sustaining sugar absorption. My co-sponsors, Drs. Diego Bohórquez, Ph.D. and David D’Alessio, M.D., are experts in neuropod cell nutrient sensing and hormone signaling in obesity, respectively. Consistent with their long- established track record of mentorship, the proposed studies and training plan will provide me with the rigorous scientific training and leadership skills necessary for a career as a physician-scientist based on gut-brain circuit manipulation as a bariatric intervention.

NIH Research Projects · FY 2024 · 2023-07

Project Summary/Abstract Disruption of mitochondrial oxidative phosphorylation (OXPHOS) is associated with the development of biochemical alterations that typically affect tissues with a high energy demand, particularly skeletal and cardiac muscle. An inherited autosomal recessive skeletal myopathy and hypertrophic cardiomyopathy has been linked to loss of function of a nuclear DNA-encoded mitochondrial protein, due to a frameshift mutation in solute carrier family 25, member 4 (SLC25A4; c.523delC, p.Q175RfxX38). SLC25A4 encodes the heart-muscle isoform of the adenine nucleotide translocator-1 (ANT1, SLC25A4), which in the wild-type state is a critical component of mitochondrial metabolism. Patients with SLC25A4 deficiency display lactic acidosis, persistent adrenergic activation, and exertional intolerance secondary to both a general skeletal muscle myopathy as well as a hypertrophic cardiomyopathy. Ultimately, myocardial thickening and cardiac dysfunction progress to end- stage heart failure necessitating cardiac transplantation. There are not currently any disease-modifying therapies available for this patient cohort. However, adeno- associated viral (AAV) mediated gene replacement therapies have emerged as a powerful strategy for disease modification of inherited monogenic disorders. The long-term goal of our research is to develop a therapeutic gene replacement strategy to treat SLC25A4 deficiency. The objective of this proposal is to further characterize the disease phenotype as well as to synthesize and evaluate the efficacy of a recombinant AAV (rAAV) vector in an in vitro model of patient-derived cell lines and organoid models. The central hypothesis of this proposal is that AAV-mediated gene replacement can ameliorate the biochemical and functional effects of SLC25A4 deficiency and can more decisively prevent disease progression. The specific aims of this proposal are: 1. Characterize the SLC25A4 deficiency phenotype in patient-derived cell lines. 2. Synthesize a recombinant AAV vector for delivery of codon-optimized SLC25A4 cDNA to skeletal and cardiac myocytes. 3. Evaluate the efficacy of AAV-SLC25A4 viral transduction in patient-derived cell lines. These experiments will improve our understanding of the molecular mechanisms underlying SLC25A4 deficiency as well as allow us to evaluate the efficacy of an AAV platform in a relevant preclinical model. Moreover, the skills I will acquire during this fellowship will help to establish me as an independent investigator and a surgeon-scientist focused on the development of translational gene replacement therapies.