Duke University

NIH Research Projects · FY 2025 · 2024-04

ABSTRACT/PROJECT SUMMARY The negative health burden of obesity leads to an estimated 500,000 deaths in the United States annually. Obesity and related metabolic disorders are caused by the excess consumption of palatable, densely caloric foods. People with obesity have diminished reward from food. Although the taste of food can elicit a rewarding feeling, the intestine is required for food reward. How the gut signals this reward to the brain is unknown. My long-term career goal is the document the physiologic regulation of food intake for better treatment of obesity. With the support of the NIH Mentored Research Scientist Development Award – K01, the objective of this proposal is to define the role of a specialized intestinal sensory cell in food reward and obesity. This proposal is built on recent fundamental discoveries that have established how nutrient choice is guided by specialized neuropod cells in the duodenum. Building on these observations, the objectives of this proposal are three-fold: (1) to establish the role of duodenal neuropod cells in reward-driven feeding; (2) map the functional brain targets of duodenal neuropod cells; and (3) determine the effects of duodenal neuropod cells on hyperphagia and obesity. Defining how reward is signaled from the gut to the brain to guide ingestive behavior will allow for novel pharmacotherapies for the treatment of obesity. Together with my mentoring team, we have designed this project to provide me with the necessary research and professional training for me to excel as an independent investigator at the intersection of gastroenterology and neuroscience.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT Coronavirus disease 2019 (COVID-19)-associated nephropathy (COVAN) is a severe form of kidney disease resulting from collapse of the glomerular tuft and manifesting clinically with proteinuria and high rates of kidney failure. More than 90% of COVAN cases are associated with two coding variants in the apolipoprotein L1 (APOL1) gene. These variants are mainly found in persons with Black, African American, or Hispanic ancestry. The objective of this proposal is to identify patient-specific determinants of APOL1-mediated COVAN penetrance. Our published and preliminary data suggest that: 1. APOL1 is robustly expressed in the glomeruli of patients with COVAN but not in healthy controls. 2. COVID-19-induced cytokines upregulate pathogenic APOL1 variants via JAK-STAT signaling in human kidney organoids and, in turn, cause podocyte injury. 3. Cytokine-induced podocyte injury is blocked by inhibition of JAK/STAT/APOL1 axis. 4. Expression of variant APOL1 protein is sufficient to cause dose-dependent cytotoxicity in vitro. Consistent with these discoveries, others found that expression of APOL1 variants caused glomerulosclerosis in transgenic mice. Based on these published and preliminary data, we hypothesize that maladaptive crosstalk between glomerular cells and immune cells drive increased expression of APOL1 and the pathogenesis of COVAN. Moreover, that patients who develop COVAN either express higher levels of pathogenic cytokines (those cytokines known to induce APOL1 expression), or the glomerular cells of these individuals have an enhanced and maladaptive response to the same level of cytokine. To test this hypothesis, we will leverage unique patient-derived kidney organoids and immune cells of persons with biopsy-proven COVAN (collected after patients have recovered from infection). We propose two aims: 1.) Delineate the divergent biological responses of kidney organoids derived from COVAN patients versus controls after (i) infection with SARS-CoV-2 or (ii) treatment with COVID-19 induced cytokines. 2.) Identify differences in cellular response of immune cells of COVAN patients versus controls after (i) infection with SARS-CoV2 or (ii) treatment with COVID-19 induced cytokines. Identifying host- factors that regulate APOL1 and immune-glomerular interactions in COVAN will provide novel biomarkers and therapeutic targets for modulating APOL1 expression to treat COVAN and other forms of APOL1 nephropathy, helping to mitigate disparities in kidney disease. Execution of these scientific aims and completion of the career development activities of this proposal, along with the experienced mentorship and strong institutional support, will prepare me for a career as an independent physician scientist equipped for research into APOL1- mediated, inflammatory-mediated, and viral-mediated nephropathies.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT A substantial part of our diet and health regime involves bitter food and medicine. Bitter molecules bind bitter taste receptors (Tas2rs) and activate a sensory pathway that elicits an aversive taste sensation in the oral cavity, mainly to avoid ingestion of toxins. Tas2rs are also found throughout the digestive tract, and bitter stimuli in the gut affect feeding behavior through gut peptide secretion and stimulation of sensory regions in the brain through the vagus nerve. Yet, it remains to be determined how sensory signaling from ingested bitter molecules are transduced from gut to brain. Our lab discovered a population of specialized gut enteroendocrine cells, called neuropod cells, that detect luminal nutrients in through apical sensors and transduce sensory signals to the brain in milliseconds, via the vagus nerve. This gut sensory pathway regulates feeding behavior in real-time. The hypothesis of this application is that neuropod cells detect bitter signals through Tas2rs and transduce them onto vagal neurons. Preliminary findings show that isolated small intestinal neuropod cells are enriched for a subset of murine Tas2rs, compared to all other intestinal epithelial cells. Expression of these Tas2rs is dynamic, as it decreases in response to bacterial endotoxins. Therefore, neuropod cells may act as gut sensors for bitter compounds to communicate the valence of bitter molecules, which range from toxic to medicinal. Specific aim 1 will determine whether neuropod cells are activated by bitter stimuli through Tas2r signaling. Expression of Tas2rs and their signaling molecules will be characterized throughout the gut and activation of neuropod cells in response to bitter molecules will be determined by calcium imaging. The requirement of Tas2rs for bitter signaling in neuropod cells will be determined through targeted knockouts of Tas2rs. Specific aim 2 will determine whether neuropod cells signal luminal bitter stimuli onto vagal neurons. The signaling molecule will be identified by release assays in intestinal organoids and single neuropod cells in response to bitter molecules, and the sensory pathway will be determined by measuring vagal activation by electrophysiology, in response to gut infusions of bitter molecules. Finally, pharmacological and optogenetic inhibition approaches will determine the requirement of neuropod cell signaling for this sensory pathway. This work will uncover how bitter stimuli in the gut are conveyed to the brain, bringing forth a new aspect of chemosensation and gut-brain communication for a variety of foods and medicine that are essential for our health.

NIH Research Projects · FY 2026 · 2024-04

1 Abstract Sepsis is the leading cause of hospital mortality in the US and imposes immense health and economic burdens. Optimizing clinical outcomes of sepsis hinges upon early detection, accurate classification of patient phenotype, and prompt treatment. However, the understanding of sepsis at a pathophysiologic level is limited, making it challenging for clinicians to effectively diagnose and treat sepsis patients. With technological advancement, mas- sive electronic health records (EHRs) and registries have been generated by the routine collection of data from medical and daily activities. These data provide exceptional opportunities for generating insights into improved clinical decision-making, prognosis, and personalized medicine treatment strategy for sepsis among many other diseases. On the other hand, EHR data are often high-dimensional, sparse, longitudinal, and include abundant temporal information. Such complex structures pose great barriers to the use of traditional statistical and machine learning methods. Although EHR data are often stored in relational databases, they can be represented by an order-3 tensor, i.e., an array with three directions representing different subjects, features, and time points that often vary greatly across subjects/features. This tensor-based representation presents a fresh perspective for analyzing EHR data, offering a new opportunity to improve sepsis care. In this proposal, we aim to improve the progress assessment, patient phenotyping, and treatment planning of sepsis through an integrated tensor-based modeling framework to tackle the challenges associated with high- dimensional EHR data and their complex temporal structure. The specific tasks include: a) developing an iterative timeline registration algorithm to enhance the assessment of the progress of sepsis by leveraging the tensor structure of EHR data; b) devising a tensor-based method for phenotyping using longitudinal high-dimensional EHR data; c) creating a novel method to estimate the potential outcomes and treatment effects of time-varying treatment regimes through low-rank tensor completion. The developed tools will become Sepsis Pulse, a module to be incorporated into an existing digital workflow known as Sepsis Watch within Duke Hospital’s healthcare system.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT One of the great achievements in medical sciences is the improved survival rate among children diagnosed with cancer. Although estimates indicate the 5-year survival rate of children diagnosed with a malignancy is near 80%, most of these individuals prior to the age of 40 demonstrate indices of physical limitation normally associated with the elderly population. Among the treatments received by pediatric cancer patients is multimodal fractionated x-ray irradiation and chemotherapy. Both induce genotoxic stress that eliminates proliferative cancer cells; however, multifarious direct and indirect effects of radiation and chemotherapy can negatively impact normal tissue growth and maintenance especially in actively growing populations. Phenotypes observed earlier in pediatric cancer survivors is the accelerated onset of frailty indices such as neuromuscular decline. In a murine model, we find weeks after image guided pediatric fractionated x-ray irradiation of hindlimbs reduced skeletal muscle fiber size, impaired neuromuscular function, fibrosis, and activation of stress related p53 gene expression. Among the p53 regulated genes elevated weeks after pediatric fractionated radiation is Growth Differentiation Factor 15 (GDF15), a regulator of food intake and body weight in response to stressors such as cancer therapies. Single cell RNA sequencing (scRNASeq) analysis revealed radiation induced GDF15 expression was restricted to muscle resident endothelial cells. Also, we identified a subpopulation of neuromuscular junction associated muscle resident mesenchymal progenitor cells expressing the receptor tyrosine kinase Ret that is implicated in mediating GDF15 activity. We find in our murine model of multimodal pediatric cancer treatment and survivorship, deficits in body weight gain and exacerbation of neuromuscular related phenotypes observed with fractionated radiation alone. Therefore, our long-term goal is to rigorously characterize the mechanisms whereby treatment related GDF15 expression impacts neuromuscular related phenotypes in our murine model of multimodal pediatric cancer therapy and survivorship. Ideally, such insight would be used to uncover interventions to attenuate pediatric cancer treatment related neuromuscular decline that increases morbidity and burdens survivors. To accomplish our objectives, we will utilize, assessment of skeletal muscle integrity, neuromuscular function, imaging analysis, mouse genetics, scRNAseq analysis, flow cytometry, gene expression analysis, pharmacological treatments, and measures of body weight and food intake. The specific aims of this proposal are 1) determine if endothelial cell specific loss of p53 or GDF15 knockout impacts pediatric cancer treatment related fibrosis and neuromuscular deficits, and 2) examine whether Ret activity regulates pediatric cancer treatment related fibrosis and neuromuscular deficits.

NIH Research Projects · FY 2026 · 2024-04

ABSTRACT The mammalian gut harbors complex communities of microbes that interact and compete with each other while also influencing the physiology of the host. Discerning the mechanisms by which major members of these microbial communities colonize the gut is a fundamentally important research goal. One major member is the gut bacterium Phocaeicola vulgatus (Pvu), a gram-negative anaerobe and one of the most abundant bacterial species found in the gut of humans and other mammals. Pvu has been associated with inflammatory bowel disease and several metabolic disorders, and can also infiltrate and colonize established gut microbial communities following a single introduction. However, there is a gap in understanding of the molecular tools that Pvu deploys to colonize and invade established niches in the gut. Our long-term goal is to understand what makes Pvu such an unusually efficient colonizer of the mammalian gut. Our preliminary studies have identified an operon specific to the Phocaeicola genus that is required for fitness within the mouse gut in a manner strongly dependent on microbial competition, but the specific functions of the genes in that operon remain unknown. I will address this gap by defining the requirement for the genes in this operon on Pvu physiology in vitro and on in vivo gut colonization amidst competition with other microbes. This will be accomplished through in vitro studies involving tagging proteins encoded in the operon for immunohistochemistry, lipid binding assays, and chromatin immunoprecipitation sequencing. Additionally, in vivo studies using germ-free and specific-pathogen free mice will be performed to understand how the specific genes in the operon influence Pvu's colonization efficiency and biogeography in the gut. The expected outcomes of these studies include identification of new mechanisms used by Pvu to effectively colonize the mammalian gut as well as fundamental new insights into Pvu physiology. These results will vertically advance the field by providing new understanding of Pvu's colonization strategies which could eventually lead to the development of new probiotic platforms and strategies for controlling gut microbiome composition in the context of inflammatory bowel diseases and metabolic disorders.

NIH Research Projects · FY 2026 · 2024-04

Project Summary Impaired emotion regulation (ER) is a risk factor for a wide range of neuropsychiatric conditions and safeguarding its development could have important implications on mental health. Pre-pregnancy obesity has long been associated with indicators of impaired ER in children – e.g., elevated mood symptoms, increased negative emotional intensity, and increased rates of psychiatric disorders with ER deficits such as depressive and anxiety disorders. Despite these well documented associations, mechanistic studies in humans that link pre-pregnancy obesity with offspring ER development are scarce. Our proposal aims to address this research gap. We will examine the overarching hypothesis that pre-pregnancy maternal obesity negatively influences offspring ER development through its effects on the maternal gut microbiome and metabolome. Our innovative design includes longitudinal assessments of the maternal microbiome and metabolome, repeated assessments of ER- related brain systems in the offspring using fetal and infant MRI scans, and behavioral assays of ER in the offspring through the first 24 months of life. Using these multimodal assessments and longitudinal design, we will test whether (a) the maternal gut microbiome during gestation is influenced by maternal obesity; (b) the maternal gut microbiome is associated with maternal metabolites within tryptophan and tyrosine metabolic pathways; and (c) maternal metabolites in turn correlate with ER development measured with brain (fetal and infant MRI) and behavioral correlates. Our study is uniquely positioned to address these aims leveraging an ongoing human birth cohort study (HOPE cohort, supported by Duke University) of mother-infant dyads enriched for obesity (50% of enrollees with pre-pregnancy BMI>=30, confirmed by medical record). The HOPE cohort includes an extensive battery of biospecimens and clinical/demographic measures with pregnant women enrolled during the first trimester gestation and their children followed into the first few years of life. With over 1 million children born yearly in the US to mothers who experienced pre-pregnancy obesity, the ER developmental impact of pre-pregnancy obesity is a substantial and mounting concern – and disproportionately impacts low income and minoritized populations. Mechanistic research in humans that links pre-pregnancy obesity with offspring ER development and psychiatric risk is critical to advancing prevention efforts.

NIH Research Projects · FY 2026 · 2024-04

Abstract: Infections caused by Mycobacterium tuberculosis (Mtb) have historically been the leading cause of death from a single infectious agent and still result in 1.5 million deaths annually. One of the hallmark characteristics of the disease is an aggregate of immune cells called the granuloma, where macrophages develop epithelioid character, expressing epithelial cadherin (E-cadherin) and other canonical markers of epithelial cells. This structure restrains the bacteria within but is unable to eradicate the infection despite exposing the pathogen to pH changes, hypoxia, and antimicrobial peptides. The unique, lipid-rich cell envelope of mycobacteria protects them from these stressors while simultaneously modulating the host immune response. Thus, targeting bacterial cell envelope components that are specific to the granuloma is a potential avenue for future drugs and therapeutics. Using the zebrafish-Mycobacterium marinum (Mm) model to recapitulate key aspects of TB disease, the proposed aims will uncover novel bacterial elements with importance in mycobacterial granulomas. I will explore how specific cell envelope components impact both mycobacterial survival and host dynamics in mycobacterial granulomas. I will assess how the bacteria sigma factor SigE (σE) impacts survival within the granuloma through cell envelope-mediated interactions with the host immune response. We have identified SigE as a potential modulator of bacterial survival in the granuloma, which will help better understand the host-pathogen interactions that occur in the granuloma. In addition, the knowledge of bacterial factors that allow mycobacterial persistence in granulomas can unveil potential drug targets and therapies to treat tuberculosis.

NIH Research Projects · FY 2026 · 2024-03

5R13AI184067-02 Abstract. The heavy burden of communicable diseases on the African continent is largely made up of HIV, Tuberculosis (TB) and malaria. This trio of diseases alone is creating societal and economic instability in most African countries across all age groups, and particularly in children. Most recent estimates by UNAIDS of the global burden of HIV are that 39 million people are currently living with HIV-1 infection, 25.6 million of whom reside in sub-Saharan Africa (SSA). The implementation of anti-retroviral therapy for people living with HIV (PLWH) comes with complications of costs and iatrogenic effects of drugs, and it is not a long-term solution to stop the epidemic in SSA. HIV prevention remains critical to turn the tide of HIV infections and an effective countermeasure against HIV-1 is desperately needed. More recently, passive immunization clinical trials have explored infusion of broadly neutralizing antibodies as an alternative HIV infection prevention strategy to pre-exposure prophylaxis with anti-retroviral drugs. Similar, new and innovative efforts are required to develop prevention strategies that would mitigate the HIV-1 co-morbidities of TB and malaria. In accordance with the 2019 NIH NOT-OD-20-018 that supports research training of the workforce, in this application, we propose to train early-stage scientists in Africa to fill the gap in ongoing efforts to build scientific literacy and skills for career development to the next generation of scientists. As the NIH is sponsoring several basic research projects as well as clinical trials in Africa to explore the appropriate countermeasure strategies, we believe that the proposed Infectious Disease in Africa: Training Symposium for the next generation of scientists (IDA) will lead to increased scientific knowledge and will help building future scientific leaders on the continent who will complement the U.S. task force in optimizing prevention strategies against infectious diseases. Building on the previous 11 IDA symposia, we now propose a further set of symposia, where the overarching aims are: 1) To provide cutting-edge knowledge in the fields of countermeasures against HIV and implementation for malaria and TB prevention. 2) To provide events that can enhance career development of the next generation of scientists, including grant writing, poster and oral presentation skills, networking and community engagement. The IDA training symposia will take place in Stellenbosch (Cape Town, South Africa) and each will leverage on the most recent reported results of the ongoing passive immunization and vaccine efficacy clinical trials for HIV-1, TB, and Malaria. Our efforts will comply with the NIH criteria for unbiased selection of participants.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Respiratory disease is the third leading cause of death in the industrialized world. Pulmonary fibrosis (PF) is a progressively debilitating and terminal disease in which the normally pliable tissue of the lung is gradually replaced with rigid scar tissue. PF is an especially difficult respiratory disease, as there are no effective treatments and patients die within an average of three to five years of diagnosis. Current models suggest that alveolar injury and ineffective repair underlie the pathogenesis of PF and other lung diseases. However, the mechanisms by which injury in the alveolar epithelium contributes to fibrotic disease remain unclear. Single cell transcriptomics data demonstrate that a transitional state exists between the well characterized alveolar type 2 (AT2) and type 1 (AT1) epithelial cells. Furthermore, marker analysis of this transitional state in human PF lungs suggests that accumulation of transitional cells occurs in regions with dense myofibroblasts in fibrotic lungs. It is unclear what molecular mechanisms regulate the state of alveolar epithelial cells, but data from our lab has shown that TP53 is transiently upregulated in normal differentiation of AT2 to AT1 cells. This proposal details approaches to uncover the role of TP53 in regulating alveolar epithelial cell states, and to determine the impact of an alveolar epithelial transition state on pulmonary fibrosis. Understanding the mechanisms that drive these transitional states and their potential role in PF disease pathogenesis offers new avenues for developing novel therapies for fibrotic and other lung diseases.

NIH Research Projects · FY 2026 · 2024-03

Cardiovascular (CV) therapeutics remain dangerously understudied in children: of 1055 FDA pediatric label changes under BPCA and PREA since 1998, only 29 were in CV drugs, and >90% of the sickest children with CV disease in intensive care units are exposed to ≥1 off-label drug, a practice known to worsen outcomes. Modern approaches to pediatric drug labeling are based on pharmacometric models to evaluate drug exposure response relationships, and when appropriate correlate them with adult findings. A lack of pediatric CV patient oriented scientists trained in these methods is the primary obstacle to more widespread labeling of CV drugs. With my unique background in pediatric cardiology and critical care, biostatistics, clinical pharmacology, and innovative early-phase clinical trial and real-world data utilization, I am exceptionally well positioned to close this training gap in junior CV patient-oriented scientists. Now in my 10th year on faculty at Duke and the Duke Clinical Research Institute (DCRI), I built an NIH, FDA, and foundation funded research portfolio of early-phase pharmacokinetic (PK)/ pharmacodynamic trials, regulatory compliant pharmacometric modeling and simulation, and innovative real-world-data utilization to study pediatric CV drug development. I systematically leveraged my portfolio to create opportunities for training and mentoring for 23 trainees to date. With the support of this K24, I will expand my mentorship and training program to a larger number of trainees, and obtain formal skills in mentorship, novel modeling techniques, and innovative real-world-data applications. I have developed and secured funding for an innovative, point-of care, electronic health record data enabled multi-drug platform PK/PD trial, OPTIC, designed specifically for training and mentorship. In this platform trial, each trainee owns a single CV drug studied within a multi-drug protocol which I oversee and will receive hands-on training from protocol writing through enrolling of subjects and data analysis for their drug. Coupled with an expectation for formal didactic coursework towards a master’s or PhD degree in quantitative sciences at Duke or the University of North Carolina (UNC), my trainees will receive rigorous training to prepare them as independently funded patient-oriented investigators focused on pediatric CV clinical pharmacology. In addition to my own funding, the proposed training program is supported by an exceptional research environment at DCRI including the NICHD funded Pediatric Trials Network focused on pediatric drug development, and my leadership roles as Director of the DCRI Pharmacometrics Center, and Program Director of a joint Duke-UNC T32 training program in clinical pharmacology. DCRI’s unique pipeline of NIH funded clinical research training programs, ranging from R38 summer programs for undergraduate students to early-career faculty K12 awards are ideal to recruit trainees. While rigorous and demanding, the feasibility of my proposed training program is supported by my success with funding and career development of current trainees including: 62 first-author publications with me, multiple early-career and NIH awards; >80% of graduates in academic positions.

NIH Research Projects · FY 2026 · 2024-03

ABSTRACT Infection with the common bacteria, Helicobacter pylori, is associated with a range of changes to the gastric mucosa, resulting in conditions which can have significant effects on a patient’s morbidity and quality of life, in addition to increasing risk of gastric adenocarcinoma. This proposal focuses on pre-malignant disease in the stomach through clinically-relevant investigation of the synergy between H. pylori and host response in patients with H. pylori infection and in gastric intestinal metaplasia (GIM). In clinical practice, H. pylori is only considered as present or absent. However, H. pylori strain-specific factors such as cytotoxin associated gene A, CagA, interfere with the host adaptive immune system to allow H. pylori colonization in gastric mucosa. In addition, H. pylori strains may carry antibiotic resistance genes that contribute to treatment failure and persistence of the H. pylori infection, a known risk factor for gastric cancer development. In this proposal, the IMAGINE: Intestinal Metaplasia And Gastritis INtErception Study, we aim to address major knowledge gaps relating to molecular information about H. pylori heterogeneity and the relationship to gastric mucosal changes and outcomes. Specifically, we will focus on generating new knowledge that will result in more effective H. pylori eradiation and new approaches to cancer interception in premalignant gastric disease through identifying factors associated with disease progression in patients with GIM. Our preliminary data suggest that in patients infected with H. pylori, higher bacterial load and antibiotic resistance are associated with increased risk of persistence. The combination of exposure to the CagA virulence factor with life stress exposures including current life-stress as well as adverse childhood experiences may be associated with risk of progression. We hypothesize that the combination of infection with virulent H. pylori strains and stress exposures synergistically contribute to the development of premalignant disease in the stomach. A strength of our research program is the establishment of proven recruitment strategies that have allowed us to build racially diverse cohorts, collecting detailed survey information as well as biospecimens, to study premalignant disease in a high-risk population. Our goal is to build knowledge of premalignant gastric disease that will help us address the starkly disparate outcomes related to H. pylori-infection in the US, given both increased rates of gastric cancer incidence and survival in Black patients. Through this work, we are also generating a large bank of gastric organoids including GIM from a diverse patient population. Our work is particularly relevant as current clinical guidelines for GIM recognize the racial differences but offer no guidance on how to incorporate this or knowledge of other risk factors into clinical Indeed, surveillance of GIM among any patients is currently not recommended and strategies for risk-stratification are lacking. Deeper understanding of the role of H. pylori-specific factors and host response may allow for personalized, precision H. pylori treatment as well as better screening and surveillance strategies for GIM, and ultimately gastric cancer interception.

NIH Research Projects · FY 2026 · 2024-03

Non-alcoholic fatty liver disease (NAFLD) is currently the leading cause of chronic liver disease, and its incidence and prevalence are expected to worsen with the growing aging population and epidemics of obesity and metabolic syndrome. Unfortunately, NAFLD pathogenesis remains poorly understood and an effective pharmacotherapy is lacking. Recent studies demonstrated that senescent hepatocytes accumulate in human NAFLD and correlate with adverse outcomes. However, the characteristics of senescent hepatocyte have not been defined, and it is unclear what molecular mechanisms drive/maintain this senescent phenotype or if/how the senescent phenotype per se effects NAFLD progression. My preliminary data from NAFLD patients, rodent NAFLD models and cultured senescent hepatocytes strongly suggest that hepatocyte senescence is involved in NAFLD pathogenesis and drives NAFLD progression. I hypothesize that preventing accumulation of senescent hepatocytes will improve NAFLD, and will test this hypothesis by pursuing three specific aims: Aim 1: Define the transcriptome, secretome and paracrine effects of senescent hepatocytes. Aim 2: Dissect the molecular mechanisms of hepatocyte senescence. Aim 3: Determine the role of hepatocyte senescence in NAFLD pathogenesis/progression. Overall, this project will evaluate a clinically relevant, innovative and impactful hypothesis by employing multiple state-of-the-art approaches (i.e. CRISPR-based screening, bioactive chemical screening, genome editing, single nuclei RNA-Seq, secretomics, etc) to comprehensively characterize the hepatocyte senescent phenotype, uncover fundamental mechanisms that regulate senescence-associated liver biology (e.g. phenotypic heterogeneity, metabolic reprogramming and state transitions) and identify novel senolytic therapies for NALFD. Successful completion of this study will provide novel molecular mechanisms for NAFLD pathogenesis and thus, establish the groundwork for future projects that exploit cellular senescence as a novel strategy to treat NAFLD and other liver diseases. Together with the comprehensive career development plan outlined herein, this project will advance my expertise in several areas, including cellular senescence, bioinformatics, secretomics, high- throughput screenings and genome editing. A seasoned mentoring team with diverse and complementary expertise, lengthy track records for training research scientists and full commitment for mentorship is established, and will guide my career development by providing mentorship and research resources, and offering professional scientific training during the award period. This award will provide me with superb professional and scientific skillsets, enabling me to be a very competitive and worthy applicant for independent academic faculty positions and future R01 applications.

NIH Research Projects · FY 2024 · 2024-03

ABSTRACT Vestibulodynia (VBD) is a chronic pelvic pain condition that affects 1 in 6 reproductive aged women, yet remains ineffectively treated by standard trial-and-error approaches. Our group has identified two distinct VBD subtypes that may benefit from different types of treatment: 1) VBD peripheral (VBD-p) subtype characterized by localized pain specific to the vulvar vestibule, and 2) VBD central (VBD-c) subtype characterized by pain at both vaginal and remote body regions. Preliminary data further demonstrate that VBD-p and VBD-c subtypes differ with respect to patient reported outcomes (e.g., physical and mental health), production of cytokines (intracellular proteins that regulate the activity of pain nerves and inflammatory processes), and expression of microRNAs (small non-coding RNA molecules that regulate gene expression). Women with VBD-p exhibit normal psychological profiles; balanced circulating pro- and anti-inflammatory cytokines; and dysregulation in microRNAs that regulate the expression of genes in estrogen pathways. In contrast, women with VBD-c report decreased functional status and increased somatization; increased pro-inflammatory but not anti-inflammatory cytokines; and dysregulation in microRNAs that regulate the expression of genes relevant to muscle, nerve, and immune cell function. Based on these data, we hypothesize that two VBD-p and VBD-c subtypes will preferentially respond to peripheral, central, or combined treatments and can be distinguished by cytokine and microRNA profiles. These hypotheses will be tested in a phase III clinical trial that evaluates diverse treatment strategies in women with VBD-p and VBD-c. Participants will be randomly assigned to one of four parallel arms: peripheral treatment with 5% lidocaine + 0.5 mg/ml estradiol compound cream, 2) central treatment with the tricyclic antidepressant nortriptyline, 3) combined peripheral and central treatments, or 4) placebo. The treatment phase will last 4 months (with a 6-week titration at treatment initiation and 2-week taper period at 4 months), with outcome measures and biomarkers assessed at 4 time points (0, 2, 4, and 6 months). First, we will compare the efficacy of treatments in alleviating pain among women with VBD-p and VBD-c using standardized tampon insertion with a numeric rating scale and self-reported pain on the McGill Pain Questionnaire. Next, we will compare the efficacy of treatments in improving perceived physical, mental, and sexual health among women with VBD-p and VBD-c using standardized questionnaires. Finally, we will measure cytokines and microRNAs in women with VBD-p versus VBD-c using multiplex assays and RNA sequencing, and determine the ability of these biomarkers to predict treatment response. Successful completion of the proposed work will provide new insights into the mechanisms that drive pain perception and treatment response in two distinct VBD subtypes, and determine the efficacy of peripheral, central, and combined therapies in reversing this pain. Such findings will readily translate to improved patient care, permitting the millions of women with VBD, their partners, and clinicians to make more informed decisions about pain management.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract Recent technological advancements have allowed for single-neuron and intracranial electroencephalographic (iEEG) recordings in freely moving humans. However, these implanted neural recording devices have not been integrated with non-invasive peripheral biochemical recordings. The emergence of an experimental platform combining mobile deep brain recordings with wearable biochemical and biophysical sensors for use in real-world settings is unprecedented. The proposed project will develop a novel platform that enables simultaneous single- neuron or iEEG, biochemical (cortisol, epinephrine), and biophysical (heart rate, skin conductance, and body and eye movements) activity to be recorded in freely moving human participants. As proof-of-concept, we will use this platform to investigate the neural and peripheral biomarker mechanisms underlying approach-avoidance behaviors during spatial navigation. Through an interdisciplinary collaboration between UCLA, Stanford University, and the Veteran’s Administration Greater Los Angeles Healthcare System (VAGLAHS), the program will have access to human participants whom will have implanted electrodes within prefrontal cortex, amygdala, hippocampus, or nucleus accumbens regions. The proposed project outcomes will empower future studies and other researchers to investigate, for the first-time, deep brain and peripheral biomarker mechanisms underlying freely moving human behavior in naturalistic and ecologically valid environments.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Candidate & Environment: The short and long-term goals of the candidate are to gain skills, knowledge, and experience necessary to become a successful independent investigator in gene therapy for neuromuscular diseases (NMDs) with respiratory pathology, such as Pompe disease. Duke University is the optimal location for the candidate to achieve these goals with a world-renowned Pompe disease clinical and research center, along with myology and gene therapy experts, who frequently collaborate and who host monthly research seminars. Research: Pompe disease is a fatal glycogen storage disease caused by mutations in the gene encoding acid α-glucosidase (GAA), which is responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in glycogen accumulation in the lysosomes of muscles (cardiac, skeletal, smooth) and motor neurons. The only FDA approved treatment is enzyme replacement therapy (ERT) of recombinant human GAA (rhGAA). ERT effectively treats cardiac muscle glycogen accumulation but cannot completely correct skeletal muscle pathology due to disrupted autophagy. In addition, ERT cannot cross the blood-brain barrier to treat motor neurons, and therefore, respiratory failure persists. Thus, to prevent respiratory distress in Pompe patients, there is a need for therapies that can clear glycogen and repair autophagy in the respiratory muscles and motor neurons. The overall goal is to identify novel adjunctive therapies to improve respiratory muscle and motor neuron pathology in Pompe disease. We propose to administer three autophagy activators which can cross the blood- brain barrier and assess their therapeutic impact on the motor unit of Pompe mice (Gaa-/-). Acute intermittent hypoxia (AIH), rapamycin, and recombinant adeno-associated virus (rAAV) gene therapy are potential therapies to treat Pompe disease, which will be evaluated across three aims. Each therapeutic has a unique and complementary ability to address autophagy, a critical component of Pompe disease cellular therapy, in key respiratory muscles and motor neurons. (1) Hypoxia is an activator of the autophagosome initiation complex. Additionally, AIH induces neuroplasticity in respiratory motor neurons in neurogenerative disorders. (2) Rapamycin is currently administered to Pompe patients as an immune suppressor, however, rapamycin also has a direct negative impact on master metabolic regulator, mTORC1, thus activating autophagy. (3) rAAV-GAA provides the deficient enzyme, thereby reducing glycogen and improving lysosome health which is a key to restoring normal autophagy. In the final aim of this proposal, we will combine rAAV-GAA therapy with AIH and rapamycin to determine the cumulative effect. Key methods of analysis include neurophysiology to assess neural output of respiratory nerves, whole body plethysmography to assess respiration, molecular proteomics analysis for autophagy pathway proteins, and immunohistochemistry to identify cellular architecture of muscle and CNS. These novel interventions are already used in clinical trials and have the potential to quickly translate to clinical care in Pompe patients suffering from respiratory distress.

NIH Research Projects · FY 2025 · 2024-02

Summary The aim of this proposal is to develop an innovative new system, including hardware assemblies and machine learning algorithms, for continuous, high-resolution 3D quantification of behavioral and eliciting stimulus dynamics in a natural mouse prey capture paradigm. The system will satisfy a critical unmet need for an easily adoptable, modern behavioral measurement technology that extends well beyond current offerings, which are difficult to set up and limited largely to measuring spontaneous animal movement in impoverished, static environments. Our system consists of a 3D convolutional neural network processing multi-perspective video recordings to provide detailed measurements of both predator (mouse) and prey (cricket) spatiotemporal movement patterns within an enclosed, compact apparatus permitting precise control over the visual environment. To reduce implementation complexity and enhance usability in other labs, the system will use only a single commercial video camera and a set of low-cost mirrors to provide the multiple perspectives required for 3D behavior tracking. By using only a single camera, we also reduce the instrument’s physical footprint, thus facilitating high-throughput studies across multiple setups. Furthermore, our 3D tracking algorithm will be built to support out-of-the-box generalization to cloned setups, meaning other labs can immediately start doing science with the instrument without laborious data labeling and training steps. As part of our system, we will also develop new methods for analyzing the rich 3D mouse and cricket data to isolate key kinematic and action variables along with comprehensive characterization of stimulus-behavior relationships. We will then investigate how these new measurements can be used to better understand retinitis pigmentosa and Parkinson’s disease. Preliminary experiments have been quite successful and illustrate the promise and power of our approach to collect large amounts of quantitative behavior data and identify new phenotypes of motor disorders. As our vision is to make as large of an impact as possible, our system and datasets will be shared openly with community to catalyze a wide range of new research into brain function and treatments for neurological disease.

NIH Research Projects · FY 2026 · 2024-02

Cardiorespiratory conditions such as the acute respiratory distress syndrome (ARDS), congestive heart failure, COVID pneumonia, and sepsis are among the most common causes of mortality and morbidity. They are also notable for high rates of persistent psychological distress symptoms including depression, anxiety, and PTSD that worsen quality of life and outcomes of the underlying conditions. Yet there are few effective strategies able to overcome barriers of limited access to mental health care. Even less is known about distress management among people from different subgroups. To fill this gap, we developed Blueprint, an adaptive coping skills training intervention, and have optimized it over years of research. We conducted a multicenter RCT (PCORI PFA 195) of a telephone- and web-based version among those recently hospitalized with serious cardiorespiratory conditions, finding that it reduced depression symptoms and improved quality of life among those with elevated baseline distress. Informed by lessons learned about intervention delivery and eligibility criteria, we next conducted a single-center pilot RCT (R34 HL145387) that targeted a broader population and tested a completely automated, self-guided, symptom-responsive mobile app version of Blueprint. We found excellent adherence and a strong effect on depression, anxiety, PTSD, and quality of life compared to control. Given these promising findings, a formal test of the Blueprint adaptive coping skills training intervention's efficacy is needed. Therefore, we propose a 5-year multicenter RCT with 6-month follow up in which 400 cardiorespiratory failure survivors with elevated symptoms of psychological distress post-discharge are randomized to either Blueprint or an Education Program control-both delivered through similar mobile app platforms. Our specific aims will: (1) Test Blueprint vs. control on symptoms of depression, anxiety, PTSD, and quality of life; (2) Determine patient-level characteristics associated with a great treatment response among sociodemographic subgroups of interest, also applying a heterogeneity of treatment effects analysis to identify other groups of clinical relevance; and (3) Ensure off-the-shelf intervention readiness for implementation by using an exploratory mixed-methods hybrid type 1 implementation framework analysis that integrates semi-structured interviews with trial participants and quantitative trial data from Aims 1 and 2. Innovative elements include a fully automated mobile health delivery system that personalizes content in response to changes in symptom trajectories, the integration of a Spanish language intervention version, and strong community engagement. This project addresses national research priorities and could advance the field with a personalizable yet population-focused therapy that could be scaled broadly and efficiently to improve patient outcomes.

NIH Research Projects · FY 2025 · 2024-02

PROJECT SUMMARY / ABSTRACT Staphylococcus (S.) sp. are ubiquitous colonizers of human skin and mucosa. Its commensal members, such as S. epidermidis, make key contributions to skin health by modulating the immune system and promoting skin microbiome homeostasis. Other species, such as S. aureus, can be asymptomatic colonizers or disease- causing pathobionts. While S. hominis, capitis, haemolyticus and other species are ubiquitous, they are little characterized vis a vis their role in the skin microbiome. Yet in our extensive surveys of healthy and diseased skin, we have found significant shifts in the species level composition of skin staphylococci. For example, healthy skin strongly favors colonization with epidermidis and hominis, while patients with congenital ichthyosis shifts dramatically to a capitis-dominated microbiome. Eczema associated with primary immunodeficiencies is also more prominently characterized by non-aureus staphylococci, including haemolyticus. Finally, in atopic dermatitis, strain-level differences in epidermidis can be associated with active disease, not only the canonical aureus. These species and strain-level differences are striking in their diversity, and we hypothesize that genetically diverse Staphylococcus species and strains differentially affect skin homeostasis by altering the inflammatory milieu and/or skin barrier. The goal of this proposal is to investigate how diverse staphylococcal species and strains drive epidermal responses. We amassed and sequenced a collection of thousands of staphylococcal isolates from human skin, and examined the transcriptional response of 3D human reconstructed skin equivalents (RHE) to colonization with 180 of these diverse species and strains, identifying species- and strain-level differences in the skin's response. Here, we will build on this preliminary data to investigate host and microbial mechanisms underlying these interactions. Selecting a subset of 30 isolates that produced signatures of interest from our RHE screen, we will examine transcriptional response in a fibroblast+RHE (F-RHE) model from multiple matched donors to dissect how increased skin complexity modifies the colonization response (Aim 1). We will use RNA-seq, scRNA-seq, spatial transcriptomics, and proteomics to isolate cell-type and layer-specific responses to colonization, as well as reconstruct likely cell-cell signaling pathways. Second, given the recently identified impact of commensal microbiota on skin barrier and immunity mediated through AhR, we will similarly investigate the ability of these staphylococci to modulate the skin barrier by producing metabolites that activate AhR (Aim 2). Using our established metabolomics and CRISPRi toolkits developed in staphylococci, we will investigate microbial mechanisms to identify likely involved metabolites. These experiments will further our goal to investigate how diverse staphylococcal species and strains drive epidermal responses, and will allow us to define mechanisms of a microbial contribution to numerous inflammatory skin diseases that affect the skin barrier.

NIH Research Projects · FY 2025 · 2024-02

CRISPR Guide RNA Exponential Directed Evolution Synergy with Machine Learning to Improve Gene Editing Efficiency Abstract: Engineered bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems consist of a single guide RNA (gRNA) and the Cas9 protein forming a ribonucleoprotein complex (RNP) that can scan and cleave DNA. The gRNA consists of a 20 base targeting region complementary to the desired DNA target site, followed by an 80 base scaffold region that binds and activates the Cas9 protein. Unfortunately, high editing efficiency only occurs on a small subset of all possible DNA targets. Editing efficiency is hindered by strong base pairing of the targeting to the scaffold region of the gRNA, and multiple groups have shown that restoring said efficiency can be achieved via low throughput testing of mutations as well as chemical modifications at predicted gRNA misfolding sites. Unfortunately, the vast majority of low efficiency gRNA targets remain unresolved, and while targeting region predictive tools to avoid low efficiency targets exist, no scaffold optimization tools that can fix said efficiency have been developed. Our lab has pioneered a novel high throughput gRNA directed evolution method that can select highly functional scaffolds from libraries of ~1E14 gRNA variants for the Staphylococcus Aureus (sa) CRISPR system, the ortholog with the leading editing efficiency used in AAV gene therapy applications. With the advantage of being encoded for delivery in a single AAV vector. My preliminary data shows improvement of gene editing efficiency via both RNP delivery and plasmid delivery, evolving hundreds of saCas9 gRNA scaffold variants with 10 to 30% variation from the wildtype gRNA scaffold. We hypothesize performing selection with clinically relevant targets will provide the best efficiency scaffolds for these targets, while also expanding our gRNA toolkit. Furthermore, using these scaffolds in a high throughput lentiviral screen, to feed into various structure and sequence-based machine learning and deep learning algorithms, we expect development of a publicly accessible efficiency prediction algorithm leading to improvement of editing efficiency on targets across the human genome. This model will aid in finding the best possible match for improved gRNA efficiency. This will free up time and resources associated with testing gRNAs while also improving the wildtype efficiency paradigm that has ruled the CRISPR gene editing field since its inception. The development of gRNA directed evolution in this Cas9 model, will serve as a proof of concept for the accelerated gRNA efficiency optimization for the use in new CRISPR systems. Furthermore, developing of Strong computational skills, especially in machine learning and deep learning, will enhance an already productive career in academia, where I aim to rapidly optimize CRISPR systems such as Cas9 guided transposons in my postdoctoral studies.

NIH Research Projects · FY 2026 · 2024-02

Project Summary/Abstract A loss of olfaction, termed anosmia, is strongly associated with Alzheimer’s Disease (AD) and often precedes the development of cognitive decline or other symptoms. Currently, we lack disease-modifying treatments for olfactory disorders or for AD. Mechanisms driving olfactory loss in preclinical AD, and its relationship to disease progression, remain unclear. To gain insights into the molecular and cellular mechanisms by which sensory system changes underlie the pathogenesis of AD, we will apply newly developed approaches to analyze human olfactory cells. We will perform olfactory function testing and obtain minimally invasive olfactory biopsies from pre-clinical or symptomatic Alzheimer’s subjects, leveraging a newly developed Alzheimer’s Disease Research Center at Duke/University of North Carolina. Samples will be processed for single cell RNA- sequencing (scRNA-seq), generating a series of data sets to be shared with the field. Using our established normal or hyposmic human olfactory single cell data sets, as well as new controls, we will compare transcriptional profiles in olfactory neurons, neuron progenitors, and associated supporting populations in early or later stages of disease. We will include clinical metadata such as sex, race and APOE-e4 status to permit sub-group analyses. Finally, we will focus attention to olfactory immune cells, as neuroinflammation is a prominent aspect of both non-AD olfactory disorders as well as AD pathogenesis. Defining the immune cell population changes in early or later disease stages and the gene expression changes in these cells, we will also perform functional tests on olfactory cells using in vitro assays. An advantage of our approach will be the ability to evaluate neural tissue from well-characterized human subjects at early or later disease stages, rather than animal models or post-mortem samples. Together, completion of these studies will identify mechanistic insights into the pathogenesis of olfactory dysfunction as well as AD progression.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY When patients present with urinary urgency, frequency and nocturia, with or without urgency incontinence, we consider this the clinical phenotype of overactive bladder (OAB). Clinical observations and data suggest that clinical OAB may be comprised of several underlying subtypes. Discerning between subtypes has been challenging but is imperative to precisely guide targeted treatments. Within the last decade, several large observational cohort studies have been developed with the goal of improving our understanding of the pathophysiology underlying benign urologic symptom disorders. The NIDDK-sponsored Multidisciplinary Approach to the Study of Pelvic Pain (MAPP) network and Lower Urinary Tract Dysfunction Research Network (LURN) have collected data on urologic pain syndromes and non-painful lower urinary tract symptoms (LUTS), respectively. Both cohorts included patients with OAB. To identify clinically meaningful subtypes, MAPP and LURN used clustering methods to mathematically classify patients into probable subtypes using clinical characteristics. Though biospecimens were collected in both cohorts, biologic data from these specimens were not included in clustering. If OAB subtypes have different pathophysiology, biologic data are likely to add important distinguishing information, and possibly be predictive of differential treatment responses. Based on clinical observations and prior clustering work, we hypothesize that the syndrome of idiopathic OAB is comprised of 5 phenotypic subtypes, including dysbiotic and metabolic subtypes that will have distinct microbiome profiles from the others. To test our hypotheses, we propose an innovative approach combining MAPP and LURN clinical datasets and already collected biospecimens. Since sex-specific factors may affect subtypes, analyses will be performed on females and males separately. Our current “one-size fits all” algorithm of OAB treatments results in repeated medical visits, high health-care costs, and marginal long-term effectiveness. There is a major gap in knowledge of OAB subtypes, which in turn hampers our ability to target treatments to subgroups where they would be most effective. The proposed analyses by experienced data scientists present a unique opportunity to leverage existing resources from well-defined prospective clinical cohorts to substantially enhance our understanding of OAB.

NIH Research Projects · FY 2025 · 2024-02

Akkermansia muciniphila is a commensal gut microbe that has been implicated in a multitude of human health conditions. A. muciniphila lives deeply embedded within the mucin layer, where it survives by consuming mucin as a source of carbon and nitrogen. This microbe has been shown to influence host cellular activity as well as protect against gut inflammation, obesity, and metabolic disorders such as diabetes. First isolated in an effort to discover mucin-degrading bacteria, this microbe has also been thought of as a potential probiotic. However, despite nearly two decades of study, the biological mechanisms by which it metabolizes mucin are unknown. Specialized metabolic machinery is required to degrade mammalian gastrointestinal mucin due to its complex structure. Recently, our lab has discovered several unique aspects of mucin metabolism that this proposal seeks to study further. Firstly, we have shown that A. muciniphila can accumulate mucin intracellularly, and this process is specific to mucin. Furthermore, we have pioneered a means to perform transposon (Tn) mutagenesis in A. muciniphila as well as subsequent high-throughput INSeq studies to assay the genetic determinants mucin utilization. These have shown that mucin metabolism occurs in several discrete steps: the binding to mucin extracellularly; its import into the cell through dedicated mucin transporters; and its subsequent degradation and metabolism intracellularly. I propose to identify genes required for mucin accumulation, as well as expand the knowledge of the glycoside hydrolases and mucin-binding proteins produced by A. muciniphila. While studies seem to suggest that this microbe has numerous health benefits, the knowledge of how A. muciniphila interacts with human host cells is predicated upon a deeper understanding of the fundamental biology and the mechanisms that enable it to thrive within the gastrointestinal mucin layer.

NIH Research Projects · FY 2026 · 2024-02

ABSTRACT Type 3 lymphocytes include group 3 innate lymphoid cells (ILC3), IL-17-producing γδ T cells (Tγδ17), and T helper 17 ( TH17) cells and are crucial for maintaining mucosal barrier defenses against extracellular bacteria and fungi. Nonetheless, dysregulated type 3 lymphocyte responses have been implicated in the pathogenesis of several autoimmune diseases, such as inflammatory bowel disease, Crohn's disease, and multiple sclerosis. ILC3, Tγδ17, and TH17 cells are defined by their shared expression of the lineage-defining transcription factor RORγt (Rorc(t)), which facilitates key inductive events during the development and differentiation of these cell types towards the type 3 lymphocyte lineage. The trans-acting factor networks that cooperatively regulate the formation of the type 3 lymphocyte cell types have been well characterized, however, the cis-regulatory elements (CREs) that control the development and differentiation of type 3 lymphocytes remain less defined Therefore, we are proposing a comprehensive assessment of the CREs controlling the expression of the Rorc(t) locus across the type 3 lymphocyte lineage cell types. To this end, we will (i) identify the CREs governing Rorc(t) expression using a high-throughput CRISPRi knockdown screen; (ii) evaluate the activity of Rorc(t) CREs using a retroviral massively parallel STARR-seq reporter assay; (iii) and determine the functional role of putative CREs for the expression of Rorc(t) in the ILC3, Tγδ17, and TH17 cell types. Altogether, the experiments outlined in this proposal will expand our understanding of the cis-regulatory mechanisms governing type 3 lymphocyte development and identity.

NIH Research Projects · FY 2026 · 2024-02

ABSTRACT Mutations that impair function of the CRB1 gene are among the most prevalent causes of inherited retinal de- generation. The events leading to degeneration when CRB1 is lost are unknown. To answer this question it is essential to understand the function of CRB1 at each site where it is expressed within developing and mature retina. This knowledge will reveal the functions of CRB1 that are most responsible for retinal pathology, thereby pinpointing therapeutic strategies to prevent pathology. The objective here is to determine how distinct CRB1 isoforms and CRB1-expressing cell types contribute to disease pathology in the Crb1null mouse model of CRB1 disease. The central hypothesis is that a photoreceptor specific isoform, known as CRB1-B, needs to be present and functional to prevent outer limiting membrane pathology and visual impairment. The rationale for this work is that systematically evaluating CRB1 function at each time and place where it is expressed will es- tablish a rational strategy for gene replacement therapy with a high likelihood of effectively halting degenera- tion. To test this hypothesis three Specific Aims are proposed. Aim 1: Determine the contribution of specific Crb1 isoforms to disease-related phenotypes. Preliminary studies defined 3 major mRNA isoforms of the mouse Crb1 and human CRB1 gene: CRB1-A, -B, and -C. To systematically test their function the PI's labora- tory has generated mouse mutant strains that remove specific isoforms, either individually or in combination. These mutants will be subjected to anatomical, electrophysiological, and behavioral analysis, thereby estab- lishing the contribution of each isoform to specific aspects of the CRB1 mutant phenotype. Aim 2: Determine how each Crb1-expressing cell type contributes to disease-related phenotypes. Conditional Crb1null mu- tant mice will be generated that allow removal or re-expression of all isoforms in a cell type-specific manner. These mice will undergo the battery of phenotypic tests noted for Aim 1 above, thereby establishing the role of each CRB1-expressing cell type in distinct aspects of disease pathology. Aim 3: Rescue disease-related phenotypes using isoform- and cell type-specific Crb1 re-expression. Preliminary data suggest a critical role for the photoreceptor-specific CRB1-B isoform in onset and rescue of disease pathology. This Aim will test the working hypothesis that restoring CRB1-B to photoreceptors will produce functional rescue in the Crb1null disease model. This work will be significant because it will clarify the molecular and cellular aspects of CRB1 expression that are required to prevent retinal pathology. Once we know the consequences for retinal pathol- ogy when specific sites of CRB1 expression are lost or restored, it will become clear which of these sites would be most beneficial to target for CRB1 replacement therapy. Thus, the proposal is expected to yield innovative new strategies for gene therapy which could be rapidly deployed to pre-clinical testing.