University Of Pennsylvania

NIH Research Projects · FY 2024 · 2020-08

Functional Interrogation of T2D-associated genes in human stem cell-derived models and mice Type 2 Diabetes (T2D) is one of the fastest-growing diseases and a leading cause of death throughout the world. A better understanding of the disease process, including characterization of both the genetic etiology and the contribution of different cell types to disease initiation, progression and heterogeneity promises to reveal new therapeutic targets. Large-scale genome-wide association studies (GWAS) of this common complex trait have driven the rapid identification of hundreds of T2D-associated loci. However, the mechanism(s) through which most of these loci influence disease susceptibility remain poorly understood. Our interdisciplinary team at Penn brings together experts in population genetics, T2D GWAS, biostatistics, metabolic tissue biology, human cellular disease modeling and T2D pathophysiology to tackle this critical knowledge gap. In collaboration with other Consortium groups, we aim to accomplish the following goals. (1) Provide the diabetes research community with a robust pipeline for mapping T2D GWAS variants to effector genes and target tissues. (2) Identify new genes and biological pathways that modulate susceptibility to T2D. (3) Define gene regulatory networks relevant to T2D with the goal of uncovering therapeutic ‘entry points’ for developing new treatments. For (1), we will prioritize “candidate effector transcripts” for downstream functional analyses by integrating multiple sources of data to gain a ‘confluence of evidence’ as to their disease relevance and tissue of action. These sources include publically available datasets, a unique collection of internal resources from the Million Veteran Program, and our own functional genomics (RNA-seq, ATAC-seq, chromatin conformation capture etc.) data generated from stem cell-derived T2D relevant cell types. For (2), we will examine the biological function of prioritized T2D-effector transcripts in human cell models of T2D-relevant tissue types using gain- and loss-of- function methods combined with a battery of physiological, metabolic, molecular phenotyping and genomic approaches. These studies include the use of induced pluripotent stem cell (iPSC) models for pancreatic b cells, hepatocytes, adipocytes and skeletal muscle cells, enabling precise genetic engineering and establishment of multiple cell types in the same genetic background. Through this process, we will identify 10 high priority candidate effector genes, which we will advance for comprehensive in vivo analyses in conditional mutant mouse models. For (3), we will perform network analyses through the integration of our multiple data sources to identify molecular memberships in broader pathways and search for pathway components that are potentially amenable for therapeutic targeting.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Cancer remains a leading cause of mortality among older adults in the United States. However, despite established guidelines supporting appropriate cancer screening in order to prevent death and adverse cancer- related outcomes, screening is often underutilized. Barriers to appropriate screening are even greater for groups facing long-standing screening disparities, such as racial/ethnic minorities and individuals with low socioeconomic status (SES). Key drivers of screening underutilization are decision-making biases facing clinicians and patients, highlighting the need for scalable solutions that are designed to address these biases and tailored to overcome barriers facing high-risk patients. Nudges, interventions designed using behavioral economic principles, improve behavior by addressing biases that lead to suboptimal decisions. Our team has extensive experience working with health systems to test and scale nudge interventions to overcome decision-making biases facing patients and/or clinicians. We have also demonstrated how these types of nudges can improve health care value and patient outcomes. Since electronic health records (EHRs) have been adopted by more than 90% of clinicians in the US, it is an ideal platform upon which to deploy large scale behavior change nudge interventions. In this study, we propose to personalize nudge interventions to clinicians and patients, with a focus on how to tailor nudges to the needs of high-risk patients and how to implement promising nudges to improve cancer screening among older adults. We will pilot this work at Penn Medicine and then implement a pragmatic trial at sites in the Penn Medicine, University Hospitals, and Sutter Health systems. In the R61 phase, we will complete the following aims at Penn Medicine: analyze EHR and claims data and identify characteristics and subgroups of patients at high-risk for not completing cancer screening (Aim 1), test the feasibility of individual nudge interventions within an EHR nudge toolkit (Aim 2), and pilot test nudge interventions to identify the most promising approaches to increase cancer screening rates for high-risk patients in each subgroup (Aim 3). In the R33 phase, we will achieve the following aims at Penn Medicine, University Hospitals, and Sutter Health: conduct a two-arm pragmatic cluster-randomized controlled trial to test the effectiveness of personalized EHR-based nudges to clinicians and patients on increasing cancer screening rates (Aim 1), evaluate if EHR-based nudge interventions reduce disparities in cancer screening rates for racial/ethnic minorities and patients with low socioeconomic status (Aim 2), and inform future design of tailored EHR interventions by examining heterogeneity of treatment response with respect to clinician and patient characteristics (Aim 3).

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY/ABSTRACT Vaccine-preventable diseases such as influenza, pneumococcal disease, and shingles lead to significant rates of illness, hospitalization, and death among older adults. In the United States, vaccination rates have been mostly unchanged for a decade with lower rates among racial and ethnic minority groups. New and scalable approaches are needed to address this important public health issue. Nudges are changes to the way choices are offered or information is framed that can have outsized effects on behavior. For example, default options are the path of least resistance and the action that takes place if no alternatives are selected. Active choice is a method that prompts a decision-making now, rather than waiting for stakeholders to recognize the need to make the decision on their own. Our groups at the University of Pennsylvania (Penn Medicine) and the University of Washington (UW Medicine) have formed behavioral design teams embedded within the operations of our health systems and have demonstrated how these types of nudges can improve health care value and patient outcomes. Since electronic health records (EHRs) have been adopted by more than 90% of clinicians in the US, this scalable technology platform is an optimal environment to implement and deploy these types of nudges. In this study, we propose to design, test, and implemented personalized nudges to clinicians and patients to target barriers among high-risk subgroups to improve vaccination rates. We will pilot this at two health systems and then implement a pragmatic trial at those health systems and sites in the VA Health System. In the R61 phase, we will focus on the following aims at Penn Medicine and UW Medicine. Aim 1: To use EHR data and analytical methods to identify high-risk groups of older adults with suboptimal vaccination rates for influenza, pneumococcal disease, and herpes zoster. Aim 2: To assess the feasibility of implementing different types of personalized nudges to clinicians and patients to target the identified groups to improve vaccination rates among older adults. Aim 3: To pilot test ways to personalize promising nudges to clinicians and patients for improving vaccination among older adults. In the R33 phase, we will focus on the following aims at Penn Medicine, UW Medicine, and the VA Health System. Aim 1: To conduct a 12-month, multisite, cluster randomized, pragmatic trial to evaluate the effectiveness of personalized nudges to clinicians and patients relative to control to improve vaccination rates among older adults. Aim 2: To evaluate the effectiveness of the intervention on reducing disparities in vaccination rates related to race/ethnicity and socioeconomic factors. Aim 3: To evaluate heterogeneity in treatment effect across clinician, patient, and practice characteristics to further tailor approaches in future intervention design.

NIH Research Projects · FY 2024 · 2020-08

The grand challenge addressed by the Penn Implementation Science Center in Cancer Control (Penn ISC3) is to apply insights from behavioral economics to accelerate the pace at which evidence-based practices for cancer care are deployed to improve outcomes for individuals with cancer. Penn offers a unique environment in which to conduct this paradigm-shifting work, given our expertise in implementation science, behavioral economics, and innovative cancer care delivery. The complementary and multi-disciplinary expertise of the three MPIs (Bei- das, Bekelman, Schnoll), coupled with existing Penn resources, including the Penn Center for Cancer Care Innovation [PC3I], the Center for Health Incentives and Behavioral Economics [CHIBE], and the Penn Imple- mentation Science Center at the Leonard Davis Institute [PISCE@LDI], represent a unique opportunity to ad- vance the quality of cancer care. The Administrative Core, led by Drs. Beidas, Bekelman, and Schnoll, will ensure that the activities of Penn ISC3 are coordinated, synergistic, and congruent with timelines. The Implementation Laboratory, led by Drs. Bekelman and Shulman, represents a diverse ecosystem that includes five hospitals and linked clinical sites, with over 200 oncologists that serve over 15,000 unique new patients annually. Our Research Program, led by Drs. Beidas and Buttenheim, will oversee the development and testing of implementation strat- egies that target patients and clinicians within our Implementation Lab and are centered on the idea of ‘nudging’ for optimal implementation and effectiveness outcomes. The Research Program includes investigators with ex- pertise in implementation science, behavioral economics, cancer care delivery research, healthcare innovation, measurement, and mixed methods. In the first two years, we propose three Signature Pilot Projects and two Methods Projects with a commitment to rapid learning that will allow Penn ISC3 to be optimally nimble. Signature Pilot Project 1 (Jenssen/Leone) tests patient- and clinician-directed implementation strategies to increase referral to tobacco cessation programs among cancer patients. Signature Pilot Project 2 (Bekelman/Patel) tests patient- and clinician-directed implementation strategies to increase higher-value bone modifying agents in patients with breast, lung, and prostate cancer. Common methods and measures are linked to allow for pooling of data and to accomplish our objectives of testing multilevel implementation strategies and mechanisms across contexts. Our exploratory and high-reward Signature Pilot Project 3 (Bekelman/Rendle) will test a patient-directed imple- mentation strategy that leverages artificial intelligence and machine learning to promote oral chemotherapy ad- herence and symptom management. Two methods projects, in support of the Projects, will advance the science of implementation methods. Methods Project 1 will develop a toolkit for the application of rapid cycle approaches (Asch/Buttenheim); Methods Project 2 will use qualitative comparative analysis to characterize multilevel con- textual variation (Barg/Rendle). The Penn ISC3 has the potential to identify novel, disseminable, and scalable ways to advance the quality of cancer care and improve the health outcomes for individuals with cancer. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT With an aging population, the impact of Alzheimer's disease (AD) on public health continues to explode. Altered daily rhythms in physiology and behavior are prominent features of AD. These altered activity rhythms are difficult to treat, disrupting the lives of both patients and caregivers. Mounting evidence suggests that these changes are more than just symptoms. Altered rhythms may contribute to AD progression and development. Many important transcripts, proteins, and metabolites oscillate with a daily cycle. Understanding these rhythms, and their influence on AD, offers the potential to identify new therapies. The translation of circadian biology to Alzheimer's care is limited. Which molecules and pathways show daily rhythms in our human brains? How do those rhythms change with AD? Do changes in molecular rhythms explain changing behavioral patterns? Can these rhythms be exploited for therapeutic benefit? To answer clinical questions, we need human data. AD brain banks provide an invaluable resource. But brain banks almost never provide the time of day when patients died, making it difficult to use these data for rhythms research. We developed CYCLOPS (CYCLic Ordering by Periodic Structure), a machine-learning tool to uncover molecular rhythms using unordered biopsy samples. Evaluating brain expression data, we showed that CYCLOPS could correctly reconstruct rhythms in brain samples and correctly predict the time of death. Here we will order cortical brain samples from control subjects and patients with AD. We will reconstruct the molecular rhythms in these human brains, identifying differences in AD patients and rhythms in known drug targets and AD disease pathways. We will analyze a subset of samples where time of death is known, comparing each subject's “internal molecular time” with the “time on the clock.” We will test the hypothesis that patients with poorly aligned molecular rhythms are more likely to have circadian behavioral disturbance. We will evaluate a measure of transcriptional rhythm strength, testing if “weaker” rhythms predict behavioral or molecular misalignment. Does AD alter rhythm generation? Does it desynchronize still rhythmic cells and brain regions? Using data from multiple brain regions sampled from the same subjects, we will evaluate intracortical circadian synchrony and compare AD patients with controls. Using single-nucleus sequencing data, we will explore the effect of AD on cell type specific rhythms and their synchrony. Finally, we will test the direct influence of important AD causing mutations on molecular clock function, measuring rhythms in isolated cells. This work will advance our understanding of circadian rhythms in AD pathology, clarify the relationship between behavioral and molecular circadian disruption, and catalyze opportunities for AD chronotherapy.

NIH Research Projects · FY 2025 · 2020-08

ABSTRACT Osteoarthritis (OA) is a leading cause of disability in adults, causing chronic progressive joint pain and tissue damage throughout the joint. No current medical treatments are effective at preventing the progressive joint deterioration, pain and disability characteristic of OA. Although it is known that inflammation and bone- remodeling are major drivers of OA pain and pathology, it is not yet clear which molecular pathways directly drive chronic OA pain and disease progression or are key targets for therapeutic development. Our lab has shown that deficiency of CD14, an inflammatory pattern-recognition receptor expressed by macrophages and other myeloid cell types, protects against OA-associated bone-remodeling and pain-related joint dysfunction after knee injury in mice. In patients, CD14 is increased in joint fluid and associated with intra-articular macrophage infiltration as well as pain severity. This receptor facilitates Toll-like receptor (TLR) signaling. TLRs are innate immune sensors which are important initial triggers of chronic inflammation in response to non-infectious tissue damage. In addition, our team has recently demonstrated that TLR-signaling is critical to development of knee OA pain in mice, via direct stimulation of pain-transmitting (nociceptive) neurons in the dorsal root ganglia (DRG) that innervate the knee. In this proposal, we will test the hypothesis that CD14 on myeloid cells including macrophages promotes OA pain-related pathology (bone remodelling and inflammation), while CD14 also augments OA pain by directly sensitizing neurons innervating the arthritic joint. We will utilize in vitro techniques and the murine destabilization of the medial meniscus (DMM) model of OA, to understand the cellular and molecular mechanisms by which CD14 drives OA pathology and inflammation in joint tissues. Specifically, in Aim 1 we will use a multi-disciplinary approach to determine how genetic deficiency of CD14 modifies inflammation and bone-remodeling during progression of OA, using the DMM model. We will determine the contribution of myeloid cells to inflammation and bone-remodeling in the model by using bone marrow (BM) chimeric mice. Lastly, we will test the effects of CD14 on differentiation of cells that drive bone-remodeling (osteoclasts) and production of inflammatory mediators of pain from myeloid cell types. In Aim 2 we will characterize effects of CD14 on TLR-mediated DRG neuronal activation and joint pain. We will use TLR stimuli with relevance to OA to evaluate DRG responses in vitro, comparing WT and CD14-deficient cells. We will additionally compare pain responses to injection of TLR-stimuli into the joint, and expect that both DRG responses and pain will be blunted in the CD14 deficient strain. Finally, in Aim 3 we will test whether pharmacological targeting of CD14 reduces OA progression and pain after DMM-induced injury. This study will then specify the molecular and cellular framework to design future anti-inflammatory therapeutics for OA aimed at CD14- and TLR-mediated mechanisms.

NIH Research Projects · FY 2025 · 2020-08

Abstract: Antiretroviral therapy (ART) has dramatically extended the lives of people living with HIV (PLWH); however, they continue to experience a plethora of co-morbid conditions including neuronal disorders and pain. Between 40 and 72% of PLWH use cannabis to mitigate anxiety, stress, ART side effects, pain, and/or for pleasure with over 55% of patients using cannabis at least daily. Interestingly, a recent study found that people who use cannabis heavily had reduced inflammatory signatures in PLWH on ART. These and a numerous other studies support the anti-inflammatory and immunomodulator effects of phytocannabinoids in a number of organ systems including heart, colon, kidney, liver and the gut; however, their medicinal use is confounded by the psychotropic activities. Efforts to separate the anti-inflammatory effects from the psychotropic effects have revealed differential activities of 3 endogenous receptors including cannabis receptor 1 (CB2) CB2 which exhibit differential tissue expression and agonism with endo- and phyto-cannabinoids. Several reports have shown that cannabinoids attenuate HIV infection and/or replication in T-cells, macrophages, dendritic cells and human fetal microglia cultured ex vivo. However, the effect of cannabinoids on HIV infection of microglia in the context of ART and the normal cellular environment of neighboring neurons and astrocytes in the CNS has not been examined. Several studies specifically implicate CB2 agonism which has been shown to have anti- inflammatory properties in the heart, gut, experimental autoimmune encephalitis and neuropathic pain via inflammasome activation. This has led us to hypothesize that Cannabinoid signaling influences HIV infection and chronic inflammation in the presence of ARV in the central nervous system by attenuating the inflammasome. In order to examine HIV infection in the context of cells of the CNS, we have developed a human induced pluripotent stem cell tri-culture model composed of iNeurons, iAstrocytes, and iMicroglia. This model recapitulates several key aspects of HIV infection in the CNS including increased cytokine production, oxidative stress response, inflammatory signaling, and integrated stress response. ARV treatment reduces HIV infection and inflammatory signaling pathways; however, a subset of pathways remain elevated despite viral suppression. We propose to further develop this model to determine the ability of cannabinoids to modulate HIV-induced inflammation and subsequent neuronal dysfunction via reducing inflammasome activation by: 1) Determining the effect of cannabinoids on chronic HIV infection and ART in the context of iMgl/iNrn/iAstr triculture. 2) Determining the effect of cannabinoids on cytokine levels, inflammatory gene expression profile, and microglial activation in iMgl/iNrn/iAstr triculture. 3) Determining the effect of cannabinoids on neurons and astrocytes in HIV infection and ART in iMgl/iNrn/iAstr triculture.

NIH Research Projects · FY 2026 · 2020-08

Project Summary Natural products (NPs) derived from filamentous fungi are essential sources of therapeutic agents, contributing pharmacophores to over 60% of FDA-approved small-molecule drugs. Despite this success, the vast biosynthetic potential of fungi remains untapped, as many biosynthetic gene clusters (BGCs) are transcriptionally silent under laboratory conditions due to complex gene regulatory networks. Accessing this chemical diversity requires innovative approaches combining advanced genome engineering with systematic exploration of fungal biosynthetic pathways. During the initial MIRA R35 ESI funding period, we achieved three key breakthroughs: the development of a multiplex base-editing (MBE) platform for simultaneous gene manipulation, the elucidation of novel enzymatic mechanisms in NP biosynthesis, and the discovery and engineering of a new class of fungal ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent anticancer activity. Building on these advances, our proposed research will pursue two complementary directions to unlock fungal chemical diversity. The first direction focuses on advancing precision genome engineering technologies. We will expand our MBE platform to engineer marker-free heterologous hosts through iterative BGC deletion, reducing metabolic interference, improving detection of minor metabolites, and enhancing precursor availability. Additionally, we will expand MBE to systematically manipulate epigenetic regulators across diverse fungal species to activate transcriptionally silent BGCs. In parallel, we will develop and optimize prime editing (PE) systems for precise, marker-free genetic modifications across diverse fungal species, with a focus on modulating key regulatory genes to activate silent BGCs. These innovations will establish scalable tools to uncover novel biosynthetic pathways and enhance their chemical output. The second research direction will comprehensively explore the untapped potential of fungal RiPPs, with a focus on the unprecedented DUF3328 oxidases. This includes developing targeted discovery platforms through bioinformatic screening of DUF3328-containing BGCs, activating cryptic RiPP clusters via in situ pathway engineering, and implementing advanced heterologous expression systems. We will conduct detailed structural studies and bioactivity evaluations, including antimicrobial and anticancer screening, to identify and optimize promising therapeutic candidates. By integrating advanced genome engineering with systematic NP discovery, this research will transform fungal biotechnology. These innovations will provide powerful tools for genome engineering, accelerate the discovery of novel therapeutic leads, and establish platforms for accessing fungal chemical diversity, driving the development of next-generation medicines to address critical human health challenges.

NIH Research Projects · FY 2025 · 2020-08

Summary/Abstract The Bugaj Lab develops precision-controlled molecular tools to better understand and control biological systems. This proposal will develop technologies that harness protein clustering for new, enabling methods to study, manipulate, and treat cells. Protein clustering plays important roles throughout cell physiology and disease, yet its potential for biotechnological probes and therapies remains unrealized. Clustering is potent because it achieves rapid and non-linear responses with minimal energetic costs. Over the past decade, optogenetic clustering probes have allowed control over protein clustering with high precision throughout a wide array of cellular processes. However, despite the high utility of optogenetic clustering, there remains predominantly one protein (A. thaliana Cry2) whose light-induced clustering is used for such probes, constraining its application due to inherent limitations, for example response time and ability to control one process at a time. Our first project will address these technological gaps by engineering a distinct photoreceptor that we recently discovered possesses a native ability to form light-induced clusters in mammalian cells, providing a complement to Cry2 with certain favorable properties, including in response kinetics. In our second project, we will develop a suite of single-component optogenetic probes that allow precise control of subcellular localization to a variety of compartments. These probes uniquely harness the ability of clustering to trigger binding through rapid increases in probe avidity. In our third project, we will develop the foundations of synthetic protein therapies that can sense and respond to endogenous protein clusters. Clustering is a hallmark of many pathologies including neurodegeneration, viral infection, and cancer, and thus protein clustering itself could be leveraged as a disease biomarker or therapeutic target. We will engineer autonomous protein systems that detect pathological protein aggregates and execute a strong, appropriate response, for instance killing a cancer cell or secreting cytokines to recruit the immune system. Successful completion of our work will establish protein clustering as a flexible new paradigm for manipulation of cellular function, resulting in broadly applicable probes for study of cell/animal behavior, as well as a new class of “sense-and-respond” smart drugs with application across a diverse array of disease states.

NIH Research Projects · FY 2024 · 2020-08

PROJECT TITLE Photoreceptor Regeneration in a Murine Model of Leber Congenital Amaurosis ABSTRACT Leber congenital amaurosis (LCA) is an early-onset, severe inherited retinal degeneration that results in childhood blindness. Gene therapy can effectively restore visual function in the Lca5gt/gt mouse model of the disease when delivered to neonatal mice before significant retinal degeneration is present. However, in moderate- and late- stage disease after photoreceptor cell death has occurred, gene therapy is no longer effective. Cell-based therapies hold great promise for regenerating photoreceptors in late-stage retinal degenerations, but progress has been limited by low efficiencies of integration and complex cellular interactions with existing retinal cells. Photoreceptor precursors represent a heterogeneous pool of diverse cells types that have the potential to differentiate into mature photoreceptor cells in the developing and diseased retina. Using unbiased single-cell RNA transcriptomics, I identified several novel populations of Crx+ photoreceptor precursors including Crx+/Nfix+ and Crx+/Slc39a1+ cells. The scientific objectives of this proposal are to regenerate photoreceptors in the Lca5gt/gt mouse model of retinal degeneration, by the transplantation of these novel populations of photoreceptor precursors and by the activation of endogenous repair pathways in Müller glia. The Aims of the proposal are 1) Subretinal transplantation of Crx+/Nfix+ and Crx+/Slc39a1+ progenitor cells in Lca5gt/gt mice, and 2) Activation of endogenous Müller glial repair pathways by gene therapy in Lca5gt/gt mice. In addition to these scientific contributions, this proposal outlines a structured, focused training plan that will equip me with the skills and expertise that will serve as the foundation for my career in developing cell-based therapies for retinal disease. The Department of Ophthalmology at the University of Pennsylvania is an ideal environment for training physician scientists in ophthalmic research, and will provide the protected time, resources, and mentorship needed for a successful transition to independence.

NIH Research Projects · FY 2024 · 2020-07

Project Summary Insomnia is among the most commonly experienced symptoms and is associated with significant distress and impairment. The assessment of insomnia is reliant on patient self-report, which is often influenced by a number of factors other than illness severity, complicating accurate diagnosis and treatment. Further, subtypes of insomnia may exist based on the presence or absence of short sleep duration. Identification of a biological ‘signature’ of insomnia that could facilitate assessment and subtyping would dramatically improve symptom management. Metabolic biomarkers have significant promise for meeting this need. Individuals with insomnia demonstrate metabolic hyperarousal compared to good sleepers. Acute disruption of sleep in the laboratory impacts the metabolome but the extent to which these findings extrapolate to chronic sleep disturbance or insufficient sleep is unknown. Our own data indicate there is a clear metabolic signature that differentiates patients with insomnia from good sleepers. The objective of this study is therefore to investigate the effects of chronic insomnia and insufficient sleep on metabolic profiles. In order to test this hypothesis we will conduct in- depth phenotyping of sleep and metabolism in 100 subjects who are in one of four groups (n=25 per group): 1) patients with insomnia and objective short sleep (<6 hours) on actigraphy; 2) patients with insomnia without objective short sleep (>6 hours); 3) habitual short sleepers (<6 hours) without evidence of insomnia; and 4) good sleepers. Home overnight polysomnography and actigraphy will be used to rule out comorbid sleep disordered breathing and confirm the presence of insomnia. All subjects will participate in a four-day inpatient protocol in the Center for Human Phenomic Science. Food intake will be provided in hourly isocaloric snacks to control for meal-induced shifts in metabolism. The first two days will be used to acclimate subjects to the environment and meals. On the morning of day 3 they will have an indwelling catheter placed for blood sampling every two hours for 48 hours with overnight polysomnography each night. During this time lighting levels will be kept constantly dim (<250 lux) to minimize the effects of light exposure on circadian rhythms. Metabolomics analysis of serum samples will be carried out using NMR and mass spectroscopy. Blood samples will also be used for melatonin and cortisol assays as standard markers of circadian rhythmicity. The global hypothesis that motivates this proposal is that chronic insomnia, insufficient sleep, and their combination are associated with distinct profiles of systemic metabolic dysregulation.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY The project will develop and pilot test a personalized medicine mobile health application, KeepCalm, that incorporates physiological stress measurement to support evidence-based practices for reducing challenging behavior in children on the autism spectrum. As much as 80% of children on the autism spectrum exhibit challenging behaviors that can have a devastating impact on personal and family well-being, contribute to teacher burnout and require frequent hospitalization. Evidence-based practices for reducing these behaviors emphasize uncovering triggers, yet parents and teachers often report that challenging behaviors surface without warning. Challenging behaviors caused by emotion dysregulation can be the most difficult to predict, as children on the autism spectrum often have difficulty communicating their distress before it results in challenging behavior. Exciting recent advances in digital technology now allow measurement of momentary emotion dysregulation, using physiological indices. Our pilot data from four separate samples demonstrate that increased heart rate predicts onset of challenging behavior in children on the autism spectrum. In order to tailor KeepCalm to end users and avoid potential barriers to its adoption, in Aim 1, we will evaluate the acceptability, feasibility, and appropriateness of app, and the needs of educational teams in managing stress in children on the autism spectrum and challenging behaviors, by conducting interviews with teachers of children on the autism spectrum, parents of children on the autism spectrum and school administrators, and conducting structured in-class observations with teachers. Through the activities of Aim 2, we will improve KeepCalm, building on our initial app prototype, in collaboration with our established technology partner, Alevio, and our established community partner, the School District of Philadelphia. We will do this through 1) exploratory work on the specificity of heart rate increase to challenging behavior, on app clinical decision support timing, and on the association of app false positives and negatives to movement or child factors; 2) monthly advisory board meetings with expert stakeholders for app development guidance, and; 3) rapid-cycle prototyping of the app with 10 educational teams (i.e. 1-2 children on the autism spectrum, and their teacher and classroom aide, if they have one). This will allow for iterative improvement based on each user’s experience. Through Aim 3, we will test the app for usability, acceptability, feasibility and appropriateness, as well as preliminary effectiveness with 20 educational teams in a randomized waitlist field trial over a 3-month period. Successful completion of these aims will result in a novel m-health app designed to help teachers support emotion regulation, and reduce or prevent challenging behavior in children on the autism spectrum, using evidence-based strategies. These activities will lay the foundation for an R01 to evaluate the effectiveness of KeepCalm in a full-scale randomized field trial. Our proposal aligns with the strategic plan of the Interagency Autism Coordinating Committee, to maximize the potential for technology- based interventions to improve the lives of people on the autism spectrum.

NIH Research Projects · FY 2024 · 2020-07

Abstract Lung cancer is both the most common cancer worldwide and the leading cause of cancer death in the US. While radiation therapy (RT) is a highly effective treatment for many cancers, thoracic RT carries an increased risk of cardiovascular (CV) morbidity and mortality that limit critical gains in cancer control and survival. Despite the significance of this problem, we have a limited understanding of how RT results in CV toxicity, and the biologic and functional mechanisms and predictors of CV toxicity in patients. Fundamental questions include: how does RT affect mechanistic biologic and imaging markers of CV toxicity? Which cardiac radiation dose-volume parameters are associated with CV toxicity? Can baseline levels or early changes in biomarkers, imaging measures and radiation-dose volume parameters identify patients at risk of adverse CV clinical outcomes? Our preliminary data suggest thoracic RT results in inflammation, oxidative stress, microvascular dysfunction, and worse CV function in patients. We will extend these findings through detailed characterization of these pathways in a multi-center, longitudinal prospective cohort of nonsmall cell lung cancer patients from the University of Pennsylvania, Washington University, and the Brigham and Women’s Hospital treated with definitive thoracic chemoradiation for curative intent. We focus on lung cancer given the high prevalence of disease, the important role of RT in cancer control, the concomitant CV morbidity and mortality associated with RT, and the high RT doses delivered to the heart. Our overall objective is to determine if RT results in early, subclinical CV dysfunction using highly sensitive, quantitative biologic and functional measures; understand how cardiac dose-volume parameters influence these abnormalities; and develop multi-marker strategies in risk prediction. Our multi- center longitudinal cohort forms the basis of all Aims. In Aim 1, we will evaluate the changes in circulating biomarkers of CV stress, inflammation and vascular dysfunction, and to define the associations with RT dose- volume measures. In Aim 2, we will quantify RT-related changes in imaging-derived measures of CV function and perfusion, and to define the associations with RT dose-volume measures. In Aim 3, we will determine the prognostic value of biologic, imaging, and RT dose-volume measures as indicators of adverse CV outcomes. By using innovative methods in deep CV phenotyping to identify high risk individuals, we will personalize the delivery of RT and targeted cardioprotective interventions, and ultimately improve CV and overall patient outcomes. We will leverage our experiences in precision phenotyping of cancer patients undergoing cardiotoxic therapy to address a high-priority research gap in response to NIH PA 19-112.

NIH Research Projects · FY 2024 · 2020-07

PROJECT ABSTRACT Epigenetics impacts all areas of cellular physiology, and epigenetic dysregulation is pervasive in human disease. Given the inherent reversibility of epigenetic changes, this presents a great opportunity for the discovery of novel therapeutics given the recent rapid development of epigenome-modifying drugs. Intriguingly, large-scale human sequencing efforts have revealed that sun-damaged, but clinically otherwise normal human skin, can harbor frequent mutations in epigenetic chromatin modifying enzymes. These include mutations that have been typically observed in cutaneous squamous cell carcinoma (cSCC), the second most common of all human malignancies, and a major economic and public health burden. Recent data suggests that these epigenetic mutations may be important drivers of malignant clone formation in the epidermis, provoking the hypothesis that proper epigenetic function is required for both maintaining epidermal homeostasis and preventing the initiation of carcinogenesis. Remarkably, despite the high incidence of both these mutations in epigenetic modifiers and cSCC, the precise mechanisms by which disruption of chromatin modifying enzymes drives the initiation of cSCC are virtually unknown. In this proposal, we will utilize multiple model systems including human patient samples and a variety of transgenic mouse models, combined with several innovative genome-wide and functional technologies in order to define the mechanistic links between chromatin regulation, transcription, epidermal cell fate, and the initiation of epidermal carcinogenesis. Collectively, these studies promise to inform both the development and utilization of epigenetic therapies in the future.

NIH Research Projects · FY 2024 · 2020-07

A better understanding of the liver’s response to toxic injury, which includes hepatocyte proliferation, and – unfortunately – an increased risk for hepatocellular carcinoma (HCC), is a prerequisite for the development of novel clinical treatments for chronic liver disease and improved cancer prevention. Existing drug therapies for HCC such as sorafenib extend patient survival by only three months. We recently developed a massively parallel in vivo screening platform to test the impact of genetic factors such as full-length cDNAs or miRNAs on liver repopulation and tumorigenesis. We have used this screening technology to build a map of all miRNAs active in liver regeneration. Here, we propose to exploit this innovative paradigm to conduct a comprehensive evaluation of the effects of the 135 most abundant but evolutionarily conserved hepatic miRNAs on the processes of recovery from toxic liver injury and HCC tumorigenesis. In Specific Aim 1, we will determine the combined benefits of three miRNAs identified in our prior screen on liver repopulation following toxic injuries, as a step toward using miRNA-mimetic drug therapies for liver diseases. This will be accomplished through delivery of miRNA-encoding plasmids or nanoparticles singly and in all combination. In Specific Aim 2, we will determine the impact of hepatic miRNAs and miRNA combinations on HCC tumor development in vivo. To this end, we have developed two models of rapid HCC development in mice, in which we will screen our library of 135 ‘tough decoys’ (“TuD’s”), or inhibitors of miRNA action, on tumor formation. We will quantify the abundance of all TuD’s using high throughput sequencing in the tumor-loaded liver compared to the input library. TuD’s enriched in after tumor formation target miRNAs that normally limit tumor growth, and those found less abundant target miRNAs that promote tumorigenesis. We will then test the combinations of the most potent miRNA effectors on tumor formation following systemic delivery. In Specific Aim 3 we will perform a conditional screen of miRNAs that impact Sorafenib resistance to identify novel combination treatments for the prevention or treatment of HCC. Together, our powerful genetic screens promise to identify miRNA effectors that can be employed for the treatment of acute liver injury and, in combination with Sorafenib, as a more effective treatment to prevent HCC initiation and progression.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY/ABSTRACT Cardiovascular morbidity and mortality disproportionately impact lower income individuals and racial minorities. These groups are also significantly less likely to have health insurance coverage in the United States. There is growing evidence that expansion of health insurance coverage in low-income populations can lead to improvements in health outcomes. In a recently published analysis by Dr. Khatana and colleagues, that serves as the preliminary analysis for the proposed research, expansion of insurance coverage through Medicaid was associated with fewer deaths from cardiovascular disease. A possible mechanism by which this occurred is improved access to cardiovascular care, however, this has not been previously studied. This proposed research plan aims to understand whether expansion of insurance coverage impacts access to inpatient and outpatient care for cardiovascular disease, and whether it narrows disparities in care access for racial minorities. Aim 1 seeks to examine whether expansion of insurance coverage through Medicaid under the Affordable Care Act led to an increase in access to inpatient care in the setting of acute myocardial infarction or stroke, and whether this varied between non-Hispanic Black and non-Hispanic White individuals. Aim 2 will investigate whether access to outpatient care for chronic cardiovascular disease is associated with changes in insurance coverage and whether insurance expansion narrows racial disparities in outpatient access. These two aims will utilize different large administrative claims databases. Aim 3 will augment these analyses by employing qualitative research methods to interview low-income patients discharged after a cardiovascular hospitalization. Patients in the post-discharge period are especially vulnerable to adverse outcomes such as readmission or death. Therefore, uninsured and insured patients will be interviewed to understand whether insurance status is associated with differences in health seeking behaviors, adherence to medical advice and therapies, health status and outcomes. These aims will lead to a future multi-site cohort study of low-income individuals with cardiovascular disease, which will be used to prospectively investigate how changes in health insurance coverage impact cardiovascular outcomes. Dr. Khatana, an early career investigator and a fellow in cardiovascular medicine, has a long-term goal of becoming an independently funded cardiovascular health policy and outcomes researcher with a focus on how health policies impact the cardiovascular health of low- income individuals using both large administrative databases as well as qualitative methods at the individual level. These research aims are part of a comprehensive training plan and will be supervised by a mentorship and advisory team consisting of national leaders in health outcomes and policy research, and advanced statistical and qualitative methodologies, and will guide his transition to an independently funded research career.

NIH Research Projects · FY 2025 · 2020-07

Abstract The induction of plasma cells that secrete antibodies against donor tissue alloantigens is a major barrier to successful transplantation. Moreover, because many plasma cells are remarkably long-lived, the resulting antibody titers are exceptionally durable. Hence strategies are needed to eliminate long-lived plasma cells that generate allospecific antibodies. A current dominant strategy for depleting plasma cells centers on compounds that interfere with the proteasome. Plasma cells are thought to uniquely require proteasome function to survive. However, for reasons unclear, substantial numbers of allospecific plasma cells resist the action of available proteasome inhibitors (PIs). This project centers on the central hypothesis that mature long-lived plasma cells (LLPCs) are especially resistant to PI-induced cell death. Our experiments test this idea while also working towards defining how the proteasome interfaces with regulators within the endoplasmic reticulum such as the chaperone protein BiP and other cytosolic regulators of proteostasis including the autophagy regulator p62 and VCP/p97, an ATPase that controls movement of proteins from the ER into the cytosol. Specifically, we will: 1) Define the role of BiP in controlling PI sensitivity in LLPCs, 2) Define the key regulators of ER-to-cytosol protein trafficking/disposal and their role in controlling PI sensitivity in LLPCs, and 3) Identify the gene regulatory networks underlying LLPC resistance to PIs. .

NIH Research Projects · FY 2024 · 2020-07

Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) is a pathogenic, tick-transmitted bunyavirus that can cause a severe febrile hemorrhagic-like disease with case fatality rates of up to 30%. Discovered during a 2009 outbreak of febrile illness in China, the geographic distribution of SFTSV extends into Korea and Japan with recent reports of infection in Vietnam and Russia. The tick vector for SFTSV is widespread throughout Asia. Numerous domestic and wild animals are naturally infected by SFTSV suggesting a large reservoir with potential spillover to humans. There are currently no vaccines or therapeutics for SFTSV. Because of its epidemic threat the WHO included SFTSV in its 2017 recommendation “A research and development Blueprint for action to prevent epidemics” and identified SFTSV as one of 11 pathogens most likely to cause severe outbreaks in the near future and proposed development of vaccines. Here we will explore two complementary and potentially synergistic strategies for an SFTSV vaccine: a recombinant viral vector and nucleoside-modified mRNA encoding the SFTSV viral glycoproteins. Vesicular stomatitis virus (VSV) is a cytopathic virus that has been developed as a vaccine vector due to its ability to rapidly induce strong, protective antibody and T cell responses to encoded foreign antigens after a single dose. Using a VSV vector expressing the SFTSV viral glycoproteins (similar to the currently employed VSV-Ebola vaccine), we demonstrate single dose induction of a neutralizing antibody response and protection from SFTSV challenge in an IFNAR1 knockout mouse model. Separately, we show that vaccination of wt mice with a single dose of nucleoside-modified mRNA lipid nanoparticles (mRNA-LNP) encoding the SFTSV glycoproteins elicits high levels of SFTSV neutralizing antibodies that are capable of conferring partial SFTSV protection when transferred into the IFNAR1 KO model. Based upon these strong preliminary findings we propose to characterize antibody and T-cell responses in rVSV and mRNA vaccinated mice when these vaccines are used alone or in a prime-boost regimen. These studies are significant as there is limited knowledge regarding vaccines for this highly pathogenic virus (a single report) and use of rVSV and mRNA in a prime-boost vaccination has not been reported. Finally, current small animal models of SFTSV infection are limited to animals with type I IFN responses knocked out. Because these animals lack an important innate immune response mechanism that supports amplification of cellular and humoral immune responses, we will develop an immune competent mouse vaccination model using transient monoclonal antibody blockade of IFNAR1 during SFTSV challenge.

NIH Research Projects · FY 2024 · 2020-07

The goal of this proposal is to quantify the role of H3K9me3-based heterochromatin gene silencing in pancreatic beta cell development and to modulate such heterochromatin to obtain more mature beta-like cells from human embryonic stem cells. Type I diabetes (T1D) results from an autoimmune depletion of beta cells within pancreatic islets, resulting in a deficiency in insulin secretion, metabolic imbalances that impair health and lifestyle, and a dependency upon exogenous insulin. Recent studies indicate that T1D also involves beta cell de-differentiation. In 2000, the Edmonton Protocol demonstrated that beta cell transplants from cadavers could elicit exogenous insulin-free survival in severe type I diabetics. Yet success typically requires islets from two cadavers, donors are limiting, and patients can regress to insulin dependence over time. Thus, there is an unmet medical need for donor human beta cells. Extensive research over the past 20 years, including from my laboratory, has defined distinct stages of development of mouse beta cells from the endoderm germ layer and identified numerous signaling effectors. Such information has been used to guide beta-like cell differentiation in vitro from human embryonic stem cells (huESCs). Despite this progress, most laboratories experience difficulty generating robust glucose-responsive insulin secretion (GSIS) in terminal cell products. My lab, for instance, observes robust GSIS in only a limited number of beta-like organoids from huESCs, and generating GSIS-competent cells from induced pluripotent stem cells (iPSCs) is an even greater challenge. The lack of consistency from huESCs and the greater difficulty from iPSCs, which can retain an epigenetic memory of their originating cell type, suggests an epigenetic basis. While H3K9me3-based heterochromatic gene silencing has long been thought to constitutively suppress repeat elements in the genome, my laboratory has recently discovered that such heterochromatin is highly dynamic in liver and pancreatic beta cell development and that such heterochromatin also marks the genes that are the most difficult to activate in various cell reprogramming approaches. Indeed, we present evidence that H3K9me3 heterochromatin is not appropriately modulated in beta-like cells derived from huESCs. We propose two Aims to exploit these insights: Aim 1: Quantify the role of H3K9me3 heterochromatin during beta cell maturation in vivo and in vitro. Aim 2: Employ a knockdown screen of broad and specific heterochromatin modulators during huESC differentiation to beta-like cells to improve glucose-stimulated insulin secretion in vitro and in transplanted animals.

NIH Research Projects · FY 2024 · 2020-07

1 Project Summary Pediatric Intensive Care Unit (PICU) survival has increased substantially over the past three decades, rendering mortality alone an insufficient metric for pediatric critical illness outcomes assessment. Currently, a comprehensive understanding of PICU morbidity and the trajectory of recovery among PICU survivors and their families is limited. Post-intensive care syndrome (PICS) consists of new or worsening impairments in physical, cognitive, or mental health status that arise and may persist after critical illness. The characteristics of PICS in children (PICS-p) are unknown. At this time, we cannot identify modifiable risk factors for poor PICU outcomes and/or develop systematic, timely, and targeted PICU or post-PICU interventions to improve PICU morbidity without first understanding the recovery in children who commonly share the PICU experience. Here, we propose a prospective longitudinal cohort study of patients undergoing >3 days of ICU therapies (case) at one of 20 US PICUs to evaluate child and family outcomes over two years. We will compare outcomes of these PICU patients with a control group of patients who received an overnight (control) PICU stay who did not receive intensive care unit therapies as well as with published quality of life data from the general and chronically ill populations. Children and their families will be enrolled locally from each PICU, and their outcomes will be followed centrally from the University of Pennsylvania and Seattle Children’s Research Institute. Our specific aims are (1) to determine the physical, cognitive, emotional, and social health outcomes and trajectory of recovery in a population of children post-critical illness; (2) to determine the baseline health, presenting problem, and PICU factors associated with impaired physical, cognitive, emotional, and social outcomes among PICU survivors, and (3) to determine the emotional and social health outcomes in parents and siblings of PICU survivors. Our primary goal is to explicate the impact of pediatric critical illness over a two-year period of time to guide future intervention research to optimize child and family outcomes. Our overall goal is to improve the health and well-being of PICU survivors and their families. 1

NIH Research Projects · FY 2025 · 2020-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The goal of the CBI Training Program is to provide students with the intellectual and technical skills that are necessary to solve important and complex biological problems that can be most effectively addressed by studies at the chemistry-biology interface. We are focusing on the mechanistic chemistry of biomolecules that have implications for impacting human health and treatment of disease. This is a theme that is of interest to a significant population of students on campus and serves as an excellent system for the application of tools at the chemistry-biology interface to solve important biological problems. The program creates a group of chemists and biologists who can speak the same language and thereby function effectively in multidisciplinary teams. The Program involves 41 participating faculty from two separate Schools within the University of Pennsylvania, The Perelman School of Medicine (PSOM) and The School of Arts and Sciences (SAS). Participating students come primarily from two graduate groups that span institution and school boundaries, the Graduate Group in Chemistry (in SAS) and the Graduate Group in Biochemistry, Biophysics & Chemical Biology (BBCB in PSOM). To ensure that students carry out research within the scope of the training program, students are selected for the program after they have chosen a thesis laboratory and project and generally in their second year. To ensure chemistry-biology interface training and cross-fertilization between the chemistry and biology students that participate in the program, students participate in the following activities: (1) research at the chemistry-biology interface, (2) didactic course-work dealing with the chemistry and biology of proteins, nucleic acids and small biomolecules, including a literature-based component, (3) an annual student-run Chemical Biophysics mini-symposia series, (4) twice monthly student luncheons involving student research and career development presentations, (5) an annual Chemistry-Biology Interface retreat, (6) an annual mid-Atlantic Frontiers at the Chemistry-Biology Interface Symposium, and (7) a structured career development plan. The program also has effective mechanisms in place for the recruitment and retention of students in the sciences, and for student instruction in the responsible conduct of research. We request funds for a steady state predoctoral training group of ~ 40 students, including 8 who are funded primarily during years 2 and 3. Oversite of the Training Program is by the Co-PIs, Ronen Marmorstein and E. James Petersson, with input from Chemistry (SAS) and BBCB (PSOM) External Advisory Committees and Internal CBI Training Committee, comprised of roughly equal faculty representation from the two participating schools and trainee representatives, which meets every 6 months. The CBI Training Committee votes on the selection of students for funded training slot positions, ensuring that highly qualified students are appointed, and administering other functions of the program.

NIH Research Projects · FY 2024 · 2020-07

Project Abstract Identification of genetic and epigenetic changes associated with disease states can afford deep insight into the underlying molecular processes, but, particularly for diseases of the central nervous system (CNS), translating this information to new drug therapies remains a challenge. Here, we will exploit the chemistry of pharmacophores found in psychoactive drugs to study the impact of genetic variants and epigenetic modifications in addiction. Hydrazine- based drugs including monoamine oxidase inhibitors (MAOI) have a long history of success in treating CNS disorders. The hydrazine group covalently inactivates several classes of enzymes (e.g. oxidases, oxygenases, demethylases, hydroxylases) in the CNS that participate in transcriptional regulation and chromatin remodeling, thereby contributing to a broad range of biological functions and disease pathologies. I previously developed a novel chemical proteomics discovery platform (which I dubbed `RP-ABPP) by exploiting the unique reactivity (reverse polarity, RP) of this pharmacophore to create unbiased probes to target these enzyme classes by activity-based protein profiling (ABPP). Given the established ability of hydrazine drugs to reach the CNS and manipulate its biochemistry, this project will implement first-in- class, nucleophilic brain-penetrating probes using our RP-ABPP platform to discover hydrazine- sensitive enzymes disrupted in preclinical models of drug addiction. Specifically, these probes will evaluate changes to the brain during the development of dependence using electronic nicotine delivery systems (ENDS) with a newly established mouse model of inhalation exposure. The goals are to i) identify novel druggable enzyme targets that are dysregulated in nicotine dependence and ii) develop a suite of selective probes that can be used by neuroscientists as pharmacological tools to study drug abuse and other psychiatric disorders. This platform is expected to i) create new opportunities to map functional consequences of genetic mutations and epigenetic modifications in drug dependence, ii) discover new druggable enzyme activities that can be spatially mapped by imaging, and iii) ultimately create a unique opportunity for therapeutic development around a relatively underexplored chemical space.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT We propose a new program of training in cancer research for surgery residents pursuing a career in academic Surgical Oncology. The rationale is that there is a nationwide shortage of surgeon-scientists. A T32 program would enable us to train high caliber surgical trainees so that they can contribute to new cancer therapies and discoveries, improve the care of cancer patients, and mentor future trainees. T32 trainees will have already completed 3 years of postgraduate clinical training in surgery. The 2-year T32 training program will provide an intensive research experience in hypothesis formulation, experimental design, research techniques, data analysis, and oral and written presentation. After completing the T32 program, the trainees return to the clinic for the 2 final clinical years of residency. Most pursue an additional 2 years of fellowship specialization after finishing residency training. The ultimate objective of the program is to train surgeon-scientists to perform oncologic research upon completion of their surgical training and become leaders in academic surgical oncology. We propose a total of 2 mentees in the first year, 5 mentees in the second year, and six mentees per year in years 3-5. The program will have a Basic/Translational Track and a Clinical Track, which reflects the strengths of our faculty and the interests of our residents. The program will leverage the vast research opportunities and infrastructure of the Perelman School of Medicine and the University of Pennsylvania, including the Abramson Cancer Center. The program is a major priority of the Department of Surgery, which has a longstanding history of training surgeon-scientists and academic leaders. The T32 Program will be managed by the Program Director, 2 Associate Program Directors, and a Steering Committee, with input from highly experienced advisors (3 internal and 2 external). The 16 mentors are based in Surgery and 8 other departments. The mentors have active funding, a track record of mentoring, and expertise in a wide variety of disciplines, including tumor immunology, cancer cell biology, cancer imaging, and clinical cancer research. The proposed curriculum is robust and will be tailored to the specific needs of each trainee. There will be instruction in the Responsible Conduct of Research, grant writing workshops, oral presentation skill sessions, and journal clubs. T32 trainees will also have the opportunity to obtain an advanced degree, such as a Masters in Translational Medicine or Clinical Epidemiology, and even a PhD. Our department has several initiatives to promote diversity through the recruitment of women and underrepresented minorities.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Dozens of drugs have failed in clinical trials for the inflammatory lung disease ARDS (acute respiratory distress syndrome), largely due to 3 pharmacological challenges particular to ARDS: ARDS patients have multi-system organ failure, so cannot tolerate off-target drug side effects; the column of liquid covering alveoli prevents effective inhaled delivery; dozens of signaling pathways underlie ARDS, so modulating just one will not work. To overcome these 3 challenges, we designed M-LACs, which are 100-nanometer lipid spheres (liposomes), loaded with multiple drugs, and coated with targeting tags that cause them to massively accumulate in the capillaries of the alveoli (air sacs of the lungs). We have previously published on the benefits of M-LACs targeted to alveolar endothelial cells, but have long seen the need to target the other major alveolar capillary cell type, alveolar marginated neutrophils. Here we introduce new targeting tags that can massively concentrate M-LACs in alveolar neutrophils. With the new ability to target LACs to both endothelium and neutrophils, we can now answer fundamental questions in ARDS biology (Aim 1) and general pharmacology (Aim 2), while radically improving M-LACs as a therapy for ARDS (Aim 3). Aim 1: In ​ex vivo​ human lungs and ​in vivo mouse models of ARDS, we quantify the relative number of marginated neutrophils compared to naive cases, and we will measure how well neutrophils and endothelial take up M-LACs. Aim 2: We will test the “depot theory” of targeted drug delivery, which says drugs efficiently elute from targeted cells to their neighbors. We will test whether drugs meant to act in neutrophils (e.g., neutrophil elastase inhibitors) will ameliorate ARDS-like phenotypes the same or worse if targeted to endothelial cells, and vice versa. Aim 3: We will identify the principles of combination therapy. We hypothesize that the most efficacious combinations will be a pair of neutrophil- and endothelial-modulating drugs (e.g., as opposed to 2 endothelial-modulating drugs). By the end of these studies, we will have uncovered new ARDS biology and answered fundamental questions in pharmacology. Additionally, we will have created a highly optimized therapy for ARDS that we will have tested in multiple mouse models of ARDS and in human lungs.

NIH Research Projects · FY 2024 · 2020-07

Although complement is historically not suspected to be implicated in the ‘pauci-immune’ anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), recent clinical and experimental evidence has shown it to play a key role in amplifying the initial inflammation and subsequent orchestration of innate and adaptive autoimmune organ injury in AAV. Human AAV is a severe systemic autoimmune disease that affects small vessels in multiple organs, but most prominently the kidney and the respiratory tract. If untreated, AAV is fatal with an average survival of only 5 months. Current treatment regimens of AAV is limited to non-specific immunosuppression which carries significant side effects and is not always efficacious in preventing relapse. Therefore, more effective and less toxic therapeutic approaches are needed. A defining feature of AAV is the presence in patient’s blood of ANCA with specificity to one of two neutrophil cytoplasmic antigens, myeloperoxidase (MPO) and proteinase 3. How complement contributes to ANCA-mediated organ injury such as necrotizing crescentic glomerulonephritis (NCGN) and lung hemorrhage is not yet fully understood. In this project, we will use a robust mouse model of MPO ANCA disease that we have recently developed to dissect the role of complement in NCGN and lung hemorrhage. Additionally, we will use this mouse model to test anti- complement therapies to provide proof of concept for targeting specific complement proteins in the treatment of NCGN and lung hemorrhage. Our specific aims are: Aim 1. To test the hypothesis that pathogenesis of both necrotizing crescentic glomerulonephritis (NCGN) and lung hemorrhage in our mouse MPO ANCA disease model requires the interplay between MPO-specific antibodies, alternative pathway of complement, and pre- existing anti-MPO cellular immunity; Aim 2. To test the role and mechanism of action of complement proteins and effectors, including properdin, C5aR and membrane attack complex (MAC), in the development of MPO ANCA-induced NCGN and lung hemorrhage; Aim 3. To test and compare therapeutic efficacy of systemically blocking properdin, C5, C5a or C5aR in preventing and treating MPO ANCA-induced NCGN and lung hemorrhage. Our innovative mouse model of MPO ANCA disease fully recapitulates the human disease phenotype, including development of NCGN and lung hemorrhage. By using this model, we expect to shed new light on the role of complement in the pathogenesis of AAV, and validate the therapeutic potential of blocking complement in the treatment of both NCGN and lung hemorrhage, two major disease manifestations of human AAV.