University Of Washington

NIH Research Projects · FY 2024 · 2020-09

Project Summary / Abstract A major shortcoming of most efforts to understand the 4D nucleome is that they have mainly focused on in vitro cell lines, rather than on dynamic, in vivo systems. Arguably, the most important in vivo system, which also happens to be the most dynamic, is development itself, wherein the nucleome both shapes and is shaped by the initial emergence of the myriad mammalian cell types. While these in vivo dynamics are presently poorly documented and understood, recently emerged technologies offer a path forward. Here we propose to establish the University of Washington 4-Dimensional Genomic Nuclear Organization of Mammalian Embryogenesis Center (UW 4D GENOME Center), which will address these massive gaps in our understanding by generating systematic datasets on nuclear morphology and associated molecular measurements in mammalian tissues and cell types. These datasets will be generated in the context of the leading model organism for mammalian development, the mouse. Our approach focuses on following nuclear structure, chromatin and gene expression changes at a “whole organism” scale, using a combination of scalable single cell profiling and “visual cell sorting” (VCS) methods, all well-established and mostly developed in our own labs. Our goal is to generate a high- resolution 4DN atlas of mouse embryogenesis for the community. The different types of data will be integrated, including cross-species imputation to integrate with human data, as well as models and navigable maps applied to pathways relevant to mammalian development.

NIH Research Projects · FY 2024 · 2020-09

In high HIV prevalence regions, women are at high risk for HIV during pregnancy and breastfeeding. To protect women and reach elimination of mother-to-child HIV transmission, the World Health Organization recommends offering oral tenofovir (TFV)-based pre-exposure prophylaxis (PrEP) to HIV-negative pregnant and postpartum women in high-burden settings. Although most pregnant Kenyan women with HIV risk factors accept PrEP when offered, >50% discontinue PrEP within 30 days of initiation and sub-optimal adherence is common. To date, no intervention studies to improve PrEP adherence include pregnant or postpartum women. We adapted an SMS communication platform (mWACh) to send PrEP-tailored, theory-based SMS to facilitate adherence among pregnant women who initiate PrEP. In a non-randomized pilot, we found that mWACh-PrEP recipients were more likely to persist with PrEP use and to self-report high adherence. We propose a randomized trial to determine the effect of the mWACh-PrEP tool on PrEP adherence during pregnancy through the postpartum period. We will also gather data on cost and delivery using the Proctor Implementation Outcomes Framework to expedite translation into routine practice. Our overarching hypothesis is that mWACh-PrEP will improve PrEP adherence among mothers at-risk for HIV, be acceptable to patients and providers, and be cost-effective. By leveraging our team’s research infrastructure in Western Kenya, we are uniquely positioned to execute the following AIMS: Aim 1- To determine the effect of the mWACh-PrEP tool on PrEP adherence during pregnancy through the postpartum period among women who initiate PrEP within ANC-PrEP. We will conduct a 2-arm randomized trial comparing mWACh-PrEP vs standard of care (SOC, i.e. in-clinic adherence counseling) among HIV-uninfected pregnant women with high HIV acquisition risk (defined by validated risk score) who initiate PrEP. The primary outcome will be adherence at 6 months postpartum (TFV hair levels >0.038 ng/mg; consistent with 7 pills/ week). Secondary outcomes will include STI incidence, adherence cofactors, and prevention-effective adherence (time- varying alignment of adherence with risk behaviors). Exploratory outcomes will include HIV incidence and perinatal outcomes by arm. Hypothesis: mWACh-PrEP will increase PrEP adherence compared to SOC. Aim 2– Evaluate barriers and facilitators of mWACh-PrEP implementation within routine ANC. Using the Proctor framework, we assess acceptability and feasibility by conducting interviews and focus-groups with ANC-PrEP users, providers, and health planners. Hypothesis: Indications for readiness of mWACh-PrEP will be identified. Aim 3– Estimate the cost-effectiveness of implementing mWACh-PrEP within ANC-PrEP, per HIV infection and disability-adjusted life-year (DALY) averted. We use data from Aims 1 and conduct micro-costing and time-and- motion studies to estimate the cost of mWACh-PrEP from a payer perspective. The incremental cost- effectiveness ratio (ICER) per HIV infection and DALY averted compared to SOC will be calculated. Hypothesis: Incorporating data on PrEP and ANC outcomes with improve ICERs for mWACh-PrEP.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The dual impacts of the HIV/AIDS pandemic and unintended pregnancies constitute a major health burden for women worldwide and justify the development of highly-effective next-generation multipurpose prevention technologies (NGM) that combine safe, effective, and easily reversible long-acting contraception with HIV prevention. Here, we propose to adapt a memory wire copper intrauterine device (IUD) frame as a platform for intrauterine delivery of antiretroviral (AVR) drugs to provide user friendly HIV pre-exposure prophylaxis (PrEP) and contraception. We propose to evaluate ARV drug delivery systems compatible with IUD delivery for duel- protection up to three years. In Aim 1, we will formulate candidate antiviral drug/prodrug solid dispersions in matrix, reservoir and electrospun drug delivery systems and integrate these into an existing copper IUD to optimize drug loading, dissolution rate, chemical and functional stability, and stable integration with and functional attributes of the IUD. We will then evaluate the safety and pharmacokinetic effects of intrauterine delivery of candidate devices in baboons, and measure drug concentrations in the vagina, cervix, and endometrium. Finally, in Aim 3 we will use the macaque model to evaluate the protective efficacy of the drug- eluting delivery system for HIV pre-exposure prophylaxis against a repeated low-dose vaginal SHIV162P3 challenge.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Many age-related oral health problems, such as masticatory dysfunction, dysphagia, periodontal disease, and tooth loss have been associated with Alzheimer’s disease (AD). How cortical and biomechanical changes in oromotor behavior contribute to the onset and progression of AD and age-related dementias (ARD) are widely unknown. This is largely because of a fundamental gap in understanding the neuromechanical processes, at the level of large-scale activity of single neurons and neuronal networks, that underlie healthy aging. This represents an important problem because until they are understood, the cortical mechanisms underlying pathological aging in AD/ARD will remain largely incomprehensible. The goal of the proposed research is to investigate changes in the orofacial sensorimotor-cognitive neuronal network that underlie healthy age-related sensorimotor changes and how these cortical correlates are affected by absent sensory inputs to oral structures and by the presence of AD-like impairments (‘pathological aging’) in old rhesus macaques. The central hypothesis is that differential patterns of the dynamics of large-scale neural activity and connectivity in the orofacial sensorimotor cortex (OSMcx) and the ventrolateral frontal cortex (VLFcx) will help disambiguate healthy and pathological aging. This hypothesis will be tested by pursuing three specific aims: (1) to identify the neuronal correlates of healthy age- related changes in feeding behavior, (2) to identify changes in cortical representations of oral somatosensation following sensory nerve block, and (3) to identify changes in neuronal responses and cortical interactions in OSMcx-VLFcx networks following drug-induced AD-like impairments. Thus, we can evaluate and compare the added burden of sensory loss and AD-like impairments on aging. The proposed research uses an innovative approach that leverages the unique sensory innervation of the oral region by different cranial nerves and the use of a pharmacological model to induce AD-like impairments in old rhesus macaques. We will record cortical activity from multiple chronically implanted microelectrode arrays in OSMcx-VLFcx simultaneously with 3D tracking of tongue and jaw kinematics using biplanar videoradiography and the XROMM workflow (X-ray Reconstruction of Moving Morphology) while young and old subjects engage in natural feeding behavior. The proposed research is significant for (1) defining cortical, biomechanical, and immunohistological profiles of healthy and pathological aging, (2) determining potential contributing factors to the onset and progression of AD, and (3) identifying cortical regions that are vulnerable to AD. Using old rhesus macaques has direct translational value to evaluate potential avenues for pharmacological or cortical therapies for AD. The knowledge gained from the proposed study has important implications for earlier identification of individuals with chronic oral health issues who may be at risk for developing AD or ARDs. It may also inform the development of more effective interventions focused on enhancing oral health outcomes in this group and thus preventing the onset or allaying the progression of AD or ARD.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Genetic testing is rapidly emerging as a cornerstone of medicine, enabling targeted therapies for patients with both common and rare disorders, and empowering patients, families and communities with knowledge about their condition. However, despite significant advances over the past decade, including the introduction of clinical whole genome sequencing, clinical genetics remains largely limited to the study of less than 1% of the genome, the exome. This limitation is thought to underlie the fact that clinical genomic sequencing is unsuccessful in elucidating the culprit pathogenic variant(s) for the majority of patients with presumed monogenic disorders who undergo testing. It is hypothesized that a significant percentage of these patients harbor pathogenic non-coding variants that disrupt the gene regulatory architecture of known Mendelian genes, a class of variants poorly illuminated using current sequencing approaches. Directly addressing this limitation requires a wholesale revision of how genetic testing is performed and interpreted, as is outlined in this proposal. Specifically, this proposal aims to overcome this fundamental limitation of human genetics by leveraging a novel approach we recently developed for simultaneously mapping the genetic and epigenetic landscape of a sample, thereby illuminating the functional impact of non-coding genetic variants that disrupt local chromatin architecture and gene regulatory patterns – Whole Epi-Genome Sequencing (WEGS). Using this approach, we plan to directly test the hypothesis that rare non-coding genetic alterations contribute to monogenic disorders. In Aim 1, we will use the WEGS approach to characterize the gene regulatory impact of non-coding sequence, structural and epigenetic alterations in healthy individuals as well as patients with known imprinting disorders. The goal of this aim is to establish the sensitivity and power of WEGS for identifying genetic variants that disrupt local chromatin architecture and gene regulatory patterns and improve our understanding of the functional impact of non-coding genetic variation. In Aim 2, we will directly evaluate the contribution of rare non-coding genetic alterations to monogenic disorders by applying WEGS to patients with suspected monogenic disorders for whom whole exome or genome sequencing has previously been non- diagnostic. Overall, this proposal has the potential to dramatically change how we approach genomic testing and our understanding of the impact of non-coding sequence and structural variation on gene regulatory patterns and human disease.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Our bodies consist of an exquisite collection of tissues and organs that undergo constant change. From morphogenesis and homeostasis to the progression of disease, these changes are associated with both the healthy and unhealthy processes that define human life. My research program at the University of Washington is developing robust and uniquely powerful multidisciplinary methodologies to mimic, exploit, and quantify these changes, particularly as they evolve in both time and 3D space (i.e., 4D). In our lab’s first five years of existence, we have: (1) developed a suite of synthetic cell-culture platforms whose biochemical and biophysical properties can be reversibly modulated in 4D using cytocompatible photochemistries, and have utilized these platforms to regulate proliferation, migration, differentiation, and intracellular signaling at single- and sub-cellular resolutions; (2) introduced a photodegradable material-based approach to generate the first endothelialized 3D vascular networks within cell-laden hydrogel biomaterials that span nearly all size scales of native human vasculature (including capillaries); (3) reported the first modular framework for imparting biomaterials with precise degradative responsiveness to multiple environmental cues/biomarkers following user-programmable Boolean logic; and (4) established the first tools for “spatiotemporally resolved proteomics”, enabling visualization and quantification of proteins produced in vitro and in vivo within user- defined regions in 4D. The present proposal expands our group’s capabilities in each of these areas, paving the way to new therapeutic targets and treatments of disease through a fundamentally transformed knowledge of basic cell physiology. In this project, we will: (1) exploit our 4D-tunable biomaterials to recapitulate and probe cardiovascular developmental signaling in vitro, examining the manner in which precise spatial and temporal presentation of signaling proteins culminates in orchestrated differentiation; (2) employ synthetic capillaries to examine drug action and resistance, screen therapeutics, and investigate microvascular occlusion, thrombosis, and altered remodeling that occurs in many hematologic diseases (e.g., sickle cell anemia, spherocytosis); (3) develop and deploy hydrogel nanoparticles exhibiting logic-based degradative response to cancer-presented biomarkers to deliver small molecule chemotherapeutics to tumors with unprecedented specificity; and (4) extend our 4D proteomic strategies to permit optically and physiologically defined proteomic mapping in living tissue and model organisms. Critically, the methods that we are developing and implementing are cell-, tissue-, and disease-agnostic, enabling enhanced understanding of a wide variety of biological processes while laying the foundation for advances in disease diagnosis, treatment, and prevention.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT Autoimmune diseases are complex diseases arising from both genetic and environmental factors. The pathogenesis of autoimmune diseases like type 1 diabetes (T1D) is not mediated by a single cell type of the immune system. The role of noncoding variants in these diseases are largely understudied, but suggest that changes in three-dimensional chromatin architecture could be altered. Early work largely focused on the role of CD4+T helper 1 (Th1) and T helper 17 (Th17) cells, followed by T regulatory cells (Tregs). More recently, T memory stem cells (Tscm) were identified and their role in autoimmunity is just beginning to be investigated. These cells possess stem-like properties of self-renewal and differentiation. Unlike memory cells (Tmem), these cells are long-lived, providing a source of prolonged immune memory. Because of their stem cell state and ability to provide long-term memory, these cells have important implications for autoimmunity, cancer immunity and immune therapies, and reconstitution of the immune system. In the context of autoimmunity that means these cells also provide a reservoir of autoreactive and inflammatory cells that can continue disrupt immune function and contribute to chronic disease. Our own data indicate that these cells are enriched in T1D donors compared to healthy control subjects, which may be associated with disease risk alleles. The overall goal of this proposal is to identify two- and three-dimensional chromatin architecture changes that distinguish CD4+ Tscm cells from their naïve progenitor and derived cell fates and determine how changes specific T1D contribute to disease pathogenesis by profiling pure populations of cell from healthy control and T1D subjects. We will determine if T1D-associated change are prevalent in other autoimmune diseases by also profiling Tscms from RA donors. We will determine what role noncoding disease-associated variants at cis-regulatory elements (CREs) might play in this process. We proposed to integrate 2D and 3D chromatin architecture across a large cohort to precisely determine target genes and mechanisms of cell- and disease-specific gene regulation. Specifically, we will identify cell-type specific chromatin architecture in pure populations of CD4+ subtypes related to stem cell memory by employing capture HiC to map regulatory loops across three key cell types in a cohort of 50 healthy control subjects. To determine if these regulatory loops are altered in a diseased state, we will identify T1D-specific chromatin architecture to determine the role of Tscm cells in prolonged inflammation and autoreactivity. Lastly, we will identify chromatin architecture changes common in autoimmune Tscm cells by profiling an addition 50 subjects with rheumatoid arthritis. The proposed study will use innovative approaches to conduct large-scale assessment of 2D and 3D genome architecture to determine cell- and disease-specific gene regulatory mechanisms using a well-defined human cohort. The proposed study will advance our understanding of the role of memory stem cells in autoimmunity and the effects of noncoding variants on chromatin architecture in a disease-specific manner.

NIH Research Projects · FY 2024 · 2020-09

In sub-Saharan Africa (SSA), retention of HIV-infection people in antiretroviral therapy (ART) is an increasing challenge. With only 67% retained on ART in SSA, over 7.5 million people are out of care, threatening both individual health and HIV epidemic control. With significant healthcare system constraints and formidable healthcare worker (HCW) shortages, ART services in SSA struggle to meet ambitious global targets of 90% retained in care. In research settings, text-based mHealth innovations show promise to increase ART retention and, therefore, reduce viral load (VL). However, few mHealth innovations have been tested or proven effective in real-world settings with severe human and financial resource constraints. mHealth innovations that successfully retain more patients on ART, at lower cost, within large-volume public ART clinics in SSA are urgently needed. Lighthouse Trust (LT), the largest public provider of ART in Malawi, operates two large clinics in Lilongwe with the Malawi Ministry of Health (MoH), Lighthouse Clinic (LH) and Martin Preuss Center (MPC), with a combined 35,000 ART patients. 12-month retention at Lighthouse is 67%, below the 90% global target for epidemic control. Both clinics employ a real-time electronic medical records system (EMRS) and implement a resource-intensive patient retention program, Back to Care (B2C). B2C aims to trace patients who miss ART visits by >14 days. MPC has over 7800 monthly ART visits, and more than 10% of clients are B2C-eligible. B2C demand, coupled with HCW constraints, results in tracing of only 33% of target clients. Lack of, or delayed, tracing reduces ART retention. Moreover, over 50% of traced B2C clients are found on ART, 60% of whom had transferred, demonstrating significant unnecessary tracing. Poor data quality reduces B2C effectiveness. Therefore, the University of Washington’s International Training and Education Center for Health (I-TECH) and LT, with support from MoH and Medic Mobile, seek to implement an innovative, proactive, patient retention system using two-way texting (2wT) between new ART patients and staff. 2wT will resolve potential retention issues before they occur and improve data quality (e.g. identifying transfers), reducing B2C workload. Using a quasi-experimental, pre-post design, this R21 (Aim 1) will test a hybrid 2wT intervention to provide adherence support for, and visit-focused communication with, new ART initiates at MPC clinic, comparing retention (ART outcomes, visit adherence, VL) and B2C referrals among two groups of new MPC ART clients: 2wT-supported (prospective intervention) and matched pre-2wT (retrospective comparison). After demonstrating improved retention at lower cost (Aim 2) using research teams (R21: years 1-2), 2wT integration into the EMRS (R33: years 3-5) will automate enrollment and management of 2WT for new clients in both LH and MPC using routine HCWs (Aim 3), assessing the transition of research to practice. The proposed mHealth study will significantly increase ART retention in public ART clinics using routine HCWs, reducing workload and costs. This innovative approach in high-volume, public settings in Malawi sets the stage for national or regional scale-up.

NIH Research Projects · FY 2024 · 2020-09

Project Summary The post-translational modification of histone H3 lysine 4 (H3K4) by methyl groups is an evolutionarily conserved epigenetic mark that is generally associated with transcription activation in all eukaryotic cells. Early studies of the yeast model system, S. cerevisiae, have not only identified the prototype of the SET1/MLL family of methyltransferases as the enzyme responsible for H3K4 mono-, di-, and trimethylation, but also revealed a yeast Set1-centric protein complex, known as COMPASS, that stabilizes and confers catalytic activity to the enzyme. The SET1/MLL family of H3K4 methyltransferases has undergone a significant expansion in animals. Mammals have evolved a total of six distinct and functionally non-redundant family members, each of which also functions within a COMPASS or COMPASS-like complex. Remarkably, recent studies have shown that mutations or dysregulation of the six human SET1/MLL methyltransferases are associated with a spectrum of mental illnesses, including schizophrenia, autism, and intellectual disability disorders. Malfunctions of some of these family members are further linked to other human diseases such as mixed lineage leukemia and congenital heart disease. Despite their important biological roles and their high relevance to human health, a molecular and mechanistic understanding of the SET1/MLL H3K4 methyltransferases is largely lacking due to the large sizes of most SET1/MLL enzymes and the complexity associated with their assemblies and regulation. To date, most structural and biochemical studies have been focused on single domains and small fragments of the yeast and human SET1/MLL enzymes and COMPASS subunits. Many questions, such as how the SET1/MLL enzymes bind and become regulated by four common catalytic module subunits, namely RBBP5/Swd1, WDR5/Swd3, ASH2L/Bre2, and DPY30/Sdc1 (human/yeast ortholog), how the resulting complexes recognize H3K4 in the context of nucleosome and differentially catalyze mono- vs. multi-H3K4 methylation, and how the activities of COMPASS and COMPASS-like complexes are regulated by upstream signals such as H2B mono-ubiquitination remain unclear. Using a combination of structural, chemical and biochemical approaches, as well as yeast cell- based functional assays, we propose to dissect the structure and function relationship of the yeast Set1 COMPASS complex as a model system and extend this work to the clinically relevant human SET1/MLL complexes. Our proposed studies hold the promise to establish the missing framework for understanding the structural basis of the SET1/MLL H3K4 methyltransferase function and regulation in eukaryotic biology and unmasking the molecular mechanisms of various human diseases associated with their malfunction.

NIH Research Projects · FY 2024 · 2020-09

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide, affecting young adults and increasingly aging patients. Patients with TBI suffer two distinct but closely related injuries. The primary injury is caused by physical forces that disrupt the structural integrity of the brain and vasculature at the site of impact, whereas the secondary injury is ischemic and inflammatory that disseminates to the most parts of the brain and other organs such as the lungs and the heart. The transition from the primary to the secondary injury is mediated through the blood brain barrier (BBB). BBB is a highly selective semipermeable barrier of microvasculature that separates the circulating blood from the brain parenchyma and extracellular fluid in the central nervous system. It consists of not only endothelial cells and the subendothelial matrix but also other perivascular cells, i.e. peri- cytes and astrocytes, that together form the neurovascular unit. Disrupting BBB at the site of injury permits direct exchange between blood and cerebral components, leading to intracerebral and intracranial hemorrhage, and systemic inflammation and coagulopathy. Despite extensive efforts on TBI-induced cerebral and systemic inju- ries in the past, how local TBI disseminates the secondary injury remains poorly understood, largely due to the lack of a physiologically relevant 3D-model system. In this proposal, we have assembled an interdisciplinary team with expertise in microvascular engineering and vascular biology, hematology and hemostasis, TBI, and cell signaling to reconstruct human BBB. This in vitro reconstructed BBB will 1) be 3D in its microvascular archi- tect to contain cellular and matrix components of BBB, 2) allow for dynamic flow of blood or its components with defined patterns and shear stresses found in arterial and venous blood flow, and 3) permit manipulation at bio- chemical (intracellular signaling) and cellular levels (light and electron microscopy). We will use this model sys- tem to exploit roles of blood derived factors in maintaining and disrupting the BBB integrity and function, to identify intracellular signal pathways (the kinase inhibitor regression analysis) that contribute to BBB breakdown and its repairs using a systems biology approach, and to develop in field or bedside devices that evaluate the state of BBB integrity (new biomarker development) and help developing new therapeutic targets for TBI. Find- ings from this proposed study will have numerous implications in future neurovascular engineering approaches and therapeutic development.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT The goal of this project is to change the treatment paradigm for proteinuric glomerular diseases by combining therapeutics development with cell-specific delivery to enhance podocyte repair and regeneration in vivo. Podocytes, highly specialized terminally differentiated epithelial cells, are injured in the majority of glomerular diseases. As podocytes cannot self-renew, podocyte loss leads to glomerular scarring. A subpopulation of parietal epithelial cells (PECs) can serve as podocyte stem cells (`PEC progenitors'), but their regenerative potential is insufficient to overcome disease-associated glomerular damage. Enhancing productive repair of podocytes thus requires a dual synchronized approach: (i) replacing lost podocytes to increase their number, and (ii) limiting/reversing damage to the remaining podocytes. However, major knowledge gaps prevent us from achieving these goals; these include our limited knowledge on the molecular factors stimulating PEC self-renewal and podocyte regeneration/repair, as well as options methods for delivering these factors to specific kidney cell types in vivo. Our team of four expert investigators will wield complementary tools to close these knowledge gaps and produce innovative therapies. Dr. Wessely will apply Design of Experiment (DoE) approaches to identify novel combinations of molecules that increase PEC progenitors and reduce podocyte loss; Dr. Roberts will conjugate these therapeutics to VHHs (nanobodies) for delivery to PEC progenitors and podocytes; Dr. Freedman will generate gene-edited human kidney organoids to validate effects of VHHs compared to clinical data from patients; Dr. Shankland will use lineage tracing animal models of podocyte depletion and human organoids transplanted into mouse kidneys for in vivo safety and efficacy analysis. This pipeline will ultimately test the hypothesis that targeted delivery of PEC- and podocyte-specific therapeutic cargos can enhance podocyte repair and regeneration in vivo, and restore glomerular function to below the clinical disease threshold. The work will be accomplished through two Specific Aims, each with unique Milestones. The first Aim is to increase glomerular regeneration in vivo by cell targeted delivery of novel combinations of peptides and small molecules to augment podocyte progenitors of parietal epithelial cell origin. The second Aim is to increase productive repair of damaged podocytes by cell-type specific delivery of newly identified therapies. For both aims, we will employ the above pipeline to discover candidate therapeutics by DoE and cross-referenced with glomerular disease signatures from human patients. These will be combined with cell type-specific VHHs from high diversity recombinant VHH libraries to selectively deliver them to human PECs (Aim 1), or podocytes (Aim 2). Enhanced regeneration in vivo will be demonstrated in animal models of FSGS and transplanted human organoids. This process will establish a new paradigm for the treatment of kidney disease, and produce lead therapeutic candidates for further pre-clinical development and ultimately human clinical trials.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT The impact of next-generation sequencing (NGS) on gene discovery and molecular diagnostics for Mendelian conditions (MCs) is hard to overstate. However, to provide affected individuals with precise natural history, recurrence risk, and prognosis in clinical settings, identification of pathogenic variant(s)/genotypes alone is often insufficient. This is a challenge most notably for genes that cause more than one MC or ~25% of all genes that underlie MCs. In such an instance, even with a known genotype a patient's phenotype has to be compared to that of all MCs caused by variants in a gene to determine which MC, if any, is the likely diagnosis or whether patient instead has a novel condition. This comparison is increasingly difficult because delineation of the ~5,100 MCs currently known has typically been based on subjective grouping of affected individuals by phenotypic similarity. We propose to develop a quantitative framework for assessing overlap among the distributions of phenotypes due to pathogenic genotypes the same gene and apply this framework genome-wide. NGS has enabled identification of causal genotypes in hundreds of thousands of individuals with MCs, providing a sufficiently large dataset that it is now feasible to use machine learning to quantitatively and systematically identify “clusters” of co-occurring genotypes and phenotypic features for each known gene. We will refine and validate our approach by comparing differences between conventionally-delineated and quantitatively-delineated MCs and by assessing the similarity of individuals with well-studied atypical phenotypes/genotypes to quantitatively-delineated MCs. We will then apply the optimal strategy across the genome to generate a “next- generation morbid map” based on quantitatively-delineated MCs. We will also apply machine learning approaches to identify genomic properties associated with the propensity for each gene to underlie multiple MCs (i.e., the numeric contribution of each gene to the morbid map or phenotropy). This will enable a more precise and complete understanding of the genotypic and phenotypic spectrum of each MC, enable more objective diagnosis of individuals with atypical phenotypes, and more robustly identify the existence of multiple MCs among individuals with non-specific “class” phenotypes (e.g., developmental delay, autism, hearing impairment). We will make all newly developed methods publicly available via interactive and programmatic web-based tools to facilitate extension of this work to other human and model organism datasets.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT It is increasingly clear that ambient and household sources of combustion derived air pollutants threaten healthy neurodevelopment based on research conducted in high income settings. Advances describe the neurotoxic potential of traffic-derived emissions, components such as polycyclic aromatic hydrocarbons (PAHs), and physical characteristics such as ultrafine particulate (UFP) in experimental models, mechanistic studies, and observational epidemiological studies of ADHD, autism, and cognitive performance. However, in low resourced settings such as sub-Saharan Africa (SSA) where exposure magnitudes are among the highest worldwide, data and capacity are lacking. We seek to extend a highly productive 30 plus year University of Washington – University of Nairobi maternal child health research partnership to incorporate capacity building and research focused on air pollution and child neurodevelopment in urban Kenya. We leverage a unique pre conception cohort and apply both biomarkers (urinary PAH metabolites) and mobile monitoring approaches to understand air pollution exposure across the early life continuum (pregnancy/infancy) as well as describe the impact of sources from ambient and household origin. Follow up to age 3 years will determine the relationship between early life air pollution exposure and child motor, cognitive, self-regulation, and executive function skills. The design and engagement reflects 2 pilot projects awarded in the past year which yielded practical experience and extensive discussions with environmental scientists and maternal child health experts in Nairobi. The project fills current gaps in child neurodevelopmental assessments as well as exposure science and laboratory methods to promote maternal child environmental health research in SSA. The project will advance lab capacity for neurotoxic biomarkers at two key institutions, will establish innovative methods for ambient air pollution measurement, will develop a unique pre conception cohort with a biospecimen archive and detailed neurodevelopmental outcome data – each foundational components of long-term, well-executed studies of air pollution exposure and brain development across the lifespan. Our overarching goal is to establish a sustained research to practice program that connects high quality, regionally relevant research to program and policy to reduce modifiable environmental risks to healthy child neurodevelopment.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Only 56% of children with hearing loss (HL) in elementary school and only 44% in high school are reading at grade-level (Geers et al., 2008). The literacy gap between children with HL and children with normal hearing (NH) is observable early: preschoolers and kindergarteners with HL score significantly lower on early literacy measures than children with NH (Easterbrooks et al., 2008; Nittrouer et al., 2012). Identifying the mechanisms underlying emergent literacy in the preschool years for children with HL is therefore crucial to begin to close the outcome gap. The long-term goal of this research program is to validate effective interventions for preschool children with HL that close the early literacy gap between preschoolers with HL and NH. Initially, we will identify the literacy acquisition mechanisms for children with HL and then build and validate interventions that address the identified mechanisms. Toward this goal, the aims of the proposed research are to determine the developmental influences between 1) phonological awareness and alphabetic knowledge, 2) phonological awareness and receptive vocabulary, and 3) expressive morphology and receptive vocabulary for preschoolers with HL. Children's emergent literacy skills will be assessed as they develop during preschool (Lonigan et al., 2000). The obtained longitudinal data will be analyzed using latent change score modeling, useful for identifying the developmental influences among co-developing constructs (McArdle, 2009). Latent change score modeling in conjunction with longitudinal data will enable us to identify the developmental influences among phonological awareness, alphabetic knowledge, receptive vocabulary, and expressive morphology for children with HL. Identifying the unique developmental influences for children with HL relative to children with NH will lead to an improvement of theoretical models of emergent literacy to account for the unique literacy mechanisms of children with HL. Improved theoretical models will allow us to infer the malleable factors in emergent literacy development for children with HL, leading to hypothesis-driven intervention creation and improved clinical outcomes for this population.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY (See instructions): Limbal stem cells (LSCs) give rise to the entire corneal epithelium and are known to reside in the border area between the cornea and conjunctiva called limbus. Loss of LSCs or destruction of the LSC niche can result in Limbal Stem Cell Deficiency (LSCD) – a common cause of vision loss in the world. While transplantation of the autologous limbal tissues removed from the contralateral eye can cure patients with unilateral LSCD, bilateral LSCD patients have no autologous limbal tissues available. These patients often require transplantation of allogeneic donor limbal grafts; however, their success is highly variable. Moreover, the worldwide corneal donor shortage poses significant challenges for the availability of allogeneic LSCs for the treatment of bilateral LSCD patients. Thus, the overarching goal of this project is to develop cell-free LSCD therapies through the discovery of novel mechanisms of LSC maintenance and regeneration. Our lab has discovered an ATP-binding cassette (ABC) superfamily member B5 (ABCB5) as a novel LSC marker. ABCB5-positive LSCs isolated from human donors were capable of the long-term corneal restoration in pre-clinical LSCD models. Clinical trials are currently on the way to address the therapeutic potential of this stem cell population in human patients. Our most recent studies aimed to explore the cellular hierarchy within ABCB5-positive LSCs using single-cell RNA-sequencing revealed a novel LSC subpopulation that could be differentiated from the other LSC clusters by low expression levels of the cornea-specific genes. Here we hypothesized that this subpopulation possesses the most primitive stem cell characteristics with the highest regenerative potential. Further in-depth analyses revealed that these cells preferentially expressed the molecules involved in FGF, BMP, and AXL signaling cascades. We posit that these molecular pathways are essential for the maintenance of the undifferentiated LSC phenotype and can be employed for de-novo LSC induction and restoration of the LSC niche in the setting of bilateral LSCD. The two Aims of this proposal will: mechanistically dissect the role of FGF7, BMP2 and AXL in the LSC maintenance using murine and human genetically engineered experimental models (Aim 1) and will test the therapeutic potential of targeting these pathways for the treatment of LSCD in pre-clinical murine disease models (Aim 2). Successful completion of this study will further advance our understanding of LSC development, maintenance, and regulation with significant implications for clinical translation

NIH Research Projects · FY 2024 · 2020-09

Summary Growing evidence suggests that extracellular vesicles (EVs), membrane vesicles that can be secreted by most cell types to mediate intercellular communication, play important roles in the initiation and or progression of Alzheimer disease (AD). Specifically, it has been demonstrated that cell-to-cell transfer of amyloid beta (Aβ), tau, and other proteins critically involved in AD pathogenesis, as well as the prion-like propagation of AD pathology within the central nervous system (CNS) is mediated at least in part via EVs. Additionally, EVs carrying unique, disease- specific, and functionally important cargo are detectable in vivo in blood, cerebrospinal fluid (CSF) and other body fluids. More recently, we and others have demonstrated not only that EVs may cross the blood-brain barrier (BBB), though the transportation mechanism remains unclear, but also that blood-based but CNS-specific EV molecules can be a valuable source of biomarkers for neurodegenerative diseases, including AD. In this study, we will first use our advanced proteomics techniques to screen for EV surface markers specific to AD-related neuronal subpopulations or brain regions to identify more CNS- and AD- specific EV markers, and in parallel adapt our nanoparticle sorting and single-molecule quantification technologies to enable high-purity isolation of CNS-derived EVs in plasma and high-precision quantification of proteins in such EVs to address several major challenges in the current field. Using the currently known (e.g., L1CAM) and more CNS- and AD- specific, CNS-derived EV surface markers, as well as the existing and further developed EV isolation and quantification technologies, we will then compare AD-related biomarkers in L1CAM-containing EVs or those from AD-related neuronal subpopulations in blood plasma from human patients, focusing on the performance of classic AD proteins and known EV candidates, specifically, Aβ, tau, α-synuclein, and their various isoforms; additional novel targets may be studied when necessary. For the verified AD-related EV proteins, we will further examine their longitudinal changes in animal models and explore the mechanisms by which they are transported from the brain to blood (e.g., crossing BBB) in cellular and animal models and potential ways to alter them as novel future AD treatment targets. The proposed experiments will likely establish the foundation leading to an inexpensive and widely available test to aid in AD diagnosis and/or disease tracking. Additionally, the proposed set of studies is an important initial step toward elucidating a novel potential clearance pathway for potential toxic CNS protein species and ultimately it may provide critical opportunities for therapeutically addressing the pathology associated with neurodegeneration in AD.

NIH Research Projects · FY 2025 · 2020-08

ABSTRACT Hyperglycemia is the cardinal manifestation of diabetes mellitus and an established risk factor for kidney failure, retinopathy, neuropathy, and cardiovascular disease in the general diabetes population. However, with dialysis, measurement and management of glycemia is notoriously difficult, due in part to known inaccuracies of hemoglobin A1c. In addition, severe hypoglycemia is common in dialysis patients with and without diabetes. As a result, relationships of glycemia with clinical outcomes and unique determinants of glycemia are poorly defined in the dialysis population. These knowledge gaps preclude development of informed therapeutic strategies. We initiated the BLOod Sugar Sensing On Maintenance dialysis (BLOSSOM) study to better characterize glycemia in the dialysis population using continuous glucose monitoring (CGM). In our initial funding period, we observed high rates of hypoglycemia, but uncontrolled hyperglycemia was surprisingly predominant. In particular, we observed remarkably poor glycemic control among dialysis patients with treated diabetes, despite reasonable clinical hemoglobin A1c measurements, as well as substantial hyperglycemia among participants with untreated diabetes (who may assume their diabetes is no longer a problem) and among nondiabetic participants treated with peritoneal dialysis. In this competitive renewal application, we propose to extend our initial observations to better define the determinants and consequences of hyperglycemia and hypoglycemia in the dialysis population and advance appropriate application of CGM in this high-risk group. Specifically, we aim to: (1) test associations of glycemia, measured by CGM, with long-term clinical outcomes in kidney failure treated with dialysis by expanding and extending the BLOSSOM cohort and linking with a comprehensive outcome ascertainment resource; (2) determine the physiologic underpinnings of the extensive, unanticipated hyperglycemia observed in BLOSSOM by linking frequently- sampled oral glucose tolerance tests to CGM patterns in a subset of BLOSSOM participants; and (3) develop novel analytic approaches to improve CGM interpretation and application. Together, this work will advance understanding of the physiologic basis and health implications of abnormal glycemia in the dialysis population and provide a foundation for clinical application of CGM and for clinical trials of new therapeutic strategies.

NIH Research Projects · FY 2026 · 2020-08

PROJECT SUMMARY Growing evidence suggests that agouti-related protein (AgRP) neurons expressed in the hypothalamic arcuate nucleus play an important role in health and disease. In response to hormonal, nutrient, and neural-related input, these neurons, when activated potently stimulate feeding and engage a broad array of other metabolic and behavioral functions. Our recent findings suggest that rather than being a homogeneous population, AgRP neurons are a heterogeneous population that contain functionally distinct subsets of AgRP neurons that conduct distinct biological functions. Specifically, using a single-cell transcriptomics approach with RNAScope, our Co-PI, Dr. Tune H. Pers, University of Copenhagen, has shown that multiple subsets of AgRP neurons exist, and these exhibit differential responses to various physiological stimuli such as leptin and glucose. This work supports our recent findings which shows that whereas fasting activates the majority of AgRP neurons, acute cold-exposure only activates a small subset. Moreover, our findings further show that AgRP neurons are required for cold-induced increases in energy intake, but not energy expenditure, suggesting that those AgRP neurons involved in feeding are distinct from those involved in energy expenditure. We therefore hypothesize that in response to different afferent input, distinct subpopulations of AgRP neurons project to separate brain regions to mediate their biological effects. Proposed aims seek: 1) to identify and transcriptionally characterize distinct subsets of AgRP neurons and 2) to map the projections and responsiveness of transcriptionally distinct AgRP neuron subsets. This project leverages the complementary expertise and resources of the Morton and Pers laboratories to create synergy that is essential to the success of this project. Dr. Morton has extensive experience examining the role of the brain in the regulation of energy- and glucose homeostasis, while Dr. Pers has expertise in transcriptomics and computation techniques. Thus, to accomplish this, we will use a combination of mouse genetic, viral and immunohistochemical techniques along with single-cell RNA sequencing (RNA-seq) strategies and bioinformatic analysis. Together, this work will fundamentally advance our knowledge and understanding of the AgRP neurocircuits that regulate feeding, relative to other biological functions, and create opportunities to develop novel strategies for the treatment of obesity.

NIH Research Projects · FY 2025 · 2020-08

The overall hypothesis of the Triglycerides, Diabetes and Cardiovascular Disease Program Project for the next 5 years is that that abnormal assembly, secretion, composition and clearance of TRLs promotes the accumulation of highly atherogenic RLPs in atherosclerotic lesions in individuals with T2D, and that RLPs contribute to CVD risk by altering lesion cell populations and functions, thereby increasing atherosclerotic lesion progression and hindering regression. We propose that the increased risk of cardiovascular disease (CVD) associated with diabetes can be understood, prevented, and treated only by increasing our knowledge of the factors that regulate triglyceride-rich lipoproteins (TRLs) and their remnant lipoprotein particles (RLPs). TRLs and their remnants comprise a great variety of nascent and metabolically derived particles differing in size, protein composition, and lipid content, which has made it difficult to identify the mechanisms that promote atherosclerosis. We plan to continue to address this complexity by focusing on specific pathways and proteins and by using unique analytical tools; building on the knowledge, methodology and tools we have accumulated in the current funding period. We believe that our highly interactive and interdisciplinary group of investigators with extensive expertise in this area is needed to answer the question of how TRLs and RLPs promote CVD risk. The expertise of our team in different aspects pertaining to the overall hypotheses of this Program Project will continue to ensure synergy and cross-fertilization between Projects, which is likely to markedly advance research in this important and timely area. Importantly, the RLPs and proteins that control them are amenable to therapeutic intervention. We therefore believe that our projects will continue to provide new insights into the pathogenesis of CVD in diabetes and suggest new ways to target and prevent the increased CVD risk in this large population. The Program Project Grant consists of three Projects and three Core units: • Project 1: Type 2 diabetes, APOC3 and cardiovascular disease – Karin E. Bornfeldt, PhD, Project Leader • Project 2: ANGPTL3-dependent mechanisms underlying adaptations in hepatic lipoprotein production and clearance – Nathan Stitziel, MD, PhD, Project Leader • Project 3: Triglycerides, lipolysis, and vascular inflammation – Ira J. Goldberg, MD, Project Leader • Core A: Administrative Core – Karin E. Bornfeldt, PhD, Core Director • Core B: Proteomics and lipoprotein characterization core – Tomas Vaisar, PhD, Core Director • Core C: Atherosclerosis and bioinformatics core – Jenny E. Kanter, PhD, Core Director

NIH Research Projects · FY 2025 · 2020-08

ABSTRACT Effective antiretroviral therapy (ART) for people living with HIV (PLWH) has dramatically reduced mortality resulting in many surviving into middle and old age. Despite this success, PLWH experience high rates of comorbidities, multimorbidity (>1 major chronic illness), and functional decline at ages 10-15 years younger than uninfected controls. Geriatric syndromes, such as frailty and falls, are becoming more prevalent in PLWH. Thus, there is an urgent need to focus on the healthspan of PLWH rather than just mortality. Healthspan, in contrast to lifespan, is defined as the time someone is healthy not just alive. The Claude D. Pepper Older American Independence Centers (OAlCs) were established to help define aging phenotypes and advance research into the causes, prevention and treatment of functional decline with age. OAlCs have developed and validated functional assessments in aging, but lack depth and breadth of HIV expertise. In contrast, Centers for AIDS Research (CFARs) have unparalleled expertise in HIV-related basic, clinical and social/behavioral research, but lack robust resources and expertise in aging biology, geriatric clinical phenotypes and functional assessments. Our R24 project, “Developing Research At The Interface Of HIV And Aging” was in response to PA-12-064 "Network and Infrastructure Support for Development of Interdisciplinary Aging Research." Through this R24 we successfully linked CFARs and OAICs to support pilot projects in high-priority focus areas and mentor researchers at the interface of HIV and Aging. Our vision for this proposal is to build on this success deepening the ongoing linkage of CFARs and OAICs and expanding the network to include Nathan Shock Centers of Excellence in the Basic Biology of Aging (NSCs) and the McKnight Brain Institutes (MBIs). By bringing together OAICs expertise on functional decline in aging, with NSC expertise in aging biology and the mechanisms underlying function decline, with MBI expertise on age-related cognitive decline, and CFAR expertise on HIV, we create an integrated approach to advancing and accelerating investigation at the interface of HIV and aging via the following Aims: Aim 1. Provide specific training models and a brief inventory of tools to efficiently collect data to improve HIV clinical care and outcomes research. Aim 2. Using a geroscience approach, provide a platform for pilot studies to determine the links between molecular hallmarks of aging with functional decline, and the development of common comorbidities among aging PLWH. Aim 3. Develop infrastructure for evaluating interventional approaches and their application to HIV care. Aim 4. Provide educational support, implementation advice and mentoring for emerging investigators to establish/advance research programs in HIV and aging. These synergistic aims leverage and expand the infrastructure and close ties built in the R24 of HIV expertise from CFARs with the gerontology and functional assessment expertise within the OAlCs and expand them with inclusion of NSCs and MBIs to enhance and accelerate investigation at the interface of HIV and aging.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT There is a substantial and growing population of children/adolescents who are HIV exposed uninfected (HEU) in sub-Saharan Africa (SSA). There is emerging evidence that fetal exposure to maternal infections and medications may result in adverse adult/adolescent mental health outcomes; it is plausible that fetal exposure to HIV or antiretrovirals (ART) could similarly influence long-term outcomes. Despite evidence for poor growth and neurodevelopmental deficits, programs to systematically monitor neurodevelopmental and mental health outcomes among HEU at a population level are lacking. Understanding whether there are increased adverse neurodevelopmental and mental health outcomes in HEU due to fetal HIV/ART exposure has been challenged by difficulties in defining appropriate comparator cohorts, changing PMTCT treatment guidelines, and lack of validated, scale-able assessment tools. Our team has a unique track record for moving novel approaches for screening and testing from pilot to implementation, conducting large scale evaluations, and conducting neurodevelopmental and mental health assessments in HIV-affected children and adolescents. We propose parallel longitudinal and population based HEU/HUU cohorts, in urban and rural settings, spanning infants/children/adolescents age 6 weeks to 18 years to determine differences in neurodevelopmental and mental health outcomes. The R61 phase will accrue a longitudinal homogenous cohort of 2000 HEU/HUU infants exposed to Option B+ PMTCT treatment regimens, with extended follow-up in the R33 phase for a total of for 4 years, and assessed for neurodevelopmental, mental health, hearing, growth outcomes and telomere length. An approach to survey older HEU/HUU (age 3-18) for neurodevelopmental and mental health outcomes and collect maternal medical, ART regimen and timing, and viral load data from medical records will be piloted in the R61 phase and expanded to 100 HIV clinics in the R33 phase. Selected neurodevelopmental/mental health screening and diagnostic tools to be used are based on open source availability, diagnostic or screening performance, cultural appropriateness, accessibility, and clinical relevance. In the national R33 survey, cost analysis will complement clinical data; and together, will be disseminated to stakeholders at a workshop to develop a framework for an integrated HEU screening program in public health settings. This proposal leverages extensive team experience in prior longitudinal MTCT cohorts, in national child and adolescent surveys, HEU recruitment, neurodevelopmental and mental health assessment, and medical record extraction and analysis at scale, to comprehensively understand burden, mechanism and outcomes in HEU. Engaging stakeholders has the potential to enhance HEU management programmatically based on findings.

NIH Research Projects · FY 2024 · 2020-08

Project Summary Obesity continues to be a major health crisis, afflicting populations worldwide with comorbidities that include type 2 diabetes and cardiovascular disease. This has stimulated research of neural circuits regulating energy balance, and technological advances have allowed for the dissection of genetically-defined brain circuits controlling feeding behavior and physiology. Studies have revealed complex central circuits, which ultimately exist for integrating endocrine and peripheral sensory signals. The vagus nerve is a critical sensory pathway for the control of meal termination and is the only direct neural link between the gut and brain. Vagal sensory afferents innervate the gastrointestinal tract and inform the brain of the quantity and quality of food being consumed. Similar to other neural systems, the vagus nerve contains heterogeneous neuronal populations that have discrete connections and perform specialized functions. However, most of what is known about the vagus and behavior comes from non-specific surgical and chemical ablation studies that do not provide mechanistic insight about distinct neuronal populations. Hence, little is known regarding primary sensory signals directly related to satiation, and research in this area is imperative for understanding the central organization and regulation of energy homeostasis. Studies in this proposal will utilize state-of-the-art wireless technology and viral/transgenic techniques to manipulate genetically defined vagal afferent neurons innervating the stomach. We will examine the relative roles of distinct gastric vagal afferent neurons in the control of food intake, which could potentially lead to the identification of novel therapeutic targets for the safe and effective treatment of obesity. 1

NIH Research Projects · FY 2026 · 2020-08

PROJECT SUMMARY Each human nucleus contains two meters of DNA that is packaged into an organelle a mere ~10 µm diameter. Despite this dramatic difference in scale, the three-dimensional organization of the genome is non-random and plays a critical functional role in health and disease. My research group is focused on the building robust and scalable methods to study the organization of chromosomes in 3D space, the interactions they participate in with at the inter- and intra-chromosomal level, and the associated RNAs and proteins that occupy functionally relevant sites. The motivation for this work is to better understand the mechanisms by the organization and composition of genomic intervals relevant for health and disease faithfully conduct the essential DNA transactions of transcription, replication, and repair. Our previous studies focused on the development of multiplexed fluorescent in situ hybridization (FISH) methods to map chromosome structure in individual cells and the creation of biochemical screening methods to identify the molecular factors present at the anchor sites for 3D chromosome loops. To this end, we have introduced new multiplexed FISH technologies and adapted these to facilitate proximity labeling and affinity purification of factors from target genomic intervals and chromatin associated RNAs. We now propose to build on these efforts by further developing FISH technologies to support a broader range of questions relevant to chromosome biology and making efforts to speed the adoption of advanced FISH technologies, by extending our proximity labeling approach to more challenging genomic targets important for the regulation of gene expression, and by investigating the position and composition of highly repetitive DNA sequences, which we hypothesize play an integral role in maintaining the structure and stability of the genome. Collectively, the studies proposed here will produce enabling new genomic technologies, produce rich datasets that detail the molecular composition of functionally important genomic loci, and shed light on the function of disease-relevant repetitive sequences within the nucleus.

NIH Research Projects · FY 2024 · 2020-07

ABSTRACT Kidney diseases are an expanding public health problem, currently affecting 37 million people and are the 9th leading cause of death in the US, while disproportionately accounting for ~27% of Medicare expenditures. Unfortunately, the number of randomized clinical trials has been fewer than all other specialties of internal medicine with very low success rates, likely due to the structural and functional complexity of the kidney. The multicellular architecture and unusual triad of physiological processes characterized by glomerular filtration, tubular secretion, and tubular reabsorption have limited the ability of animal models to recapitulate the diversity of etiologies, mechanisms, and heterogenous manifestations of most human kidney diseases. Additionally, until recently there has been a lack of in vitro models that recapitulate critical aspects of kidney physiology, mimic the unique complexities of specific nephron segments, or assess reparative mechanisms in response to injury. In response to this critical unmet need, our group has pioneered the development of `human kidney on a chip' microphysiological systems (MPS). Our integrated approach for in vitro disease modeling includes differentiating human kidney cells and organoids from diseased patient-derived inducible pluripotent stem cells (iPSCs), CRISPR gene editing, single cell transcriptional profiling and engineered MPS platforms for both living human kidney vascular networks and tubular units. This approach has already led us to achieve new mechanistic insights into the pathogenesis of autosomal dominant polycystic kidney disease (PKD, the leading monogenetic cause of kidney failure) and potential new therapeutic pathways. In parallel, significant efforts led by us are underway in the Nephrotic Syndrome Study Network (known as NEPTUNE) and the Kidney Precision Medicine Project, NIH funded Consortia designed to address the functional heterogeneity of kidney disease by rigorous molecular, histologic and phenotypic characterization of kidney diseases. The NCATS Rare Disease Clinical Network NEPTUNE is testing the precision medicine concept by matching individual molecular profiles from patients to targeted therapy trials. We now propose to leverage these field-leading tools to inform clinical trial design and planning, accounting for human genetic and clinical response heterogeneity for PKD and Focal Segmental Glomerulosclerosis (FSGS), the form of nephrotic syndrome with the most severe patient consequences. Based on our data, we hypothesize that kidney-on-a-chip MPS will manifest patient-specific phenotypic responses in vitro commensurate with clinical trial outcomes in vivo, establishing a robust molecular and cellular basis for kidney precision medicine approaches. We have established a multidisciplinary investigative team with all the field-leading expertise needed to address all technical and experimental challenges.

NIH Research Projects · FY 2024 · 2020-07

Although preventing infection by Mycobacterium tuberculosis (Mtb) is paramount for controlling the tuberculosis (TB) epidemic, transmission in endemic settings is poorly understood. Across communities in high burden settings, there is variability in TB prevalence in the form of spatial clusters or “environmental hotspots”. Heterogeneity is also present in the infectiousness of patients with TB, leading to the concept of “individual superspreaders,” who are responsible for the majority of Mtb transmission events. The overall objective of this project is to develop novel interventions to interrupt Mtb transmission by discovering the primary drivers at the individual and community levels through evaluations of hotspots and superspreaders. We previously developed a novel Mtb cough aerosol sampling system (CASS) and a digital cough signature assessment platform and discovered immunogenetic factors associated with resistance and susceptibility to Mtb infection and TB disease. In 2015, the Kenya National TB Program, conducted a nationwide population-based TB survey, that found a high burden of TB with wide variation between surveyed communities. We now propose to investigate TB prevalence within 10 previously surveyed Nairobi neighborhoods (with varying TB burdens), develop novel tools for identifying superspreaders including aerobiology and immunogenetic profiling, and investigate biomarkers of recent transmission among household contacts (HHCs). We hypothesize that highly infectious individuals have unique digital cough and immunologic signatures, and that their HHCs have a higher frequency of persistently positive interferon-gamma release assays (IGRAs), specific transcriptional profiles, and higher rate of progression to TB disease. We hypothesize that neighborhoods with stably high TB prevalence rates are associated with demographics skewed to young men, high in-migration, decreased healthcare access, and poorly ventilated community spaces. By using innovative methods, we will gain insights into temporal trends of TB prevalence in small areas, identify factors associated with high prevalence, and develop tools to identify individuals responsible for a disproportionate amount of TB transmission. Knowledge from this study will enable targeting of resources to improve TB control.