Emory University

NIH Research Projects · FY 2025 · 2025-06

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. This T32 proposal is for the support of an interdisciplinary, pre-doctoral training program that is an integral part of the Center for Advancement of Diagnostics by Joining User-centered and Scalable Technologies (ADJUST Center) at Emory University, Georgia Tech, and Children’s Healthcare of Atlanta. The goal of this innovative graduate program is to create the next generation of inventors of point-of-care/home diagnostic technologies. Moreover, our unique training program will not only teach PhD students about diagnostic technology development but also validation, translation, and commercialization thereof, and importantly, enable our trainees to ensure that the technologies they develop serve those who need them the most. Our trainees will be recruited from an applicant pool of >80 eligible PhD students from the Joint Department of Biomedical Engineering (BME) at Georgia Tech and Emory, the interdisciplinary Bioengineering (BioE) Graduate Program at Georgia Tech, and the Department of Chemistry at Emory. BioE graduate students come from various Departments, including Electrical and Computer Engineering, Chemical and Biomolecular Engineering, and Mechanical Engineering at Georgia Tech. Our unique program will train our students on all aspects of development, assessment, and manufacturing of diagnostics, ranging from mid/high complexity clinical laboratory, point-of-care, and home-based/OTC tests and will incorporate the regulatory, verification and validation, user-centered design/human factors, and commercialization aspects thereof, but just as importantly, we will teach our trainees how to apply those technical principles under the auspices of achieving improved access to diagnostics. To that end, trainees will: 1) take foundational courses in biosensors, microfluidics/microfabrication, wearables design, AI/ML data science, medical device development, innovation/commercialization at Georgia Tech, 2) take healthcare-related courses at Emory’s Robert W. Woodruff Health Sciences Center, 3) complete “internal internships” within our diagnostics centers’ infrastructures including but not limited to, the clinical laboratories of Emory Healthcare and Children’s Healthcare of Atlanta and our center’s rapid microdevice prototyping facility, 4) complete official internships with our Centers’ medical device industry partners, 5) conduct clinical needs assessments in various communities in metropolitan Atlanta and rural Georgia, and 6) engage in unique programs integral to our diagnostics centers including “technology clinics” that assess/validate/implement new diagnostics and our “Diagnostic and Disease Discovery” clinic, a state-of-the-art, specialty referral clinic dedicated to evaluating patients with longstanding undiagnosed symptoms who might benefit from new diagnostic or biomarker technologies.

NIH Research Projects · FY 2025 · 2025-06

The goal of this project is to extend the reach and efficacy of adeno-associated virus (AAV)-based gene therapy by enlisting endogenously produced extracellular vesicles (EVs) to transport engineered transgene mRNA or protein products among cells and across tissues. The motivation for this project is that many inherited disorders are refractory to current AAV-based gene therapy for 1 of 2 reasons. First, the fraction of cells transduced by AAV when administered systemically at a well-tolerated dose is limited and for some disorders may be insufficient to rescue phenotype. Second, even if initial transduction efficiency is high, because AAV is non-replicating and predominantly non-integrating, viral genomes tend to be lost quickly from cells in high mitotic tissues by simple dilution due to repeated cell divisions as the child grows. Of note, booster shots are not a good option because of neutralizing antibodies elicited by the initial dose. We propose a conceptually simple solution to these problems: targeting intracellular transgene product – either as protein or as mRNA – to endogenous EVs so that cells containing viral DNA will be able to share their expressed transgene product locally with neighboring cells and systemically via transport through the bloodstream, including across the blood- brain barrier. Specifically, we hypothesize that EV-facilitated transfer of transgene-encoded protein and/or mRNA will increase the percentage of transgene product-positive cells in key tissues and will allow cells in low mitotic tissues that retain viral DNA to share their expressed transgene product with cells in other tissues. Here, we propose to test this hypothesis using an existing rat model of classic galactosemia administered scAAV9 virus encoding human galactose-1-P uridylyltransferase (GALT) either untagged or carrying 1 of 3 different EV- targeting tags. The results of the proposed 2-year study will inform the direction of future research and set the stage for application of this approach to gene therapy options for a large number of genetic disorders for which there is currently no effective treatment. 1

NIH Research Projects · FY 2025 · 2025-06

Prenatal psychosocial stress increases risk for a range of adverse maternal, perinatal and child neurodevelopmental outcomes. However, nationally representative samples with prospective data on maternal report of prenatal stress and verified perinatal and child health outcomes are lacking, as are investigations that consider risk and protective pathways that reflect solution-oriented research. The ECHO Cohort is ideal for advancing solution-oriented science around maternal psychosocial stress exposures, perinatal risk pathways, and child health outcomes as the protocol includes measures of prenatal stress, social support and cohesion, prenatal health behaviors (diet, physical activity, sleep, substance use), pregnancy complications and birth outcomes, and child health outcomes for a large national sample. Geocoding efforts also enable the consideration of neighborhood level measures of risk, allowing for multi-level, multi-domain analyses focused on child health risks. We seek to extend follow-up of our Atlanta ECHO Cohort participants under the ECHO Protocol and to participate in solution-oriented team science. Enrolled children are now 4 to 10 years old and will be followed with annual visits consistent with the ECHO Protocol, with a focus on child neurodevelopment as a specialized outcome area. The unique contributions of our Cohort Study site include our capacity to follow hard-to-reach families; our geographic location in a major metropolitan area in the US Southeast that experiences unique regional and area-level stressors; and our multi-disciplinary team of psychosocial, clinical, and omics investigators with expertise in (personalized) exposure assessment, maternal stress, perinatal and birth outcomes, and child neurodevelopment. Given our team’s expertise, and preliminary data that supports an association between stress, omics pathways, and neurodevelopment, we plan to lead test novel hypotheses about how prenatal stress leads to perturbations in the metabolome, epigenome and microbiome, and how these biological perturbations, in turn, predict child neurodevelopment. We will also test the role of prenatal stress experiences in children’s health outcomes in the ECHO cohort, as well as modifiable mediators (maternal prenatal sleep quality and postpartum depression) and moderators (social support) which will inform potential intervention strategies designed to improve children’s health outcomes.

NIH Research Projects · FY 2026 · 2025-06

Pulmonary hypertension (PH), an increasingly common clinical disorder, causes significant morbidity and mortality. PH is caused by increased pulmonary vascular cell proliferation resulting in pathological remodeling of the pulmonary vascular wall and increased arterial stiffness. Altered mitochondrial and metabolic function drive pulmonary vascular remodeling. This proposal focuses on the role of altered mitochondrial function in patients with PH secondary to lung disease (Group 3 PH). Because current PH therapies are ineffective in Group 3 PH patients, this proposal seeks to identify novel targets and therapies that can reduce mitochondrial and metabolic dysregulation in Group 3 PH. The proposed studies focus on the role of a novel outer mitochondrial membrane protein, mitoNEET (gene cisd1). MitoNEET regulates mitochondrial iron metabolism, bioenergetics, and cellular metabolism through binding and transfer of Fe-S clusters. Preliminary data reveal that mitoNEET expression is increased in pulmonary artery smooth muscle cells (PASMC) and platelets from Group 3 PH patients. Our data show that increases in PASMC mitoNEET are sufficient to induce PASMC proliferation and alterations in mitochondrial function in vitro and cause PH in mice in vivo. Preliminary data further show that selective mitoNEET ligands reverse PASMC derangements in vitro and attenuate PH in vivo. We hypothesize that mitoNEET-mediated mitochondrial iron loading induces mitochondrial dysregulation that promotes PASMC proliferation and that mitoNEET ligands stabilize Fe-S clusters in mitoNEET to reduce mitochondrial iron, metabolic dysregulation, and PASMC proliferation in Group 3 PH. Aim 1 will define how mitoNEET regulates PASMC mitochondrial function and proliferation in vitro using human and rat Group 3 PH PASMC, mitoNEET gain- and loss-of-function approaches, and mitoNEET ligands. These studies will provide new insights into regulation of PASMC proliferation by mitoNEET. Aim 2 will examine alterations in mitoNEET expression and function during PH pathogenesis in vivo using a rat model of Group 3 PH. Dose- and time- ranging studies of mitoNEET ligands will be examined for ability to modulate PH and vascular remodeling in vivo and to reverse PASMC derangements. Aim 3 will test mitoNEET and bioenergetic function in platelets and PASMC from Group 3 PH patients. Platelet mitochondrial function will be assessed before and after ex vivo treatment with mitoNEET ligands. This novel approach could enable further sub-phenotyping / characterization of bioenergetic dysregulation in Group 3 PH patients and permit identification of patients uniquely susceptible to mitoNEET ligands in future clinical trials. The results will provide novel mechanistic understating of mitoNEET in PASMC biology and Group 3 PH pathogenesis and permit new and potentially disease-modifying strategies for targeting aberrant metabolism in PH to reverse disease progression. The study team will leverage their expertise in pulmonary vascular, mitoNEET, bioenergetics and mitochondrial biology, drug development, and clinical trials in PH to ensure the success of the proposed studies.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY Since the birth of the first child from in vitro fertilization (IVF) in the US in 1981, more than ten million babies have been born from this technology, including more than 1.37 million babies annually worldwide. In 2018, IVF births accounted for 2.24% of all US births. Consistent research findings indicate that IVF-conceived pregnancies and IVF-conceived children are at greater risk for a spectrum of adverse perinatal outcomes. A persistent, unresolved issue remains what proportion of the adverse perinatal and child health outcomes after IVF is due to parental factors and what proportion is due to the IVF procedures. In this grant, we are using an early life-course approach to evaluate the growth and health of children born from IVF, from conception through young adulthood. The two primary aims of this proposed renewal are (1) comprehensively evaluate the role of IVF conception on birth defects, cancer, and the co-occurrence of these conditions through large-scale registry linkages and analysis of the neonatal methylome and (2) to evaluate the effect of IVF treatment parameters, specifically the use of ICSI, number of embryos transferred (embryonic and/or fetal loss as plurality at conception versus at birth), oocyte source/state-embryo state combinations, on fetal growth (size at birth) and health (birth defects, cancer, acute and chronic illness, and premature mortality), accounting for parental, socioeconomic, and environmental factors. This proposed study will include a total of more than two million children, including more than 220,000 IVFconceived children born 2004-2022, 43,000 naturally-conceived siblings, and 1.75 million naturally-conceived control children, with 27 million person-years of follow-up, averaging 13.5 years (ranging from birth to age 23), and will provide a comprehensive contemporary picture of child and young adult health after conception by in vitro fertilization.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT One million children develop tuberculosis (TB) each year and TB is the leading cause of death among children with HIV. Novel strategies to diagnose and prevent pediatric TB are urgently needed, but progress has been hindered by poor sensitivity of current microbiologic and immunologic diagnostic tools, as well as an unclear understanding of immune correlates of TB protection. We have led clinical and translational research studies to elucidate humoral immune responses to TB in children and adults and have optimized a multiparameter high- throughput assay to characterize detailed antigen-specific antibody Fc features that guide both innate and adaptive TB immune responses. We propose to leverage specimen repositories from well-characterized clinical pediatric cohorts from Kenya, South Africa, India, and a multi-center IMPAACT study to define antibody Fc features associated with protection from Mycobacterium tuberculosis (Mtb) infection among infants with and without maternal HIV exposure (Aim 1) and identify biomarkers for active TB and Mtb infection as compared to Mtb-uninfected healthy controls (Aim 2). We hypothesize that an IgM-based signature detected in early infancy will be associated with protection from Mtb infection, and that we will discover unique Mtb-specific antibody Fc glycan profiles among children with TB disease. The results of this study will have significant public health implications for children affected by HIV in TB-endemic settings, through advancing our knowledge of Mtb- specific humoral immunity that can be harnessed for innovative public health strategies, including new maternal and infant vaccines, monoclonal antibody development, and point-of-care (POC) testing for Mtb infection and TB disease.

NIH Research Projects · FY 2026 · 2025-05

Project Summary. Dysregulation of poly(ADP-ribose) polymerase-1 (PARP1) has been implicated in various neurological disorders and neurodegenerative diseases, including Alzheimer’s disease (AD) and related dementias, extending beyond its well-known application in oncology. PARP1 not only co-localizes with tau and Aβ in the brains of AD patients, but also play a pivotal role in the microglia activation and TNFα release. Therefore pharmacological modulation of PARP1 represents an attractive therapeutic approach for AD treatment. Positron emission tomography (PET) is capable of quantifying biochemical processes in vivo, and a suitable PARP1 ligand would substantially improve our understanding of PARP1-mediated cell death signaling pathway under different CNS disorders, otherwise inaccessible by ex vivo (destructive) analysis. Quantification of PARP1 in the living brain by PET would provide the assessment of distribution, target engagement and pharmacodynamic response of novel PARP1-targeted neurotherapeutics. To date, no successful examples have been demonstrated to image PARP1 in the human brain, representing a significant deficiency of our ability to study this target in vivo. Therefore, we propose to develop a novel PARP1-selective brain penetrant PET ligand that can fill this void, as the first translational imaging tool. We are among the first groups to develop PARP1-specific ligands for non-oncology PET imaging applications, including the first PARP1-selective ligand [18F]PA-823. However, this ligand was discontinued due to low-to-moderate brain penetration in rodents. In our 2nd generation, we identified a lead molecule, PA-917, which showed high binding affinity and excellent selectivity. An 18F-isotopologue of PA-917 was synthesized and preliminary PET imaging studies confirmed that we have overcome the two major obstacles for PARP1-specific ligand development by achieving: 1) substantially-improved brain penetration (≥1 SUV brain uptake) and 2) high target specificity. Though PA-917 is a promising lead molecule for the development of new PARP1-targeted PET ligands, we are not clear if it is satisfactory for PET kinetics and quantification in cross-species study for clinical translation. Further optimization for balanced binding specificity and proper brain kinetics are sought for cross-species imaging studies to achieve optimal PARP1 quantification for human use. On the basis that PA-917 serves a validated lead for medicinal chemistry optimization, as specific goals, we will design and prepare a focused library of PARP1-specific modulators with balanced binding affinity and brain kinetics, as well as amenable for radiolabeling. We will evaluate their ability to quantify PARP1 expression and changes during drug challenge in rodents and nonhuman primates, as well as autoradiography and biological validation in postmortem human brain tissues. The impact of this work is not only to develop the first successful brain penetrant PARP1-specific PET ligand for the observational/mechanistic study of neurodegenerative disease-related biological process, but also ultimately, via PET imaging validation in higher species, to advance this ligand for potential clinical translation and monitor target response and safety margins of novel AD neurotherapeutics.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY/ABSTRACT: The Emory National Primate Research Center Genomics Core (GenCore) is requesting support to acquire an Illumina NovaSeq X Plus Sequencing System. Since 2011, the EPC Genomics Core has served a diverse collection of EPC and Emory University investigators; assisted in planning, preparing, and executing projects having dynamic deep-sequencing needs. From the characterization of genome-wide DNA methylation profiles in the context of neurodegeneration, to identifying differentially expressed genes following various vaccination regimens, the GenCore has worked closely with investigators to successfully meet the demands of their research programs. Over the past several years, massively parallel deep sequencing has made significant contributions to biomedical research– particularly due to increased affordability and practicality over more conventional approaches to gene sequence and expression characterization. Our growing user base, and the increased sophistication of our users' needs, requires the GenCore to replace our existing Illumina NovaSeq 6000 with the NovaSeq X Plus, which will decrease turn-around time, decrease sequencing costs, and increase the volume of samples that can be sequenced simultaneously. The NovaSeq X Plus will bring genome-center capabilities to the EPC GenCore– investigators will have in-house access to genome-scale deep sequencing services with corresponding informatics support. Our users, minor and major, correspond to investigators leading their field, and supporting their evolving sequencing needs by acquiring an Illumina NovaSeq X Plus will undoubtedly benefit the larger scientific community.

NIH Research Projects · FY 2025 · 2025-05

PROJECT SUMMARY The combination of human induced pluripotent stem cells (hiPSC) technology with 3D organoid culture methods provides the opportunity to study human biology at an unprecedented level. However, despite the exciting potential for these 3D in vitro platforms organoids to model human disease, significant challenges have halted their broad utility. Specifically, the high variability, lack of standardization across protocols, and cost of the reagents for long-term culture have made it difficult for new labs to implement these new technologies, as well as compare and integrate results across labs. To address these challenges, we previously established the Organoid Hub at Emory University, a unique state-of-art initiative with the overarching goal of innovating, streamlining, and advancing the procedures for maintaining, differentiating, and utilizing hiPSCs and organoids through three main principles: standardization, scalability, and accessibility. This Organoid hub is staffed by highly trained personnel dedicated to optimizing and standardizing iPSC-derived organoid cultures. Here, we propose the acquisition of a Molecular Devices CellXpress.ai automated organoid cell culture system and its integration with the Organoid Hub. Automating organoid culture will significantly enhance the Organoid Hub's capabilities, enabling high-throughput and standardized production of various organoid types, including brain, gut, cardiac, and kidney organoids. This technology will reduce hands-on time, minimize human error, and ensure consistent and reproducible results across experiments. Furthermore, it will lower the financial and labor barriers for researchers, promoting wider adoption of advanced 3D culture methods. The integration of the CellXpress.ai system into the Organoid Hub will also foster collaboration and innovation within and beyond the Emory community. By offering a fee-for-use service, the Hub will provide researchers access to cutting-edge automated organoid culture, facilitating groundbreaking research in human cellular biology and disease modeling. This proposal highlights the critical need for this advanced system to enhance reproducibility and standardization, and expand the scientific reach of the Organoid Hub, ultimately advancing our understanding of human biology and disease.

NIH Research Projects · FY 2025 · 2025-05

Project Summary/Abstract The overarching goal of this proposal is to contribute to the fundamental understanding of the spatiotemporal transmission dynamics and control of vaccine-preventable childhood infections. The central scientific premise motivating this project is that integrative modeling approaches, effectively combining traditional mechanistic models and machine learning techniques, have the greatest potential to address the persisting methodological challenges in fully characterizing and accurately predicting the complex spatiotemporal transmission dynamics of childhood infections. We hypothesize that such integrative approaches can overcome the limitations in the inferential and predictive capabilities of existing models, precisely disentangling and predicting the influence of the underlying drivers of transmission dynamics. As such, they can be leveraged to define the most effective control strategies, including vaccination. Three specific aims are proposed. Aim 1: Develop and validate a highly interpretable and predictive modeling framework by deeply integrating graph neural networks with compartmental models. We deliberately develop a highly flexible and generalized graph neural network-based approach to incorporate the underlying network of infection transmission among interconnected populations (or a metapopulation) such as a collection of neighboring towns. We propose new inferential approaches leveraging Approximate Bayesian Computation to facilitate the integration. Aim 2: Develop a comprehensive software platform to facilitate implementations of our models developed in Aim 1, and to promote and catalyze the development and adoption of machine learning modeling research in the community of infectious disease modeling. Aim 3: Test our developed modeling framework using rotavirus and measles as examples and illustrate how our trained models can be used to guide efficient adaptive vaccination and control strategies for rotavirus and measles in the US. Rotavirus and measles are example types of vaccine-preventable diseases, endemic and re-emerging, respectively. Results of this project will also broadly lend valuable insights into future studies (e.g., designing effective vaccination programs) for other vaccine-preventable diseases (VPDs), such as RSV and norovirus which are expected to become VPDs soon.

NIH Research Projects · FY 2025 · 2025-05

Project Summary: Central to HIV-1 assembly, maturation, and entry are two structurally distinct protein lattices formed by the viral protein Gag. The immature lattice is formed by the uncleaved Gag protein. Maturation is brought about by retroviral protease cleavage of Gag, and subsequent assembly of the liberated CA domain into a mature lattice that encloses the genome and viral enzymes like RT and IN (the Capsid Core). Following fusion and release of a Capsid Core into the cell cytoplasm, a series of interactions with cellular proteins can result in “docking” to a nuclear pore. Premature disruption of the CA lattice can restrict infection. A structural understanding of these states and transitions is key to the design and optimization of antivirals that target capsid. The work proposed here will leverage single-particle cryo-Electron Microscopy and cryo-Electron Tomography and Subtomogram Averaging to interrogate the structural details of immature HIV-1 assembly, and the mechanistic properties of mature Capsid. This work will employ continued development and application of novel sample preparation for cryo-EM, and data processing approaches to determine the tertiary and quaternary structure of these assemblies. Samples biochemically assembled in vitro from purified proteins will be studied to gain a precise understanding of the molecular determinants of these processes. Virions from cells will be examined to understand how these molecular determinants act in the native virus. Cells will be used to investigate how these actions relate to the cellular environment. Time-resolved sample preparation will be applied to samples at specific initiation states of assembly, which may represent novel targets for inhibition. Specific Aim 1: DEVELOPMENT OF SINGLE PARTICEL CRYO-EM TO STUDY THE IMMATURE AND MATURE GAG AND CAPSID LATTICES. Assembly and budding of HIV-1 is a tightly regulated process involving protein-protein, -RNA, -membrane, and -cellular cofactor interactions. A more detailed understanding of the initiation of assembly and the transition to a budded virus may reveal targets to block the release of the virus. Application of cryo-EM to investigate these dynamic processes has provided, and will continue to provide, important insights to HIV-1 biology. My lab will work to be a leader in this area by developing new and novel approaches to sample preparation and structure determination as outlined in this aim. Specific Aim 2: STRUCTURAL, BIOCHEMICAL AND CELLULAR INTERROGTOIN OF LEN BINDING TO HIV- 1. The mature Capsid Core contains all the necessary viral components for establishing a new infection. Following release into the cytoplasm, Capsid interacts with (or hijacks) a series of cellular proteins to facilitate trafficking to, and docking with, a nuclear pore. Compounds that bind to these capsid-cellular co-factor binding sites are an attractive and successful antiviral strategy. We will study the molecular details and effects of Lenacapavir, the first-in-class capsid targeting compound, on immature assembly, capsid stability, and infectivity.

NIH Research Projects · FY 2026 · 2025-05

Project Summary This project will test the hypothesis that signaling through Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways is a key mechanism mediating the effects of inflammation on corticostriatal circuits related to motivation and motor activity as well as symptoms of anhedonia and psychomotor slowing in patients with major depression (MD). The role of JAK/STAT pathways will be examined using a FDA-approved JAK inhibitor, baricitinib, and an innovative biomarker-driven, brain-targeted approach. MD is a disabling disorder, with over 1/3 of MD patients failing to respond to conventional antidepressant therapies. Thus, the elucidation of novel pathways to pathology and related treatment targets is imperative. One pathophysiologic pathway thought to contribute to symptoms of depression and particularly anhedonia is inflammation. A significant proportion of patients with MD exhibit elevated biomarkers of inflammation in the peripheral blood and central nervous system (CNS), e.g., C-reactive protein (CRP) and inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor, which are in turn associated with antidepressant nonresponse. A rich literature demonstrates that inflammation affects dopamine (DA) in key brain circuits involving the striatum and prefrontal cortex that are critical for motivation and motor activity. Our group has established that MD patients with higher endogenous inflammation (as indexed by peripheral blood CRP and inflammatory cytokines) have low functional connectivity (FC) within DA-rich corticostriatal reward and motor circuits in association with anhedonia and psychomotor slowing, which is improved by drugs that increase DA. Interestingly, our published and preliminary data indicate that JAK/STAT signaling is a key transcriptional pathway that is reliably associated with inflammation’s effects on DA and motivation and motor circuits and related symptoms. Thus, small molecule JAK inhibitors like baricitinib may be an optimal strategy to target these immune pathways in MD, especially given known advantages of these agents over anti-cytokine antibodies including notably CNS penetrance. Indeed, baricitinib was found to reverse the inhibitory effects of IL-6 on DA neurons derived from human induced pluripotent stem cells through inhibition of JAK/STAT3 signaling, while also blocking inflammation and reducing depressive-like behavior in a laboratory animal model. These data are intriguing given evidence from laboratory animals that IL-6 has direct access to the DA-rich ventral striatum through breaches in the blood brain barrier in the context of stress. Of note, STAT3 was recently identified as a top upstream mediator of the molecular pathology of MD. Given the wealth of clinical and translational evidence regarding the role of JAK/STAT pathways in the pathophysiology of MD, this project will examine whether JAK inhibition with baricitinib can improve inflammation-related deficits in reward and motor circuits (Aim 1) in association with improved motivation and motor function (Aim 2) in MD patients enriched for high CRP, while exploring additional CNS and peripheral immune pathways (Aim 3) that may inform avenues for future work.

NIH Research Projects · FY 2025 · 2025-05

Intrinsic brain activity exhibits time-varying, non-localized states which are altered in psychiatric and neurological disorders. Intrinsic activity is dominated by a few whole-brain spatiotemporal patterns that occur repeatedly over time (quasiperiodic patterns or QPPs). In particular, research has linked QPPs to attention and focus, traits that are affected by disorders such as attention deficit/hyperactivity disorder (ADHD) and major depressive disorder (MDD). However, QPPs are whole-brain phenomena detected with functional magnetic resonance imaging (fMRI), which makes them difficult to interpret in terms of underlying neural activity. Hints of similar repeated spatiotemporal patterns have been observed in wide field optical imaging (WOI), which can image neural activity directly. We hypothesize that QPPs are surrogates for faster repeated patterns of neural activity detected with WOI, and that behaviorally-relevant brain states can be defined by the relative incidence of a few QPPs. The observation that large-scale dynamics of intrinsic brain activity are altered in psychiatric and neurological disorders suggests that interventions that reverse these alterations and restore normal whole-brain dynamics may prove therapeutic. We will therefore attempt to purposefully affect the relative expression of QPPs and underlying neural brain states as a demonstration that they can be manipulated in a predictable manner. Based on our prior work, we hypothesize that a multisensory flicker, a strong sensory stimulus, can alter the relative expression of QPPs. We will test these hypotheses using a state-of-the-art method for simultaneous WOI of fluorescent calcium indicators and whole-brain fMRI in unanesthetized mice. Specific aims are 1) Use simultaneous WOI and fMRI to determine whether the distribution of QPPs reflects underlying patterns of neural activity during behaviorally-relevant states and 2) Determine whether multisensory stimulation can alter the expression of WOI neural patterns and fMRI QPPs. Fluctuations in mood, focus, arousal levels, etc, are universal in healthy humans and often symptomatically affected in mental disorders. These attributes vary on relatively slow time scales and are likely to arise from systems-level brain states rather than localized changes in neural activity. We anticipate that the relative expression of a few spatiotemporal patterns will be sufficient to characterize natural changes in brain state over the course of a scan, in this case produced spontaneously by a shift from quiescence to activity or deliberately by the application of sensory stimulation. Because QPPs can be detected noninvasively in healthy humans, if they prove adequate as a surrogate for whole-brain configurations of neural activity, they will be valuable for characterizing brain states related to mental attributes and their alteration in disorders. They may also serve as guides for the design of modulatory approaches and markers of successful intervention.

NIH Research Projects · FY 2026 · 2025-05

Project abstract Heparin has long been used as an anti-coagulant in the management of coronary artery disease, deep vein thrombosis, pulmonary embolism, and in the prevention of thrombosis during cardiopulmonary bypass and extracorporeal membrane oxygenation (ECMO). A small fraction of patients on heparin (0.1% - 5.0%), will progress to having heparin-induced thrombocytopenia (HIT) which can lead to significant morbidity, particularly thrombosis, bleeding, and amputation, and in some cases death. The current gold standard assay to detect HIT is complex, requires C14-serotonin, and can only be performed in several reference laboratories in the US. Therefore, patients awaiting definitive diagnosis of HIT are on inferior anticoagulants with increased cost and bleeding risk. Hence there is a need for rapid and accurate detection of HIT. The central hypothesis of this project is that the mechanical forces generated by platelets provide a physical biomarker of platelet activity and may serve as a diagnostic marker of HIT. This hypothesis is supported by strong rigor of prior research as well as strong preliminary data. Platelets mechanically contract with significant force after their initial activation, contributing to the strength and stability of platelet aggregates as part of normal blood coagulation. Therefore, our approach is to develop and test a new molecular sensor that can detect the mechanical forces generated by platelets. To address this need, we have assembled an interdisciplinary team including Dr. Salaita who is a biophysical chemist, Dr. Roman Sniecinski a recognized expert in perioperative coagulation, and Dr. Cheryl Maier an expert on immune coagulation disorders including HIT. The team developed a new technology named MCATS (Mechano-assisted Cas12a Tension Sensor) that can detect the forces generated by human platelets using a conventional fluorescence plate-reader. Preliminary data shows that MCATS can detect platelet dysfunction including HIT using 5 uL volume samples of platelets in a 1 hr time window. The MCATS technology is the center-piece of this proposal and represents the focus of the approach. One goal of this application is to investigate the signaling link between HIT activation and platelet forces. Another goal is focused on optimizing the MCATS technology to enhance the signal obtained from human platelets. Finally, we will validate and benchmark MCATS against that of standard laboratory tests for HIT such as ELISA and SRA, which are the current standard of care for diagnosing HIT. If successful, then our planned work will lead to the development of mechanical assays to detect HIT in a rapid and broadly accessible manner.

NIH Research Projects · FY 2025 · 2025-05

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a global pandemic since first emerging in 2019. Since, Pfizer, Inc. (USA) developed Paxlovid™, an FDA-approved antiviral containing the protease inhibitor nirmatrelvir (NIR) that has seen moderate success in treating SARS-CoV-2 infections. The main protease (Mpro) of SARS-CoV-2 cleaves the viral polyproteins to release proteins essential for replication. NIR mimics the Mpro consensus sequence, thus covalently blocking Mpro activity and viral replication. However, resistance mutations to NIR have started to develop as its use increases globally. Understanding how such substitutions confer resistance and balance the impacts on fitness will enable the strategic design of the next generation of Mpro inhibitors. My preliminary data and published experiments have identified drug resistance mutations (DRMs) in Mpro that confer significant resistance to NIR including E166V. Further, I have demonstrated that the E166V substitution remains susceptible to GC376, a feline coronavirus protease inhibitor, and PF-00835231, developed during the SARS-CoV-1 epidemic. E166V also severely decreases viral fitness and requires compensatory mutations such as L50F to rescue fitness. Despite this fitness cost, E166V and L50F/E166V were both observed in patients treated with Paxlovid™ during the clinical trial, and recent case studies also identified the L50V/E166V combination. It is unclear how E166V decreases fitness and why mutations at Leu50 restore fitness. Structural work from other labs and my preliminary data indicate Mpro forms an active homodimer with the N-terminus from the opposite protomer interacting with Glu166. Loss of these interactions due to the E166V mutation are likely to disrupt Mpro dimerization and, consequently, activity. Of note, dimerization has not been studied in the context of drug resistance, and the role of Leu50 mutations in restoring fitness is poorly understood. Building upon my previous work, I will analyze E166V and L50V/E166V using virological, biophysical, and structural techniques. The goal of my project is to characterize mechanisms of NIR resistance, determine how DRMs impact Mpro activity and fitness, and investigate strategies for overcoming NIR resistance. I hypothesize that DRMs alter the intermolecular interactions in the active site resulting in decreased binding of inhibitors and Mpro dimer formation. Aim 1 will characterize the effect of the selected substitutions on Mpro resistance to NIR, GC376, PF-00835231, and a novel inhibitor shown to inhibit E166V Mpro (NIP-22c) using a virus-like particle (VLP) assay (Aim 1.1) and elucidate the mechanism of and strategies for overcoming NIR resistance using biochemical (Aim 1.2) and crystallographic studies (Aim 1.3). Aim 2 will investigate the effect of E166V and L50V on viral replication efficiency in cells (Aim 2.1) and on Mpro dimerization in vitro using biochemical methods (Aim 2.2). This project will provide valuable insights into mechanisms of drug resistance, impacts on viral fitness, and strategies for overcoming NIR-resistant SARS-CoV-2 Mpro informing the design of next-generation antivirals targeting Mpro and future development of pan-coronavirus antivirals. Completing this project will train me in techniques and skills essential for my future career as an independent scientist.

NIH Research Projects · FY 2026 · 2025-05

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by motor neuron degeneration and has been linked to mutations in the KIF5A gene, which encodes the motor protein KIF5A. The ALS-associated KIF5A variants result in exon 27 skipping (KIF5AΔE27), generating a novel C- terminal peptide. This study comprehensively investigates the molecular mechanisms underlying KIF5AΔE27's role in ALS pathogenesis through three specific aims. Aim 1 focuses on determining the gain of function in transgenic mice expressing KIF5AΔE27. Preliminary data demonstrate early neurite aggregates and mild motor deficits in these mice, suggesting age-dependent motor deficits and pathological abnormalities. We also aim to identify age- and disease-dependent molecular pathway alterations in the spinal cord. Aim 2 delves into cargo transport deficits and altered RNA metabolism caused by KIF5AΔE27 in induced pluripotent stem cell-derived motor neurons. Our proteomic analyses have revealed a range of proteins with altered binding to KIF5AΔE27, particularly those associated with mitochondria and RNA granules. Aim 2A focuses on identifying altered cargo transport, while Aim 2B explores mRNA metabolism defects by profiling mRNA transcripts and splicing patterns. Aim 3 investigates the molecular determinants and toxicity of KIF5AΔE27 aggregates. We aim to elucidate the role of active transport processes in KIF5AΔE27 aggregation in Aim 3A, and in Aim 3B, we will explore whether these aggregates contribute to neurotoxicity while investigating their regulation by altered interacting proteins. These multidisciplinary approaches offer a comprehensive examination of KIF5AΔE27 in ALS, potentially yielding critical insights into disease mechanisms and innovative therapeutic strategies.

NIH Research Projects · FY 2026 · 2025-04

SUMMARY - Overall Transmission of respiratory infection in the context of both seasonal epidemics and infrequent pandemics leads to widespread health and economic disruptions. While strategies developed to limit transmission could be very impactful, their evidence-based design is hampered by gaps in understanding of the fundamental biological and physical processes that underlie transmission. Our multidisciplinary team will address these gaps through a well- integrated program that will examine influenza A virus (IAV) in a controlled human infection model (CHIM). Through three program-wide Specific Aims we will pursue hypotheses related to both biological and physical drivers of transmission. Aim 1 will seek to define the dynamics of viral load, immunological responses and infectious aerosol production in humans infected with seasonal IAV. Owing to the well-defined time of infection and opportunity for frequent longitudinal sampling, the CHIM system is uniquely suited to obtain a high-resolution picture of infection dynamics. These features of our study design will be leveraged to document the dynamics within each infected individual of viral amplification, innate responses, adaptive responses, and infectious aerosol production. Emphasis will be placed on capturing heterogeneity both across individuals and across anatomical sites within an individual. Aim 2 will examine the role of aerosols in mediating IAV infection and onward transmission. While we expect to obtain insight into multiple potential modes of transmission, two aspects of our program will focus on the spread of infection through the air: a subset of participants will be exposed to aerosolized IAV and the respiratory aerosols generated by all participants will be evaluated. These aspects of our approach will reveal the infectious potential of virus-laden aerosols and the quantity and characteristics of such aerosols produced by infected human hosts. Aim 3 will investigate the complex relationships between host responses, viral replication, and expulsion of infectious virus into the environment. Through the collection of diverse samples and datasets from each study participant, and integration of the information obtained through statistical and mechanistic modeling, we will seek to define the drivers of specific infection outcomes, with primary emphasis on susceptibility to and potential for transmission through expulsion of infectious virus into the air. The experimental data, computational models and conceptual understanding attained through this research are expected to open new lines of inquiry and to directly enable an evidence-based transformation in public health strategies and biomedical interventions to control influenza.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Chromosome 22q11.2 deletion syndrome (22qDS) is a rare genetic disorder caused by microdeletions of 1-3 MB on chromosome 22 leading to haploinsufficiency of ~50 protein-coding genes and numerous non-coding genes. Genes in the 22q11.2 region encompass a variety of functions, including miRNA processing, mitochondrial metabolism, and protein trafficking which shape cellular function through distinct pathways. At the clinical level, 22qDS manifests in cardiovascular, immune, and nervous system defects. Strikingly, ~25 % of 22qDS patients develop schizophrenia (SCZ) with clinical symptoms mirroring idiopathic SCZ. In particular, while several lines of evidence show that inflammation and microglial hyperactivation contribute to SCZ development through overt synaptic pruning during adolescence and idiopathic SCZ patients display chronic peripheral low- grade inflammation and microglial activation, 22qDS patients also show signs of dysregulated immune homeostasis with elevated plasma pro-inflammatory cytokine levels, particularly in individuals with psychosis. Microglia are brain residing immune cells that play the central role in brain development and inflammation. However, how microglial functions are impaired in 22qDS and how their dysfunction contributes to 22qDS- associated neuropsychiatric disorders remain elusive. In this study, we will comprehensively assess the impact of 22qDS on microglial function by using 22qDS patient-specific induced pluripotent stem cell (iPSC) models. We have successfully generated iPSCs from a cohort of 22qDS patients with age- and sex-matched controls (HC) and differentiated them into microglia (iMG). Our preliminary data suggest that 22qDS iMG have dysregulated mitochondrial metabolism, reduced ribosome biogenesis and display an exacerbated response to inflammatory stimulation. We hypothesize that diminished expression of genes located on 22q11.2 leads to microglial hyperactivation, partially through the miRNA dysregulation, leading to impaired neurodevelopmental and neuronal dysfunction. We will comprehensively assess the impact of 22qDS on microglial function. We will determine the molecular and cellular impacts of 22q11.2 deletions on microglia functionality and mechanistically interrogate the function of individual 22qDS genes (Aim 1). We will also assess the role of miRNA dysregulation in 22qDS microglia (Aim 2). We will further determine the impacts of 22qDS iMG on neuronal development and function by using microglia-integrated cortical organoids (Aim 3). The proposed experiments will provide important insights into the role of neuroinflammation in 22qDS and lead to identification of potential therapeutic targets that likely can inform on idiopathic SCZ as well.

NIH Research Projects · FY 2026 · 2025-04

Abstract Optimization of voice therapy is a public health imperative, crucial for the social, emotional, occupational, and economic well-being of the 23 million Americans experiencing voice disorders at any time. Behavioral voice therapy, the primary treatment for over 80% of patients with hyperfunctional voice disorders, faces high attrition rates and lacks long-term effectiveness data. One problem with most voice therapy models is they often fail in generalizing treatment techniques to daily life, leading to prolonged treatment times and high dropout rates. Hierarchical voice therapy models, while ubiquitous, have not been empirically validated for long-term outcomes and may contribute to these challenges by delaying the application of therapeutic skills to real-life settings. In contrast, we introduced the first non-hierarchical delivery method, Conversation Training Therapy (CTT), which delivers treatment components in patient generated conversation from the onset, aiming to immediately integrate improved voice quality into everyday communication. The primary component of CTT is clear speech, which directs the speaker to use crisp, clear consonants and precise articulation. Patients focus on the sensation of articulating speech sounds while talking. Unlike other voice therapy approaches that focus on a set of phonemes (e.g. nasals in resonant voice), the stimuli in CTT are not phonemically restricted, enabling patients to practice at the conversational level from the outset and improve accuracy with practice over time. Understanding the impact of specific treatment components and their proposed mechanisms of action on treatment targets, particularly long-term outcomes, is critical to advancing clinical voice care and comparative effectiveness research. The non-hierarchical delivery method and use of clear speech distinguish CTT from other voice treatments. Thus, the effects of these components on voice treatment outcomes are priorities for investigation. Isolating the hierarchy by comparing a non-hierarchical delivery method to a reproducible hierarchical delivery with the same components, and investigation of the effects of clear speech on dysphonic speakers are necessary steps to determine how these components influence outcomes. If delivery method (hierarchical vs. non-hierarchical) and clear speech components are critical mechanisms of change, this knowledge will inform future voice therapy comparative effectiveness trials, contribute to the refinement of existing treatments, and aid in the development of novel treatment approaches. This proposal aims to rigorously test patient outcomes associated with non-hierarchical delivery of CTT compared to hierarchical delivery, and determine the relationship between clear speech, voice quality, and voice outcomes. The importance of this proposal lies in its potential for both practical and theoretical impact beyond any specific therapy approach for voice rehabilitation. This research further aligns with the NIDCD 2023-27 strategic plan, aiming to establish evidence-based practices that enhance health outcomes across voice therapies.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT This MPI R01 proposal seeks to advance our understanding of how a novel neoadjuvant therapy impacts the immune system in patients with solid tumors, using refractory melanoma as a model. This work stems from a series of discoveries by our team, led by basic T cell immunologist (Dr. Paulos) and translational scientist (Dr. Lesinski) in the context of a neoadjuvant, window of opportunity clinical trial (NCT03769155) in patients with resectable metastatic melanoma. These patients were treated with a novel agent called pepinemab—an antibody that blocks semaphorin 4D (SEMA4D) signaling—in combination with nivolumab and/or ipilimumab—as a ‘neoadjuvant’ presurgical therapy only being conducted at our institution (Winship/Emory). We found that most patients (7/8) given pepinemab/nivolumab/ipilimumab have not recurred after treatment nearly 4 years later (ongoing response). Our data reveals that type I conventional dendritic cells (cDC1) and M1 macrophages were increased in tumors of patients given potent αSEMA4D-based combination therapy, suggesting they play a key role as antigen presenting cells. Also, B cells and CD4+ T cells infiltrated tumors of patients treated with αSEMA4D/immune checkpoint blockade (ICB), versus those receiving standard of care nivolumab or surgery alone. Our teams’ results suggest SEMA4D blockade and ICB strengthens cDC1 and M1 macrophage induction in the tumor. This in turn enhances B-T cell interactions that drive adaptive immune responses and can safely elicit efficacy in melanoma, overcoming αPD-1 resistance. In Aim 1, using unique surgical tissue specimens from this trial, we will gain insight in how blocking SEMA4D impacts cDC1 and macrophages, positing their infiltration into the tumor is enhanced when combined with ICB, along with increased proximity of B cells to stem-like T cells in tumor specimens. These data will be compared to that from surgical samples of patients given neoadjuvant nivolumab/ipilimumab (from Co-I Dr. Wargo) to test our idea that αSEMA4D uniquely acts on myeloid cells to impart ICB treatment efficacy. In Aim 2, using genetically diverse murine neoadjuvant models, we will mirror our clinical trial and explore the role of distinct immune cell populations including B cells, T cells, cDC1 and macrophages in eliciting durable immunity via antibody depletion or using conditional knockout mice. Finally, our work reveals the inhibitory checkpoint LAG3 is induced on TIL of patients unresponsive to αSEMA4D/PD-1 therapy and is induced in mice by this therapy. In Aim 3, we will use relevant mouse models to determine if LAG3 blockade can serve as a more tolerable and efficacious alternative to αCTLA-4 that can be targeted to overcome αSEMA4D/PD-1 resistance, in turn mediating primary and protective immune responses against tumors. Overall, the proposed research will uncover the mechanism by which αSEMA4D-containing regimens sustain durable immunity against advanced melanoma and how this target can pair with LAG3 ICB for future clinical translation. This work will have impact across solid tumors as a future approach to rescue patients \nonresponsive to emerging systemic immunotherapies.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY At fertilization, histone modifying enzymes drive massive maternal epigenetic reprogramming that is vital for appropriate embryonic and postnatal development. In C. elegans and mouse, the lysine specific demethylase 1 (LSD1/KDM1A) acts as a repressor during this reprogramming by removing H3K4me1/2 and preventing the inappropriate inheritance of active transcriptional patterns to the progeny. In mouse, our lab has shown that LSD1 is required maternally to erase H3K4me1/2 at fertilization, enabling the switch from maternal to zygotic transcription. The maternal loss of LSD1 results in embryonic arrest at the 2-cell stage, stemming from a failure to repress maternal/oocyte genes, suggesting that maternal LSD1 epigenetic reprogramming is essential for embryogenesis to proceed. However, it is unclear how histone modifying enzymes are regulated during maternal reprogramming and whether defects in this reprogramming can lead to inherited disease. To address these questions and bypass the 2-cell arrest, we developed a hypomorphic allele of Lsd1, that predominantly affects the binding of LSD1 to its maternal partner CoREST. Data from this new model show that progeny from mothers with maternally hypomorphic Lsd1 exhibit increased perinatal lethality, as well as developmental delay, craniofacial abnormalities, and potential behavior defects. This suggests that the maternal function of LSD1 is CoREST dependent and that partial loss of LSD1 can lead to inherited phenotypes. de novo mutations in LSD1/KDM1A and in Kabuki Syndrome, which is caused by mutations in related histone methylation enzymes, lead to neurodevelopmental disorders, characterized by developmental delay, craniofacial defects and behavioral abnormalities. Intriguingly, the overlap in phenotypes of the human patients and our hypomorphic Lsd1 progeny raises the exciting possibility that a defect in maternal reprogramming may contribute to disease in these patients. To determine how partially compromised maternal LSD1 reprogramming leads to perinatal lethality, developmental delay, and craniofacial abnormalities, I will utilize our new mouse model in which LSD1 is partially compromised only maternally. Using this mouse model, I will determine if compromised maternal Lsd1 at fertilization leads directly to phenotypes in the progeny by examining changes in gene expression and inappropriate H3K4me2 retention. I will also investigate whether compromised maternal Lsd1 at fertilization leads indirectly to phenotypes by examining DNA methylation. Combining the analyses of these aims will give me a wholistic view of how compromised maternal LSD1 reprogramming gives rise to phenotypes observed in patients with mutations in histone modifying enzymes. Understanding this etiology is essential if we are to develop therapeutic treatments in the future.

NIH Research Projects · FY 2026 · 2025-04

Project Summary Steroid hormones mediate crucially important aspects of development, metabolism, inflammation, immune function, and behavior. Many effects of steroids are due to activation of their classical nuclear receptors to control transcription, but steroids can also control numerous cellular processes via rapid stimulation of G protein signaling. For most steroids, though, the receptors mediating these rapid non-genomic actions have not yet been identified. In preliminary studies, the adhesion G protein-coupled receptor GPR123 (also known as ADGRA1), which is highly expressed in the brain and known to regulate metabolism, was screened for potential stimulation by steroids and several hits were identified. We propose to build on these screens and pharmacologically characterize steroid binding by GPR123, including assessment of signaling bias by steroid ligands through both G protein- and arrestin-mediated signaling pathways. We also propose to elucidate the structural determinants of steroid activation of GPR123. These studies will provide new insights into the rapid non-genomic actions of steroid hormones and furthermore lay the groundwork for the development of GPR123- targeted therapeutics that might be useful in the treatment of metabolic disorders and other human diseases.

NIH Research Projects · FY 2026 · 2025-04

Project Summary/Abstract Carotid webs (CaW) are intraluminal projections into the internal carotid artery bulb that represent the underlying etiology in many ‘cryptogenic’ stroke cases in younger patients. Although CaW only cause minor stenosis, these lesions are associated with thromboembolism that is resistant to standard antiplatelet therapy, and hence have a high rate of recurrent stroke risk. Due to the lack of knowledge of the hemodynamic parameters underlying CaW-induced thromboembolism, there is a no consensus on how to manage patients with CaW. This is particularly important in incidentally discovered CaW. The goal of this project is to define the specific, quantitative values for hemodynamics parameters in the region where a clot forms. To achieve this goal, we will use a unique dataset where we have 30 subjects where imaging by computed tomography angiography (CTA) was done when a clot was present on the web and when clot was absent. Combining these CTA images with computational fluid dynamics (CFD) simulations we can determine specific, quantitative hemodynamic parameters at the exact location where a clot formed. Our preliminary studies using CFD simulations suggest that CaW produce low shear rates, which are associated with red clot formation. Prior work on CaW has been limited by sample size or over-simplification of CaW geometries. We propose to use a databases of CaW patients from 5 centers to obtain a robust sample size. The use of geometries based on CTA will make the simulations patient-specific. Successful completion of the project will significantly advance our understanding of the mechanism of thrombogenesis and stroke in these patients as well as provide hemodynamic metrics for a clinical classification system of CaW that provides patient-specific stroke risk.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Radiotherapy (RT) is used for locally advanced head and neck cancer either as primary/definitive therapy or in the adjuvant (post-surgical) setting to improve local control rates. However, RT may occasionally improve distant tumor control due to immune stimulation. Tumor regression outside the radiation field (distant effect) is termed the abscopal effect. RT mediates this, in part, by acting as in-situ vaccine, liberating tumor antigens, and generating an inflammatory milieu that enhances the CD8+ T-cell anti-tumor response. RT can synergize with immune checkpoint blockade (ICB) to enhance the abscopal effect, but this has rarely been seen in the clinical setting. Inhibitor of apoptosis proteins (IAPs) regulate the extrinsic and intrinsic apoptotic pathways, which can be induced by radiation. IAP antagonists can enhance these apoptotic pathways and are under active study as anti-cancer therapeutics. We have shown that the efficacy of RT + IAP antagonism can be further enhanced with the addition of anti-PD-1 ICB. Our prior preclinical work in head and neck cancer models showed that the when the IAP antagonist tolinapant was added to RT, local tumor control was enhanced, and T-cell stimulation was noted in the tumor draining lymph node (TDLN). This combination also enhanced several other components of the anti-tumor immune response, including antigen presentation, immunogenic cell death, and dendritic cell activation. Using biospecimens from our first-in-human clinical trial of tolinapant + RT (without platinum chemotherapy), we have seen dramatic increases in circulating, activated T cells in 50% of patients. These results suggest that tolinapant + RT can induce systemic anti-tumor immunity. RT stimulates a tumor-specific stem-like CD8+ T cell subset within the tumor and TDLN, which is critical for responses to ICB and abscopal responses. IAP antagonism can also promote T-cell costimulation, which is important for reinvigoration of stem-like CD8+ T cells. Thus, we hypothesize that tolinapant will enhance the RT-stimulated abscopal response by stimulating stem-like CD8+ T-cells. In Aim 1, we will use innovative, orthotopic abscopal murine models of oral cancer to determine whether tolinapant can enhance anti-tumor immune responses outside of the radiation field by increasing stem-like CD8+ T cells within the TLDN. In Aim 2, we will take a more in-depth look at blood and tissue biospecimens from our innovative clinical trial to determine if IAP antagonists combined with RT, in the absence of platinum chemotherapy, can induce systemic anti- tumor immunity. We will perform single-cell RNA sequencing on blood samples of patients who have had increased CD38+HLA-DR+ T cells in the blood and compare results with samples from patients who have not had these increases. Spatial transcriptomics will be used on baseline tissue samples to determine what tumor cell and immune cell features are associated with robust peripheral immune responses. With multiple large clinical trials of RT + IAP antagonism under development for HNSCC, these studies will be critical for advancing the field and improving our understanding of how IAP antagonists can be best utilized to enhance the effects of RT.

NIH Research Projects · FY 2025 · 2025-04

Abstract Prevention of HCV infection remains an important public health objective even with the recent adoption of highly effective antiviral therapies. Importantly, treatment with direct acting antivirals (DAAs) does not prevent reinfection in those who have successfully been treated. A vaccine to prevent HCV persistence is needed to stem an emerging epidemic of infection in adolescents and young adults who inject drugs that have limited access to screening and treatment. Only twenty five percent of acute HCV infections resolve spontaneously. Resolution does, however, sharply reduce the risk of persistent infection upon re-exposure to the virus. Memory CD4+ T helper and CD8+ cytotoxic T cell responses contribute to accelerated clearance of a second infection. However, the role of neutralizing antibodies during reinfection is less clear. Importantly, vaccines to generate an equivalent T cell response failed to thwart off the rate of persistence in naïve recipients. Here, we will test the central hypothesis that memory CD4+ T cells contribute significantly to HCV reinfection outcome by promoting expansion of HCV-specific B cells and production of broadly neutralizing antibodies that contribute to viral clearance. Unique features of this proposal are (i) the use of longitudinal samples from the Montreal cohort of high- risk people who inject drugs. Participants are monitored prior to, during, and after HCV infection and then subsequently followed again when they have been re-exposed to HCV a second time after resolving their primary infection. (ii) We have assembled a team of investigators in Cairo, Egypt who will assist us with recruiting and treating subjects undergoing DAA treatment. Egypt has the highest prevalence of HCV infection in the world. In 2014, Egypt's government has made DAA treatment affordable and 2.5 million subjects have started treatment. Understanding the immune responses in these two cohorts will provide us with valuable information to develop an efficacious vaccine regimen that would emulate the successful responses generated during a natural challenge with HCV. Three highly interactive Projects are proposed. Project 1 (N. Shoukry, PI) will compare the frequency, breadth, function, and phenotype of CD4+ T cells in HCV reinfections that either resolve or become persistent. Project 2 (A. Grakoui, PI) will use new state of the art technology to isolate and characterize antigen-specific B cells during HCV reinfection and post DAA treatment. Project 3 (R. Amara, PI) will develop vaccine modalities to induce both a CD4+ T helper response and a robust antigen-specific antibody response in blood and liver using DNA, modified vaccinia Ankara (MVA) and protein-based vaccines against HCV proteins in non-human primates.