Emory University

NIH Research Projects · FY 2025 · 2025-07

Systemic inflammation plays a central role in HIV pathogenesis- one that persists even when viral replication is controlled by anti-retroviral therapy. This persistent inflammatory state means it is important to better measure and understand inflammation in people living with HIV (PLWH), especially those who engage in pro- inflammatory behaviors such as stimulant use (SU). Unfortunately, current methods of collecting biospecimens to measure inflammation rely on invasive techniques (e.g., blood draws), highly variable sampling techniques with low concentrations of analytes (e.g., dry blood spots), and easily contaminated samples (e.g., saliva). Human sweat, however, provides a novel way to circumvent many of the current issues in inflammatory biomarker collection. Containing inflammatory biomarkers in similar proportions as human blood, sweat has been shown to be highly correlated with serum blood levels of immunological biomarkers in populations across the lifecourse. Sweat-derived inflammatory biomarkers collected via sweat patches, which are worn on the arm or abdomen for 24 hours, are non-invasive, fairly temperature-stable, and more convenient than many existing methods. However, no studies have so far trialled the use of sweat-derived biomarkers collected remotely, in PLWH, or in people who use stimulants. In this study, we aim to demonstrate the feasibility, acceptability, and validity of remote self-collection of sweat-derived biomarkers for the first time. To do this, we will recruit a sub- sample of N=100 participants enrolled in the American Remote Collection HIV Epidemiology Study (ARCHES; 5UG3DA058304), an ongoing, longitudinal cohort of N=1000 men living with HIV (n=700 of whom use stimulants). Building on our previous pilot of in-person, sweat-derived biomarker collection, we will examine the feasibility and acceptability of remotely collected, sweat-derived biomarkers compared to two common collection methods: dry blood spot (DBS) and blood (Aim 1). We will then test the validity of these specimens by comparing within-persons correlations of inflammatory cytokines collected via sweat, DBS, and blood (Aim 2). This cost-effective study is well-aligned with the NIDA Strategic Plan framework and stands to have a sustained and powerful impact on HIV science by trialing an inexpensive and accessible method of inflammatory biomarker data collection, developing methods to do so remotely, and assessing its validity compared to currently accepted methods. If funded, this ASTART application will provide the formative data for PI Metheny to apply for additional R-level funding designed to achieve his long-term goal of understanding how stress, violence, substance use, and ART adherence influence systemic inflammation. It will also provide other scientists with the initial data to begin extending sweat-derived biomarker collection to other research areas.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Glaucoma, a major cause of irreversible blindness, damages the retinal ganglion cell axons that carry visual signals to the brain. The exact mechanisms and primary sites of axonal injury remain debated, challenging clinicians to predict disease progression. Research suggests the lamina cribrosa of the optic nerve head (ONH), where axons exit the eye, as a key injury site, though theories also consider the neuro-retinal rim and peripapillary retina. This complexity underscores the need for deeper insights into glaucoma's pathophysiology. Our recent work has highlighted the critical role of biomechanics in glaucoma pathophysiology. Changes in intraocular pressure (IOP) cause distortion of the neural tissues within the ONH. The tissue-level distortions represent an insult to the axons with several components, including stretch, compression, bending, and torsion. These insults, when excessive, prolonged, or combined with other risk factors may lead to axonal injury. Given that each ONH is unique and subjected to a distinct biomechanical environment, the variability could explain why injury sites differ among individuals. However, these phenomena have never been directly observed in vivo in humans. Our proposal aims to leverage innovative tools from Engineering, AI, and advanced imaging (optical coherence tomography or OCT) to make these observations possible. By monitoring patients longitudinally, we intend to unveil which mechanical insults to axons are most likely to influence vision loss progression, identify where these insults occur, and explore variations across glaucoma subtypes and demographics. For this grant, we hypothesize that the deformations of individual ONH axon-bundles following an acute but gentle IOP insult reflects axonal health and could serve as diagnostic/prognostic biomarkers for glaucoma; further the axon-level deformations will help identify the most likely primary site of axonal injury across different glaucoma subgroups and demographics and point to the mechanisms underlying axonal damage and vision loss. The following three aims are proposed. Aim 1. To map the human ONH axonal paths in 3D from a single OCT scan of the ONH. Aim 2. To leverage AI to assess whether axonal deformations could serve as diagnostic and prognostic biomarkers for glaucoma. Aim 3. To leverage explainable AI to provide insights into axonal injury mechanisms by identifying the types of axon-bundle deformations and their locations that putatively influence visual field loss patterns and progression; and by assessing how age, glaucoma severity, and sub-types influence the patterns of these insults. Our project aims to apply Engineering and AI tools to precisely characterize, for the first time, insults at the axon- bundle level. This initiative not only holds the potential to enhance clinical practices for diagnosing and prognosing glaucoma but also deploys explainable AI to pinpoint potential injury sites across various glaucoma subtypes and demographics. Such advancements could significantly refine our understanding and management of glaucoma, ultimately paving the way for innovative therapeutic approaches tailored to individual patient needs.

NIH Research Projects · FY 2025 · 2025-07

Tuberculosis (TB) is a leading global killer among infectious diseases and the leading cause of death among people with HIV. Among the estimated 9 million TB survivors each year, up to half are left with impaired lung function and chronic respiratory symptoms. More than four times as many quality-adjusted life years (QALYs) are lost to post-TB lung disease (PTLD) as are lost to TB mortality. Although there has been growing recognition of PTLD in recent years, there are no known interventions to prevent or treat this devastating outcome. This knowledge gap is due in large part to a fundamental lack of data on mechanisms driving PTLD. Lung damage from TB is often viewed as an inevitable consequence in those who present “too late;” however, recent studies by our group and others suggest an alternative paradigm where much of PTLD can be prevented by giving host-directed therapies during TB treatment. Defining the biological pathways that drive PTLD and the populations at risk will provide a rare opportunity to address one of the most common global causes of chronic lung disease in people with and without HIV. In the Inflammation and Fibrosis in Pulmonary TB (INFIN-TB) study, we will test the hypothesis that pulmonary neutrophilic inflammation (Aim 1) and profibrotic activity (Aim 2) occurring early during TB treatment increase the risk of PTLD. We will enroll a prospective cohort of 250 people, 125 with HIV and 125 without HIV, with newly diagnosed, drug-susceptible pulmonary TB and will follow them for 12 months, from the time of TB diagnosis and treatment initiation until 6 months after completion of TB treatment. To ascertain relevant pathophysiology from the site of disease, we will collect airway samples at multiple time points in addition to comprehensive measurements of lung function and high-resolution CT scans. For Aim 1, we will determine the association between sputum levels of matrix metalloproteinase (MMP)-8, a matrix-degrading enzyme released by neutrophils, and the risk of PTLD, as measured by formal lung function testing. For Aim 2, we will determine the association between sputum levels of transforming growth factor (TGF)-β, a master regulator of fibrosis, and PTLD. Secondary analyses will determine whether HIV modifies the relationship between neutrophilic or profibrotic activity and PTLD, and will include additional biomarkers of neutrophil and profibrotic activity in both sputum and exhaled breath condensate, in addition to direct assessment of collagen deposition in the lungs using a novel collagen-binding PET probe. By focusing on the complementary pathways of neutrophil-mediated lung damage and profibrotic repair and remodeling, and then connecting activity in those biological pathways to clinically significant impairments in lung function among TB survivors, this study will be the most comprehensive study of PTLD to date. The knowledge gained from this study will directly inform future mechanistic and therapeutic studies with the goal of reducing rates and severity of PTLD and improving long-term outcomes for millions of TB survivors each year.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Histone proteins are the basic packaging unit of DNA. They comprise a hetero-octamer protein complex around which DNA is wound, and are compacted into chromatin. Histone proteins also play a critical role in regulating gene expression; however, missense mutations in genes encoding histone proteins, referred to as oncohistones, drive cancers. Although the majority of characterized oncohistones occur in histone H3, mutations in other histone genes can also drive cancer. Histone residues that support post-translational modification have also been shown to regulate DNA damage repair. Missense mutations to these residues could disrupt these pathways and potentially impact how cancer cells respond to DNA-damaging therapeutics, such as radiation. Through the analysis of publicly available tumor genomic data, I have identified five histone H2B variants that recur in patient tumors. Like the most extensively characterized H2B oncohistone, H2BE76K, these variants occur in various cancer types, including solid cancers and hematological malignancies such as Non-Hodgkin Lymphoma (NHL), making human mammary epithelial cells and Ramos cells, a transformed cell line modeling NHL, relevant models in which to characterize the functional impact of these mutations. I hypothesize that novel cancer-associated histone H2B variants can drive oncogenic phenotypes and alter cellular responses to DNA damage. To examine the oncogenic potential of cancer-associated H2B variants, I have engineered human cell lines expressing these H2B variants and have designed a research strategy implementing several interdisciplinary approaches that will characterize H2B variants as putative oncohistones, and elucidate their functional impact in the DNA damage response. The overarching goal is to test whether cancer-associated H2B variants may contribute to oncogenic processes and explore how these H2B variants may impact cellular DNA damage response, gene expression, and chromatin accessibility in response to DNA damage. These goals will be accomplished though two Specific Aims. In Aim 1, I will characterize H2B variants’ impact on cancer hallmarks in vitro by assessing cell proliferation, colony formation, migration and invasion comparing cells expressing wildtype H2B to cells expressing the variants I have defined. I will then examine the ability of H2B variants to drive tumorigenesis using in vivo murine xenografts. In Aim 2, I will examine how H2B variants influence cell viability after exposure to DNA damaging agents such as irradiation and characterize DNA damage repair. I will then examine gene expression and chromatin accessibility changes in cell lines that score in in vitro assays in the presence and absence of DNA damage. My exciting preliminary data reveal that several of these potential oncohistone mutants can drive pro-invasive phenotypes, providing proof of principle that the potential oncohistone mutations I have identified confer oncogenic phenotypes. Completion of these studies will test the model that additional H2B variants function as oncogenic drivers and provide mechanistic insight into their cancer-driving properties, which may unmask possible therapeutic targets for H2B oncohistone-driven cancers.

NIH Research Projects · FY 2025 · 2025-07

Neuronal transport is essential for neuronal function and is often impaired in neurodegenerative diseases. We and others have identified impairments in mRNA transport and local translation in neurological diseases, including spinal muscular atrophy (SMA), Fragile X syndrome (FXS), amyotrophic lateral sclerosis (ALS) and most recently, myotonic dystrophy (DM1). Our recent research identified that the kinesin-3 motor, KIF1C, associates with MBNL1, an RNA binding protein (RBP) known to depleted in DM1. We characterized domains involved in MBN-KIF1C interactions involved in mRNA localization in neurons. This discovery has motivated the present research to explore a potential new link of RNA localization to heterospastic paraplegia type 58 (SPG58), a neurological disease caused by the aberrant function of KIF1C, although mechanisms are poorly understood. Our major goal is to test the hypothesis that KIF1C interacts with many RBPs to transport mRNAs in neurons and that these mechanisms are altered in SPG58. A critical gap is lack of a human neuronal disease model to investigate possible impairments in mRNA localization. We have recently developed human iPSC CRISPR induced KIF1C knockout cells that will be differentiated into motor neurons. We propose to use this neuronal disease model, together with a variety of imaging and biochemical methods, to identify a set of RBPs and their mRNA targets that depend on KIF1C. In Aim 1, we will identify RBP and mRNA cargoes specific to KIF1C. Aim 1A will test the hypothesis that KIF1C binds mRNAs through different RBP adaptors and domains and that this binding is altered by KIF1C SPG58 pathogenic variants. We will use mass spectrometry to broadly identify pathology-related RBP targets. Aim 1B will identify mRNAs mislocalized in neuronal processes of human neurons lacking KIF1C. We will bioinformatically compare the datasets obtained in Aim 1A and Aim 1B to identify specific KIF1C mRNP cargoes dysregulated in SPG58. In Aim 2, we will test the hypothesis that altered dynamics of KIF1C transport affects mRNA localization in neurons and are involved in HSP. In Aim 2A, we will perform live imaging of KIF1C, its pathogenic variants and known RBPs and mRNAs cargos in human motor neurons to evaluate KIF1C transport dynamics. In Aim 2B, we will likewise validate mRNA targets identified in Aim 1. Additionally, we will overexpress selected pathogenic Kif1C variants and evaluate rescue phenotypes of mRNP cargo localization. The proposed studies will uncover disease specific targets of KIF1C in a new human disease model and delve into the mechanism of dysregulated mRNA transport in SPG58 and its effect on neuronal function. This research will provide broader mechanistic insight into several neurological diseases with implications for development of therapeutic strategies.

NIH Research Projects · FY 2025 · 2025-07

Fragile x syndrome (FXS), the most common form of inherited intellectual disability and monogenic cause of autism, is caused by loss of FMRP, the fragile x messenger ribonucleoprotein-1. FMRP is a mRNA binding protein known to regulate mRNA translation, including local protein synthesis important for synapse development and function. FMRP often acts to repress translation, which results in elevated expression of many proteins, including cytoskeletal proteins and components of the postsynaptic density. We have previously shown that FMRP represses translation of PSD-95 mRNA at synapses. Loss of FMRP in FXS models also results in dysregulated surface expression of membrane proteins, although underlying mechanisms remain unclear, as several do not appear to be direct FMRP targets. Previously we showed that FMRP depleted neurons have reduced surface expression and enhanced rate of AMPA receptor endocytosis, which is a likely driver of the enhanced mGluR-LTD. Other studies from our lab and others suggest that FMRP may play a broad role to regulate the dynamic trafficking of numerous membrane surface proteins, but underlying mechanisms are not known. A critical gap is lack of understanding of underlying mechanisms for if and how FMRP might directly and/or indirectly regulate membrane protein surface expression to control synapse development and function. We conducted an unbiased mass spectrometry analysis following surface biotinylation and streptavidin pulldown analysis of membrane labelled and associated proteins from control and FMRP depleted cortical neurons (DIV21) from mice. A surprising result was the increased expression of four subunits of the Clathrin-Associated Adaptor Complex Protein-2 (AP2) in FMRP depleted neurons compared to controls. As several data sets of FMRP binding targets have identified Ap2 subunit mRNAs, these results suggest that FMRP may repress translation, resulting in increased levels of AP2 subunits in FXS. We hypothesize that elevated nascent synthesis of AP2 subunits leads to enhanced endocytosis of several membrane proteins to alter synaptic development and function in FXS. Aim 1 will test the hypothesis that FMRP is a negative regulator of the synthesis of AP2 subunits and that elevated levels of AP2 subunits at synapses are responsible for the reduced surface expression of AMPA receptors in FXS. Aim 2 will test the hypothesis that elevated levels of AP2 subunits are also responsible for the reduced surface expression of other membrane proteins identified in our screen that are relevant to FXS and other neurodevelopmental brain disorders. Viral shRNA knockdown and pharmacological strategies will be used to reduce or inhibit the elevated levels of AP-2 subunits in FXS. As an alternative and innovative approach, we will use a new CRISPR-Cas9 TKI method to introduce SEP tags on endogenous GluA1/2 subunits. This research has implications for development of therapeutic strategies that target AP-2 to correct for altered membrane protein surface expression contributing to impairments in synaptic development in FXS.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT The CACNA1A gene encodes the α1A subunit of the P/Q-type voltage-gated calcium channel Caᵥ2.1 which facilitates presynaptic neurotransmitter release in neurons. Loss-of-function (LOF) and gain-of-function (GOF) variants in CACNA1A are both associated with developmental and treatment-resistant epileptic encephalopathy in patients. How do variants with divergent effects on Caᵥ2.1-mediated calcium influx converge to produce overlapping epileptic symptoms? Electrophysiology studies from mouse models have revealed LOF variants selectively disrupt inhibitory neuronal function while GOF variants selectively affect excitatory neurotransmission. We propose that cortical excitatory/inhibitory balance is disrupted in both cases, with LOF leading to weakened inhibition and GOF leading to excessive excitation. Selective disruption of inhibitory neurons in LOF and excitatory neurons in GOF have led to the hypothesis that cell types may functionally compensate by upregulating other calcium channels in LOF while downregulating calcium channels in GOF. However, molecular mechanisms underlying this compensation—including specific channels affected and the degree to which they successfully rescue calcium influx and normal neuronal activity—appear species-specific and cell type-specific. No published study has examined consequences of CACNA1A mutations on expression of genes beyond other calcium channels. To bridge these gaps and improve our understanding of how cortical hyperexcitability emerges in human CACNA1A-related epilepsy, I will characterize cell type-specific functional activity and gene expression in CACNA1A mutations with a 3D, human-derived in vitro model of the developing cortex. Utilizing forebrain assembloids containing cortical cell types from engineered LOF, GOF, and unedited isogenic control lines, this proposal compares function and gene expression of excitatory and inhibitory neurons between genotypes. Aim 1 utilizes calcium imaging to assess calcium transient rates and calcium influx of excitatory and inhibitory neurons alongside network-level measures of excitability. Aim 2 employs single-cell RNA sequencing to ask how gene expression of excitatory and inhibitory neurons is differentially affected in LOF and GOF relative to controls. Together, these experiments may identify expression changes in calcium channels and novel pathways in Aim 2 that explain cell type-specific functional abnormalities in LOF versus GOF revealed in Aim 1. The central hypothesis is that excessive activity and insufficient downregulation of calcium channels in excitatory neurons leads to hyperexcitable cortical networks in LOF while reduced activity and insufficient upregulation of calcium channels in inhibitory neurons leads to hyperexcitable cortical networks in GOF. Completion of these aims will provide novel insights into compensatory mechanisms, functional abnormalities, and etiology of epileptic phenotypes in CACNA1A for the first time in a human-derived model system—potentially informing novel therapeutic targets and variant classification criteria.

NIH Research Projects · FY 2025 · 2025-07

Current estimates suggest that 17.8 million women are infected with HIV-1 and that it is the leading cause of death in women of reproductive age. However, many studies of HIV-1 transmission and pathogenesis to date have focused on a single sex and are thus unable to directly compare disease course and outcomes between men and women. The initial experiments outlined in this grant utilize samples from a cohort of subtype C HIV-1 acutely infected Zambian men and women that allow for direct comparison of viral, transcriptional and immunologic characteristics between the sexes in individuals with a common genetic background. Paradoxically, even though CD4+ T cells from acutely infected women are significantly more highly activated (CD38+) than in men, women have consistently lower viral load than men, both in the earliest stages and chronic phase of infection. On the other hand, while women exhibit similarly effective levels of viral suppression on antiretroviral treatment (HART), they do bear a greater burden of non-AIDS comorbidities than men. These observations likely result from a complex interaction between a number of viral, hormonal and immunological factors, including the increased production of type I interferons (IFN) in women which can simultaneously cause immune activation as well as restriction of viral replication. In order to understand the molecular basis of these sex-based differences, we propose three Specific Aims: Aim 1: Assess sex-specific differences in immunological and transcriptional profiles of CD4+ T cells in early infection. Aim 2: In ART-suppressed women and men, define the landscape of immune cell activation, the nature of the latent reservoir, and its potential for reactivation in the presence and absence of sex hormones. Aim 3: Define the mechanism and cell source of sex hormone modulation of viral replication in vitro. The proposed experiments will fill a significant gap in our understanding of the mechanisms underlying observed differences in HIV-1 disease course and comorbidities between men and women, an important question at a time when sex differences are clearly defining distinct disease outcomes in various disease settings.

NIH Research Projects · FY 2026 · 2025-07

PROJECT ABSTRACT This application investigates the role of electrostatic forces in the interaction of membrane proteins and charged membrane lipids. Specifically, we will examine the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and the Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5- bisphosphate (PIP2). PIP2 is necessary to open ENaC. However, there is a problem with a simple model of ENaC and PIP2 association by lateral diffusion in the membrane. Given the abundance of PIP2 and ENaC and the diffusion constant of PIP2 in the apical membrane, the mean time it would take PIP2 to find an ENaC channel by random diffusion would be 6.3x10 2s or about once in 10 minutes. But, in cells expressing ENaC, the channel opens about every other second. We hypothesize that normal channel activity requires MLP-1 associated with the inner leaflet of the cell membrane. MLP-1’s strongly positively charged effector domain sequesters PIP2 electrostatically. We also hypothesize that MLP-1 and functional ENaC channels are associated with specific membrane domains known as lipid rafts. PIP2 within these domains stabilizes MLP-1 and ENaC while increasing opening (Po) of individual ENaC. ENaC in these domains can be destabilized by PIP2 degradation. To investigate MLP-1-ENaC-PIP2 interactions, we will (1) examine the unusual electrostatic interaction of ENaC and MLP-1 with PIP2 in the membrane. (2) Investigate ENaC stability by determining if ENaC is present in PIP2- rich lipid domains and determining if MLP-1 stabilizes ENaC in these lipid domains. (3) Determine if cytoskeletal interactions maintain MLP-1 and ENaC in the PIP2-rich lipid domains by using FRAP and STED FCS before and after latrunculin or cytochalasin E disruption of the cytoskeleton. 4) Examine the phenotype of renal principal cell-specific, MLP-1 knockout mice using single channel measurements in isolated, split-open collecting ducts.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Shigella is a leading cause of diarrhea and associated mortality among children under 5 years of age in low- and middle-income countries. Targeted antibiotic treatment of Shigella diarrhea may effectively improve outcomes across diverse settings, but current syndromic treatment guidelines and an inability to diagnose Shigella at the point-of-care result in underuse of active antibiotics for most cases. At the same time, overtreatment of viral and parasitic diarrhea episodes contributes to antimicrobial resistance. The goal of this project is to increase appropriate treatment of Shigella while limiting antimicrobial resistance by characterizing the contributions of inappropriately treated Shigella to poor diarrhea outcomes in a high mortality setting, evaluating novel biomarker-based strategies to identify Shigella, and designing population-level interventions to target antibiotic treatment to Shigella and decrease total antibiotic use. The hypothesis of this study is that appropriate identification and treatment of Shigella could prevent poor diarrhea outcomes and reduce antibiotic use for diarrhea among children in low-resource settings. We will enroll a prospective cohort of children hospitalized with diarrhea in Haydom, Tanzania and estimate the impact of Shigella on death and rehospitalization, diarrhea duration, and child growth. We will further evaluate whether those impacts are mitigated by treatment with antibiotics expected to be active for Shigella. Next, we will evaluate the performance of fecal inflammatory biomarkers to identify shigellosis cases in the cohort, incorporating the biomarkers into clinical prediction algorithms for Shigella diarrhea. We will further assess biomarker generalizability using previously collected samples and existing data from a cohort that is representative of children presenting to care with diarrhea in Dhaka, Bangladesh. Finally, we will model the impact of population-level interventions to target antibiotic treatment to Shigella on diarrhea outcomes and antibiotic use to determine which strategies would be most effective to prevent the poor outcomes of Shigella and reduce antibiotic use for diarrhea overall. The hypothetical interventions will target Shigella based on etiology, clinical syndrome, fecal inflammatory biomarkers, and/or time of the year, and will be pre-specified or optimized using machine learning. The research team includes leading epidemiologists in enteric infectious diseases with expertise in molecular diagnostics, large field studies in Haydom, and epidemiologic methods in causal inference and machine learning. This study will address key knowledge gaps around antibiotic treatment decisions for diarrhea in low-resource settings, specifically by characterizing outcomes that could be prevented by the targeted treatment of Shigella and by evaluating resource-efficient strategies to identify shigellosis at the point-of-care. This work will build an evidence-base for precision public health interventions that could reduce global diarrhea morbidity and mortality while limiting antimicrobial resistance.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY: Centrosomes are multifunctional organelles that organize the microtubule cytoskeleton required for mitotic spindle formation, enabling chromosome segregation and safeguarding genomic stability. During interphase, centrosomes also contribute to intracellular trafficking, cell polarity and migration, and ciliogenesis. Centrosomes undergo cell cycle-dependent oscillations in composition and organization to modulate their activity and diversity of functions. Prior to mitotic onset, centrosomes increase their microtubule-organizing activity through a process called centrosome maturation involving the recruitment of centrosomal proteins to the pericentriolar material. RNA is also recruited to centrosomes before mitotic entry. Our research contributes to an emerging model whereby local RNAs and on-site translational control influence centrosome activity. While RNA localization to centrosomes is conserved across metazoans, we still lack a complete understanding of which RNAs localize to centrosomes, how they get there, and their biological functions. The overarching goal of our research program is to investigate how posttranscriptional mechanisms instruct centrosome activity. The proposed work examines how local RNA directs centrosome function by determining the basis for cell cycle-dependent oscillations in RNA enrichment, conserved regulatory paradigms, and the unbiased discovery of novel regulators. To achieve these goals, we utilize genetically tractable models and multidisciplinary approaches. These studies will provide new insights into how centrosomes regulate their activities and may inform how centrosome dysfunction contributes to developmental disorders, neurodegeneration, and cancer.

NIH Research Projects · FY 2025 · 2025-07

Abstract As of 2023, nearly 40 million people worldwide are living with human immunodeficiency virus (HIV), of which over three-fourths have access to antiretroviral therapies (ARTs). Although access to therapies improves an individual’s expected lifespan and quality of life, resistance-associated mutations (RAMs) can develop in the virus in response to treatment. As a result, novel antivirals are needed to expand upon current options that are available. Recently, the HIV capsid has become the newest FDA-approved antiviral target with the release of Gilead Sciences’ lenacapavir (LEN), as the capsid is essential in both early and late stages of the viral replication cycle. Unfortunately, administration of LEN – a picomolar-potency drug – has led to the early emergence of RAMs that severely deplete the drug’s potency. To overcome the burden of LEN-resistant RAMs, novel capsid effectors (CEs) have been developed by our laboratory in collaboration with a team of medicinal chemists. Three of these CEs target the highly reported LEN-resistant mutant CAM66I (over 80,000-fold resistance) with higher efficacy than LEN. I hypothesize that the unique structures of these novel CEs allow these compounds to work against LEN-resistant CAs, including CAQ67H, CAN74D, and CAQ67H/N74D, distinctly from LEN. First, biochemical and biophysical assessments of each CE•CA interaction will be done using biolayer interferometry, thermal shift assays, CA Tube assembly assays, and native mass spectrometry (Aim 1). Additionally, the in cellulo potency and cytotoxicity of each CE during infection with HIV – with and without these CA mutations – will be determined using cell-based antiviral assays (Aim 2). Finally, insight into the structural differences owing to the CEs’ activities will be provided by X-ray crystallography studies to solve high-resolution crystal structures of each CE•CA complex (Aim 3). For all assays, comparisons will be made between WT and mutant CAs and between these CEs and LEN to understand mechanistic differences among each CE•CA combination. Together, these three aims will detail insight into methods of circumventing LEN resistance that can be used to optimize novel CEs with potential to be used as ARTs. As a result, these studies will allow us to continue combatting drug-resistant HIV through structure-based drug design, with the goal of suppressing the HIV epidemic.

NSF Awards · FY 2025 · 2025-07

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). Non-Technical Description: Halide perovskites semiconductors have recently attracted considerable attention for devices such as solar cells and light-emitting diodes. However, their poor stability results in devices with short lifetimes, precluding widespread use. One particular challenge is that movement of ions alters the electronic properties of perovskites. Thus, understanding ion transport is essential to solve the stability problem. The PI will develop novel structures from two-dimensional perovskites and quantify how ions diffuse and migrate under heat, light, and electrical bias. Understanding these fundamental questions will help researchers design perovskites with enhanced stability. This project also provides interdisciplinary training to undergraduate and graduate students. They will gain critical-thinking and problem-solving skills needed for careers in materials science, chemical and electrical engineering, and semiconductor technology. Technical Description: Accurately quantifying ion transport in halide perovskites is challenging. The current understanding is based primarily on conventional charge transport studies, where the contribution from electrons and co-movement of cations and anions is difficult to separate. Furthermore, ionic diffusion studies have been limited to polycrystalline thin films where grain boundaries and defects play critical roles. In this project, novel epitaxially grown 2D perovskite heterostructures, a microscopically well-defined diffusion couple, will be fabricated and used as a platform to probe ionic migration and diffusion. This project will establish a comprehensive family of lateral and vertical 2D halide perovskite heterostructures; quantify ionic diffusion and migration under different external stimuli; elucidate the ionic diffusion and migration mechanism; identify the most promising materials stabilization strategies; and finally demonstrate proof-of-concept devices with enhanced stability. The education objective of the project is to build an interdisciplinary materials science program at Purdue University that transcends traditional boundaries between science and engineering disciplines to educate our next-generation scientists and engineers. Specifically, new curriculum and new teaching methods that cut-across traditional boundaries for undergraduate and graduate education will be developed. Raising public awareness of materials science and semiconductors is another important objective of this project. Finally, an impactful global network of science and education through partnership with developing countries (e.g., Colombia and Indonesia) will be built. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2025-07

Myopia, or nearsightedness, is increasing at an alarming rate and reaching epidemic levels worldwide. Despite optical correction, myopia increases the risk of developing blinding diseases so there is a critical need to understand the underlying pathways that regulate its progression for effective prevention. Spending time indoors is associated with more myopia progression whereas spending time in bright, outdoor environments is protective. However, an effective explanation is still lacking for how these environments directly influence myopigenesis because different environments are comprised of many visual cues, all of which have associations with myopia. Importantly, all visual scenes are composed of bright and dark features and the visual system encodes these features using the ON and OFF retinal pathways, respectively, through which all visual information is processed. Broad disruption of ON pathways can influence myopia development but accumulating evidence suggests that OFF pathways, or the signaling balance between the two pathways, may also be important. Additionally, ambient light level is strongly associated with myopia progression and modulates ON and OFF visual signaling differently. Currently, it is unknown how ON and OFF pathway signaling is affected in myopic eyes or the role of ON/OFF signaling balance in myopia susceptibility across light levels. This proposal will address these gaps in knowledge to increase our understanding of the visual pathways that regulate myopia development. Preliminary data suggests that experimentally-induced myopia or exposure to myopigenic stimuli affects ON and OFF pathways very differently, potentially altering ON/OFF retinal balance. Therefore, the hypothesis that disrupted ON/OFF signaling balance initiates and drives myopia development will be tested. First, single-cell electrophysiological recordings from ON and OFF pathways will be performed in isolated retinas and compared between myopic and non-myopic eyes to determine which pathways are most impacted. Second, ON and OFF retinal pathway balance will be disrupted in different lighting conditions while monitoring myopia susceptibility. In Aim 1, quantification of ON and OFF signals from inner retinal neurons will be obtained in dim and bright ambient light conditions. In Aim 2, quantification of ON and OFF ganglion cell receptive field signals will be obtained to determine the extent of myopic changes in retinal output neurons. In Aim 3, ON/OFF retinal balance will be disrupted in multiple lighting conditions with exposure to ON or OFF visual stimuli or pharmacological agents. Myopia susceptibility and ocular biometry will be monitored throughout the disruption. The expected outcomes of this proposal will provide powerful insight into what visual pathways are implicated in myopia progression and may help inform which visual environments are optimal to preserve retinal signaling balance for myopia interventions. This proposal aligns with the mission statement of the National Eye Institute by investigating mechanisms underlying visual disease that may aid in the development of new preventative methods.

NIH Research Projects · FY 2025 · 2025-06

Current unimodal approaches to attain precision medicine have been difficult to adopt at scale due to cost of tests, and variable clinical output that results in undertreatment or overtreatment of some patients. Multimodal approaches for precision medicine are advancing rapidly due to the growing availability of varied data sources and advanced AI systems, with demonstrated ability to capture health complexity of many patients. However, multimodal predictive models are limited by lack of standardized approaches to tackle data heterogeneity, including limited options for handling missing data. Our proposal aims to address these gaps by developing novel digital phenotypes through the integration of multimodal datasets—imaging, pathology and electronic health records (EHR). Leveraging advanced techniques in radiopathomics extraction, data fusion, novel graph architectures, and unsupervised clustering, we aim to discover biologically relevant patient populations to improve risk stratification and treatment optimization. To achieve this, we will first develop an open-source fusion framework for multimodal data integration. This robust framework will incorporate tools for data harmonization, radiopathomics feature extraction, multimodal data embedding, and graph neural networks. We will apply this framework to discover biologically relevant digital phenotypes for breast and prostate cancer, and train phenotypespecific risk prediction models which will be robustly validated at 4 external sites. Our proposal emphasizes an open-source AI framework that incorporates privacy and accountability throughout the model development process with a goal of ensuring reproducibility and scale across different dataset types and diseases. We will adopt an iterative model co-development approach, engaging multidisciplinary stakeholders, including physicians, collaborators, and industry partners, to continuously refine our data and models. This co-design process ensures that our models remain clinically relevant to actualize the promise of multimodal data for precision medicine.

NIH Research Projects · FY 2025 · 2025-06

Project Summary Lung cancer causes about 20% of all cancer deaths making it the most lethal cancer. For central lung cancer (CLC), even the most conformal stereotactic body radiotherapy (SBRT) treatment plans inevitably overlap with organs at risk (OARs) such as the lung, heart, esophagus, and central airways. This overlap incurs substantial physical doses to these OARs, resulting in up to a 35% risk of fatal morbidity and guarded prognosis. Conformal FLASH proton therapy holds great promise for toxicity reduction while maintaining tumor control, by combining the advantages of (1) zero physical dose for OAR beyond Bragg Peak in proton SBRT, (2) 50% biological effective dose (BED) reduction to OAR in ultra-high dose rates (UHDR), which is delivery of a hypo fraction dose at a dose rate ≥40 Gy/s and (3) 30% more BED and greater immune response with more elevated linear energy transfer (LET) in tumor. CLC needs all three advantages because OAR toxicities hinder physical dose escalation. Its clinic translation needs UHDR in OARs and high LET in tumor but has two challenges: (1) if not optimized, elevated LET in OAR can lead to an elevated OAR toxicity (2) quality assurance (QA) to ensure the micro timing structure and microspatial LET, two critical factors impacting FLASH has not yet been developed. Our integrated physical optimization (IPO) considers dose, dose rate and LET in the objective function. This grant aims to establish an innovative platform of Simultaneous Intensity Energy Modulation and Compensation (SIEMAC) optimization to inversely solve the variables (intensity and energy) of the IPO objectives and a QA system that combines micro-timing/dose rate with micro dosimetry/LET. ADVACAM protype detector and commercial 2D strip-segmented ionization chamber array (SICA) can meet the QA needs. Aim 1: SIEMAC platform for CLC conformal FLASH. We will compare SIEMAC optimized plans with IMPT plans using ten CLC patients involving different OARs (lung, heart, esophagus, and central airways). Based on our preliminary data, the criteria are a >20% reduction in OAR BED or a >20% reduction in the irradiated volume for heart at 5 Gy and lung at 20 Gy, relative to IMPT, maintaining target coverage. We will reoptimize these plans for pulsed cyclotron and synchrotron beams to evaluate the impact of pulse structures over average dose rate. Aim 2: QA platform to validate Monte Carlo (MC) calculated UHDR and LET. We will perform MC simulation, 3D print example passive energy modulation filters for three SIEMAC optimized FLASH fields. We will use ADVACAM prototype detector to measure LET spectra and micro timing and SICA to measure dose rate under conformal FLASH. Measured dose rates should agree within 5% of SICA. Measured LET spectra agreement with MC simulation should be <5% via Bhattacharyya distances, a dissimilarity meter. Innovation: The first test of SIEMAC to test its feasibility for CLC and QA scalability in clinic. Impact: We will have an IPO treatment planning platform an immunotherapy conformal FLASH clinical trial of CLC and QA platform for conformal FLASH to be adopted across different proton FLASH-capable machines.

NIH Research Projects · FY 2026 · 2025-06

PROJECT ABSTRACT This project will develop, test and apply new statistical methods to address analytical challenges associated with three key research questions in environmental epidemiology: (1) how to utilize exposure data at fine spatial resolution in health analysis, (2) which individual- and area-level factors render an individual more at-risk to harmful environmental exposures, and what are the joint health effects of environmental mixture exposures? We propose methods that adopt state-of-the-art and developing ideas from functional data analysis, Bayesian analysis and machine learning, while grounding the work on established environmental health study designs, current analytic practices and real-world data sources. In Aim 1, we will develop Bayesian scalar-on-quantile- function regression models to estimate associations between aggregated health outcome and population-wide exposure distribution. This is motivated by the increasing availability of spatially-resolved exposure products. We will treat exposure quantile function as a functional covariate to better characterize health effect associated with shifts in different parts of the exposure distribution. In Aim 2, we will extend Bayesian additive regression trees (BART) models to estimate heterogeneous associations to handle matched case-control data that arise from the case-crossover design. BART offers a strategy to simultaneously consider multiple effect modifiers due to its ability to capture nonlinear and complex interactions. We will also develop tools to visualize and summarize heterogeneous associations. In Aim 3, we will develop Bayesian Gaussian process regression to estimate joint effects environmental exposure mixtures. We will characterize the exposure-response surface by providing a more computationally practical and robust framework via random Fourier feature approximation. In this framework, we will also examine how to perform variable selection/dimension reduction, handle missing data, and distinguish differences between subgroups. In Aim 4, we will create R packages to handle continuous, binary, count, and clustered outcomes to facilitate adaptation of these methods by the wider scientific community. Each methods development area will be paired with motivating and ongoing epidemiologic studies. These include (1) estimating short-term health effects of wildfire-related air pollution and ambient temperature on emergency department (ED) visits in multiple U.S. states, (2) identifying heterogeneous effects of temperature on heat-related ED visits due to individual comorbidity, medication, and residential built environment using electronic health data from Emory Healthcare, and (3) estimating associations between various environmental exposures and adverse pregnancy outcomes in deeply phenotyped African-American mother-child dyads and among deliveries at Emory Healthcare. Overall, this project will provide timely analytic methods motivated by methodological gaps, emerging data sources and research priorities in environmental health. Our Bayesian approaches to distributional covariate, tree-based models for matched case-control data, and fast Gaussian process regression may also be appealing and relevant in other biomedical and public health research areas.

NSF Awards · FY 2025 · 2025-06

Large language models (LLMs) have reshaped artificial intelligence (AI) research and implementations. In the healthcare domain, extensive enthusiasm has been witnessed on the exploration of LLMs in answering medical questions, extracting clinical information, and assisting clinical decisions. Studies have also revealed the limitations of LLMs regarding their lack of knowledge, fuzzy inference, and hallucination. Knowledge graphs (KGs) have been widely studied due to their advantages in storing accurate, explicit and easily-modifiable knowledge. In the healthcare domain, various medical KGs have been used to support basic science research, pharmaceutical research, clinical decisions, and policy making. However, healthcare data are notoriously noisy and complex, where datasets about specific concepts and conditions come from various sources and include multiple data modalities. While pioneering studies have started to explore the combination of KGs and LLMs for healthcare, most of these studies have focused on applying existing methods and do not aim to fundamentally improve the KGs and LLMs towards solving various healthcare problems. In this project, the research team aims to comprehensively investigate and address the data, model and application challenges in healthcare, through Knowledge Graphs-Large Language Model Co-Learning (KG-LLM Co-learning), a systematic framework that will provide most (if not all) major functionality needed to build high-quality KGs that integrate complex healthcare data, enhance LLMs to obtain reliable healthcare models, and leverage multi-agent systems to account for data privacy, human values and broader factors in healthcare applications. The proposed KG-LLM Co-learning framework includes several transformative technical innovations. First, the investigator's team will develop novel LLM-based methods for constructing comprehensive healthcare KGs, where they will study: (1) ontology-infused prompt designs for unifying existing healthcare KGs collectable from different sources, (2) structure-oriented retrieval augmented generation to continuously improve the healthcare KG based on evolving biomedical literature, and (3) alignment-based instruction tuning to further enrich the healthcare KG based on multi-modality patient data. Second, the investigator's team will design novel usage of KGs to obtain reliable healthcare LLMs, where they will focus on (1) enhancing the planning capability of LLMs through providing biomedical knowledge from KGs, (2) enhancing the reasoning capability of LLMs through enabling biomedical neural symbolic rule learning over KGs, and (3) enhancing the grounding capability of LLMs through enforcing post-hoc biomedical error detection based on KGs. Third, the investigator's team will explore a new way of integrating data and models through the collaboration of LLMs in a flexible federated multi-agent system, which can rigorously facilitate (1) protection of data privacy, (2) alignment with human values, and (3) consideration of broader health factors. Finally, the investigator's team will conduct comprehensive evaluations regarding the important healthcare applications of risk prediction, treatment suggestion and disease subtyping based on de-identified patient data from publicly available databases, Emory Hospitals and the NIH Bridge2AI CHoRUS Consortium, through collaborations with healthcare professionals and clinical experts. If successful, the studies will fill critical gaps between AI advances and healthcare, informing methodology and technology designs in data management and AI/Machine Learning communities, while potentially generating new biomedical and disease knowledge for improving healthcare. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2025 · 2025-06

Electronic Health Records (EHRs) have revolutionized modern healthcare by providing comprehensive digital repositories for patient data. The use of deep learning and the emerging foundation models has further enhanced the potential of EHRs, enabling high-precision tasks in digital medicine. However, modeling EHR data to effectively support clinical decision-making is susceptible to both adversarial attacks and privacy breaches. The project’s novelties are its focus on addressing adversarial robustness and privacy concerns in modern EHR systems by tackling two key challenges: (1) the complex correlations in EHR data, including cross-feature, temporal, and cross-modality correlations, and (2) the security and privacy vulnerabilities introduced by the increasing use of pre-trained models in healthcare. The project's broader significance and importance are in safeguarding patient data and enhancing the overall security and privacy of medical infrastructures. The project’s intellectual contributions include a comprehensive framework of attack strategies to assess system vulnerabilities and defense mechanisms to enhance robustness and privacy. Specifically, it explores: (1) robustness with adversarial attacks and defenses that leverage EHR data correlations, as well as backdoor attacks exploiting pre-trained models and defenses utilizing test-time model fine-tuning; (2) privacy with partial knowledge attacks that exploit data correlations and countermeasures for both partial and correlated source protection, as well as privacy amplification attacks using pre-trained models and corresponding defenses by private fine-tuning and model editing; and (3) synergy between robustness and privacy with empirical evaluations and certifiable approaches for transferring robustness into privacy protections and vice versa. Real-world validations are conducted using predictive models for sepsis and critical care syndromes to ensure practical applicability. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2025-06

In the quest to identify a widely applicable approach to cure HIV, broadly neutralizing antibodies (bNAbs) have been tested in nonhuman primates (NHPs) and humans, including newborns and children. While encouraging, these studies have not shown a consistent reduction in the size of the persistent viral reservoir compared to antiretroviral therapy (ART) alone. Whether these immunotherapies can be optimized to sufficiently limit reservoir formation and contribute to ART-free virologic control therefore remains a gap in knowledge. The Objective of this proposal is to perform a systematic analysis of immunotherapy optimization strategies. We focus on the unique setting of postnatal infection of infants, as over half of new HIV infections in children now occur through breastfeeding. The strategies to be tested leverage three different mechanisms to enhance bNAbs: i) reactivating virus expression from latently-infected cells for bNAb recognition, ii) promoting the ‘vaccinal effect’ of bNAbs through innate immune stimulation, and/or iii) improving bNAb binding to infected cells. Our Central Hypothesis is that the impact of bNAb therapy in the first weeks of perinatal infection can be enhanced through targeted virus and host directed adjunctive treatments. In Aim 1, we will define how non- canonical NF-κB pathway activation with bNAb administration during early ART impacts reservoir establishment. In Aim 2, we will evaluate if TLR7/8 stimulation with bNAb administration during early ART enhances antiviral immune responses thereby limiting reservoir formation. In Aim 3, we will determine whether bNAb immunotherapy during early ART is improved by the attachment inhibitor fostemsavir (FTR), that maintains gp120 in a ‘closed’ conformation. The bNAbs to be used in all in vivo experiments are N6-LS and PGT121, based on their distinct target sites on the HIV Env (N6: CD4-binding site; PGT-121: V3 glycan), with the goal of maximizing binding to circulating virions and virus-infected cells. Both PGT121 and N6 have potent neutralizing capacity, with N6 being exceptionally broad. We will define the impact of these approaches in an infant rhesus macaque model of postnatal infection on viral load decay, intact reservoir size, virologic control after ART interruption, and antiviral immunity. The proposed research builds on our expertise with infant NHP models to deeply interrogate immunotherapeutic approaches aimed towards cure of perinatal HIV infection. Testing three different strategies simultaneously will vastly improve our understanding of immunotherapy in infants and incorporating shared control groups makes the best use of a limited and expensive animal resource. The experiments proposed here will provide new evidence regarding the mechanisms of HIV/SIV reservoir establishment and how this process may be perturbed in a model of perinatal infection. It is our mission to turn these discoveries into clinical trials to advance research towards a cure for children with HIV.

NIH Research Projects · FY 2026 · 2025-06

PROJECT SUMMARY HIV and bacterial sexually transmitted infections (STI), including gonorrhea, chlamydia, and syphilis, are disproportionately concentrated among men who have sex with men (MSM) and transgender women (TGW). Doxycycline post-exposure prophylaxis (doxy-PEP) taken as a single 200mg dose <72 hours after sex is a new intervention that can significantly reduce the risk of bacterial STI including a >70-80% reduction in chlamydia and syphilis and >50% reduction in gonorrhea among MSM and TGW with and without HIV. However, significant concerns remain about the potential for substantial harms associated with increased use of doxy- PEP including antimicrobial resistance and effects on the microbiome that could influence long-term acquisition of comorbidities, such as obesity. However, absent from the conversation on the risks/benefits of doxy-PEP to date are the known direct anti-inflammatory and immunomodulatory effects of doxycycline, independent of its antimicrobial effects. Among people with HIV (PWH), chronic inflammation, in part mediated by damage to the gut mucosa and microbial translocation, is thought to contribute to increased morbidity through accelerated acquisition of comorbidities. Our group has previously characterized the altered gut immune environment among MSM with HIV and the contribution of bacterial STI to rectal mucosal inflammation and perturbations in the microbiome among MSM with HIV. In addition, our group has extensively characterized a pro-inflammatory rectal mucosal immune environment among MSM without HIV engaging in receptive anal intercourse that may be associated with HIV acquisition. Given the critical role that inflammation likely plays for MSM both with and without HIV, the potential for anti-inflammatory effects of doxy-PEP, and our preliminary data suggesting minimal effect of doxy-PEP on the gut microbiome, this begs the provocative hypothesis: Could doxy-PEP play a beneficial role in reducing systemic and gut inflammation with minimal harmful effects on the gut microbiome and resistome among MSM and TGW with and without HIV? To address our hypothesis, we will enroll a mechanistic clinical trial of MSM and TGW (n=75 with HIV on antiretroviral therapy; n= 75 without HIV on pre- exposure prophylaxis) and randomize 2:1 to take 200mg of doxycycline at least 3 times/week for 12 weeks vs. observation with biologic sampling. We will sample blood and rectal mucosal secretions and tissues before and after doxy-PEP use that will allow us to fully characterize the effects on systemic (specific aim 1) and rectal mucosal (specific aim 2) inflammation and the microbiome and resistome (specific aim 3) at high resolution and determine whether we can ‘flip the script’ on the potential harms of doxy-PEP for this high need population. Completion of the proposed aims will allow for a comprehensive assessment of the anti-inflammatory and microbiome/resistome effects of doxy-PEP and could ameliorate concerns for widespread implementation of this new intervention.

NIH Research Projects · FY 2025 · 2025-06

The overall goal of this proposal is to evaluate the safety and therapeutic potential of a novel strategy towards a cure of HIV infection. The proposed treatment combines soluble IL-15/IL-15Rα-Fc with a TGF-β pathway blockade and therapeutic vaccination to enhance/restore function of anti-viral immunity that will lead to sustained viral remission following anti- retroviral therapy (ART) interruption against HIV, using the SIV/Rhesus macaque (RM) model. Persistence of viral reservoirs and dysfunctional anti-HIV immunity represent two major obstacles that must be addressed to achieve sustained viral remission via blocking viral reactivation in the absence of ART. ART is highly effective in controlling virus replication but does not significantly improve T cell function and fail to eliminate viral reservoirs. Anti-viral CD8 T cells are critical for the control of HIV/SIV replication, yet these cells are largely excluded from secondary lymphoid organs which host a significant fraction of viral reservoirs during ART, primarily in T follicular helper cells (Tfh) that reside in B cell follicles and germinal centers (GC). Thus, novel therapies that that not only restore/enhance function of both anti-viral CD8 T cells and NK cells, but also promote follicular homing of these cells while viral reservoirs are being reactivated will significantly enhance their clearance within lymphoid tissues, thereby contribute to sustained viral remission following analytical ART interruption (ATI). Our preliminary data demonstrate that galunisertib (GAL) treatment improves anti-retroviral bioavailability in human lymphoid endothelial cells and cytokine (IL-15/IL15Rα-Fc) treatment in vivo markedly enhances the magnitude, proliferation and cytotoxic potential of SIV-specific CD8+ T and NK cells with follicular homing potential, which is critical for the reduction of reemerging viremia post ART interruption. In addition, in vitro stimulation of chronically infected PBMC with IL-15/IL15Rα-Fc therapy plus GAL treatment resulted in higher proliferation anti-viral CD8 T cells and NK cells. Importantly, in vivo administration IL-15/IL15Rα-Fc plus GAL treatment in ART treated macaques enhanced antiviral CD8 T cell and NK cell function. Given these highly encouraging results, we propose to comprehensively test the effects of IL-15/IL-15Rα-Fc therapy plus GAL treatment either alone or in combination on different T and NK cell subsets during chronic SIV infection under the umbrella of ART (Aim 1), and investigate how these changes affect viral reservoirs under ART and viral control after ART interruption (Aim 2). Last, we will combine the optimal cytokine therapy with vaccination to further enhance the magnitude and breadth of SIV-specific CD8 T cell responses that we hope will further improve the therapeutic benefit (Aim 3). These studies will advance our knowledge about how IL-15/IL-15α-Fc therapy plus GAL differentially influence the reservoirs and function of different subsets of T and NK cells during chronic SIV infection and ART, and what immune mechanisms are induced by dual therapy and vaccination, and how these contribute to control of chronic SIV/HIV infection in vivo.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY/ABSTRACT: In ongoing experiments, we show that early-life antibody-secreting cells (ASCs), or plasma cells (PCs), appear spontaneously in the spleen, not the bone marrow (BM), before birth. The splenic ASCs develop from transcriptionally distinct fetal B-progenitors absent in adults. The splenic ASCs develop spontaneously in utero and secrete public natural antibodies (NAb) highly shared across individuals. Notably, these naturally occurring splenic ASCs secrete public NAbs with specificities against self, commensals, and common pathogens, including encapsulated bacteria known to cause life-threatening infections in infants. Finally, the naturally occurring ASCs upregulate survival genes similar to the long-lived plasma cells (LLPCs), suggesting they persist throughout adulthood. These findings highlight the relevance of the spleen, separate from the BM, in supporting the development and survival of B cells, ASCs, and NAbs that are protective against common pathogens. We validated these findings using humanized mice and showed that fetal B-progenitors give rise to long-lived mature B cells that survive >1 year and spontaneously differentiate into ASCs in the mouse spleen, not BM, and secrete public NAbs that recapitulate the specificity of the shared clones in the human fetal spleen. Here, we will characterize early-life B-cell and ASC development and function (and compare them to their adult counterparts) to uncover the mechanisms of adaptive immunity operating at birth. This will provide a new framework for understanding human immune responses at different ages and reveal new targets to modulate such responses. In three Specific Aims, we will test our hypothesis that the human immune system comprises at least two developmentally and functionally distinct lineages (or layers) of B cells and ASCs— early-life and adult. Aim 1 will focus on the B-cell development at different hematopoietic organs (liver and BM) and determine the main cytokines and metabolic dependencies that regulate B cells in early life versus adulthood. Aim 2 will characterize the phenotype and functions of the mature B-cell subsets and their spontaneous differentiation to ASCs in the spleen before birth. This aim will reveal the mechanisms of B-cell activation and metabolic reprogramming that lead to effector B cells, ASCs, and NAbs in early life. Finally, Aim 3 will focus on the antigen specificity and effector functions (glycosylation profile) of the public NAb repertoire that spontaneously emerge in the spleen before birth. We will clone the top (> 500) antibody repertoire shared across individuals to test their specificity against common pathogens, commensals, and self-antigens and search for these clones in the adult spleen. These studies will greatly extend our knowledge of the B-cell and antibody responses before birth and establish the human spleen, separate from the BM, as a unique reservoir for the development and survival of public B cells, ASCs, and functionally glycosylated NAbs against self, commensals, and pathogens known to cause life-threatening infections in children.

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

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.