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NIA - National Institute on Aging

PROJECT SUMMARY Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that remains without effective treatment. Mounting evidence suggests that neuroinflammation plays a critical role in AD progression, yet the contribution of astrocytes—a major glial cell type involved in brain homeostasis—remains poorly defined. Recent discoveries from our lab have identified varicose projection (VP) astrocytes as a novel, reactive astrocyte phenotype associated with neuroinflammatory conditions, including AD. Unlike traditional astrocytes, VP astrocytes display distinct morphological features (long processes with evenly spaced varicosities), are enriched in the cortex and white matter of AD brains, and can be experimentally induced in vitro by pro-inflammatory cytokines. Importantly, the VP astrocyte phenotype is reversible upon cytokine withdrawal, highlighting its potential as a targetable cellular response in AD. This AREA R15 project aims to elucidate the role of VP astrocytes in AD pathology using human-induced pluripotent stem cell (hiPSC)-based models and human postmortem brains. The project will engage undergraduate researchers at Towson University in Dr. Falcone’s lab and within a novel neuroscience Course- Based Undergraduate Research Experience (CURE). Here, we propose to investigate the role of VP astrocytes in AD pathology through two aims: (Aim 1) Define the molecular signature of VP astrocytes in AD-associated neuroinflammation using Patch-seq transcriptomic profiling and validation in human postmortem brain tissue; (Aim 2) Assess the functional impact of VP astrocytes on neuronal degeneration using hiPSC-derived neuron-astrocyte co-cultures, testing cytokine withdrawal and pharmacological inhibition of key inflammatory pathways. By integrating cellular, molecular and anatomical approaches in human-based models, this project will reveal the mechanistic contribution of VP astrocytes to AD, laying the groundwork for novel therapeutic strategies while training the next generation of biomedical scientists.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Defects in cellular proliferation can lead to a variety of developmental disorders as well as cancer. This dysregulation is achieved through deleterious mutations of tumor suppressor genes (TSGs). However, tumor suppressors can be non-genetically inactivated, which is difficult to detect with standard genomic techniques. While some TSG somatic mutations have been identified in hematologic malignancies, there is a critical gap in knowledge for the role of TSG silencing in leukemogenesis. This study will examine how TSGs are inactivated at the post-transcriptional level in acute myeloid leukemia (AML) and how their restoration is a novel therapeutic avenue for leukemic elimination. I found that the transcriptional repressor ZBTB7A is post-transcriptionally silenced in AML, blocking differentiation in the myeloid lineage. This is achieved through alternative polyadenylation, resulting in an isoform with a longer 3’UTR that leads to deadenylation. I was able to restore ZBTB7A expression through epigenetic targeting of upstream negative regulators as a proof of concept of TSG re-activation. During the training (K99) phase of this award, I will uncover the exact RNA-binding proteins mediating downregulation of ZBTB7A, and if this is part of a larger pro-AML regulatory network blocking differentiation. To this end, we are employing a novel long read sequencing method termed Pull-a-Long Sequencing (PL-Seq) in collaboration with Dr. Pedro Miura, to uncover the roles of ZFP36 and ELAVL family members on the transcriptional stability of ZBTB7A. ZBTB7A has two paralogs in mammals, ZBTB7B and ZBTB7C. ZBTB7B has been implicated in hematopoietic differentiation of the lymphoid lineage, and the DNA-interacting residues of its zinc fingers are highly conserved to ZBTB7A. During the independent (R00) phase, I will apply the insights and tools acquired during the training phase to identify the contribution of ZBTB7B paralogs with ZBTB7A, and ascertain if they are synergistic in mediating myeloid differentiation. In my preliminary data, I have utilized a Rosa26LSL-Cas9-eGFP mouse model to delete Zbtb7a in the murine hematopoietic system and found that loss of Zbtb7a results in hematopoietic stem and progenitor (HSPC) expansion, as well as a bias in myeloid differentiation. With the guidance of Dr. Jennifer Trowbridge, an expert in mouse modeling of the hematopoietic system, I will more fully characterize Zbtb7a hematopoietic specific knockout mice as well as Zbtb7b knockout mice. Together, this will open multiple lines of inquiry into a family of transcriptional repressors and the role of non-genetic inactivation of tumor suppressors. My proposed study will generate novel tools and testable hypotheses that will lay a strong foundation for my independent research program, where I will continue to characterize non-genetic mechanisms of AML regulation as novel means for therapeutic intervention.

NIA - National Institute on Aging

Sporadic Alzheimer’s disease (sAD) affects millions worldwide, yet the earliest molecular events that drive its progression remain poorly understood. Hallmark pathologies—amyloid-β (Aβ) plaques and tau neurofibrillary tangles (NFTs)—develop decades after subtle cellular changes have already begun. One of the strongest emerging biomarkers of preclinical AD is soluble phosphorylated tau at threonine 217 (pT217Tau), which appears in cerebrospinal fluid and plasma years before overt symptoms or brain pathology. Endosomal enlargement, a robust early abnormality linked to Aβ42 generation from amyloid precursor protein (APP), is also present in at-risk individuals. However, the spatial and temporal relationship between early-stage tau hyperphosphorylation, amyloid pathology, and endosomal changes has never been directly visualized in human or nonhuman primate brains. This gap limits the development of early diagnostic tools and therapies. The overall goal of this project is to define the earliest molecular convergence points between soluble pT217Tau, APP/Aβ42, and endosomal abnormalities across human postmortem tissue, aging nonhuman primates, and human induced pluripotent stem cell (hiPSC)–derived neurons. This multiscale approach is uniquely enabled by (1) postmortem human tissue spanning Braak stages I–VI, (2) perfusion-fixed rhesus macaque brains that preserve soluble phosphorylation states and native ultrastructure, and (3) mechanistic testing in hiPSC-derived neurons including SORL1-deficient lines, which model genetic risk for endolysosomal dysfunction. Aim 1 will define the spatial relationship between pT217Tau, endosomal enlargement, and APP/Aβ42 in early human AD stages using quantitative immunofluorescence across vulnerable (entorhinal cortex, hippocampus, dorsolateral prefrontal cortex) and resilient (primary visual cortex) regions. Aim 2 will examine age-related associations between pT217Tau, APP/Aβ42 and endosomal abnormalities in rhesus macaque cortex, applying dual-label immuno- electron microscopy to achieve nanoscale resolution of tau–endosome interactions. Aim 3 will determine whether promoting tau hyperphosphorylation in hiPSC-derived neurons is sufficient to induce endosomal abnormalities and altered APP/Aβ42 trafficking, with particular emphasis on retromer dysfunction in SORL1- deficient cells. By combining observational and experimental systems, this project will directly test the hypothesis that soluble pT217Tau—but not fibrillar tau—is preferentially associated with APP/Aβ42-containing endosomes in neurons of vulnerable cortical regions, and that this phenotype can be recapitulated in vitro. The proposed studies are highly innovative in their integration of human, nonhuman primate, and cellular models, and in their use of cutting-edge nanoscale imaging to capture disease processes at unprecedented resolution. Results will illuminate the etiology of sAD pathogenesis, identify the earliest cellular events linking tau and amyloid pathology, and reveal new targets for early intervention. Ultimately, this work has the potential to transform how we detect and treat sAD by focusing on the earliest and potentially reversible stages of the disorder.

NIGMS - National Institute of General Medical Sciences

Project Summary: Nutrients are important regulators of cell states and cell fate transitions. In this context, Vitamin C (VitC), an essential nutrient, is an important regulator of extracellular matrix (ECM) synthesis and of multiple cell fate transitions, including somatic cell to induced pluripotent stem cell (iPSC) reprogramming. However, the molecular mechanisms underlying these effects remain poorly understood. Prior studies have largely attributed VitC function to the regulation of Fe2+/α-ketoglutarate-dependent dioxygenases (αKGDDs). Yet, several studies report only minimal VitC-dependent changes in collagen hydroxylation, and our preliminary data indicate that VitC- dependent enhancement of iPSC reprogramming occurs independently of αKGDD activity. Instead, we discovered that VitC rapidly activates the oxidative pentose phosphate pathway (PPP), increasing NADPH and ribose-5-phosphate (R5P) levels, and enhances nucleotide sugar synthesis. We also found that VitC increases total ECM glycosylation, including N-linked glycosylation of collagen I (Col I) and P4HA1, the prolyl hydroxylase required for collagen secretion. We hypothesize that VitC-dependent metabolic rewiring promotes glycosylation- dependent ECM synthesis, and regulates transcriptional programs to facilitate iPSC reprogramming. The proposed research aims to understand the molecular mechanisms by which VitC regulates ECM synthesis and cell-fate transitions. AIM 1 will elucidate the molecular mechanisms of VitC-dependent metabolic rewiring and provide training in genetic manipulation and metabolite tracing techniques to enable mechanistic studies of metabolic pathway regulation. AIM 2 will determine the role of VitC-driven metabolic changes in regulating ECM synthesis, and in regulating transcriptional programs during iPSC reprogramming, and provide necessary training in single-cell RNA-seq approaches to define how metabolic and ECM changes influence transcriptional trajectories and cell fate transitions. Finally, the independent phase of this award, AIM 3, will elucidate the mechanisms by which VitC-dependent metabolic rewiring regulates ECM protein glycosylation and secretion. This work will reveal novel pathways linking nutrient-dependent metabolic rewiring to ECM remodeling and cell state regulation, with broad implications for understanding ECM-related diseases such as fibrosis and for nutrient-driven control of cell fate. The mentored phase of this award provides the opportunity to work with leaders in the fields of cell-fate regulation and metabolism, to acquire the training needed to achieve my long- term goal of becoming an independent investigator at the interface of both fields. Together, the proposed research, mentorship team, and resources provided for career development at University of California, Los Angeles, will provide the training needed to achieve the defined career goals.

Dell Technologies Foundation

Supports youth development and STEM education programs, with focus on underrepresented groups in technology careers.

Deloitte Foundation

Supports education and workforce development through grants to universities and nonprofits focused on accounting, business, and STEM.

NIAID - National Institute of Allergy and Infectious Diseases

Multiple sclerosis (MS) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) are immune-mediated demyelinating diseases that impact close to 5 million people worldwide. White matter plaque formation leads to acute inflammation, chronic demyelination, and axonal loss. These inflammatory lesions predominantly consist of T lymphocytes and reactive microglia, which lead to the breakdown of the protective myelin sheath coating on axons and the destruction of oligodendrocytes. This degradation leads to the release of myelin oligodendrocyte glycoprotein (MOG) and other proteins expressed on the outermost later of the myelin sheath into the lesion microenvironment. With the recent explosion of biologics and immunotherapy, new treatment options have become available for MS but remain limited for MOGAD. However, while these therapies are effective at reducing the frequency of relapses, there are risks associated with long-term systemic immunosuppression and in many patients, their disease eventually progresses. Therefore, the overall goal of this proposal is to develop novel, safe, and effective treatments and strategies that can specifically modulate the immune response in MS and MOGAD. Mesenchymal stem cells (MSCs) are characterized by their immunosuppressive and regenerative properties. As such, adoptive transfer of MSCs has been investigated as an independent therapeutic option in immune-mediated diseases and was recently FDA approved in pediatric graft-versus-host disease. MSCs mediate their immunosuppressive capabilities at least partly through the secretion of immunosuppressive cytokines, such as interleukin (IL)-10. IL-10 is a potent anti-inflammatory cytokine that inhibits the synthesis of IL-2 and interferon-ɣ, two cytokines crucial for T cell development and activation. IL-10 is also reported to have neuroprotective effects by inhibiting astrocyte activation and preventing accumulation of the excitatory neurotransmitter glutamate. Previous studies have implicated that engineering MSCs with designer chimeric antigen receptors (CARs) results in enhanced trafficking to sites of inflammation and augmented immunosuppressive capabilities. This leads to our central hypothesis that engineering MSCs to express MOG-targeting CARs that secrete IL-10 (MOG-CAR-IL10) can ameliorate treatment-refractory MS and MOGAD. This hypothesis will be tested by pursuing two specific aims: Aim 1 will study trafficking of MOG-CAR-IL10 to the central nervous system in mouse models of T-cell mediated demyelination and Aim 2 will determine the immunosuppressive effects of MOG-CAR-IL10 on MS in preclinical models. By the completion of this work, we expect to have defined how MOG-CAR-IL10 interacts with T cells in MS and MOGAD and to have developed novel targeted cellular engineering therapies to ameliorate treatment-refractory MS and MOGAD more effectively. This proposal will allow me to acquire cross-disciplinary training in (i) mouse modeling, (ii) vector design and cell engineering, (iii) MSC-immune cell functional interactions, and (iv) translational science.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Background: The combination of hypomethylating agent (HMA) and the BCL2 inhibitor venetoclax (venHMA) has become the new standard of care for newly diagnosed older patients with acute myeloid leukemia (AML). However, one third of patients do not respond and the remainder of patients inevitably relapse on therapy in the absence of stem cell transplant. Biomarkers to predict response to venHMA are limited and clinical relapse mechanisms remain poorly characterized. No proven therapy exists for the majority of patients who relapse after treatment with venHMA. Rationale: To develop effective therapies to prevent and treat relapse, improved understanding of the molecular mechanisms underlying primary and secondary resistance to venHMA is required. A comprehensive study of samples from AML patients who are treated in both clinical trial and realworld settings is the best mechanism by which to derive clinically relevant biomarkers of response and resistance. Methods: We will perform integrated bulk and single cell molecular characterization of samples from patients treated on a large multi-center clinical trial of venHMA in older patients with AML to develop biomarkers of clinical response and determine mechanisms of acquired resistance. We will perform confirmatory sequencing studies in patients similarly treated in a real-world setting. Moreover, we will perform mechanistic validation studies of putative clinical resistance mechanisms in genotype-matched AML cell lines in vitro and in vivo in patient derived xenografts (PDXs). The use of in vivo PDX models will allow us to validate resistance mechanisms and test a clinically actionable therapeutic strategy of RAS inhibition in the most patient-relevant model system. Expected Results: We anticipate that this work will uncover critical insights about clinically relevant mechanisms of response and resistance to venHMA. The overall goal of this work is to develop clinically relevant biomarkers to guide the treatment of AML patients and to facilitate the development of new treatment strategies for refractory/relapsed AML, a group of patients with no current standard of care.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Influenza A viruses (IAV) continue to impose a major global health burden. The recent spread of highly pathogenic avian H5N1 across continents and into United States dairy cows and wildlife increases its potential for zoonotic adaptation and pandemic spread and necessitates the development of effective vaccines. However, responses to novel IAV antigens are shaped by immune imprinting, a phenomenon where pre-existing immunity to prior IAV antigens biases the response toward cross-reactive epitopes at the expense of generating de novo, more protective antibodies against the new antigens. As most adults have been exposed to IAV, it remains unclear how much of an antibody response elicited by H5 hemagglutinin (HA) vaccines reflects imprinted recall versus de novo protective responses. The proposed research addresses these gaps through two complementary aims. In Aim 1, we will develop serological assays that distinguish and quantify vaccine-induced H5-specific versus cross-reactive imprinted antibody responses across different prior IAV experience. In Aim 2, we will characterize the B cell populations and repertoire induced by H5 HA vaccines and assess the functionality of generated monoclonal antibodies to determine whether vaccine antigen design can preferentially target neutralizing epitopes on the H5 HA and engender more protective responses. We propose to mutate the conserved stem epitope on H5 HA to disrupt binding by B cell receptors on cross-reactive memory B cells and reduce imprinting. Collectively, this project will address a fundamental barrier to effective H5 vaccination and provide broadly applicable insights for improving vaccines against emerging IAV in antigen-experienced populations. As part of this fellowship training plan, I will pursue both scientific and clinical activities to expand my intellectual inquiry, technical expertise, and scientific communication skills towards my goal of excelling in a postdoctoral fellowship and eventually becoming an independent physician-principal investigator in the field of viral immunology. My fellowship training plan at WashU in St Louis, one of the nation’s leading Medical Scientist Training Programs, integrates strong mentorship from leading immunology and virology experts as my thesis advisor/sponsor and collaborators, rigorous genomics coursework, manuscript writing, and presentation at local and international research meetings. This environment will provide me the mentorship, resources, and scholarly and research opportunities to accomplish my aims and position me to succeed in my long-term goal of leading an independent laboratory and caring for patients.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary The brainstem relays information between the brain and the spinal cord, regulating vital autonomic functions. Like other tissues, the brainstem is vulnerable to malformation and disease. Yet, despite its importance, little is known about its development, making it challenging to understand brainstem pathology. Many of the neuron populations that regulate vital brainstem function arise from a homogenous progenitor pool that expresses the proneural transcription factor Atonal homolog 1 (Atoh1). Atoh1 is functionally relevant for driving migration, however, the mechanisms that regulate progenitor proliferation and prime cells for differentiation are unknown. This incomplete understanding of brainstem development has made it challenging to develop accurate in vitro models of the brainstem, thereby hindering studies aimed at elucidating disorders and disease. Recent transcriptomic mapping of embryonic mouse hindbrain development has revealed significant expression of Notch signaling genes. Notch signaling is a key regulator of cell fate decisions across neurogenesis, and it is known to regulate proneural genes such as Atoh1. Importantly, the development of in vitro models often relies on small molecule-based approaches that direct stem cell fate by mimicking native signaling environments. Yet, the specific role of Notch signaling in Atoh1 lineage development remains poorly defined, making it challenging to utilize this pathway to model development in vitro. This proposal will investigate how Notch signaling influences Atoh1 lineage progression by integrating computational transcriptomics, stem cell differentiation, and synthetic biology. The specific aims of this project are to: (1) define transcriptomic patterns of Notch signaling during brainstem development and predict regulatory function through in silico perturbation modeling; (2) engineer a novel multi-reporter stem cell line to visualize real-time Notch ligand dynamics during Atoh1-directed differentiation; and (3) modulate Notch activity in vitro to assess the impact of ligand induction on Atoh1 fate decisions. Together, this work will clarify how Notch signaling shapes Atoh1 lineage progression and establish tools to visualize and manipulate Notch signaling in vitro. These insights will provide foundational knowledge for improving in vitro brainstem models and for probing neurodevelopmental disorders linked to brainstem dysfunction.

NCI - National Cancer Institute

Title: Develop a human pluripotent stem cell-derived preclinical model for NUT Carcinoma Project Summary: NUT Carcinoma (NC) is a devastating cancer with no effective treatment. A deeper understanding of its oncogenesis mechanism is vital for developing treatments that improve its prognosis. Although NC cases are strongly associated with Nuclear Protein in Testis (NUTM1) fusion genes, predominantly BRD4::NUTM1 (70% of cases), their oncogenic functions have been under debate. Using one of the first two NC genetically engineered mouse models we created, we demonstrated that inducing an endogenous chromosome translocation that forms the Brd4::Nutm1 fusion gene in progenitor cells in tissues as distinctive as oral mucosa, thyroids, lungs, and pancreas can induce carcinomas recapitulating human NC. Our results provided the long-awaited proof of NUTM1 fusion genes as the oncogenes for NC. Our new GEMM provided a critical tool to deepen our understanding of the molecular mechanisms of NC oncogenesis and develop effective treatments. However, the 90 million years’ evolution distance between humans and mice posed two significant challenges for translational studies of NC using the mouse model: · Due to the evolutionary divergence of protein and sequence structure, targeting agents including CRISPR-CAS9-based gene therapy agents and NUTM1-degrading molecular glues cannot be effectively tested using mouse model. · Due to the relatively loose evolution constraints on regulatory sequences, the genetic regulatory network (GRN) controlled by the NUTM1 fusion genes could differ between the two species. This could hamper the effective identification of BRD4::NUTM1 targets for future therapeutic development. To overcome these challenges, we propose to develop a human pluripotent stem cell (hPSC) derived NC model. We will use a genetic design demonstrated in our GEMM to build human PSC cell lines for modeling NC. To gain access to the progenitor cells of respiratory epithelial tissues, from which lung NCs that account for more than 50% of reported human cases likely originate, we will use human PSC-derived teratoma in immunocompromised mice as the platform to generate NCs. We will first create and characterize the human PSC-NC model (Aim 1) and then use this model to demonstrate the BRD4::NUTM1 dependency and thus the proof-of-principle of the effectiveness of NUTM1-targeted therapy for NC. (Aim 2). Overall Impact. Our project will provide a critical human-relevant in vivo preclinical model for studying NC. It will provide a proof-of-principle demonstration of NUTM1-targeted therapy. Our study will also provide a novel generalized road plan for developing in vivo human-relevant models for fusion gene-driven cancers.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Hematopoietic stem cell (HSC) mobilization from the bone marrow to the peripheral blood is essential for bone marrow transplants, a life-saving treatment for hematological malignancies such as leukemia, lymphoma, and multiple myeloma. Poor mobilization remains a major clinical challenge, particularly in older patients (>60 years old), who represent the majority of those diagnosed with blood cancers yet often exhibit diminished responses to mobilization treatments. Age-related changes to the bone marrow microenvironment, specifically changes in microenvironmental stiffness, are believed to contribute to these mobilization failures. It has been recently measured that the bone marrow contains unique stiffness values in each identified sub-niche and observations in age-related stiffening has been reported; however, challenges with accurately measuring these values in vivo limits our understanding on how these gradients change with age. Existing 3D bone marrow models fail to capture the nonlinear stiffness gradients observed in vivo; therefore, the long-term objective of this proposal is to improve clinical predictions of a patient’s ability to successfully mobilize HSCs for a transplant. To achieve this objective, we will engineer a heterogenous, multi-niched bone marrow model with methacryloyl gelatin (GelMA) bioinks and extrusion bioprinting technologies to decouple the effects of young and aged stiffness environments on HSC mobilization. We expect the precision and automation of this approach will more accurately recapitulate the spatially transient stiffness environments of the native sub-niches. This research will target two major knowledge gaps: 1) how nonlinear gradients and age-related changes in microenvironmental stiffness influence HSC migration and phenotype, and 2) how to improve the ability to predict a patient’s ability to mobilize HSCs for more effective and personalized transplant strategies. The overarching hypothesis of this project is that age-related stiffening is a key microenvironmental cue which restricts HSC mobilization to the peripheral blood; and the mobilization of HSCs encapsulated in in vitro biofabricated models with physiomimetic stiffnesses of young and aged bone marrow sub-niches can predict the mobilization of in vivo HSCs to the peripheral blood. I will test this hypothesis through two specific aims; 1) assess the mobilization behavior of HSCs in response to GelMA stiffness gradients, and 2) correlate in vivo mobilization behavior in young and aged mice with in vitro behavior using bioprinted bone marrow models. We expect to identify the role of transient nonlinear stiffness gradients and age-related stiffness changes on HSC mobilization behavior. Furthermore, this work will improve strategies for predicting patient-specific mobilization outcomes for patients with hematological malignancies.

NIMH - National Institute of Mental Health

Abstract Despite antiretroviral therapy (ART), HIV-associated brain injury (HABI) persists in over half of people with HIV (PWH), manifesting as chronic cognitive impairment. While HIV-1 primarily infects microglia, driving central nervous system (CNS) neuroinflammation, current preclinical models do not recapitulate the chronic, suppressed infection characteristic of the ART era. Furthermore, they do not capture complex patient genetics and multicellular, glial and neuronal, interactions in a scalable and efficient manner. To address this need for more physiologically relevant models, we propose the development of an AI-guided triculture platform comprising major CNS cell types. This platform will use induced pluripotent stem cell (iPSC)-derived microglia, astrocytes, and neurons, using both morphological profiling and other omics-based profiling to model HABI under ART suppression. AI/machine learning (ML)-driven analysis of cellular morphology, combined with multi-omic data integration, will facilitate rapid classification and prediction of microglial functional states and their impact on neuronal health. Leveraging Modulo's established triculture system, previously successful in yielding therapeutic candidates for amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) currently in Investigational New Drug (IND)-enabling studies, we will construct a scalable HABI model under ART suppression. Our objectives are to (1) develop and validate an HIV-infected, ART-suppressed triculture platform, utilizing AI/ML-driven morphological profiling to classify HABI-specific microglial states; and (2) comprehensively characterize this model through neuroinflammatory profiling, behavioral correlates, and integration with publicly available HABI patient datasets. We hypothesize that our combined computational lab-based triculture system can effectively model HABI pathophysiology under ART conditions, enabling both rapid disease state classification and identification of therapeutic targets. Through the integration of experimental and computational approaches, this platform will provide insights into HABI mechanisms and accelerate therapeutic development. We will disseminate this model to the scientific community through publication and collaboration. Connecting in vitro modeling with patient outcomes offers a powerful tool for investigating neuroimmune dysfunction in HIV and related neurological disorders. Successful implementation will yield a platform for modeling neuroHIV under ART suppression, advancing our understanding of disease mechanisms and facilitating the discovery of novel therapeutic strategies for PWH with cognitive impairment.

NIDCR - National Institute of Dental and Craniofacial Research

SUMMARY. The programmed death receptor 1 (PD-1) drives immune escape in head and neck squamous cell cancer (HNSCC), whose incidence is rising due to human papillomavirus (HPV). PD-1 inhibitors (PD-1i) are in use in many cancers including both resectable HPV-neg and recurrent/metastatic (R/M) HPV-pos and -neg HSNCC, but most patients do not respond. Limitations in studying immunotherapy include the scarcity of models providing key aspects of human adaptive immunity, such as 1) systems where the tumor and immune system are partially or fully matched, and 2) thymic education necessary for T cell recognition to occur. In recently reported work we reprogrammed peripheral blood mononuclear cells (PBMCs) and cancer-associated fibroblasts (CAFs) from melanoma and HNSCC patients into induced pluripotent stem cells (iPSC). Next, we developed a novel protocol to differentiate patient iPSCs into functional isogenic multicellular stem cell derived thymic organoids (sTOs) by combining TEPs, mesenchymal cells and hematopoietic lymphoid progenitor cells generated from the same iPSC line. sTOs can educate developing T cells, both via positive and negative selection. The key advantage of sTOs is that they can be used both as organoids to generate T cells for testing in vitro and can also be implanted in humanized mice (HM) to test immunotherapies in vivo. The sTO produces T cells that are educated isogenically; after activation and exposure to PD-1i and other immune checkpoint inhibitors (ICI) these T cells can be co-cultured with autologous cancer cells to generate organoids (iSpheres). This cancer and T cell interaction tests immune-directed therapies in a patient specific manner, and by using controls that are not autologous (both cancer and T cells) the contribution of thymic education can be accurately dissected. Then we will produce cohorts of thymectomized HM, with HLA-matched cord blood derived HSCs, that will be transplanted with sTOs and patient-derived xenografts (PDX) from the same patient. This results in functional maturation of thymic tissue in vivo, education of developing T cells, peripheral migration, and tumor infiltration in an isogenic manner. We recently published results showing a delay in tumor growth in HM bearing sTO, but not in HM with mouse thymus, which we hypothesize is due to improved T cell education in matched thymi, a proof of principle observation. We have a large, developing collection of patient-matched PBMCs, CAFs, iPSC, cancer cell lines and PDX from HNSCC patients that will be used to develop a first set of ~10 in vitro and in vivo testing tools. We will then test this approach prospectively on models generated from ~10 patients receiving PD-1i. This plan has the potential of identify rational ICI selection strategy and allow testing the value of thymic education in cancer immunology. Neoantigen (neoAg) profiling of PD-1i-susceptible vs resistant cases to identify neoAg that are immunogenic adds a mechanistic layer. This project will focus on creating in vitro and in vivo patient-derived tools with T cell education to test mechanisms of therapy response in HNSCC, and will provide innovative mechanistic insights at the immunologic and molecular level of immune response.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

ABSTRACT Early stage pediatric faculty members, even those who have potential for success as academic investigators based upon substantial experience in basic research, will require further intensive training and mentoring in basic research to successfully embark upon an independent research career. Our proposed Child Health Research Career Development Award (CHRCDA) Program entitled “Developing Diverse Physician-Investigator Leaders for the Future of Child Health” will provide a cutting-edge research opportunities, combined with a carefully constructed mentoring and career development pathway, to enable our Scholars to emerge as leaders among the ranks of pediatric academic investigators. The basic biomedical research training community at the University of California San Diego (UCSD) has a long-established reputation of excellence, and UCSD Pediatrics now ranks in the top five in NIH research funding of all Pediatric Departments in the country. Notably, the last 5-10 years have witnessed an impressive academic expansion within of the UCSD Department of Pediatrics and the research it conducts, coupled with the formalization of its partnership with Rady Children's Hospital San Diego, the largest Children's Hospital in California. The key objectives of our CHRCDA are as follows: (1) To increase the number Pediatrician-Scientists engaged in basic research as applied to children's health; (2) to attract outstanding young pediatricians to UCSD and to facilitate their career development under the guidance of world class, established investigator-faculty mentors; and (3) to cultivate the early careers of women and minority investigators in children's health. With close input from the program pioneers in our Department, CHRCDA Scholars will participate fully in the program of UCSD National Center for Leadership in Academic Medicine (NCLAM), a longstanding and highly successful junior faculty mentoring program in UC Health Sciences that provides workshops and longitudinal mentoring in all facets required for successful advancement in academic medicine, and diversity enrichment modules including the Border Health and Doc-for-A-Day programs. All CHRCDA program faculty mentors are highly regarded scientific investigators, each leading a vibrant and cutting-edge basic or basic/translational research program of strong relevance to pediatric medicine. Research training opportunities for CHRCDA Scholars are organized into six research themes of five members each, which an integral role in the program structure, curriculum and mentorship approach: (1) Genomics, Big Data & Systems Biology; (2) Infection, Immunity & Inflammation; (3) Organ Physiology & Metabolism; (4) Neuroscience & Brain Development; (5) Human Microbiome & Child Health; and (6) Developmental & Stem Cell Biology. A guiding philosophy of this CHRCDA will be to support the greatest possible number of young physician-scientists within this Program, and an additional year of Department-funded support has been added to three years of K12 support to create a vibrant program with one new Scholar per year and four fellows total in the steady state.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY Chemical vesicants such as sulfur mustard (SM) and nitrogen mustard (NM) act as alkylating agents causing severe skin injuries characterized by blistering, necrosis, and persistent pathology. Both pose significant threats to civilians and warfighters, yet no approved therapeutics exist for managing vesicant-induced skin injuries. This project addresses this critical medical gap by developing innovative multifunctional wound dressings engineered to simultaneously target cell membrane damage, chronic inflammation, and secondary infection—the primary pathological mechanisms underlying vesicant-induced skin trauma. Our therapeutic approach centers on MG53, a TRIM family protein with demonstrated efficacy in cell membrane repair and wound healing. Beyond its membrane-protective functions, MG53 enhances regenerative capacity in diabetic wounds by revitalizing hair follicle stem cell activity and exerts potent anti-inflammatory effects through NF-κB pathway modulation. Building on these mechanistic insights, we will develop transformative wound dressings that integrate a novel rapid-gelling antimicrobial hydrogel with recombinant human MG53 (rhMG53) protein. We hypothesize that this synergistic combination will dramatically accelerate wound healing and tissue regeneration following chemical vesicant exposure. The goal of this project is to engineer and optimize multifunctional wound dressings combining rapid-gelation antimicrobial hydrogel technology with rhMG53 protein for vesicant-induced cutaneous injuries. Successful completion will yield breakthrough multifunctional wound dressings with integrated tissue repair, anti-inflammatory, and antimicrobial capabilities. These shelf-stable therapeutic platforms can be strategically stockpiled and rapidly deployed as essential medical countermeasures against chemical vesicant exposure, addressing both immediate clinical needs and national security preparedness requirements.

NEI - National Eye Institute

Project Summary/Abstract Vision loss is a devastating medical problem as it can lead to reduced productivity, lower quality of life, and loss of independence. Glaucoma affects over four million Americans and 76.0 million people worldwide, making it the second leading cause of irreversible blindness globally. The disease is characterized by retinal ganglion cell degeneration with consequent loss of the axons that connect the eye to the brain and progressive damage to the optic nerve. Pharmacological and surgical interventions that lower intraocular pressure can slow or even stop retinal ganglion cell degeneration. However, many patients do not seek medical attention until the disease is advanced and others continue to experience disease progression despite treatment, ultimately resulting in the widespread loss of retinal ganglion cells and profound vision loss. Unfortunately, the human retina has minimal regenerative capacity and cannot replace lost retinal ganglion cells, making vision loss in these patients permanent. The candidate’s long-term career goals are to advance our understanding of gene regulatory networks directing retinal ganglion cell differentiation during development and to apply these insights to formulate strategies for regenerating retinal ganglion cells from dormant progenitor cells in the adult retina. The proposed career development and training plans will allow the candidate to acquire further expertise in retinal development and regeneration. By learning additional cutting-edge experimental techniques, the candidate will also enhance the scientific rigor and impact of their research program. In the first specific aim, the candidate will investigate the role of transcription factor Pou2f2 in retinal ganglion cell development using conditional gene deletion, as well as gain-of-function by in vivo electroporation of postnatal progenitors. In the second and third specific aims, the candidate will perform an in vivo screen of more than 40 candidate transcription factors to identify a combination capable of reprogramming Müller glia into retinal ganglion cells. Further studies will focus on characterizing induced retinal ganglion cell morphology, laminar position, axon extension, electrophysiology and gene expression. Adaptive optics will be used to longitudinally image the reprogramming process in vivo. Lastly, the candidate will investigate survival and circuit integration of these newly generated retinal ganglion cells in mouse models of glaucoma. Because retinal ganglion cells are widely used as a model for axonal regeneration in vertebrates, these studies have broader implications for regeneration of central nervous system neurons and pathways. The candidate will conduct the proposed research in collaboration with co-mentors Dr. Yvonne Ou and Dr. Xin Duan, and the other members of the advisory committee. Experiments will take place in Rock Hall, where the candidate has dedicated laboratory space in close proximity to all collaborators. The UCSF Department of Ophthalmology is a leading center for vision science research, providing the candidate with access to NEI P30 funded core facilities and extensive university-wide resources. The candidate will also benefit from interactions with the broader neuroscience and stem cell research communities at UCSF.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Sex-based differences in ventricular cardiac pharmacology are well known but remain poorly predicted and understood. In clinical populations, women have a prolonged QT interval when compared to their male counterparts. The female sex-specific prolonged QT phenotype is thought to be linked to disproportionately higher rates (2-4X) of life-threatening drug-induced arrhythmias, such as drug-induced long QT and torsade’s de pointes. This sex-dependent response is often credited to higher expression and function of potassium channels in males and higher calcium handling ability in females. However, previous research into each of these causes is conflicting, often confounded by model-to-model variability and genetic heterogeneity. Additionally, differential sex hormone expression, such as estrogen and testosterone, has been tied to these responses, making the roles of chromosomal sex versus the role of sex hormones difficult to isolate in current pre-clinical model systems. Recently, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC- CM) have emerged as a promising model to predict adverse drug effects. However, a major limitation with these models is the genetic heterogeneity required between male and female lines, making novel discoveries difficult to claim. To address this gap, the proposed project uses isogenic hiPSC-CMs derived from a patient with mosaic Klinefelter Syndrome (XXY) to systematically investigate sex-based differences in the myocyte electrophysiological response to antiarrhythmic drugs. Ventricular XX and XY iPSC-CM were validated thorough flow cytometry, imaging and functional data. Using optical fluorescence assays, it was found that this model system recapitulates the significantly prolonged AP phenotype in XX-CM, which aligns with the prolonged QT interval seen in clinical female populations, both at baseline and under drug perturbation when compared to their male counterparts (Aim 1). This project aims to further quantify this relationship utilizing patch clamping, optical assays, in conjunction with hormonal supplementation (Aim 2) to further understand the role of sex as a complex variable in cardiac pharmacology. By establishing this platform, we aim to predict whether novel reagents have a sex dependent likelihood to become proarrhythmic, informing de novo drug development. The long-term goal is that the introduction of this model system in both cardiac and eventually other tissue types will provide fundamental knowledge about sex differences within human systems to enhance patient health, develop new solutions to reduce illness, and increase quality of life.

OD - NIH Office of the Director

Project Summary Accidental exposure to ionizing radiation (IR) poses a significant risk for cardiovascular morbidity, a leading cause of mortality among irradiated populations. However, the mechanisms driving long-term cardiovascular risks remain poorly understood. IR disrupts immune homeostasis, exacerbating chronic inflammation and accelerating pathological remodeling. The current multi-PI U01 proposal seeks to address this knowledge gap by developing an innovative extracorporeal system composed of human stem cell derivatives to identify biomarkers and medical countermeasures (MCMs) for radiation-induced delayed cardiac remodeling (RidCR). In Milestone 1, we will establish a 3D cardiac-immune co-culture system using iPSC-derived cardiomyocytes, endothelial cells, fibroblasts, and macrophages to simulate the immune-competent 3D microenvironment. Optimized culture conditions will be validated in 3D engineered cardiac tissues (EHTs) for physiological and inflammatory responses. In Milestone 2, we will perform multi-omics analyses on irradiated EHTs and parallel animal models to identify molecular signatures of RidCR via multi-omics and functional assessments of tissue contractility. In Milestone 3, AI/ML will integrate the generated datasets to predict candidate MCMs, which will be validated in vitro and tested in vivo using a protracted irradiation mouse model. Comprehensive evaluations will assess the efficacy of the lead MCM in mitigating RidCR.

NINDS - National Institute of Neurological Disorders and Stroke

Project summary/Abstract Variants in the ubiquitin like modifier activating enzyme 5 (UBA5) result in an ultra-rare autosomal recessive disease with neurological presentations. UBA5 patients present with infantile spasms, failure to thrive, hypotonia, developmental delay, microcephaly, intellectual deficit, loss of motor skills and seizures. Most of the patients die in childhood. Current standard of care for UBA5 deficiency is focused on managing the clinical signs with standard anti-seizure medications or surgical procedures and physical therapies, but there is no treatment. Compound heterozygous mutations in UBA5 causes impairment in a ubiquitin-like post-translational modification pathway called Ubiquitin-fold modifier 1 (UFM1). UBA5 is an E1 activating enzyme on UFM1 pathway. The role of the UBA5 and UFM1 system in the central nervous system (CNS) has not been studied. This stems from lack of a viable mammalian model for UBA5 deficiency. Our team has identified the first viable Uba5 mouse model that carries patient mutation, exhibits an overt phenotype, and recapitulates presentations of UBA5 deficiency in patients including smaller body size, motor, cognitive and gait abnormalities. Our team has postmortem tissues of UAB5 patients, their clinical course, MRI and EEG records. The first neuropathological characterization of postmortem UBA5 patient brain indicates the shared features with Uba5 mice. To determine the top adeno associated virus (AAV) vector candidate for efficacy studies in Uba5 mouse, we developed four UBA5 expressing constructs and showed their 1) efficacy in restoration of expression and function of UBA5 in UBA5 Knockout HEK293T cells and 2) durability, safety, and cell type tropism in a one-year study in wild type mouse. The top candidate, AAV9-JeT-UBA5, restored motor, cognitive and most aspect of gait abnormalities in Uba5 mouse model treated by neonatal intracerebroventricular (ICV) treatment. However, weight of treated Uba5 mice did not get normalized. We hypothesize that gradual loss of transduced cells in liver prevented long term weight gain normalization. Since the overarching goal of this project is to develop a transformational AAV gene therapy to treat our symptomatic UBA5 patient cohort at UMass Chan, we need to address the therapeutic imperfections and develop biomarkers. We will perform CNS and periphery wide gene therapy of JeT-UBA5 in pre and post symptomatic Uba5 mouse to determine the therapeutic window and feasibility of gene therapy to rescue or modify disease course. We will use 1) AAV9 capsid for combined CSF and periphery wide gene delivery in Uba5 mouse and 2) a new blood-brain barrier penetrant capsid (BI-hTFR1; interact with human Transferrin Receptor (TFRC)) for very efficient gene delivery to CNS and periphery by systemic injection. We will perform an in-depth characterization of the Uba5 mouse model and its humanized version, expressing TFRC, with clinically relevant outcomes measure (MRI and EEG) and compare them with patient findings (Aim 1). Four gene therapy approaches will be performed in Aim 2. In Aim 3 metabolomic based biomarker discovery will be performed and the best gene therapy approach to normalize transcriptomic profile of Uba5 mouse of will be determined.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that establishes lifelong latency and poses a serious threat to immunocompromised individuals and developing fetuses. A major barrier to developing effective antiviral therapies is our limited understanding of the mechanisms governing HCMV latency, due in part to challenges in modeling this process in physiologically relevant human cells. This R03 project proposes to establish adipose-derived stem cells (ADSCs) as a novel, scalable, and biologically relevant in vitro model of HCMV latency. We will define the conditions under which ADSCs support latent infection and compare their transcriptional program to established latency models using RNA sequencing. This research will advance our understanding of HCMV latency and provide a critical tool for future studies aimed at developing therapies to prevent viral reactivation in vulnerable populations.

NCI - National Cancer Institute

PROJECT SUMMARY Immunotherapies such as bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR) T cells have shown significant promise in treating hematologic malignancies. However, therapeutic responses vary between patients and across the different subtypes of leukemia and lymphoma. A key challenge in optimizing these therapies is the lack of in vivo preclinical models that accurately reflect both the patient's immune and cancer cell biology. Current patient-derived xenograft (PDX) models lack functional immune systems, while humanized mouse models typically involve healthy donor immune cells paired with cancer cells from a different donor. These immunologically mismatched, or allogeneic, models fail to replicate autologous immune-cancer cell dynamics and the effect the cancer microenvironment and therapy have on immune cell function. To overcome these limitations, we aim to develop innovative autologous humanized PDX models using leukemia and immune cells derived from the same patient. We will collect paired bone marrow (BM) samples from pre-B acute lymphoblastic leukemia (B-ALL) patients at diagnosis and remission. Hematopoietic stem and progenitor cells (HSPCs) from the remission BM will be expanded and transplanted into immunodeficient mice to generate humanized mice with intact immune systems. These mice will then be engrafted with diagnostic leukemia cells, creating PDX models with autologous immune and leukemia cells. In parallel, a second cohort of models will be developed using autologous peripheral blood mononuclear cells (PBMCs) and leukemia cells from the same patient. We hypothesize that these fully patient-derived autologous models will provide deeper insights into immune-leukemia interactions and enhance preclinical testing of immunotherapies. To test this hypothesis, we will pursue three specific aims. First, we will establish autologous PBMC- and HSPC- humanized models using samples from B-ALL patients with diverse genetic and risk subtypes. We will evaluate disease progression and immune responses longitudinally in these models. Second, we will assess the efficacy of a CD3/CD19 BiTE and CD19 CAR-T cells in these autologous models. Third, we will analyze the immunophenotypic and transcriptional profiles of cells from autologous models, allogeneic models, and patient samples before and after immunotherapy treatment to validate the translational relevance of the models. These studies aim to create robust, patient-specific models that can be used to test and optimize immunotherapies, ultimately improving their clinical impact in the treatment of hematologic malignancies.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Immunotherapy has revolutionized the treatment of many tumors. However, most GBM patients have not, so far, benefited from immunotherapeutic treatment. With the goal of exploring ways to boost anti-GBM immunity, we’ve developed a B-cell-based vaccine (BVax) that consists of 4-1BBL+ B cells activated with CD40 agonism, BAFF and IFNγ stimulation. BVax migrate to key secondary lymphoid organs and are proficient at antigen cross-presentation, which promotes both the survival and functionality of CD8+ T cells. A combination of radiation, BVax, and PD-L1 blockade conferred tumor eradication in 80% of treated tumor-bearing animals. We have been successful at generating GBM patient-derived BVax that activated autologous CD8+ T cells, which shows a strong ability to kill autologous glioma cells. This demonstrates that BVax can be produced from patient’s peripheral blood. Our preliminary data obtained under the parental 5R37CA258426 proposal showed that BVax promotes the expansion of clones that differ from CD8 T cells activated by dendritic cells (DC) and the proliferation of stem-like TCF-1+ CD8 T cells. In addition, we provided solid evidence that BVax produces antibodies that react to tumor-associated antigens and inhibit tumor growth. Our central hypothesis is that the BVax have unique properties as antigen-presenting and antibody- producing cells. More specifically, BVax might present a different set of antigens to CD8 T cells. In addition, BVax monoclonal antibodies (mAbs) might have a potential therapeutic effect. This research proposal aims to deep-dive into the immune mechanisms underlying this protection and prevention of tumor growth. We will focus on two processes: antigen presentation and activation of CD8+ T-cell memory formation (Aim 1) and the characterization (sequencing and cloning) of single-BVax monoclonal Ab production (Aim 2). Overall, our study provides a novel alternative to current immunotherapeutic approaches that can be readily translated to the clinic.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT There are two subgenera of βCoVs, Merbecovirus and Sarbecovirus, that cause severe disease in humans, for which broadly protective medical countermeasures are needed. While conserved sites of vulnerability on the virus Spike, called the receptor binding domain (RBD) and stem helix (SH), have been noted for merbecoviruses (MERBs) and sarbecoviruses (SARBs), antibodies to these epitopes tend to be infrequently elicited during infection or with current treatments. Our scientific premise is that nanoparticle (NP) S fragment immunogens elicit high titers of broadly neutralizing antibodies by focusing the immune response on subdominant epitopes. We proposed here to employ our innovative NP design technology to elicit broadly protective immunity against both subgenera of viruses. In rigorous preliminary studies, our RBD NP elicited neutralizing antibodies and protected mice against MERB MERS-CoV and four different SARBs. Also, our RBD NPs elicited neutralizing antibodies against 11 different SARBs, 5 different MERBs, and protected monkeys against SARB and MERB infection. Our central hypothesis is that neutralizing antibody breadth can be increased to cover many viruses within a βCoV subgenera by simultaneously eliciting a polyclonal, high-affinity antibody response to RBD and SH on CoV S. To determine the extent that polyclonal responses are needed, Specific Aim 1 will determine the antibody clonality and immunogenetics features that afford broad MERB and SARB neutralization. No known antibodies that bind to RBD and neutralize both MERBs and SARBs have been identified to date. The work in this aim will define the antibodies that mediate neutralization of each subgenus of viruses in order to understand the structural and genetic basis for how the neutralization breadth of these antibodies is limited within or across subgenera. In Specific Aim 2, we will further optimize our NP immunogens to increase the breadth of antibody response they elicit. Guided by the information about what viral sequences are resistant to the current induced antibodies, new immunogens will be designed using computational structural and bioinformatic approaches. These new immunogens will be tested for the ability to elicit broader neutralizing antibodies in mice than a commercial drug and our earlier immunogen designs. In Specific Aim 3, we will define the Ab responses that correlate with protection against SARBs and MERBs. Successful immunogens will be examined for their ability to elicit protective immunity against both Merbecoviruses and Sarbecoviruses in innovative mouse challenge models uniquely available to this team. Upon completion of this study, its impact will be the elucidation of new design approaches to target and protect against large subgenera of viruses rather than a single virus. Also, the final immunogen designed here, can be manufactured in the future to provide a countermeasure against deadly viruses for which there is no current treatment.