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Friends of STEM Technology

Youth participants will attend a Science Technology Engineering and Math summer camp where they will have two opportunities to enhance their video skills. They will learn the technical design process all programmers use to create video games. They will also assist in creating, writing and producing a basic video that will be viewed in a public venue upon completion.

NHLBI - National Heart Lung and Blood Institute

Summary The overarching goals of this proposal are to identify pathways necessary for the maturation of pre-hematopoietic stem cells (pre-HSCs) into long-term, multi-lineage adult-repopulating HSCs. Our primary focus is on the transforming growth factor beta (TGFβ) and/or bone morphogenetic protein (BMP) signaling pathways, based on our discovery that a negative regulator of these pathways, mothers against decapentaplegic homologue 7 (SMAD7), is important for efficient pre- HSC to HSC maturation in mouse embryos. Current stepwise protocols for producing long-term multi-lineage adult-repopulating HSCs from induced pluripotent stem cells (iPSCs) modulate TGFβ and BMP signaling at early stages of the culture, but not during the later pre-HSC to HSC maturation stage. We will use a quantitative ex vivo culture system to systematically interrogate which components of the TGFβ/BMP signaling pathway must be inhibited, and when, to improve HSC maturation from SMAD7-deficient pre-HSCs. We will also determine the molecular changes that occur over the course of pre-HSC to HSC maturation using clonal barcoding single-cell RNA sequencing to identify pre-HSCs that have undergone successful HSC maturation and develop transcriptional profiles of HSC-forming pre-HSCs throughout culture. Finally, we will apply the knowledge we gain by studying this process in mouse embryos to optimize the generation of HSCs iPSCs.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Human immunodeficiency virus (HIV) targets the immune cells and weakens our defense against many infections and cancer. Without treatment, HIV infection advances to acquired immunodeficiency syndrome (AIDS). Although combination antiretroviral therapy has dramatically improved clinical outcomes, the rapid development of drug-resistant HIV strains limits the selection of effective therapies available for patients. Thus, there is an urgent need to identify alternative viral targets for inhibition. An essential yet poorly understood step in HIV replication is the Rev-response element (RRE)-mediated nuclear export of viral RNAs. During HIV infection, partially spliced and unspliced viral RNAs need to be exported from the nucleus to the cytoplasm for viral protein synthesis and virion assembly. This process depends on a specific interaction between the viral protein Rev and RRE present in the incompletely spliced viral RNAs. Multimeric Rev proteins bind to the RRE structure and recruit the nuclear export complex for cytoplasmic translocation of the viral RNAs. Despite the essential function in HIV replication, the RRE-Rev complex is currently an unexploited target in HIV chemotherapy, largely due to the lack of the structural information on the full-length RRE. Using a tRNA-scaffold approach, we previously determined the crystal structure of RRE stem-loop II, the initial Rev protein binding site. In this proposal, we will (1) determine the structure of the full-length RRE, (2) test the sequential and cooperative mechanism of Rev assembly, and (3) develop antisense oligonucleotide and de novo protein inhibitors that disrupt RRE-Rev interaction. Since RRE-Rev interaction is absolutely required for viral RNA export and virion assembly, targeting this complex has strong potential to suppress HIV replication and reduce viral burden in HIV- 1 infected individuals.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract The development of effective vaccines against complex pathogens like HIV-1 requires immunogens that elicit broadly neutralizing antibodies (bnAbs). Traditional vaccine development strategies, including live- attenuated or subunit vaccines, and newer approaches leveraging genomic data, have not consistently induced bnAbs against HIV-1. Challenges stem from the unique structural and functional properties of bnAbs, such as improbable somatic mutations, autoreactivity, and long heavy-chain complementarity determining region-3 (HCDR3) loops. While priming immunogens have shown promise in selecting bnAb precursors, the production of mature bnAbs remains elusive, even in models engineered to ensure the presence of the relevant B-cell receptors. Our preliminary findings highlight the critical role of the transition state between unbound and bound antibody-antigen interactions in determining HIV-1 neutralizing antibody affinities. These transient structural states, which influence affinity maturation, neutralization breadth, and viral evolution, are poorly understood. Current methodologies primarily focus on static structural determinants of binding, neglecting the dynamic encounter complexes and intermediates that govern antibody-antigen association. This proposal aims to elucidate the association transition states and related intermediates for V2 apex- and CD4- binding site-directed bnAbs. Using structural and kinetic analyses, we will determine how variations in HIV-1 Env influence these transition states and how bnAbs overcome association barriers posed by Env sequence diversity. By dissecting the residue-level processes involved in antibody-antigen association, we will identify critical factors driving bnAb maturation and viral escape mechanisms. Using this information and large affinity datasets based on antibody clone HIV-1 Envelope interactions, we will develop cutting edge artificial intelligence/machine learning models to design immunogens with favorable affinity gradients for maturing bnAbs. Our work will address significant gaps in the understanding of antibody-antigen association, transitioning from phenomenological models to precise structural definitions of these poorly understood states and will directly use this information to inform immunogen development.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV) are leading causes of severe respiratory illness in infants, the elderly, and immunocompromised individuals, yet effective treatments and vaccines remain elusive. This critical unmet medical need stems from a limited understanding of the structural and molecular mechanisms governing how these viruses assemble and spread within host cells. This proposal will leverage cutting-edge cryo-electron tomography (cryo-ET), cryo-correlative light and electron microscopy (cryo-CLEM), and cryo-focused ion beam (cryo-FIB) milling, complemented by molecular biology and biochemical approaches, to provide unprecedented in situ structural insights into these processes. We hypothesize that distinct, yet conserved, molecular interactions between viral proteins and host factors orchestrate the precise formation of infectious virions, and disrupting these interactions represents a viable therapeutic strategy. Aim 1 will elucidate the high-resolution structural architecture of RSV and HMPV virion assembly directly within infected cells. We will employ cryo-FIB milling, cryo-ET, volume segmentation, and sub-tomogram averaging to determine the precise spatial organization and structures of viral components, identify structural intermediates at the plasma membrane, and investigate the involvement of host cellular machinery and membrane microdomains. Aim 2 will define the molecular mechanisms governing the critical nucleocapsid-matrix-envelope-glycoprotein linkages essential for RSV and HMPV assembly. This involves mapping protein-protein interfaces and characterizing conformational changes using cryo-EM/ET and biochemical assays, followed by functional validation of targeted mutations to assess their impact on virion formation and infectivity. Aim 3 will characterize the structural basis and functional differences between RSV and HMPV F proteins and their impact on virion morphogenesis. We will determine native high-resolution cryo-ET structures of F proteins, including HMPV F in complex with host receptors. Cryo-CLEM will visualize the localization and clustering of F protein during assembly and assess how mutations affect attachment, fusion, and virion infectivity. Collectively, this research will provide fundamental mechanistic understanding of pneumovirus assembly and dissemination in situ, directly informing the rational design of next-generation antivirals and other broadly protective therapeutics to combat these significant global health threats.

NIAID - National Institute of Allergy and Infectious Diseases

Hepadnaviruses are partially double-stranded DNA viruses that replicate by protein-primed reverse transcription. This family includes duck hepatitis B virus (DHBV) with which hepadnaviral reverse transcription was discovered and human hepatitis B virus (HBV) that kills 1,100,000 people annually. HBV therapy primarily employs nucleos(t)ide analog drugs that target the viral reverse transcriptase (RT) activity. Reverse transcription is catalyzed by the 4-domain viral polymerase (P) which has protein priming, RT, and ribonuclease H (RNase H) activities. The TP and spacer domains are unique to the hepadnaviruses. Reverse transcription starts with chaperone-mediated binding of P to the ε stem loop on the viral pregenomic RNA (pgRNA). Reverse transcription is primed by a tyrosine in P’s terminal protein domain (TP), templated by ε. The RT synthesizes the first strand of the viral DNA, and the RNase H destroys the pgRNA to permit synthesis of the second DNA strand. P is a monomer, and the covalent linkage between P and the DNA persists throughout reverse transcription. Hepadnaviral protein-primed reverse transcription differs greatly from retroviral reverse transcription, but its enzymology is poorly understood even though HBV P is a major drug target. This knowledge gap is in part due to the inability to determine the structure of P. We recently predicted the structure of P and validated the model. This revealed a novel fold in which the TP domain that primes reverse transcription is cupped over P’s catalytic core of P, with the priming tyrosine on a loop over the RT active site. This model makes mechanistic predictions regarding reverse transcription and provides guidance for how to test the hypotheses. Premise: The molecular model of P enables in-depth mechanistic analyses of P structure, nucleic acid binding, and DNA priming by the enzyme for the first time. Aim 1. What P sequences are needed for ε binding and priming? We will define the minimal active form(s) of P for RNA binding and DNA priming, and identify residues of P that contact ε and are essential for priming. Aim 2. What are the structural alterations to P associated with the shift from the priming-incompetent to priming-competent state? We will define how the TP domain binds to the catalytic core of P, explore P’s conformational shifts during priming, and determine how key RNA binding motifs are exposed during ε binding. Aim 3. How do conformational dynamics of P contribute to ε binding and priming? We will probe how P’s flexibility affects ε binding and DNA priming using molecular dynamics plus pharmacological and mutational analyses. We will test effects of mutations affecting RNA binding and DNA priming on viral replication in cells. This study will fill major gaps in our understanding of hepadnaviral reverse transcriptase enzymology by defining the interactions holding P in its novel conformation, how P binds to ε, and how enzyme flexibility contributes to the early phases of reverse transcription. It will also provide key information needed to develop non-active site inhibitors of HBV P to improve therapy for HBV patients.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Modern medicine's greatest pharmaceutical achievement has been the development of antibiotics, which revolutionized healthcare by enabling complex surgical procedures with substantially reduced infection risks. However, we now face an impending medical catastrophe as antimicrobial resistance threatens to usher in an era where these life-saving drugs become ineffective. Throughout history, discovering compounds effective against double-membrane bacterial pathogens has presented significantly greater challenges than targeting single-membrane Gram-positive organisms. This difficulty stems from the distinctive asymmetric outer barrier that double-membrane bacteria possess alongside their inner cellular boundary. Since most antimicrobial targets lie beyond this outer barrier, therapeutic compounds must successfully traverse it to exert their effects. Unfortunately, this outer structure demonstrates exceptional efficiency at preventing small molecule passage, presenting a formidable obstacle for researchers. The initial golden period of antimicrobial discovery capitalized on naturally occurring compounds that could be easily identified through conventional screening approaches. However, these traditional methods have yielded diminishing returns over recent decades, making new discoveries increasingly elusive. The future of antimicrobial development may be transformed by our expanding repositories of protein, genetic, and metabolic information, which could unveil promising therapeutic targets. Both academic institutions and pharmaceutical companies might harness these datasets to engineer small molecule therapeutics with enhanced potency and selectivity. To achieve this goal, we seek to formulate foundational principles that define the molecular characteristics governing cellular penetration in bacteria, similar to Lipinski's Rule of Five framework.

National Institutes of Health

The NIH Research Education Program (R25) supports research education activities in the mission areas of the NIH. The over-arching goal of this R25 program is to support educational activities that foster a better understanding of biomedical, behavioral and clinical research and its implications. To accomplish the stated over-arching goal, this FOA will support creative educational activities with a primary focus on research experiences for high school or undergraduate students or science teachers during the summer academic break. The proposed program needs to fit within the mission of the participating IC that the application is being submitted to and should not have a general STEM focus (see below and Table of IC-Specific Information and Points of Contact).

National Institutes of Health

The NIH Research Education Program (R25) supports research education activities in the mission areas of the NIH. The over-arching goal of this R25 program is to support educational activities that foster a better understanding of biomedical, behavioral and clinical research and its implications. To accomplish the stated over-arching goal, this FOA will support creative educational activities with a primary focus on research experiences for high school or undergraduate students or science teachers during the summer academic break. The proposed program needs to fit within the mission of the participating IC that the application is being submitted to and should not have a general STEM focus (see below and Table of IC-Specific Information and Points of Contact).

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Cardiovascular disease remains a leading cause of morbidity and mortality, despite significant advances in surgical techniques and medical therapies. The development of novel diagnostic, treatment, and prevention strategies requires an in-depth understanding of cardiovascular pathophysiology, paired with advanced engineering and computational expertise. However, a critical gap exists in formal training opportunities for medical students to integrate surgical and engineering skills, contributing to a growing deficit of cardiovascular surgeon-scientists. To address this need, we propose the Surgery, Technology and Engineering Mentorship for Medical Students (STEMS) short-term training program at Washington University in St. Louis. This program is founded on the premise that early engagement of post-baccalaureate medical students will inspire their pursuit of academic careers in cardiovascular surgery while accelerating the development of innovative technologies for diagnosing, treating, and preventing cardiovascular diseases. Research topics will include critical areas such as cardiac arrhythmia, coronary artery disease, peripheral vascular disease (arterial/venous), aortic aneurysmal disease, and neurovascular disease. With a diverse and accomplished faculty mentorship network, robust institutional support, and established multi-disciplinary research programs, the STEMS training program will equip trainees with synergistic skills in translational research and engineering to address complex challenges in this field. Graduates of this program will emerge with foundational knowledge to pursue impactful future academic and research pursuits in the diagnosis, management, and prevention of cardiovascular diseases.

NEI - National Eye Institute

PROJECT SUMMARY Millions of elderly individuals suffer from visual impairment due to dry age-related macular degeneration (AMD), for which effective treatments are limited. In advanced stages of dry AMD, the gradual loss of retinal pigment epithelial (RPE) cells leads to the death of overlying photoreceptors, causing progressive vision loss and eventual blindness. Transplantation of healthy RPE to replace lost/diseased RPE cells have shown in rodent studies to rescue rod and cone photoreceptors, improve retinal electrophysiological responses, and enhance visual thresholds over extended periods. However, these studies have used xenogeneic (across species) cell transplants, which require immune suppression (IS) to prevent the host’s immune system from rejecting the transplanted cells. Similarly, allogeneic (same species) RPE cell transplants in non-immune suppressed models, including pigs and nonhuman primates, are typically rejected within three weeks. Although IS appears currently necessary and sufficient to protect transplanted cells, it raises significant safety concerns, especially for elderly AMD patients who may experience toxic side effects from long-term or indefinite IS use. Additionally, IS introduces challenges such as patient compliance and the risk of rejection with suboptimal dosages. Current Phase I/II clinical trials using allogeneic RPE cells combine multiple IS medications to prevent rejection, but the majority of adverse effects stem from the IS regimen rather than the cell therapy itself. To address the complications of IS, the NIH has initiated an autologous RPE cell trial, despite the high logistical and cost barriers to broad implementation. In contrast, our approach focuses on developing a scalable allogeneic cell-based therapy that can avoid immune rejection, providing greater access, efficiency, and lower costs. We have demonstrated feasibility of this approach in multiple settings including short-term RPE cell transplants in the eye in non-immune suppressed NHPs. In the proposed studies, we will generate multiple lines of allogeneic induced pluripotent stem cells (iPSCs) from nonhuman primates (NHPs) and engineer them to lack expression of class I and II major histocompatibility complexes and to overexpress the “don’t eat me” signal, CD47. We will then optimize the differentiation of these modified iPSCs into RPE cells for transplantation studies in both normal and diseased NHP retinas. Transplantation studies will include short and long-term survival and in diseased retinal conditions to replicate acute version chronic rejection in normal and diseased retinal environments. Finally, we will optimize the use of a safety switch to enable selective removal of cells in the subretinal space should that ever be necessary. These studies will demonstrate the potential of gene-modified RPE cells to evade immune rejection while maintaining a high safety profile and will help identify factors in retinal disease environments that may affect the survival of transplanted RPE cells. Successful completion of these aims will lay the groundwork for translating these studies toward clinical application.

NCI - National Cancer Institute

Abstract H-FIRE is an emerging, minimally invasive focal ablation technique that utilizes low energy, microsecond-long electric pulses delivered locally within targeted tissues for several minutes. Tumor cell death is rapidly induced through a loss of cellular homeostasis and is not dependent on thermal effects. Due to its non-thermal mechanism, H-FIRE has been shown to preserve important tissue components such as the extracellular matrix, blood vessels, and nerves, as well as the immunogenicity of tumor antigens compared to other ablation modalities. H-FIRE can ablate predictable volumes of tissue without the need for neuroparalytics as is required for its FDA-approved predecessor, and safe and effective H-FIRE treatment has been demonstrated in rat glioma models and canine patients with spontaneous brain tumors. Key advantages of H-FIRE for GBM treatment include i) glioma and glioma stem-like cell specific ablation resulting from tuned H-FIRE pulses; ii) enhancement of ablation through combination with a targeted molecular adjuvant; iii) reversible BBB breakdown and enhanced delivery of molecular therapeutics from the dissipating H-FIRE field well beyond the tumor margin; iv) electrical feedback for monitoring and modeling treatment; and v) activation of innate and adaptive anti-tumor immune responses from intracranial H-FIRE. These advantages motivate the central hypothesis for this effort, specifically that H-FIRE combined with the cancer adjuvant immunomodulatory drug, indoximod, will overcome key drivers of therapy resistance by combining effective ablation of a core tumor mass with enhancing delivery and efficacy of a small molecule drug that is normally blocked from entry by the BBB. The project has two aims. In Aim 1, pulse parameters will be evaluated on their ability to completely and selectively ablate malignant tissue in comparison to normal brain tissue, to corroborate preliminary in vitro data using a more relevant in vivo glioma rat model. In Aim 2, the in vivo synergy resulting from the combination of H-FIRE with indoximod will be quantified using an orthotopic rat GBM model. Computational models and in vitro experimentation are highly adaptable, allowing real-time data collection and analysis. However, fully replicating the intricate brain environment and the complete surgical procedure involving the skull can only be accurately assessed using an in vivo animal model. In selecting the species and strain, female and male Fischer rats were chosen because no lower species model is available that uniquely support the electrode configuration for in vivo brain electroporation studies. A mammal is needed for adequate analysis of tissue ablation. Additionally, species matching will allow us to make comparisons to previously completed studies. These aims will lead to independently optimized H-FIRE protocols for i) ablation of a tumor bulk resulting in effective anti-tumor immune stimulation and ii) reduction of barriers to the efficacy of indoximod adjuvant therapy. Intraoperative MRI-guided electrode insertion will allow for highprecision electrosurgery and fine-tuned BBB disruption and brain cancer treatment efficacy in preparation for treating canine patients, with the expectation to translate those results to human patients

NIA - National Institute on Aging

Project Summary Alzheimer’s disease (AD) is the leading cause of dementia, affecting over 6.7 million people in the United States, yet the mechanisms underlying its pathology remain incompletely understood. In virtually every case of AD, extracellular amyloid-beta (Aβ) plaques and intracellular Tau neurofibrillary tangles co-occur, suggesting significant interplay between these two proteins. Although a direct link between Aβ plaque formation and Tau tangle deposition is still being elucidated, recent evidence indicates that soluble, intracellular Aβ oligomers can bind Tau and promote its aggregation. Both Aβ and Tau are intrinsically disordered proteins that undergo liquid– liquid phase separation (LLPS), forming protein-dense biomolecular condensates that can either facilitate physiological function or propagate pathological aggregation in neurons. Our preliminary data show that Tau condensates can adopt either a physiological state, recruiting tubulin to stabilize microtubules and resist aberrant aggregation, or a pathological one, where Aβ recruitment triggers fibril formation. Accordingly, we seek to characterize how Tau:Aβ interactions within condensates drive pathological aggregation, clarifying the kinetics of oligomeric species formation and identifying potential therapeutic targets. Additionally, we will determine whether reinforcing functional Tau:tubulin interactions can prevent or reverse the formation of pathogenic Tau:Aβ assemblies. In Aim 1, I will define how Aβ alters the structural properties of Tau condensates to drive pathological aggregation. Specifically, I will employ advanced biophysical techniques, including fluorescence lifetime imaging microscopy (FRET-FLIM) and microfluidic modulation infrared spectroscopy (MMS), to investigate whether Aβ accelerates Tau oligomerization within condensates and promotes β-sheet–rich, aggregation-prone structures. I will also delineate how these protein assemblies within condensates differ in kinetics and stability from analogous reactions occurring in dilute conditions. In Aim 2, I will determine whether tubulin can rescue Tau from this Aβ-driven pathology by reinforcing physiological Tau:tubulin interactions. Using an optogenetic system in neuronal cell lines, I will manipulate tubulin levels genetically and pharmacologically to test how tubulin enrichment affects Aβ-driven Tau aggregation, as measured by high-molecular-weight oligomers and hyperphosphorylated Tau. I will extend these findings to patient-derived induced pluripotent stem cell (iPSC) neurons to validate whether tubulin stabilization similarly protects against endogenous Tau pathology in a disease-relevant context. Upon completion of these studies, I will have established a mechanistic framework for how intracellular Aβ accelerates Tau aggregation via condensates, and how tubulin might mitigate this transition. These insights will contribute to our understanding of early-stage AD pathology and may inform future therapeutic strategies aimed at preserving or restoring physiological condensate function.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

Synthetic Genetic Controller Circuits for Transcription Factor-Directed Differentiation PI: Domitilla Del Vecchio1,2,3 co-I: James J. Collins2,3,4,5 co-I: Thorsten Schlaeger6 1Department of Mechanical Engineering, MIT; 2Department of Biological Engineering, MIT 3Synthetic Biology Center, MIT; 4Broad Institute of MIT & Harvard; 5The Wyss Institute 6 Stem Cell Transplantation Program, Boston Children’s Hospital PROJECT SUMMARY The ultimate goal of this project is to create synthetic genetic circuits that accurately control the level of cell fate- specific transcription factors (TFs) autonomously in response to cell state changes. The underlying hypothesis is that the level and timing of expression of critical TFs dictates the efficiency of cell conversion protocols and the quality of produced cells. Here, we focus on the differentiation of human induced pluripotent stem cells (hiPSCs) into hemogenic endothelial cells (HECs) from which all hematopoietic stem and progenitor cells (HSC/HPCs) arise. Current methods to derive definite HECs (dHECs), which have the potential to produce adult-type lymphoid cells and HSCs, remain largely inefficient and are also difficult to execute and scale, and, as a consequence, exhibit high degrees of variability in out- comes between different labs, hiPSC lines, and even between replicate experiments.These problems hamper analysis of the underlying developmental processes and pose formidable obstacles to clinical translation of hiPSC-derived blood cell products since ensuring the safety and cost-effectiveness of the product necessitates high differentiation efficiency and consistency. Prior work has demonstrated that SCL (S), LMO2 (L), GATA2 (G), and ETV2 (E) TFs, when expressed in mesodermal cells, activate dHEC gene regulatory networks (GRNs) across species but also that efficient forward programming to dHECs requires discovery and subsequent implementation of both optimal expression levels and tim- ing for the TFs. Yet, conventional methods for TF-mediated cell fate programming generally rely on indiscriminate overexpression with little control on cellular TF levels and without cell state sensing. This is largely due to our inability to precisely control TF profiles during cell fate programming, and this limitation has prevented discovering optimal tra- jectories and subsequently enforcing them. Here, we propose synthetic genetic controller circuits that overcome this hurdle. In Aim 1, we create genetic circuit designs that set TF levels and use them in an efficient in vitro differentiation protocol to discover the optimal combination of S, L, G, E levels and timing. In Aim 2, we develop a circuit architecture, based on a novel TET1-enabled positive feedback system, to prevent epigenetic silencing of genetic circuits once de- livered to hiPSCs. In Aim 3, we make our genetic controller circuits enforce autonomously the optimal SLGE TF levels found in Aim 1 in response to the hiPSC-to-mesoderm transition. We achieve this by a new autocatalytic ADAR-based RNA sense-and-respond system, which senses the mesoderm marker Brachyury (TBXT) and enforces user-defined TF levels in response to it. We anticipate that this process, by being autonomous as opposed to manual and by enforcing optimal TF trajectories, will result in a more efficient, repeatable, and robust hiPSCs to dHECs conversion protocol, thereby helping fill the gap to clinical translation. Although in this project we tailor the genetic circuit designs to controlling SLGE TFs after sensing mesoderm-specific transcripts, the designs can be readily modified to express different TFs in response to any other cell type- or state-specific transcript. Therefore, we believe that the synthetic biology technology that we will establish will have broad impact on any other cell fate programming as well as on cell-or gene-therapy projects where expression levels and timing, as well as resistance to silencing, are important.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ABSTRACT Although allogeneic hematopoietic cell transplantation (HCT) remains the only curative option for many hematologic malignancies, its effectiveness is often undermined by prolonged immune deficiency following myeloablative conditioning. This leaves patients vulnerable to infections and relapse—the two leading causes of post-HCT mortality. This proposal investigates a novel, clinically translatable strategy to accelerate immune reconstitution through the adoptive transfer of ex vivo- generated T cell precursors (PreTs). These PreTs are derived from donor hematopoietic stem and progenitor cells (HSPCs) using a Good Manufacturing Practice-compliant Notch ligand-based platform. Preliminary data demonstrate that PreTs efficiently mature into T cells in vivo and promote de novo thymopoiesis, leading to functional antiviral and antitumor immunity without inducing graft- versus-host disease (GVHD) in murine allogeneic HCT models. In addition, PreT therapy enhances the generation of donor HSPC-derived innate immune cells, including NK cells, neutrophils, and monocytes, suggesting a broad immunoregenerative role that extends beyond adaptive immunity. The hypothesis of this project is that PreTs prevent infection and relapse by restoring T cell immunity alongside two T cell-independent mechanisms: (1) lineage plasticity, whereby PreTs give rise to NK cells with effector function; and (2) reprogramming of donor HSPCs to enhance their function and myeloid output. These mechanisms will be tested using fate mapping, single-cell multiomics, and functional assays in murine HCT, infection, and relapse models. This project addresses a central challenge in HCT: the lack of therapies that broadly accelerate immune recovery without exacerbating GVHD. It also provides a strong foundation for the candidate’s transition to research independence. Dr. Jennifer Tsai, MD, PhD, is a physician-scientist at Memorial Sloan Kettering Cancer Center (MSK). This K08 award will support her development through targeted training in molecular immune- oncology, single-cell immunogenomics, and translational research. Her career development plan includes close mentorship from Drs. Joseph Sun and Omar Abdel-Wahab, both of whom have outstanding records of mentoring scientists and physician-scientists to research independence. Their mentorship will be accompanied by formal coursework, computational training, and guidance from a multidisciplinary advisory committee with complementary fields of expertise and strong track records of collaboration. Along with exceptional mentorship, a highly engaged advisory team, and robust institutional support at MSK, this K08 award will position Dr. Tsai to launch an R01-funded career advancing next-generation cellular immunotherapies for transplantation and cancer.

NHLBI - National Heart Lung and Blood Institute

Tachycardia, or abnormally fast heart rate, is an important risk factor for cardiovascular morbidity and mortality. Prolonged tachycardia is known to induce cardiomyopathy in patients who have no prior structural heart diseases. Moreover, transient tachycardia, frequently observed in heart failure patients, can exacerbate the cardiovascular outcome. However, very little is known about the molecular drivers underlying tachycardia-induced cardiac dysfunction. This gap in our knowledge hinders the development of more effective heart failure treatment, especially for patients with hard-to-control tachycardia. This K99/R00 proposal will leverage recent advances in induced pluripotent stem cell (iPSC), tissue engineering, and multiomics technologies to uncover the molecular signaling pathways critically involved in the pathology of tachycardia-related heart disease. The applicant, Dr. Chengyi Tu, has established and validated an in vitro tachycardia platform using engineered heart tissue (EHT). In Aim 1, Dr. Tu will perform metabolomic and transcriptomic profiling of EHTs with or without tachypacing. To validate the physiological relevance of the EHT model, canine samples from tachypacing-induced heart failure will also be profiled. Preliminary data from the EHTs and the canine samples coherently indicate that the disruption of glycolysis homeostasis may underly the impairment of cardiac function by tachycardia. Metabolomics analysis shows that tachypacing in EHTs resulted in a selective accumulation of glycolysis intermediates such as glyceraldehyde 3-phosphate (GA3P) and 3-phosphoglycerate (3PG). Interestingly, promotion of fatty acid metabolism accelerated the recovery of cardiac contractility in tachypaced EHTs. Based on these novel results, Aim 2 will focus on elucidating how different glycolysis intermediate metabolites affect the function of cardiomyocytes, which has yet to be systematically examined. Lastly, Aim 3 (R00 phase) will employ state-of-the-art mass spectrometry workflow to screen for novel binding targets of glycolysis intermediates in cardiac cells, and examine the potential therapeutic benefits of manipulating these targets. This K99/R00 proposal will be guided by an excellent mentoring team with diverse expertise, including mentor Dr. Joseph Wu (iPSCs and cardiac biology), co-mentor Dr. Sanjiv Narayan (arrhythmia), advisors Dr. Michael Snyder (genetics and multi-omics), Dr. Yuqin Dai (metabolomics), Dr. Stanley Qi (CRISPR interference) and Dr. Beth Pruitt (bioengineering), as well as collaborators Dr. Fabio Recchia (canine model) and Dr. Donald Bers (cardiac physiology). To sum up, the completion of the proposed study will significantly advance our mechanistic understanding of how tachycardia adversely affects the heart, thereby creating new opportunities for therapeutic interventions. The proposed training will significantly strengthen and expand Dr. Tu’s research expertise, providing substantial momentum to his transition toward an independent cardiovascular researcher.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. Our studies in humanized mice and non-human primates (NHPs) have demonstrated that hematopoietic stem cells (HSPCs) based CAR therapy could provide life-long engraftment and production of functional CAR-T, CAR-NK and CAR-Macrophages (CAR-M), resulting in significantly reduced viral rebound after ART withdrawal. These studies underscore both the feasibility and efficacy of HSPCs-based CAR therapy. However, major challenges remain to achieve sustained viral remission in the absence of ART with current engineered immunity approaches. Mounting evidence has shown that environmental factors, such as metabolic regulation, innate signaling and chronic inflammation greatly impact in vivo function and persistence of engineered immune cells. Here we propose to investigate pharmacological interventions to enhance metabolism of engineered cells, improve effector functions, reduce immune suppression, prevent/restore immune exhaustion, and enhance memory formation of engineered CAR cells in vivo. Building on our extensive work on CAR engineered immunity, innate signaling and immune metabolism, we will 1) improve CAR-MQ, CAR-T and CAR-NK effector function and enhance expansion of CAR T cells by modulate immune metabolism with lactase targeting enzymes; 2) promote CAR-T cell persistence, memory formation and prevent exhaustion by targeting mTOR (mammalian target of rapamycin) pathway; and 3) Optimizing CAR-cell function by temporal integration of metabolic and immunoregulatory modulators. To minimize off-target effects and toxicity, we will leverage our established nonocapsule platform to deliver these pharmacological modulators specifically to CAR-expressing cells. We hypothesize that targeted immune modulation will maximize the in vivo function and persistence of multilineage CAR cells, providing a robust strategy towards a functional HIV cure.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary / Abstract Type 1 Diabetes (T1D) results from progressive destruction of pancreatic β-cells driven by autoimmune attack and intrinsic stress. While much attention has focused on immune-mediated mechanisms, the contribution of β- cell–intrinsic stress pathways to disease progression remains incompletely understood. Emerging evidence implicates ChREBPβ (Carbohydrate-Responsive Element-Binding Protein β), a stress-inducible transcription factor, as a central integrator of metabolic and inflammatory signals that impair β-cell identity and survival. Although ChREBP has been studied in the context of glucose metabolism and Type 2 Diabetes, its pathological role in T1D remains unexplored. Our preliminary data demonstrate that ChREBP and its target genes are upregulated in β-cells from autoantibody-positive (AAb+) and T1D donors, as well as in NOD mice, suggesting early activation in disease pathogenesis. ChREBPβ overexpression induces apoptosis, oxidative stress, and β-cell dedifferentiation. We also developed a novel small molecule, Compound 43, which functions as a “molecular glue” that stabilizes the ChREBPα–14-3-3 interaction, thereby suppressing ChREBPβ expression and protecting β-cells from stress- induced damage, and we demonstrate here that this stabilizer is able to protect human β-cell identity and function under cytokine-induced toxicity. This proposal aims to define the contribution of ChREBPβ to β-cell dysfunction in T1D and evaluate the therapeutic potential of Compound 43 in mitigating this process. In Aim 1, we will characterize the impact of ChREBPβ activation on β-cell stress and survival under inflammatory and ER stress conditions using human islets. We will assess gene expression, apoptosis, UPR activation, and lipid metabolism using transcriptomic and lipidomic profiling. In Aim 2, we will test whether Compound 43 can protect human islets and stem cell– derived β-cells from cytokine- and ER stress–induced dysfunction, using functional, metabolic, and transcriptomic readouts. This study will establish ChREBPβ as a previously unrecognized contributor to early β-cell failure in T1D and provide preclinical validation for a new pharmacologic approach that targets β-cell resilience, rather than immune modulation. These findings will offer a new conceptual and therapeutic framework for preserving β-cell function in the earliest stages of T1D, directly aligning with the mission of the Human Islet Research Network (HIRN) to understand and prevent β-cell failure.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Dilated cardiomyopathy (DCM) is one of the most common hereditary heart conditions, yet effective targeted therapies remain lacking. Extensive myocardial fibrosis underscores the critical role of non-myocyte dysfunction, particularly fibroblasts, in disease progression. Concurrently, immune activation and macrophage infiltration are increasingly recognized in DCM, implicating inflammation as a key driver of adverse remodeling. However, the mechanisms by which inflammatory signals activate fibroblasts remain poorly defined. This multi-PI R01 integrates patient-specific induced pluripotent stem cells (iPSCs), 3D cardiac organoids embedded in engineered disease-specific niches, CRISPR-based high-throughput screening, single-cell RNA sequencing (scRNA-seq), bioinformatics, and in vivo DCM mouse models to investigate and therapeutically target this axis. In Aim 1, we will test how inflammatory signals from DCM iPSC-derived cardiomyocytes (iPSC-CMs) initiate tri-cellular crosstalk with macrophages (iPSC-MΦs) and fibroblasts (iPSC-CFs), promoting fibroblast activation through key signaling and feedback loops. In Aim 2, we will evaluate how immune–fibroblast crosstalk contributes to fibrotic remodeling and cardiomyocyte dysfunction using single-cell, monolayer co-culture, and 3D cardiac organoid platforms embedded within engineered fibrotic niches. In Aim 3, we will map immune–fibrotic networks using scRNA-seq and genome-scale CRISPRi/a screens, followed by high-throughput drug screening and validation of prioritized targets, in both iPSC-based systems and in vivo DCM mouse models. Together, this study will define the cellular and molecular mechanisms of immune–fibroblast crosstalk in genetic DCM and accelerate the development of precision antifibrotic therapies.

NIA - National Institute on Aging

Aging with co-morbid conditions (i.e., obesity, hypertension) increases the risk of developing heart failure with preserved ejection fraction (HFpEF), and related cardiovascular morbidity and mortality for which there are few approved treatments. Importantly, HFpEF has become the most common form of HF and is characterized, in part, by cardiac fibrosis and reduced capillary density (i.e., rarefaction). Capillary rarefaction is associated with cardiac fibrosis in HFpEF and cardiac fibrosis is independently predictive of cardiac mortality in adults >70 years of age. LARP6 (La Ribonucleoprotein 6, Translational Regulator), an RNA binding protein, is implicated in pathologic fibrosis via binding to a unique 5' stem loop (5'SL) structure in collagen mRNA resulting in mRNA stabilization and increased translation, and excess collagen production/deposition. Our preliminary data provide the first evidence of LARP6-dependent cardiac fibrosis and dysfunction in response to chronic cardiac stress. Specifically, disruption of the LARP6-collagen mRNA interaction in a genetic (unique knock-in mouse model where the 5'SL region is mutated to prevent LARP6 binding; 5'SL mutant mice) and an interventional (the use of C9, a small molecule inhibitor of LARP6-collagen mRNA interaction) model each prevented cardiac fibrosis and contractile dysfunction following chronic β-adrenergic stimulation. Moreover, disruption of LARP6- collagen interaction promotes pro-angiogenic LARP6 signaling exhibited by increased capillary density and vasculogenic gene signatures in the heart. Lastly, cardiac LARP6 expression is increased in aging. Based on these `proof of concept' findings, we hypothesize that LARP6 signaling is a druggable target for the treatment of co-morbid aging-associated HFpEF. To test this hypothesis, we will utilize a `three-hit' mouse model of co- morbid aging-associated HFpEF to examine the following Specific Aims: Aim 1 will delineate the therapeutic potential of targeting LARP6-collagen interaction in cardiac fibrosis and dysfunction in lean and HFpEF 5'SL mutant mice/littermate controls (genetic) as well as lean and HFpEF C57BL/6J mice treated with C9 or vehicle (interventional). Cardiac morphology, function, and fibrosis in vivo by magnetic resonance imaging coupled with ex vivo analysis of fibrosis by staining and atomic force microscopy and assessment of LARP6 signaling will serve as major endpoints. Utilizing the same genetic and interventional models (5'SL mutant mice and C9), Aim 2 will elucidate the therapeutic potential of LARP6-Vegfa manipulation on capillary rarefaction and cardiac vasculogenic signaling in co-morbid aging-associated HFpEF. Capillary density, cardiac vascularity, analysis of cardiac pro-angiogenic mediator expression, delineation of the cardiac myocyte and non-myocyte transcriptome, and capillary sprouting in a tissue culture model are major endpoints. Together, the proposed conceptually innovative and translationally significant studies will provide novel evidence that disruption of LARP6-collagen interaction is a viable therapeutic target to attenuate cardiac fibrosis and enhance vascularity in co-morbid aging-associated HFpEF for which there are currently few approved treatments.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease that destroys insulin-producing beta cells in the pancreas, requiring insulin injections and blood glucose monitoring, which do not replicate the precise glycemic control of functional beta cells nor prevent disease progression and complications. In long-standing T1D, most beta cells are destroyed, which requires the replenishment of beta cell mass to restore insulin production. This can be achieved by regenerating endogenous beta cells or differentiating human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into beta cells for transplantation, combined with therapies to prevent autoimmune destruction of the newly formed beta cells. Different therapeutic windows exist for intervention or a potential cure, particularly in early-onset or newly diagnosed T1D where significant beta cells remain, presenting an opportunity to intervene and preserve these cells, potentially delaying or preventing further beta cell loss. Recent findings suggest that beta cell injury or stress may significantly contribute to immune- mediated beta cell loss in T1D, thus therapies aimed at reducing beta cell stress and injury may avert immune targeting during the progression to overt T1D. Combining beta cell therapies with immune modulation may yield better outcomes, yet the specific targets and pathways for effective treatment remain unclear. Renalase (RNLS) has emerged as a promising therapeutic target in T1D, being associated with T1D in genome-wide association studies (GWAS) and linked to beta cell protection. Loss of RNLS function in beta cells reduces endoplasmic reticulum (ER) and oxidative stress, immune cell infiltration, and natural killer (NK) cell activation, preventing autoimmune destruction. Designing a better strategy for targeting RNLS enzymatic activity may offer a potential therapeutic strategy for T1D by providing beta cell protection against stress and autoimmunity in humans. RNLS, known as an oxidase similar to monoamine oxidases (MAO), can be bound by some MAO inhibitors. We found that one of the FDA approved MAO inhibitors, Pargyline, is able to bind to RNLS and protect pancreatic beta cells from stress and autoimmune destruction. Apparently, pargyline may not be specific or potent enough for RNLS. Therefore, developing a more specific and potent RNLS inhibitor is crucial. Utilizing structure-based drug design, the goal is to create a new class of compounds for early or preventive treatment of T1D. This research includes uncovering RNLS's role in beta cell metabolism and immune interactions, characterizing RNLS structure and enzymatic function for robust assay development, and evaluating RNLS inhibitors by biochemical, cell, and animal model-based assays. The expected outcome is the development of potent and selective RNLS inhibitors that enhance beta cell survival and function, reduce stress and autoimmune destruction, and demonstrate safety and efficacy in humanized mouse models, providing promising therapeutic options for T1D by protecting beta cells from stress and immune attacks at an early stage.

NEI - National Eye Institute

Project Summary Mitochondria play a crucial role in maintaining neuronal health and function. Dysfunction of these organelles leads to various neurological diseases. Retinal ganglion cells (RGCs), the primary output neurons in the retina, are particularly vulnerable to mitochondrial damage. The two most common hereditary optic neuropathies characterized by RGC degeneration, Autosomal Dominant Optic Atrophy (ADOA) and Leber Hereditary Optic Neuropathy (LHON), both stem from mitochondrial dysfunctions. ADOA arises from mutations in OPA1, which regulates inner mitochondrial membrane fusion, while LHON is caused by mutations in the complex I subunit genes encoded by the mitochondrial genome. Currently, no effective treatments exist for either condition, underscoring a critical unmet need to unravel the disease mechanisms and develop therapies to safeguard RGCs from degeneration. The project’s significance lies in investigating the role of SARM1, a trigger of neurodegeneration, in mitochondria-induced RGC degeneration. Our lab has built a novel ADOA mouse model carrying the pathogenic Opa1R290Q/+ mutation. This model recapitulates key features of human ADOA, including mitochondrial fragmentation, aberrant glutathione redox, age-related RGC degeneration, and declines in RGC function. We found that knocking out Sarm1 in these ADOA mice nearly completely protects against all the degenerative phenotypes, suggesting that SARM1 activation drives RGC death in ADOA. Given the similarities between ADOA and LHON, we hypothesize that the same mitochondria-SARM1 pathway also contributes to LHON pathology. Therefore, the central hypothesis of the project posits that mitochondria-induced SARM1 activation leads to RGC death in both ADOA and LHON, and inhibiting SARM1 represents a promising therapeutic approach. Aim 1 of the proposal aims to identify specific mitochondrial defects triggering SARM1 activation in OPA1 mutant RGCs, and elucidate the underlying mechanisms. Aim 2 seeks to establish a dominant-negative SARM1-based therapeutic approach in ADOA mice. During the R00 phase in Aim 3, I will characterize a LHON mouse model and examine whether Sarm1 KO provides protective effects. I have assembled an advisory committee to provide conceptual and technical guidance as I pursue this study. Furthermore, I have also formulated a comprehensive training and career development plan to be executed during the grant period. This integrated proposal, encompassing the research plan and mentoring activities, will provide me with a solid foundation to embark on an independent academic career.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Hashimoto’s Thyroiditis (HT) is a prevalent autoimmune disease affecting 15% of the population and characterized by chronic autoimmune attack on thyroid follicular cells. Over time, this persistent autoimmune attack results in thyroid gland failure and the requirement for lifelong hormone replacement, a hallmark shared with other chronic autoimmune diseases such as Type 1 Diabetes Mellitus (T1DM) and Addison’s Disease. Despite its prevalence, the mechanisms driving the unrelenting autoimmune attack in HT remain poorly understood. Identifying factors underlying the persistent autoimmune response, may identify new therapeutic targets to halt disease progression in HT and similar chronic autoimmune disorders. We have identified a population of TCF7+ stem-like progenitor CD8 T cells within the thyroid tissue of HT patients, analogous to those seen in chronic viral infections and Type 1 Diabetes, that sustain autoimmune attack by replenishing the pool of terminally differentiated cytotoxic effectors. Using single cell RNAseq and TCRseq of human thyroid specimens from individuals with HT, our preliminary data further demonstrate the transcriptional transition and clonal expansion of TCF7+ progenitor CD8 T cell to effectors with killing ability within the thyroid. In addition, our preliminary data suggest that tertiary lymphoid structures (TLSs), organized collection of immune cells within the thyroid, provide a microenvironment that promotes autoimmunity, driven in part by CD4 T follicular helper (Tfh) cells and IL-21, a cytokine implicated in CD8 T cell differentiation. Using a mouse model of HT, we demonstrate that IL-21R deletion protects against thyroid autoimmunity, suggesting that TLS-associated factors may drive the conversion of progenitor CD8 T cells into cytotoxic effectors. Thus, our overarching hypothesis is that TCF7+ CD8 T progenitor cells sustain autoimmune persistence in HT, while TLS- associated signals promote their differentiation into cytotoxic effectors, perpetuating disease progression. In Aim 1, we will define the role of TCF7 in maintaining stem-like CD8 T cells by genetically deleting TCF7 in an HT mouse model and using WNT pathway agonists to assess its regulatory function. In Aim 2, we will investigate TLS-driven CD8 T cell conversion by assessing the deletion of CD4 Tfh cells and IL- 21 signaling in HT progression using mouse models. We will leverage spatial transcriptomics data previously collected by our lab from HT thyroid specimens, to determine how TLS localization influences CD8 T cell differentiation. These studies will uncover fundamental mechanisms of chronic autoimmunity in HT, providing potential therapeutic targets to disrupt persistent autoimmune attack and yielding broader insights into T cell– mediated autoimmune diseases.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary: PFA ependymoma is a rare and aggressive pediatric brain tumor with a poorly understood molecular mechanism. Unlike many cancers, PFA ependymoma exhibits very few genetic alterations. Instead, it is thought to be driven primarily by epigenetic dysregulation. A key player in this disease is the EZH1/2 inhibitory protein EZHIP, which is normally expressed only in germ cells. EZHIP is aberrantly expressed in PFA ependymoma, where it disrupts the function of Polycomb Repressive Complex 2 (PRC2), a master epigenetic regulator of developmental gene repression through deposition of the trimethylated histone H3 lysine 27 (H3K27me3) repressive histone mark. EZHIP-mediated dysregulation of PRC2 involves both enzymatic inhibition and physical stalling of PRC2 on CpG island (CGI) chromatin, leading to a global loss of H3K27me3 levels, an epigenetic hallmark of PFA ependymoma. PRC2 itself is a highly dynamic and intricate complex that assembles into two functional variants, PRC2.1 and PRC2.2. These two variants share a core composed of the catalytic subunits EZH1/2, along with EED, SUZ12, and RBBP4/7, and differ by incorporating distinct accessory subunits. PRC2.1 includes PHF1/MTF2/PHF19, EPOP, and PALI1/2, while PRC2.2 features AEBP2 and JARID2. Our preliminary data reveal intriguing molecular crosstalk between EZHIP and multiple PRC2 components, suggesting potential competitive or cooperative interplay. The ability of EZHIP to inhibit PRC2 partly stems from its mimicry of the oncohistone H3K27M, which harbors a lysine-to-methionine mutation that causes diffuse midline glioma, another devastating brain tumor in children, where PRC2 activity is also globally suppressed. However, the precise, EZHIP-specific mechanisms behind PRC2 dysregulation in PFA ependymoma remain largely unexplored. Our work aims to uncover these elusive mechanisms using a powerful combination of structural biology, biochemistry, and genomics approaches. Ultimately, we aim to identify therapeutic strategies that disrupt the pathogenic EZHIP–PRC2 crosstalk and restore the normal H3K27me3 epigenetic landscape. Specifically, in Aim 1, we will determine the structural and biochemical mechanisms underlying the enzymatic inhibition of the PRC2 core complex by EZHIP. In Aim 2, we will elucidate the molecular basis of EZHIP-mediated stalling of PRC2 on CGI chromatin, involving PRC2 functional variants. In Aim 3, we will explore an exciting mechanism-based therapeutic strategy to overcome PRC2 enzymatic inhibition and chromatin stalling induced by EZHIP.