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NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Autoimmune diseases impact ~25 million people in the United States and are increasing in prevalence. Autoimmune diseases are driven by a failure of immunological tolerance that triggers aberrant immune responses against self-antigens that can impart debilitating morbidities and even death. There are no cures for autoimmune disease and current treatments non-specifically blunt immune responses against both self- and non-self-antigen, requiring life-long treatment compliance that leaves patients more susceptible to infection and malignancy. We propose to develop a develop and test a new strategy for targeted treatment of autoimmune diseases that harnesses the intrinsic immunoregulatory properties of extracellular vesicles (EVs). EVs are secreted by all cell types as a mechanism for promoting transfer of molecules between cells and have been implicated in the maintenance or induction of immunological tolerance through their ability to deliver diverse immunoregulatory cargo. We hypothesize that EVs derived from immunosuppressive cell sources and engineered to deliver autoantigens can be employed as a tolerogenic vaccine (i.e., inverse vaccine) that promotes antigen-specific T cell tolerance that abrogates autoimmune disease. Towards this end, we have devised strategies for exogenous loading of peptide antigens onto EV surfaces, thereby enabling coordinated delivery of antigens and immunosuppressive EV cargo to antigen presenting cells (APCs), resulting in the presentation of autoantigen in a potently tolerogenic context. While our EV-based tolerogenic vaccine platform – tolEVax – is amenable to EVs isolated any cell source and can be applied to several autoimmune diseases, we will focus on engineering of EVs derived from mesenchymal stem cells (MSC-EVs) and will test our approach in a model of multiple sclerosis. We propose to establish tolEVax as a promising strategy for promoting immune tolerance and treating autoimmunity through two Specific Aims. In Aim 1, we will load MSC- EVs with peptide antigens, evaluate effects on antigen biodistribution and uptake by APCs, and characterize effects on antigen-specific CD8+ and CD4+ T cell responses to model antigens. In Aim 2, we will evaluate the capacity of tolEVax to inhibit autoreactive T cell responses and self-antigen mediated inflammation and pathology in a model of multiple sclerosis. We expect these studies to identify MSC-EVs as potently tolerogenic antigen nanocarriers, to provide new insight into how EVs modulate adaptive immune responses, and to demonstrate the efficacy of tolEVax as a potential treatment for MS. Overall, this research will result in a platform technology that addresses the unmet need for effective antigen-specific immunotherapies for autoimmune disease by exploiting the inherent and multimodal immunosuppressive functions of EVs.

NIA - National Institute on Aging

Engineering FlowGERO-Chips: Flow reversal-enabled long-term vessel-chips for modeling geroscience Age is a major risk factor for cardiovascular disease (CVD) where a significant contributor to vascular dysfunction is the age-associated impairment of arterial hemodynamics, specifically adverse retrograde or oscillatory blood flow, known as flow reversal. Current preclinical in vitro models, such as microphysiological systems incorporating cells from Hutchinson-Gilford Progeria Syndrome (HGPS) patients, have some limitations because they represent a pathological state from the initial time, often cannot form a fully confluent lumen, and typically remain stable for only a few days, thus failing to capture the progressive dynamics of aging. To overcome these limitations, we propose the development of FlowGERO-Chips, a New Approach Method (NAM) designed to replicate human aged arteriolar blood vessels and assess treatments relevant to geroscience. The FlowGERO- Chip is innovative in three key ways: it utilizes normal induced pluripotent stem cell (iPSC)-derived endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in a multicellular architecture; it is stable and supports long-term (up to 90-day) culture; and it enables the time-dependent assessment of EC-VSMC crosstalk under clinically-relevant flow reversal conditions. For Aim 1, we will engineer the FlowGERO-Chip with a cylindrical lumen of iPSC-ECs surrounded by extracellular matrix-embedded iPSC-VSMCs, mimicking in vivo vessel structure. Using the custom-engineered Hemadyne perfusion system, which can accurately reproduce pathological flow reversal waveforms, we will expose the chips to normal versus reversed arterial periodic flow for up to 90 days. We anticipate that flow reversal will accelerate a shift towards a senescent phenotype, characterized by increases in senescence associated biomarkers. For Aim 2, we will validate the FlowGERO- Chips by administering a telomerase (hTERT) mRNA therapy, which has been shown to reverse vascular senescence in vivo, either alone or in combination with the senolytic cocktail Dasatinib + Quercetin. We will monitor longitudinal changes in senescence-associated secretory phenotype (SASP) markers, expecting that these interventions will reverse or delay the EC and VSMC senescence induced by reversed/retrograde flow waveforms and extend the viability of the chip. In summary, this platform will provide a robust tool for predictive studies and preclinical drug discovery related to cardiovascular aging.

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

Project summary The human endometrium is a uniquely regenerative tissue, undergoing cyclical remodeling without fibrosis ap- proximately 450 times in a woman's lifetime. This remarkable regenerative capacity is essential for reproductive health but is often disrupted in disorders such as endometriosis, Asherman's syndrome, uterine fibroids, and unexplained infertility. These conditions, which collectively affect millions of women and represent hundreds of billions of dollars in economic burden, are associated with impaired regulation of the stem cell niche and epithe- lial–stromal interactions. Despite its clinical importance, the mechanisms governing endometrial regeneration and stem cell niche formation remain poorly understood due to the lack of suitable human models. This exploratory R21 proposal seeks to address this critical biotechnological gap by developing an innovative human pluripotent stem cell (hPSC)-derived endometrial organoid platform that closely recapitulates the phys- iological interactions and developmental trajectories of the endometrium. Unlike biopsy-derived organoids, which lack developmental plasticity and are difficult to genetically manipulate, our hPSC-derived platform fol- lows the developmental trajectory of the Müllerian duct, allowing the spontaneous emergence of the endometrial epithelium and its associated stem cell niche. This provides an unprecedented platform to investigate endome- trial homeostasis, regeneration, and disease mechanisms. To establish this system, we will define the differentiation trajectory from Müllerian duct progenitors to endo- metrial organoids using single-cell RNA sequencing, computational lineage inference, and machine learning- driven tissue crosstalk analysis. By benchmarking against human reproductive single-cell atlases, we will refine key signaling inputs—including RA, BMP4, and Wnt—to optimize epithelial-stromal interactions and identify transitional states where stem-like populations emerge. We will then apply a kinome-wide CRISPRi screen to uncover genetic regulators of endometrial stem cell specification and maintenance, leveraging a validated sgRNA library to systematically inhibit kinase activity during niche formation. Pharmacological validation of top candi- dates in both hPSC-derived and patient-derived endometrial organoids will confirm functional relevance, while single-cell RNA sequencing of inhibitor-treated organoids will reveal how lineage trajectories are altered in re- sponse to kinase inhibition. By integrating cutting-edge stem cell biology, large-scale functional genomics, and machine learning-driven sig- naling analysis, this project will generate a scalable, physiologically relevant platform for dissecting human en- dometrial stem cell niche regulation. Our novel microphysiological platform will fuel future hypothesis-driven research and lay the groundwork for therapeutic strategies in reproductive medicine and regenerative biology. 1

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Chronic inflammation is a central factor in a major fraction of all human disease burden. In chronic inflammation, immune cells mediate a dysregulated inflammatory response that persists despite the removal of the initial insult, suggesting stable, steady-state dynamics between immune and tissue cells. Macrophages are key players in this process, mediated by their status as central effectors of tissue inflammation and their high plasticity for both inflammatory and anti-inflammatory states, as well as tissue-specific functionality. This process of transition from a healthy to a chronically inflamed tissue steady state involves competition between trafficking and invading macrophages from the periphery vs. resident ones, conversion between inflammatory states, and crosstalk with neighboring tissue cells that is hypothesized to be tissue-specific. Existing tools for studying chronic inflammation are limited in their ability to provide a mechanistic understanding of these processes. They use overly high doses of inflammatory agents, which cause non- specific responses with little predictive value. The macrophages used are not authentic resident cells; they lack the correct epigenetic, immune effector, and metabolic characteristics, and their polarized states are poorly defined. Furthermore, these models fail to account for tissue-specific inflammatory responses, leading to inaccurate, non-physiological results that misrepresent the therapeutic window for chronic diseases. The overarching goal of this project is to develop tools that allow us to manipulate and study inflammatory state space in complex human tissues with a high degree of control. Aim 1 focuses on assembling immunocompetent human brain, liver, and adipose microtissues from pluripotent stem cell-derived progenitors. We will quantify resident macrophage expansion versus monocyte infiltration during inflammation initiation while benchmarking tissue structure and immune responsiveness. Aim 2 will establish advanced optical and genetic tools to continuously track transitions between basal, inflammatory, and reparative tissue states. We will combine fluorescence and label-free imaging techniques (e.g., fluorescence-lifetime and second harmonic generation) with engineered genetic reporters to generate high-density time-series data, identifying links between macrophage functions and inflammation divergence. Aim 3 will solve the problem of nonphysiological inflammatory cues by creating stable and inducible genetic switches to control macrophage activation states orthogonally and in situ within human microtissues. We will assess how these cells influence tissue structure, signaling, and the trajectory of inflammatory state, as well as test whether chronic tissue remodeling can be driven or reversed through specific pathway regulations. The successful development of this platform will provide the first human system to distinguish the roles of resident and infiltrating macrophages, track their real-time impact on tissue trajectories, and define the requisite physiological parameters needed to stabilize or reverse chronic inflammation.

NIGMS - National Institute of General Medical Sciences

ABSTRACT Dysregulated inflammation is a central driver of a significant fraction of all human diseases, including chronic metabolic conditions, tumor metastasis, autoimmune disorders, and aging. Despite numerous advancements in tissue engineering and disease modeling, our ability to accurately capture cell-mediated tissue inflammation in vitro that faithfully mimics in vivo human physiology remains limited. Here, we propose to combine human pluripotent stem cell (hPSCs) and genetic engineering tools to create immunocompetent tissue models that replicate the natural behavior of tissue-resident macrophages with on-demand control over inflammatory states. Specifically, we suggest an immunoengineering strategy to generate human microtissues in a dish containing bona fide resting tissue-resident macrophages. We aim to accomplish this using a “progenitor- based assembly” tissue engineering approach, where myeloid progenitors derived from hPSCs are combined with their developmentally matched tissue and vascular counterparts. Target model systems for this research will include skeletal muscle, liver, and adipose microtissues, as these are all known to exhibit dysregulated inflammation in the context of metabolic diseases—a globally pressing clinical need. Along with strategies to generate and characterize these tissues, we propose to establish bioprocessing procedures to enhance the scale and ability to cryopreserve key progenitors that will enable the dissemination of these tools to labs focused on tissue inflammation research but lack expertise in tissue engineering or stem cell biology. With the development of these microtissues, we then aim to develop and employ genetic engineering tools to overcome known limitations in conventional controllable gene induction systems that are not functionally compatible with hPSCs and their derivatives. Using these tools and a novel lineage tracing and retrieval approach, we further propose to develop multi-lineage CRISPR/Cas9 screens that will identify cell-mediated inflammatory mechanisms that modulate neighboring metabolic tissue cells – the core essence of immunoregulation. Additionally, we will employ these approaches to drive cell-mediated inflammatory and anti-inflammatory states within tissues, overcoming challenges associated with the limited efficacy of in vitro macrophage via recombinant cytokines. Lastly, we will explore using our tool sets to evaluate adoptive hPSC-macrophage transfer strategies as a test bed for immunotherapy development. As part of this effort, we will test hypotheses regarding the “open niche” dependency for successful transfer, the long-term fate and durability of macrophage phenotypes after transfer, and develop additional, more therapeutically relevant genetic tools to modulate these processes. This research will ultimately deepen our understanding of tissue–macrophage biology, create new tools for studying immunoregulatory processes, and develop putative therapeutic strategies for metabolic and inflammatory diseases. Collectively, we aim to greatly expand our understanding of macrophage biology and establish transformative new tools for stem cell-derived tissue modeling and regenerative immunotherapy.

NHLBI - National Heart Lung and Blood Institute

Project Summary: Cardiovascular diseases (CVDs) such as myocardial ischemia/reperfusion (I/R) injury lead to extensive cardiomyocyte death and subsequent reduced cardiac function. Due to the human adult heart’s limited regenerative capacity, there is a significant need for exogenous cardiac function restoration post-I/R. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a favorable cell source to restore myocardial function. While promising, the therapeutic potential of hPSC-CM transplantation is hampered by low cell survival and inadequate integration with host myocardium. Thus, our lab has developed nanowired human cardiac organoids composed hPSC-CM, primary human adult cardiac fibroblasts, endothelial cells, stromal cells, and electrically conductive silicon nanowires (e-SiNWs). Our in vivo data shows that organoids are capable of robustly engrafting and providing functional recovery (69% Fractional Shortening recovery) in I/R injured rat hearts, while using 10% of the cell dose (1E6, 1x106 cells/rat) that other hPSC-CM transplantation studies used (1E7, 1x107 cells/rat). Additionally, to alleviate major histocompatibility class (MHC) mismatching to provide a translational platform for hPSC-CM delivery, our lab developed isogenic cardiac organoids, composed of hPSC-CMs, -cardiac fibroblasts (hPSC-cFBs), and -endothelial cells (hPSC-ECs) from a single hiPSC cell line. The goals of this proposal are to 1) investigate the impact of e-SiNW geometry on isogenic organoid function, and 2) demonstrate the therapeutic efficacy of optimized nanowired isogenic cardiac organoids in a rat I/R injury model. The central hypothesis of this proposal is that engineered nanowire surface geometry will enhance interactions between host myocardium and transplanted hPSC-CMs within isogenic cardiac organoids, resulting in efficient engraftment and significant functional recovery. The innovation of this proposal is engineering e-SiNW surface roughness to optimize the efficiency of myocardial engraftment, and thus the functional recovery of I/R-injured hearts. My long-term goal is to leverage translational engineering and informatics to develop a clinically viable cardiac cell therapy heart repair. Accordingly, we will pursue the following two aims: 1) investigate the effects of e-SiNW geometry on isogenic cardiac organoid function, and 2) determine the therapeutic efficacy of optimized nanowired isogenic cardiac organoids to treat I/R-injured rat hearts and investigate e-SiNW graft-host interactions using spatial transcriptomics. The proposed research would provide a translational platform for cardiac repair with efficient engraftment.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Total transplantation of the immune system via bone marrow transplantation is a critical treatment for various conditions, yet it faces significant challenges, most notably Graft versus Host Disease (GVHD). This project proposes a transformative approach to understand and mitigate GVHD, especially in pediatric patients. We aim to develop a groundbreaking multi-tissue Organotypic Culture Model (OCM) that integrates gut, liver, and blood components to replicate the complex immune interactions occurring during acute GVHD with intestinal and hepatic involvement. This model will provide a physiologically relevant platform to study donor-recipient immune responses in bone marrow transplantation, overcoming the limitations of current in vitro models. Central to our approach is the creation of patient-specific OCMs using tissues from pediatric bone marrow transplant recipients. This personalized methodology allows for an in-depth understanding of individual GVHD responses and the potential for tailored therapeutic strategies, thus advancing precision medicine in transplant immunology. Specifically, we will first develop a gut-liver-immune OCM platform to study the impact of HLA- mismatched donor-recipient immune interactions using HLA-matched and mismatched “donor” blood and characterizing the recipient's acute immune response to simulated transplantation and gut ulceration. Second, we will develop a gut OCM of pediatric bone marrow transplantation and rejection by performing deep multi- omics profiling of gut and liver biopsies and peripheral blood from patient undergoing bone marrow transplantation samples and then develop OCM models of pediatric aGvHD to replicate salient features of immune activation and tissue damage. Finally, we will develop a computational model of the donor-recipient immune networks to create “immunologic digital twins” and characterize the drivers of successful transplantation and GvHD. These models will integrate multi-omic data, including single-cell RNA sequencing, to simulate and predict transplant outcomes, including the risk and severity of GVHD. This approach represents a significant stride in preclinical translation modeling, offering a powerful tool for preemptive intervention and personalized treatment planning. The project is underpinned by a unique and transformative interdisciplinary team comprising experts in OCM technology, computational modeling, and clinicians specializing in pediatric GVHD. This collaboration ensures a comprehensive, bench-to-bedside approach, facilitating rapid translation of our findings into clinical practice. Our work promises to not only deepen the understanding of GVHD pathophysiology but also to revolutionize the management and treatment of this condition in bone marrow transplantation. By integrating innovative OCM platforms with advanced computational modeling and patient-centric research, we aim to deliver a new approach available for the studies of transplant immunology and the potential to improve patient outcomes significantly.

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

Project summary Infertility affects approximately 15% of the global population, and current assisted reproductive technologies fail to address the needs of several populations, including individuals who cannot produce viable gametes and same-sex couples seeking biologically related children. In vitro gametogenesis (IVG), and specifically, producing egg cells (oocytes) from stem cells, offers a promising solution for these underserved groups. The reconstituted ovary (rOvary) system, currently only possible in mice, combines embryonic day (E) 12.5 ovarian somatic cells with developmentally younger E9.5 stem cell-derived primordial germ cell-like cells (PGCLCs). However, only 1-3% of PGCLC-derived oocytes achieve developmental competence, presenting a significant barrier to clinical translation. This developmental mismatch between PGCLCs and their somatic environment may contribute to this low efficiency. My preliminary data shows that E12.5 primordial germ cells (PGCs), which comprise 5% of germ cells in the rOvary, achieve key developmental milestones more effectively than PGCLCs. Specifically, PGCs complete meiotic prophase I earlier, establish timely interactions with supporting granulosa cells and produce significantly larger oocytes and follicles than PGCLCs. To address these limitations, I will: 1) Compare the progression of PGCs and PGCLCs through meiotic prophase I using immunofluorescence and single-cell RNA sequencing; 2) Assess oocyte-granulosa cell communication during follicle formation through protein expression dynamics and transcriptional profiling; and 3) Implement an established protocol using retinoic acid and BMP2 to mature PGCLCs to an E12.5-like state before rOvary formation, potentially enhancing their developmental potential and reducing chromosomal asynapsis. This research will be conducted at UCLA under the mentorship of Dr. Amander Clark, a world-renowned expert in stem cell biology and in vitro gametogenesis. Success in this project could significantly improve the efficiency of in vitro oocyte generation, advancing our understanding of germline development and the potential clinical applications of IVG technology. This work represents a crucial step toward expanding reproductive options for individuals facing infertility and broadening access to biological parenthood for currently underserved populations.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Cervical spinal cord injury (SCI) results in debilitating deficits, including life threatening damage to the phrenic motor circuit controlling the diaphragm – the primary muscle of respiration. There are no therapies availblable for people with SCI that repair damaged circuits. While transplanting human pluripotent stem cell (hPSC)-derived neurons can promote repair post-SCI, outcomes remain incomplete and inconsistent. Evidence from our team and others shows that ventral spinal progenitors, including V2a excitatory interneurons, integrate into injured circuits and improve respiratory function, while dorsal progenitors alone fail to do so. However, dorsal interneurons contribute to motor plasticity and may complement ventral transplants in a reparative strategy, suggesting that repair requires rebuilding both dorsal and ventral components of phrenic motor network and ensuring reliable host-donor-host connectivity. The central hypothesis of this project is that 1) ventrally and dorsally derived donor cells have distinct, complementary roles in network repair, and 2) that neuromodulation can enhance their integration to damaged networks. In Aim 1, we will determine the anatomical and functional integration of dorsal, ventral, and combined dorsal and ventral hPSC-derived transplants using viral tracing, 3D tissue clearing, and terminal diaphragm electromyography. In Aim 2, we will test whether daily chemogenetic neuromodulation strengthens donor activity, improves host-donor-host connectivity, and enhances respiratory recovery, using chronic diaphragm telemetry, plethysmography, and molecular (single nucleus RNA sequencing) profiling. By pairing engineered donor populations with neuromodulation, these studies directly address the critical challenge of variability in transplant outcomes. Results will establish a blueprint for consistent, circuit-specific repair of the phrenic network and accelerate the translation of interneuron-based cell therapies to restore breathing after cervical SCI.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY The kidney is responsible for maintaining an overall state of homeostasis, which it achieves by filtering a large volume of plasma through millions of epithelial units termed nephrons. It is estimated that 1 in 7 adults in the US suffer from chronic kidney disease (CKD), resulting from impairment of these functions. A seminal event in the development of every nephron is the formation of a connection with a collecting duct that establishes a patent pathway for the drainage of fluid into the collecting system and eventually out of the kidney. Human kidney organoids derived from pluripotent stem cells (hPSCs) are an innovative and powerful tool to study renal pathophysiology and to potentially inform new therapeutic avenues, but they suffer from several major limitations. One paramount deficiency is the lack of collecting ducts and failure of their nephrons to drain into a collecting system. To address this problem, we have recently developed a novel platform in which nephrogenic mesenchyme is combined with ureteric bud (UB) progenitor cells to produce an integrated system of collecting ducts within the organoids. Like in vivo development, the nephrons connect with the UB through a controlled fusion event with the distal GATA3+ segment that establishes a continuous passage for fluid flow. Currently, nephron connectivity in this system is limited by a relatively low efficiency of distal specification, and we hypothesize that finetuning the molecular processes underlying fusion will enable more complete integration of nephrons with collecting systems. In this proposal, we will apply this integrated organoid model to address major gaps in our mechanistic understanding of distal nephron patterning and fusion. In Aim 1, we will investigate how WNT and NOTCH signaling interactions coordinate and synergize to control nephron segmentation, and these mechanisms will be applied to enhance nephron-UB fusion in the organoid model. Aim 2 will delineate the cell- autonomous mechanisms underlying distal nephron fusion. Preliminary data show that expression of GATA3 is sufficient to induce fusion-competent distal tubules. Here we will build on this understanding by dissecting the genetic mechanisms downstream of GATA3 and how they regulate this unique cell biological property. Collectively, this work will generate novel and important insights into the mechanisms underlying nephron fusion, and simultaneously it will lead to further advancement in our state-of-the-art organoid model by informing methods to optimize nephron-UB connectivity. We anticipate that these approaches will yield seminal improvements in the structural organization of hPSC-derived kidney organoids, which will translate to progress in the functional capabilities of these tissues and their utility as models for investigation of renal pathophysiology and potentially for developing new therapies for CKD.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Therapies for insulin-dependent diabetes remain inadequate. Islet cell transplantation using donor islets or stem cell-derived tissue could revolutionize treatment, but preventing immune rejection of the transplanted beta cells remains a critical unsolved issue. Current approaches focus on editing hypoimmune islets or redirecting T regulatory suppressor cells, two exciting but challenging strategies. As a novel and more flexible approach, we recently developed synthetic suppressor T cells that use engineered synNotch receptors to sense local disease signals and, in response, induce the targeted expression and delivery of customized immune suppressive payloads. These synthetic suppressor cells are capable of locally targeted immune suppression, without systemic suppression. Our goal is to design optimal therapeutic suppressor cells that can protect transplanted islets by locally blocking immune rejection and promoting tolerance. The flexibility of this platform allows us to systematically explore what payloads and other cell modifications result in the most effective and durable immune protective behaviors. Our aims are: Aim 1. Systematically screen combinatorial suppressive payloads to identify those that yield synthetic suppressor cells that can effectively block key components of cell-mediated rejection (CD4+, CD8+, CD4+Th17, NK, B) and humoral mediated rejection (e.g. complement). Combinations will then be tested for ability to protect islets in several in vivo immune rejection models. Programs will be triggered by synNotch receptors that detect antigens specifically on the beta cell surface, including targets like GLP1R. Aim 2. Introduce modifications to synthetic suppressor cells to increase their survival and persistence, including versions of growth and survival cytokines designed to function in a fully autocrine manner (i.e. cause synthetic suppressor cells to survive without inducing proliferation/inflammation via other immune cells). These survival signals will be induced in a local manner via Islet-sensing synNotch receptors. Suppressor cells with these modifications will be tested for improved persistence in islet immune protection. Aim 3. Engineer synthetic suppressor cells to take up residence in islets by engineering them with chimeric synthetic cell adhesion molecules that recognize islet antigens. Together, these aims should yield optimized suppressor cells that are strongly retained in islets, durably persist only in islets, and produce combinatorial suppressive payloads that prevent immune rejection. This work will yield a better fundamental understanding of immune rejection and the requirements for immune protection and would yield approaches to local immune suppression relevant to solid organ transplantation and autoimmune disease.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT The advent of genome editing provides the opportunity to treat genetic diseases at their root cause. However, clinical translation remains limited by the challenge of delivering genome editors efficiently and specifically to target cells in vivo. Delivery vehicles are needed to protect genome editors, engage cell-surface receptors and release active enzymes into target cells. Inspired by viral tropism, we developed Enveloped Delivery Vehicles (EDVs), lentivirally derived lipid vesicles that encapsulate CRISPR–Cas9 ribonucleoproteins and display fusogens or engineered antibody fragments on their surface. EDVs enable receptor targeting, but how antibody fragment density and binding affinity control uptake, biodistribution and genome editing remain poorly defined. Building on our laboratory’s expertise with EDVs and CRISPR-Cas9 mechanistic biology, our goal is to define how antibody fragment presentation on EDVs influences their ability to target hematopoietic stem cells (HSCs), a clinically important cell population for treating blood and immune disorders. HSCs are a compelling target for blood cancers, because myeloid malignancies such as acute myeloid leukemia and myelodysplastic syndromes originate from malignant HSCs. Our central hypothesis is that an optimal range of ligand density and affinity (i.e., avidity) maximizes selective uptake and genome editing in HSCs while minimizing off-target uptake by bystander cells. We will test this hypothesis through two aims: 1) Quantify how EDV avidity affects uptake and genome editing in human HSCs ex vivo. 2) Quantify how EDV avidity affects biodistribution and genome editing specificity in humanized mouse models. Mouse models are indispensable for delivery vehicle development because invertebrate, in vitro, organoid, and computational systems cannot recapitulate the immune, spleen, liver, and bone marrow environments or the biophysics of blood flow and tissue perfusion that govern the biodistribution and elimination of delivery vehicles. Unlike prior approaches that focused solely on maximizing antibody affinity or surface presentation, this proposal systematically dissects how density and affinity interact to determine avidity, uptake, and editing efficiency. Completion of this project will produce new targeted delivery vehicles for HSC editing and establish quantitative rules for tuning delivery vehicle avidity to maximize potency and specificity. These results will be broadly applicable to other delivery platforms and accelerate the development of safe and effective in vivo genome editing therapies.

NEI - National Eye Institute

Project Summary Corneal diseases pose a significant public health challenge in the United States, often leading to vision impairment and decreased quality of life. Mesenchymal stem cell (MSC) delivery to the cornea after a severe injury has shown promise by accelerating repair and significantly suppressing inflammation. However, a major bottleneck in developing MSC therapy for corneal repair is the lack of effective delivery methods. Moreover, optimizing the dosage and timing of MSC therapy is crucial for achieving therapeutic outcomes while minimizing side effects. MSCs must also survive and integrate into corneal tissue to exert their therapeutic effects. To date, MSCs have been delivered via surface injection, fibrin gel, or as a sheet on an amniotic membrane. However, these methods are limited by poor MSC survival and/or rapid matrix degradation. To address these issues, we propose the development of adhesive hydrogels that can effectively encapsulate and release MSCs in a sustained manner while having similar biomechanics as the corneal tissue. Our platform composed of a single hybrid polymeric structure with tunable variables to generate two distinct mechanical properties and degradation rates: 1) a soft/controlled degradable adhesive hydrogel to function as a bandage containing MSCs that release secreted factors for promoting corneal epithelial regeneration and 2) a strong/highly adhesive hydrogel that can adhere to corneal stromal defects and simultaneously serves as a stromal replacement while providing a platform for the delivery of MSCs to promote repair of stromal injuries/ulcerations. Our proposed biomaterial is a photocurable adhesive composite hydrogel based on chemically modified gelatin and hyaluronic acid (HA), encapsulated with MSCs. First, gelatin will be dual-functionalized with methacrylic anhydride (MA) and phenylboronic acid (PBA) to control mechanical properties and promote tissue adhesion. The incorporation of methacrylate HA derivatives in the hydrogel will also control the viscosity of the prepolymer and improve its mechanical properties. The physical properties of the resulting hydrogels, such as stiffness, swelling ratio, and degradation rate, which affect MSCs differentiation, will be tuned by varying polymer ratios, degree of polymer functionalization, final polymer concentration, and crosslinking time. We will first optimize the mechanical properties of the proposed hydrogels, and their degradation rates will be tuned to achieve a rate supporting MSCs growth and proliferation (Aim 1). We will then assess the in vitro epithelial proliferation and MSCs differentiation using in vitro models developed in our labs (Aim 2). Finally, we will test the in vivo efficacy of MSC-laden hydrogels using two animal models: a corneal epithelial wound healing model and a corneal stromal injury model (Aim 3). Based on our preliminary data, we anticipate that successfully achieving the Specific Aims of this project will result in a novel treatment that enhances MSC survival and retention by providing a 3D environment resembling the corneal extracellular matrix. The treatment is expected to improve visual outcomes, seal and repair stromal injuries, facilitate re-epithelialization, and reducing the healthcare system burden.

NIGMS - National Institute of General Medical Sciences

The University of West Alabama (UWA), a rural, predominantly undergraduate, and resource-limited institution, seeks funding to acquire the Attune™ CytPix™ (BRV6Y Package) Flow Cytometer, a state-of-the-art instrument designed to provide high-throughput, high-precision flow cytometry analysis. This equipment is essential for advancing our research capabilities in cellular analysis, enabling us to perform detailed image-assisted flow cytometry with unprecedented accuracy and efficiency. It combines up to 14 fluorescence channels and integrated brightfield imaging, allowing simultaneous detection of cell surface markers, intracellular proteins, apoptosis, cytokines, DNA content, and fluorescent reporters. The need for this instrument is both urgent and foundational. UWA lacks a flow cytometer, limiting faculty and students' ability to perform high-throughput cellular analysis. Ongoing research in host-microbe interactions, immunophenotyping, phagocytosis, genome size variation, and stress signaling is hindered by this gap, restricting data generation, undermining student training, and preventing competitive NIH proposals. Most faculty are early-career investigators, and UWA has never received NIH funding and has minimal funding from NSF. Acquisition of the Attune CytPix will transform UWA's research by providing essential infrastructure for studies in plant stress, fungal symbiosis, amphibian immunity, and microbial resistance. It will enhance NIH-aligned Course-Based Undergraduate Research Experience (CURE) programs like SEA-PHAGES and Molecule to Microbe, engaging over sixty undergraduates annually in hands-on research and supporting thesis projects in the M.S. in Conservation Biology program through advanced training in fluorescence-based cell analysis. The instrument will operate as a shared core facility, accessible across departments and to partner PUIs in the region. Its integration into the Department of Biological and Environmental Sciences' Strategic Plan for STEM Growth ensures long-term sustainability through dedicated faculty oversight, institutional maintenance support, and a centralized scheduling and usage tracking system. By enabling advanced data acquisition, fostering collaborations, and strengthening faculty competitiveness for R15 and R01 grants, this proposal directly advances NIH's mission to seek fundamental knowledge about the nature and behavior of living systems and to apply that knowledge to enhance health, lengthen life, and reduce illness and disability.

NCI - National Cancer Institute

Project Summary/Abstract The long-term goal of this proposal is to improve anti-CD19 chimeric antigen receptor T cell (CART19) therapy for patients with relapsed or refractory large B cell lymphoma (r/r LBCL). Unfortunately, 50%-60% of patients with r/r LBCL receiving FDA-approved autologous CART19 therapy (tisa-, axi- or liso-cel) will relapse, often due to poor in vivo expansion and persistence of the CART19. IL-7, IL-15, and IL-21 are critical homeostatic regulators of T cells and promote stem cell memory phenotypes. NT-I7 is a long-acting version of human IL-7, while HCW11-006 is a long-acting multimeric protein containing IL-7, IL-15, and IL-21. While our recent Phase 1b trial (NCT05075603) confirmed that NT-I7 is safe 21 days post-CART19, this timing coincided with the CART19 contraction phase, resulting in minimal expansion. In Aim 1a, we will conduct a new Phase 1b trial evaluating early (Day 10) and repeated (Day 31) dosing of NT-I7 following axi- or liso-cell infusion in r/r LBCL patients. Aim 1b will involve extensive correlative studies to analyze the impact of NT-I7 on (1) serum cytokines, (2) CART19 and endogenous T cells, (3) other non-T cell subsets, and (4) the tumor microenvironment (TME) using flow cytometry, single cell RNA-sequencing, and multiplexed TME profiling. We hypothesize that initiating a repeated NT-I7 dosing schedule during CART19 expansion (Day 10) is safe, promotes superior in vivo CART19 persistence/expansion and improves clinical outcomes for r/r LBCL patients. However, because NT-I7 may not provide durable remissions, we will perform an unbiased CRISPR screen to identify genes enhancing NT-I7-treated CART19 function (Aim 1c). In Aim 2, we will evaluate the impact of NT-I7 or HCW11-006 (IL-7/IL-15/IL-21) on CART19-mediated toxicities (CRS/ICANS) and efficacy using autologous (Aim 2a) and allogeneic (Aim 2b) preclinical mouse models. We hypothesize that early, repeated cytokine administration sustains CART19 proliferation, persistence, and tumor killing without exacerbating toxicity. Using immunocompetent and “humanized” NSGS mice, we will compare an early regimen (days 1 and 21) against the clinical schedule (days 10 and 31). Since new approaches are needed to overcome host-mediated allogeneic CART rejection by CD2-expressing T cells and NK, we will test if CD2 epitope editing of allogeneic CART19 confers resistance to CD2 antibody-mediated lymphodepletion and enhances their persistence and efficacy in murine allogeneic or xenogeneic hosts with LBCL. Preclinical mouse models are essential to capture the complex dynamics of autologous and allogeneic CART19, including trafficking, TME remodeling, and CRS/ICANS, which in silico and in vitro methods cannot replicate. By preserving an intact vascular system and host immunity, these models provide a robust foundation for clinical trials designed to improve LBCL survival while minimizing toxicities.

NCI - National Cancer Institute

Abstract This project focuses on macromolecular prodrug-based delivery of a topoisomerase I inhibitor, SN22, structurally enhanced to overcome tumor defense mechanisms in order to achieve durable suppression of high-risk neuroblastoma (NB), the most common and deadly solid tumor of childhood. The intensive, multimodality treatment currently used clinically fails in over half of high-risk NB patients: 50-60% experience a relapse with no curative salvage treatment options. Centered on developing and optimizing a drug delivery strategy against the aggressive disease not responding to conventional therapies, with a particular focus on a high-risk form of multiple drug-resistant NB with increased “stemness” driven by a MYCN protooncogene and its downstream target, ABCG2 (an ABC drug efflux pump suppressing chemosensitivity and promoting tumorigenicity), this project will evaluate an approach integrating polymer-linked prodrug design and structural optimization of the cargo to improve delivery, extend drug residence in the tumor, and reverse drug resistance. Guided by our past work and the results of our preliminary studies toward this project, we hypothesize that prodrug-mediated delivery of SN22 will potently suppress growth of aggressive, pre-therapy and chemorelapsed NB tumors by enhancing drug uptake and extending tumor exposure to therapeutically effective drug levels and by taking advantage of the inactivation-resistant molecular design of this agent. This hypothesis will be tested by pursuing the following specific aims: Aim 1 studies will focus on in vitro evaluation of a series of prodrug constructs on NB cells derived at relapse from MYCN-amplified high-risk NB tumors; Aim 2 studies will examine tumor uptake, biodistribution and elimination of the prodrugs in orthotopic xenograft NB models; Aim 3 experiments will comparatively evaluate antitumor efficacy of prodrug- mediated delivery in models of newly diagnosed and recurrent MYCN-amplified NB in comparison to a new syngenetic model of disseminated (MYCN-driven) high-risk disease. Through optimizing the design and performance of SN22 prodrugs using a panel of clinically relevant models recapitulating distinct types and phases of aggressive NB, this research is expected to have a strong impact on the field by addressing several barriers to the translation and clinical implementation of macromolecular prodrugs and by paving the way to improved clinical management of drug-resistant NB and other high- risk cancers showing minimal or no response to conventional therapies and currently lacking effective treatment options.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with a median survival of only 15–18 months even with therapy. The poor prognosis reflects the tumor's rapid growth, cellular heterogeneity, and resistance to current treatments. Oncolytic virotherapy has emerged as a promising adjunct, and Zika virus (ZIKV) has unique potential given its natural tropism for glioblastoma stem-like cells. While attenuated ZIKV strains have shown preclinical efficacy in reducing tumor burden, challenges remain regarding safety, incomplete tumor clearance, and limited control over viral replication. To address these limitations, we propose to develop a novel optogenetic gene circuit, BiViSTA (Bioluminescent Viral Sensor and Transcriptional Activator), that detects ZIKV infection and couples it to therapeutic gene expression. BiViSTA leverages a split- luciferase sensor that reconstitutes upon ZIKV NS5 protein binding, producing bioluminescence that activates a light-sensitive transcription factor to drive pro-apoptotic or immunogenic transgenes. This approach uniquely links viral infection to targeted tumor cell destruction, creating a self-amplifying and tumor-restricted therapeutic system. We will pursue three Specific Aims: Aim 1 will engineer and optimize BiViSTA constructs responsive to ZIKV NS5 and validate their function in permissive human cell lines. Aim 2 will integrate BiViSTA into 2-D and 3-D GBM culture models to test selective induction of pro-apoptotic genes and assess enhanced ZIKV-mediated killing. Aim 3 will evaluate tumor-specific activation and therapeutic efficacy in an orthotopic xenograft mouse model of GBM, including rigorous safety assessments. Our multidisciplinary team combines expertise in synthetic biology, neuro-oncology, and viral pathogenesis, supported by strong preliminary data demonstrating a “plug and play” optogenetic system. The proposed work is highly innovative as no existing technology dynamically couples virus infection to therapeutic gene expression in real time. Successful completion will open the door to a transformative strategy that will improve the safety and efficacy of oncolytic virotherapy for GBM that can integrate nonpathogenic ZIKV pseudovirions. Beyond GBM, BiViSTA represents a broadly adaptable platform for treating virus-associated cancers and for dynamically controlling gene expression in basic and translational research.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary/Abstract The small intestine, central to nutrient absorption like amino acids and lipids, houses highly responsive Lgr5+ intestinal stem cells (ISCs) in the crypt bottom. Over the past decade, our group and others have utilized the mouse intestine to investigate how dietary interventions (fasting, high fat diet, mitochondria pyruvate shuttle inhibition, and high cholesterol diet) impact ISC fate decisions. Although much focus has been on ISCs, the small intestine is a complex environment that includes a variety of non-epithelial cells including resident immune cells that coordinate ISC function and maintenance. In particular, tissue-resident immune cells produce and secrete the cytokine interleukin-22 (IL-22), which is known to be a critical regulator of epithelial homeostasis. Amino acids constitute many nutrients in various foods. However, little is known about how specific amino acids impact ISC proliferation and intestinal immune-stem cell interactions. My research has uncovered that the amino acid cysteine controls ISC function through two mechanisms: 1) by directly activating PPAR-CPT1A-HMGCS2 mediated ketogenesis in ISCs via mTORC1 suppression, and 2) by indirectly boosting IL-22 production by CD8β+ T cells through activation of epithelial Coenzyme A (CoA) biosynthesis. The aims of my proposal focus on elucidating both stem cell-intrinsic and extrinsic mechanisms by which cysteine enhances ISC-mediated repair after injury. Specifically, I plan to: 1) determine how cysteine regulates ISC self-renewal and differentiation in intestinal homeostasis and injury through the control of ketogenesis; 2) determine the cysteine metabolic pathways that contributes to ISC mediated repair after injury; 3) determine how CD8β+ T cells mediate the cysteine response in ISC-mediated repair after injury. This career development K99/R00 award will be essential to my training and provide significant support as I transition to an independent investigator. It will protect me to receive the comprehensive education and training through the robust MIT/Harvard system, coupled with the exceptional research resources, fruitful partnerships will uniquely position me to embark on an unparalleled journey as a rising independent investigator in stem cell metabolism research.

NIAID - National Institute of Allergy and Infectious Diseases

ABSTRACT Tuberculosis (TB) remains a leading cause of mortality in people living with HIV (PLHIV), with 161,000 deaths in 2023 despite widespread use of combinatorial antiretroviral therapy (cART). Although cART effectively suppresses HIV replication, it does not eliminate the markedly elevated risk of Mycobacterium tuberculosis (Mtb) reactivation in co-infected individuals. This persistent susceptibility is thought to stem from incomplete immune reconstitution, including impaired TH1/TH17 balance, reduced CD4⁺ effector memory T (TEM) cells, and ongoing immune activation in the lung. Our preliminary studies in an established Mtb/SIV rhesus macaque model demonstrate that cART alone fails to restore IL-21 and STAT1 signaling, both critical for immune control of TB and HIV. IL-21, a pleiotropic cytokine produced by CD4⁺ T cells, regulates TH1 and TH17 responses and supports macrophage function. Prior work shows that IL-21-IgFc therapy in SIV-infected macaques is safe and preserves mucosal immunity. We propose that IL-21-IgFc, when administered during early cART, can enhance immune reconstitution, reduce viral persistence, and improve TB control. The central hypothesis of this study is that adjunctive IL-21-IgFc therapy will restore key immune pathways in the lung, limit viral reservoirs, and improve macrophage function—thereby preventing Mtb reactivation in SIV- infected macaques. To test this, we will pursue two specific aims: • Aim 1 will determine how IL-21-IgFc therapy during early cART impacts pulmonary immune reconstitution and Mtb-specific responses. We will assess T cell subsets, STAT1/STAT3 signaling, granuloma integrity, and transcriptional signatures using flow cytometry, scRNAseq, and spatial transcriptomics. Additionally, we will perform cross-species transcriptomic comparisons between NHP and human PBMCs from cART-treated and untreated cohorts to identify conserved immune signatures and pathways associated with protection or disease progression. • Aim 2 will evaluate the effect of IL-21-IgFc therapy on viral persistence and macrophage function. We will measure SIV reservoirs in lung tissue, quantify macrophage proliferation, and assess tissue pathology to determine the impact of IL-21 on HIV-associated innate immune dysfunction. Impact: These studies will elucidate mechanisms by which IL-21 enhances lung immunity and limits pathogen persistence in TB/HIV co-infection. Findings will guide development of IL-21-based immunotherapies, a promising host-directed strategy to improve outcomes in high-burden populations where TB and HIV remain syndemic.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

Abstract Laboratory automation is reshaping scientific discovery by enabling high-throughput, precise, and scalable experimentation. Yet, unlike fields such as manufacturing, transportation, and agriculture—where robotics has driven rapid advances in innovation—laboratory automation has lagged behind. Most commercial lab robots are still built to mimic human pipetting rather than enabling experiments that were previously impossible to execute manually. Closed, proprietary platforms prevent researchers from fully leveraging their computational skills and severely limit training opportunities for students and early-stage investigators. This project addresses those gaps by expanding PyLabRobot (PLR), a free, open-source, Python-based framework for designing, simulating, and executing complex liquid handling protocols across a wide range of robotic systems. PLR is software- and hardware-agnostic, supports complete virtual simulation, and can be run in the cloud, making it accessible to anyone with basic Python skills—no expensive equipment required. By enabling users to develop and debug protocols entirely in simulation, PLR allows students, educators, and researchers to build automation expertise before ever stepping into a physical lab. This significantly lowers the entry barrier and ensures that trainees enter the workforce ready to contribute immediately to automation-driven projects. Python’s ubiquity across STEM and engineering makes PLR a natural training tool for modern science. In a recent graduate-level class piloting PLR in full simulation mode, students with no prior robotics background programmed highly sophisticated workflows running from CRISPRi screening, AlphaLISA and TR-FRET assays, small molecule synthesis, FISH staining, and cell line manufacturing—demonstrating both the accessibility and real-world relevance of the platform. These examples highlight PLR’s potential not just to replicate existing workflows, but to unlock new experimental designs that would be infeasible with manual techniques. This R03 proposal supports three focused aims: (1) Create a web-based repository of validated, sharable protocols and documentation to enable reproducibility, benchmarking, and AI-driven method development; (2) Integrate PLR into cloud platforms like Google Colab and develop a dedicated IDE for real-time visualization, debugging, and seamless protocol execution; (3) Build a digital twin simulation engine that models labware, deck layouts, and liquid handling to support predictive optimization before execution. This project is supported by letters of support from the entire PyLabRobot development team and multiple industry stakeholders, including leading automation companies. By removing the barriers that have long constrained laboratory robotics programming, this work will accelerate discovery, lower costs, and build a highly skilled U.S. workforce ready to lead in bioengineering, automation, and advanced manufacturing.

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

PROJECT SUMMARY Enteric neuropathies are serious conditions caused by abnormalities in the enteric nervous system (ENS), an extensive neural network that spans the entire gastrointestinal tract and controls gut functions. Reduction in number of neuronal nitric oxide synthesis (nNOS) expressing enteric neurons is a hallmark of many human gastrointestinal diseases, including esophageal achalasia, gastroparesis, and Chagas disease. Despite the prevalence of these conditions and the morbidity they cause, current treatment options are significantly limited and do not directly address the underlying pathophysiology. Cell therapy represents a novel, curative approach to alleviating enteric neuropathies by replacing the absent or injured neurons. Although we have achieved significant successes in our preclinical cell therapy studies using animal models of enteric neuropathy, an enteric neural cell therapy approach specifically tailored for nNOS-deficient diseases would be a significant advance. Recent studies, and our own preliminary experiments, have demonstrated that enteric neural stem cells (ENSCs) generate two neuronal classes that subsequently differentiate into specific phenotypes and that this process includes phenotype switching. This led us to hypothesize that manipulation of this enteric neurochemical plasticity (ENCP) would allow us to optimize ENSC therapy by generating therapeutically relevant, disease- specific neuron subtypes. By utilizing optimized ENSC culture conditions, cell transplantation techniques, transcriptomic analysis, and genetic manipulation, we propose to test our hypothesis that enteric neuronal subtype plasticity occurs postnatally and can be leveraged for cell therapy application. We will also elucidate the role of the transcription factors, Pbx3 and Tbx3 in postnatal ENCP. The proposed studies will advance our understanding of the mechanisms underlying cell commitment in the ENS and how this knowledge could be leveraged to optimize the therapeutic efficacy of cell therapy applications in the future.

National Aeronautics and Space Administration

Awards will be made as cooperative agreements to accredited Minority Serving Institutions (MSIs) partnered with non-profit organizations in the United States that are eligible to apply for this NASA Research Announcement (NRA). The period of performance for an award is up to 2 years. Prospective proposers are requested to submit any questions in writing to NASAMUSIC@nasaprs.com no later than 10 business days before the proposal due date so that NASA will have sufficient time to respond. Proposers to this NRA are required to have the following, no later than the due date: 1) a Data Universal Numbering System (DUNS) number, 2) a valid registration with the System for Award Management (SAM) [formerly known as the Central Contractor Registry (CCR)], 3) a valid Commercial And Government Entity (CAGE) Code, 4) a valid registration with NASA Solicitation and Proposal Integrated Review and Evaluation System (NSPIRES) (this also applies to any entities proposed for sub-awards or subcontracts.) Consult Appendix H Section H.3.1 for more eligibility information. Consult Appendix H Section 2.2 regarding teaming requirements and partnership guidelines. The goal of NASA MUSIC is: to provide strategic effort that will leverage research and contract relationships of MSIs and NASA through relationships developed by non-profit organizations that may include collaboration of subject matter experts and access to NASA research facilities; An effort to improve STEM education and research at MSIs; A funded activity that seeks to build institutional capacity of MSIs; An activity to support long-term sustainability of STEM research at MSIs. MUSIC seeks to address the agency goals and objectives through: Increasing the institutional awareness of NASA competitive resources that can build the capacity of MSIs to offer and conduct STEM undergraduate and graduate research with a focus on NASA opportunities. Assembling MSIs and their stakeholders with common interests, and challenges then provide common tools for MSIs to increase efficiency and optimize resources including opportunities to develop formal and informal partnerships. Connecting MSI administrators and university STEM leaders to cutting-edge initiatives at NASA that can increase interest in securing research and contracting opportunities while supporting NASA s policy to achieve an Agency-wide goal of providing one percent of total contract value of prime and subcontracting awards to MSIs. https://www.hq.nasa.gov/office/procurement/regs/1826.htm To achieve these goals, MUSIC seeks to increase university program capacity about practical uses of research to drive institution sustainability through the following targets: Advance the understanding of MSIs on how to effectively develop institutional administrative support by competing at the university level for funding opportunities, which will result in successful application to, and management of these funding opportunities (including those at NASA). Extend MSI s capabilities by: A. Leveraging the MSIs research capabilities with NASA research to develop Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) projects that develop and demonstrate innovative technologies that fulfill NASA needs and have significant potential for successful commercialization. B. Increasing the preparation of undergraduate and graduate science, technology, engineering, and mathematics faculty with opportunities to participate with NASA researchers and missions through grants and contracts. To achieve these goals and objectives, NASA solicits proposals from MSIs to implement the NASA MUSIC; to engage MSIs in authentic STEM experiences related to NASA missions; and to inspire and captivate learners utilizing NASA s unique assets to develop a keen interest in STEM. Every institution that intends to submit a proposal to this NRA, including the proposed prime award or any partner whether an education institution, other non-profit institutions, and other organizations that will serve as sub-awardees or contractors, must be registered in NSPIRES. Electronic submission of proposals is required by the due date and must be submitted by an authorized official of the proposing organization. Such registration must identify the authorized organizational representative(s) who will submit the electronic proposal. All principal investigators and other participants (e.g. co-investigators) must be registered in NSPIRES regardless of submission system. Potential proposers and proposing organizations are urged to access the system(s) well in advance of the proposal due date(s) of interest to familiarize themselves with its structure and enter the requested information. Electronic proposals may be submitted via the NASA proposal data system NSPIRES or via Grants.gov. Organizations that intend to submit proposals via Grants.gov must be registered 1) with Grants.gov and 2) with NSPIRES. Additional programmatic information for this NRA may develop before the proposal due date. If so, such information will be added as a Frequently Asked Question (FAQ) or formal amendment to this NRA and posted on http://nspires.nasaprs.com. It is the proposer s responsibility to regularly check NSPIRES for updates to this NRA.

NIA - National Institute on Aging

Project Summary Alzheimer’s disease (AD) currently affects 5.8 million Americans and 13.8 million people over the age of 65 are expected to develop the disease by 2030. Aging is the single greatest risk factor for developing AD, something that is a growing concern as the US population continues to age at an unprecedented rate. Both normal aging and AD are accompanied by cognitive decline, yet our understanding of the cellular and molecular mechanisms underlying this process is incomplete and remains a critical unsolved challenge. One aspect of cognition that is especially vulnerable to aging is memory updating, the ability to modify existing memories with new information. To address this, we have developed a paradigm called the Objects in Updated Locations (OUL) task to investigate the mechanisms that support memory updating in young and old rodents. Using OUL, our preliminary research found that age-related impairments in memory updating may stem from disrupted co- allocation, or overlap, between the neuronal ensembles supporting the original and updated memories. Our preliminary work further suggests that the repressive histone deacetylase 3 (HDAC3), an epigenetic inhibitor of gene expression, might contribute to age-related updating impairments; blocking HDAC3 improves memory updating in old mice. Here, we hypothesize that abnormal HDAC3-mediated epigenetic repression in the old hippocampus disrupts proper co-allocation of the OUL update, preventing successful memory updating in old mice. We will test this hypothesis in two specific aims. In Aim 1, we will investigate how inhibiting HDAC3 systemically or within the dorsal hippocampus affects both memory updating and hippocampal co-allocation in young (3-m.o.) and old (18-m.o.) mice. In Aim 2, we will determine the extent to which activating or inhibiting neurons in the original memory ensemble can overcome age- and HDAC3-mediated effects on memory updating and co-allocation. Together, these aims will identify important epigenetic and cellular mechanisms underlying the effects of age on memory updating. More broadly, this work will identify basic biological mechanisms that underlie age-related impairments in memory updating, a critically understudied aspect of the aging process. Thus, this work represents an important step toward developing therapeutics to treat or prevent cognitive decline in both normal aging and AD.

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

PROJECT SUMMARY/ABSTRACT The major cell types of the placenta are derived from trophoblast stem cells undergoing a choreographed allocation of cell fates in response to signaling cues from the developing fetus and the uterine environment. Early defects in the differentiation of trophoblast stem cells can lead to abnormal placentation which is thought to underlie a range of adverse pregnancy conditions including miscarriage, fetal growth abnormalities, preeclampsia, and stillbirth. The long-term goal of this project is to uncover the critical regulatory mechanisms of human trophoblast cell fate specification to improve health outcomes of these pregnancies. In this proposal, we will generate insights into the epigenetic events that control the formation of the syncytiotrophoblast cells of the mouse labyrinth and human chorionic villi. These structures separate the maternal and fetal blood and orchestrate the exchange of oxygen, nutrients, and fetal waste. The first aim focuses Polycomb Repressive Complex 1.1 (PRC1.1), an epigenetic silencing complex which is thought to facilitate differentiation by repressing stem cell genes and maintain lineage fidelity by repressing genes that promote alternate lineages. The PRC1.1 component BCOR (BCL6 CORepressor), interacts with DNA-binding factors BCL6 and KDM2B to recruit PRC1.1 to its genomic targets. BCOR is an X-linked gene with essential roles in placental development in mice, is mutated in human syndromes involving low birth weight and intrauterine growth restriction, and women with a heterozygous null mutation in BCOR can experience recurrent miscarriages. Extraembryonic deletion of Bcor mice leads to a reduced labyrinth and an expanded junctional zone. We will use PRC1.1 as an entry point for understanding how epigenetic changes in the genome control differentiation of trophoblast stem cells by profiling cell types with single nuclei RNA-seq and ATAC-seq and changes in H3K27ac, H3K27me3 and DNA methylation with or without BCOR loss in all trophoblast cells or specific placental cell subtypes. The second aim will elucidate the roles of eight candidate DNA-binding factors that may control gene regulatory networks in differentiating trophoblast cells. Using inducible CRISPR/Cas9 genome editing of human trophoblast stem cells we have found that loss-of-function mutations in four of these candidates (ELF5, FIRRE, IKZF4, and ZBTB7C) alter trophoblast differentiation. We will use single nucleus RNA-seq and genome-wide profiling of H3K27ac, H3K27me3, and single nucleus ATAC-seq in autonomously differentiating 3D cultures of wild-type and knockout cell lines. We will also determine the immunolocalization and genome-wide occupancy of these factors in 3D cultures and in vivo placenta samples using authenticated antibodies. Our functional genomic approaches will identify new genes involved in trophoblast differentiation that provide diagnostic and therapeutic targets for preclinical studies. Together these aims will reveal how cell fate specification in the developing placenta is controlled by the coordinated action of epigenetic modifiers and DNA-binding factors through modifications to chromatin structure.