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U.S. National Science Foundation

The Louis Stokes Alliances for Minority Participation (LSAMP) program invests in the Nation's colleges and universities to aid student success to create a new generation of STEM discoverers for the national STEM enterprise. The program takes a comprehensive approach to the STEM Learning Ecosystem to impact STEM student development and retention. LSAMP is an alliance-based program, whereby a group of institutions of higher education (IHEs) work together to diversify the nation's science, technology, engineering, and mathematics (STEM) workforce by increasing the number of STEM baccalaureate and graduate degrees awarded to persons from LSAMP populations. LSAMP populations are defined as persons from groups underrepresented in the STEM enterprise: Blacks and African-Americans, Hispanic and Latino Americans, American Indians, Alaska Natives, Native Hawaiians, and Pacific Islanders. The LSAMP program provides funding to alliances that implement comprehensive, evidence-based, innovative, and sustained strategies that ultimately result in the graduation of well-prepared, highly competitive students from LSAMP populations who pursue graduate studies or careers in STEM, while also supporting knowledge generation, knowledge utilization, assessment of program impacts, dissemination activities and dissemination of scholarly research into the field. Projects supported by the LSAMP program include: --Alliance Development Grants (ADG) support the conceptualization and development of new B2B and new SPIO alliances. (New) --Bridge-to-the-Baccalaureate (B2B) alliances facilitate the successful transfer of students from LSAMP populations to four-year institutions in pursuit of STEM baccalaureate degrees. --STEM Pathways Implementation-Only (SPIO) alliances are designed for new and reconstituted alliances. These projects focus on building and strengthening strategies and approaches to assist Institutions of Higher Education (IHEs) increase STEM baccalaureate degrees to LSAMP populations and facilitate entry into STEM graduate degree programs. --STEM Pathways Research Alliances (SPRA) are designed for well-established alliances. These projects serve as models of excellence in STEM broadening participation by (1) steadily increasing STEM baccalaureate degrees to LSAMP populations and facilitating entry into STEM graduate degree programs; (2) producing and disseminating new scholarly research on the broadening participation of LSAMP populations (or underrepresented and underserved populations in STEM disciplines and the nation's STEM workforce) and, (3) holistically assess the state of institutionalization and sustainability of the alliance. --Bridge to STEM Graduate Degrees in National Priorities (BD-Master's) projects support cohorts of six graduate students pursuing a M. S. degree in STEM national priority areas, providing financial support (stipends and cost of education) and support to help develop and maintain academic and research skills that enable participants to successfully persist in STEM graduate degree programs at Master's comprehensive-degree producing institutions only. (New) --Bridge to STEM Graduate Degrees in National Priorities (BD-Doctoral) projects support cohorts of twelve graduate students pursuing a Ph.D. degree in STEM national priority areas, providing financial support (stipends and cost of education) and support to help develop and maintain academic and research skills that enable participants to successfully persist in STEM doctoral degree programs. --STEM Networking Incentives and Engagement (NETWORKS) projects provide support to incentivize the creation and participation of LSAMP populations in STEM networks. (New)

U.S. National Science Foundation

The Major Research Instrumentation (MRI) Program (MRI Program Website) serves to increase access to multi-user scientific and engineering instrumentation for research and research training in our Nation's institutions of higher education and not-for-profit scientific/engineering research organizations. An MRI award supports the acquisition of a multi-user research instrument that is commercially available through direct purchase from a vendor, or for the personnel costs and equipment that are required for the development of an instrument with new capabilities, thereby advancing instrumentation capabilities and enhancing expertise for instrument design and fabrication at academic institutions. MRI instruments are, in general, too costly and/or not appropriate for support through other NSF programs. MRI provides support to acquire critical research instrumentation without which advances in fundamental science and engineering research may not otherwise occur. MRI also provides support to obtain next-generation research instruments by developing instruments with new capabilities that open new opportunities to advance the frontiers in science and engineering research. Additionally, an MRI award is expected to enhance research training of students who will become the next generation of instrument users, designers and builders. An MRI proposal may request from NSF up to $4 million for either acquisition or development of a research instrument. Each performing organization may submit in revised "Tracks" as defined below, with no more than two (2) submissions in Track 1 and no more than one (1) submission in Track 2. For the newly defined Track 3, no more than one (1) submission per competition is permitted. As a result, it is now possible for an institution to submit up to four MRI proposals within the Track limits as described above. Track 1: Track 1 MRI proposals are those that request funds from NSF greater than $100,000[1] and less than $1,400,000. Track 2: Track 2 MRI proposals are those that request funds from NSF greater than or equal to $1,400,000 up to and including $4,000,000. Track 3: Track 3 MRI proposals are those that request funds from NSF greater than or equal to $100,000[1] and less than or equal to $4,000,000 that include the purchase, installation, operation, and maintenance of equipment and instrumentation to conserve or reduce the consumption of helium. Institutions may submit no more than one Track 3 proposal. Submission of a Track 3 proposal does not impact limits that apply for Track 1 and Track 2 proposals. Cost sharing requirements for new awards in the MRI Program are waived for a period of 5 years beginning with the FY 2023 MRI competition. Institutional submission limits for Track 1, Track 2 and Track 3 proposals remain. The MRI Program especially seeks broad representation of groups, institutions, and geographic regions that are underrepresented in STEM disciplines. Proposals from women, underrepresented minorities, persons with disabilities and early-career PIs are encouraged, as are proposals that benefit early-career researchers and proposals with PIs from geographically underserved regions, including EPSCoR jurisdictions. Additionally, proposals are encouraged from under-resourced institutions, including from emerging research institutions, where MRI can significantly build capacity for research. ___________________________ [1]Track 1 proposals requesting funds from NSF less than $100,000 will be accepted only from: a) eligible performing organizations requesting instrumentation supporting research in the disciplines of mathematics or social, behavioral and economic sciences; or b) non-Ph.D.-granting institutions of higher education requesting instrumentation supporting research in any NSF-supported disciplines.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Diabetes is a tremendous healthcare burden, with costs of hundreds of billions a year in the US. Diabetes has 2 major forms, type 1 diabetes (T1D) and type 2 diabetes (T2D). While these diseases have distinct causes (immune dysfunction or metabolic dysfunction), end stage disease in both leads to loss of beta cell mass and glucose homeostasis. Protocols have been developed to generate beta-like cells from human pluripotent stem cells (PSC)s which are currently in clinical trials as a cellular therapy for T1D and T2D. A beta cell therapeutic faces several hurdles to become a long-term cure for diabetes. First, it must survive transplantation which includes a hypoxic environment prior to graft vascularization and survive the inflammatory environment induced by the transplant surgery. For long term survival, the graft needs to be robust and survive diabetic conditions before the graft achieves full functionality and for use in T2D patients, needs to be able to function and overcome insulin resistance by secreting sufficient insulin long term. Lastly, immune attack of the graft by the host immune system needs to be prevented. Immune attack is being addressed by many groups in the scientific community via multiple strategies. This proposal is focused on generating the most robust stem cell derived beta cell that can survive and function long term in challenging conditions present in diabetic patients. To do so, we are leveraging naturally occurring hyperactive mutations that have been found in the HNF1A gene and which protect individuals from T2D. HNF1A is a transcription factor important in beta cell development and function. Loss of function mutations in HNF1A is the most common cause of monogenic diabetes and genome wide association studies have linked this gene to both T1D and T2D. Multiple studies of loss of function HNF1A mutants have demonstrated defects in insulin secretion, glycolysis, mitochondria function, and response to cellular stress. We hypothesize that the HNF1A hyperactive mutants will have superior function and response to cellular stress. We will test this hypothesis by generating a panel of hyperactive HNF1A mutants previously found in the human population in human PSCs. These mutants will be tested both in vitro and in vivo utilizing rodent xenotransplant studies. A second focus of this proposal is to acquire a deeper understanding of the mechanisms in how HNF1A loss of function can influence diabetes pathogenesis. HNF1A can influence both beta cell development but also plays a role directly in beta cell function. To focus on its role in beta cells, we are utilizing genetically engineered human PSC lines where HNF1A can be degraded specifically after development into beta cells. We will use this system to study how acute loss of HNF1A impacts beta cell function, survival, and response to cellular stressors important in diabetes pathogenesis. This proposal will give new insights into beta cell function and responses to cell stress which is critical for diabetes pathogenesis and developing cellular therapies for this disorder.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

PROJECT SUMMARY/ABSTRACT Directly genome editing cells inside the body could treat numerous genetic diseases, including sickle cell disease. However, genome editing of most cell types outside of the liver, such as hematopoietic stem cells, is limited by inefficient delivery. Only a small subset of cells inside of a tissue are accessible to delivery vehicles and editing enzymes. To overcome this delivery problem, my central objective is to enable cells initially receiving delivery vehicles to transiently produce and distribute genome editing enzymes to neighboring cells in vesicles. This allows editing activity to spread beyond initial delivery. My proposal builds on my postdoctoral research studying the delivery mechanisms of Enveloped Delivery Vehicles (EDVs). EDVs are lentivirus-derived lipid vesicles engineered to package CRISPR-Cas9 ribonucleoproteins that can be targeted to specific cell types using surface-displayed fusogens and antibodies. I hypothesize that transient, local production of EDVs in vivo will amplify genome editing efficiency by increasing the concentration of genome editing enzymes and enabling their spread across cells. This hypothesis will be tested through three specific aims: (1) develop single nucleic acid molecules encoding EDVs, (2) establish methods to target EDV production and uptake to specific cell types, and (3) deliver EDV-encoding plasmids to amplify editing in vivo. In preliminary work for this proposal, I showed that hydrodynamic injection of EDV-encoding plasmids into mice amplified genome editing efficiencies compared to Cas9 only plasmid controls. Successful completion of this proposal will generate fundamental insights into propagating genome editing effects beyond cells initially reached by delivery vehicles starting with hematopoietic stem cells as a model therapeutic cell type. This approach could broadly transform biological therapy delivery by overcoming low delivery efficiencies through localized amplification and spread of therapeutic macromolecules. The training acquired through this proposal in primary cell culture, bioinformatics, and next-generation sequencing will bolster my readiness to lead an independent research program. I will be mentored by Dr. Jennifer Doudna, a global leader in genome editing technology, and leading experts and clinicians in primary cell engineering, hematopoietic stem cell biology and virology in the California scientific community. During the mentored phase of this project, I will hone my scientific and professional skills to become a scientific leader. I will engage in structured professional development activities and actively present my research at leading conferences to facilitate a successful transition to an independent academic research position. The vibrant and collaborative environment provided by UC Berkeley and the Innovative Genomics Institute offers an outstanding environment to complete my training and start my independent scientific career.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

PROJECT SUMMARY/ABSTRACT Janus kinase inhibitors (JAK}---including tofacitinib, baricitinib, and upadacitinib-have emerged as promising oral treatment options for patients with rheumatoid arthritis who experience an inadequate response to conventional therapies. However, major safety concerns, including increased risk of cardiovascular events, venous thromboembolism, and serious infections, have prompted FDA black box warnings and remain major barriers to widespread use. These safety concerns largely stem from potential signals in integrated analyses of premarketing studies and the ORAL Surveillance trial, which reported a dose-dependent elevated risk of cardiovascular and thromboembolic events with tofacitinib compared to tumor necrosis factor (TNF) inhibitors. However, the ORAL trial has left some critical questions unanswered-specifically, whether the observed risk elevation is attributable to a specific JAK inhibitor toxicity, a protective effect of TNF inhibitors, or both; or how the safety profile of specific JAK inhibitors may compare to approved alternatives, such as interleukin-6 inhibitors. Approved JAK inhibitors have differences in selectivity and target binding affinity, which may translate into differences in adverse health outcomes. As a pan-JAK inhibitor, tofacitinib targets multiple JAK isoforms (JAK1, JAK2, JAK3), while newer agents such as upadacitinib and baricitinib exhibit greater selectivity for JAK1. The current FDA black box warning applies to all JAK inhibitor class members, but comparisons among specific class members are lacking. Moreover, all currenUy approved JAK inhibitors are metabolized via cytochrome P450 3A4 (CYP3A4), making them susceptible to clinically significant drug-drug interactions {DDls). Co-administration with moderate or strong CYP3A4 inhibitors, such as diltiazem, can substantially increase plasma JAK inhibitor concentrations, and potentially increasing the risk of adverse events. In this R01, we propose a comprehensive examination of the safety of JAK inhibitors among patients with rheumatoid arthritis. We will use the estimand framework and target trial emulation methods to compare cardiovascular, thrombotic, and infectious risks between JAK inhibitors and other commonly used therapies, overall and by agent. We will also evaluate how co-prescribed CYP3A4 inhibitors modify these risks. Our findings will directly inform clinical guidelines and prescribing decisions for patients with rheumatoid arthritis.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Broadly neutralizing antibodies (bnAbs) that target the CD4 binding site (CD4bs) on the HIV envelope glycoprotein (Env) are an attractive target for an HIV vaccine and antibody-based therapy for HIV protection. However, eliciting CD4bs bnAbs in a vaccine is challenging, and current CD4bs bnAbs are insufficient to provide long-term HIV suppression. This difficulty in CD4bs bnAb-mediated treatment stems from Env mutations near the CD4bs referred to as “loop D,” including the gain of a glycan at N276 that has yet to be dependably overcome in vaccine regimens. Although improvements in antibody isolation have provided mechanistic and evolutionary insight into some CD4bs bnAbs, it remains poorly understood how CD4bs bnAbs evolve breadth and potency from their naïve precursors, complicating attempts to elicit them via vaccination. To address this issue, high-throughput deep mutational scanning (DMS) techniques will be used to characterize the effect of all possible single-amino acid mutations at each position in the variable domains of the VRC01 and IOMA CD4bs bnAbs that represent two stereotyped mechanisms of Env CD4bs recognition. VRC01 and IOMA variant libraries will be expressed on the cell surface of yeast. Using this yeast-surface display (YSD) platform, both VRC01 and IOMA variant binding affinities will be measured against various Env proteins using fluorescence-activated cell sorting and high-throughput sequencing (FACS-seq), facilitating the measurement of the binding properties of these bnAb libraries in parallel. Aim 1 will determine the mutations in CD4bs bnAbs that enable HIV Env N276 glycan accommodation. DMS, YSD, and FACS-Seq will be performed on libraries of mature VRC01 and its naïve precursor to determine their binding affinities to two closely related donor-autologous Env proteins that contain and lack the N276 glycan. These experiments will reveal routes to overcome common barriers in CD4bs bnAb development and illuminate how the next generation of immunogens will better elicit broad and potent bnAbs. Aim 2 will map the mutational landscape that confers CD4bs bnAbs breadth and potency. DMS, YSD, and FACS-seq will be performed on mature VRC01 and IOMA to measure the impact of all single mutations on binding to a previously established panel of 12 heterologous Env strains that comprise a reference of global Env diversity. These results will illustrate how mutations in these antibody scaffolds confer binding to divergent Env strains versus promoting strain-specific binding, fully defining the functional constraints and flexibilities of these CD4bs bnAb classes. The results of this proposal will illustrate a new approach to investigate the evolution and engineering of HIV CD4bs bnAbs, setting the stage for the development of improved immunogens and therapeutic antibodies against HIV. More broadly, the methodological innovation herein will provide a new platform for antibody engineering for the treatment and prevention of various infectious diseases.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY In this application, we seek to continue and enhance the MARC U*STAR program at the University of Rhode Island (URI). This program has had a major positive impact on the career development of our high-achieving underrepresented (UR) STEM students, with 70% (11 of 16) of our trainees matriculating into competitive graduate programs in the biomedical sciences to date. The program has also been instrumental in creating a diverse community of student biomedical scientists at URI, united by a strong sense of belonging and an enthusiasm for scientific discovery. Our institutional self-assessment provides compelling justification for an undergraduate research training program for underrepresented students at URI: 4-year graduation rates for underrepresented (UR) students (33%) continue to lag behind that for well-represented (WR) students (49%). Furthermore, rates of matriculation into advanced biomedical degree programs for UR students (15%) also continue to lag behind that for WR students (20%). Moreover, we have a robust pool of UR STEM students and a diverse pool of actively funded faculty committed to supporting the career development of UR students. Our program goals are to; 1) Develop students who are knowledgeable and well trained in the biomedical sciences, who can readily employ rigorous research methods and critical thinking to solve important scientific problems, and 2) Create a community of student scholars who enjoy a strong sense of belonging, thereby promoting a sustainable culture of undergraduate research excellence at URI. Our approach to achieving these goals will be informed by lessons learned over the course of the previous funding period; we will continue to cultivate a culture of inclusive mentoring excellence across campus by promoting and facilitating mentoring training for faculty and graduate student research mentors; we will use evidence-based approaches to monitor and support trainee mental health and wellness; we will emphasize strong foundations in quantitative and computational competencies and instill a pervasive culture of rigor and reproducibility in experimental design among our trainees. With these enhanced strategies, we expect that 1) 90% of our trainees will graduate with a baccalaureate degree in a STEM field within a 4-year period, and 2) 75% of our trainees will matriculate into PhD or MD-PhD degree programs in biomedical research fields within three years of graduating from the program, ultimately advancing the diversification of the nation's scientific workforce.

Massachusetts Executive Office of Education

STEM education initiative grants for MA to expand science, technology, engineering, and mathematics programs in K-12 and postsecondary institutions.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Mesenchymal stem/stromal cells (MSCs) are potent regulators of immune cells, and their immunosuppressive function is being actively investigated for a number of therapeutic applications. In particular, it has been demonstrated that MSCs can inhibit the proliferation of effector T cells and induce regulatory T-cell differentiation for treating graft versus host disease (GvHD). A few MSC products have been approved by regulatory agencies in countries outside of the U.S. However, MSCs have not always shown consistent efficacy in GvHD clinical trials. This is in part due to the challenges of generating MSCs with high therapeutic potency. The overarching goal of this project is to develop MSC therapies with enhanced immunosuppressive efficacy for GvHD treatment by identifying and providing optimal microenvironment mechanical cues in MSC production. Mechanical cues from cell microenvironment play important roles in regulating cell behavior. For example, studies have shown that matrix stiffness directs cell activity and fate such as migration, proliferation, and differentiation. However, matrix or material stiffness only describes their static, elastic mechanical property. Instead of being simply elastic, natural extracellular matrix (ECM) and living tissues are viscoelastic, exhibiting stress relaxation over different characteristic time scales (stress relaxes at different rates). We have developed a hydrogel system that can recapitulate the stiffness and viscoelastic behavior of different types of tissues. Using the hydrogels as culture substrates, we discovered that matrix stress relaxation, in addition to stiffness, is an important mechanical factor regulating cell–ECM interactions and directing MSC activities including spreading, proliferation, differentiation, and in vivo bone regeneration. In collaboration with Dr. Kyung Sung at FDA, we recently found that substrate stress relaxation also regulates MSC's immunosuppressive capacity and their ability to inhibit T cell proliferation; Interestingly, MSCs retained their mechanical “memory” even after being extracted from the hydrogels (preliminary data). In light of these new findings, we hypothesize that biomaterials with tailored stress relaxation properties can provide inducing mechanical cues in MSC production to enhance MSC's immunosuppressive efficacy for GvHD treatment. We will test this hypothesis in the following specific aims: Aim 1: Elucidate the molecular mechanisms by which matrix stress relaxation regulates the immunosuppressive capacity of human MSCs (hMSC) derived from bone marrow. Aim 2: Compare the effect of matrix stress relaxation on hMSCs derived from different donors and tissues, and examine the influence of stiffness in the fast stress relaxing environment. Aim 3: Evaluate the efficacy of hMSCs primed by viscoelastic hydrogels with different stress relaxation properties for GvHD treatment in an animal model. Successful completion of these aims will have significant impact in understanding how matrix mechanical cues regulates the immunosuppressive capacity of hMSCs, with the findings potentially leading to better treatment for GvHD.

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

Project Summary Infertility is a complex disease that impacts approximately 1 in 10 couples of reproductive age in the United States. However, the pathogenesis underlying many of these cases is unknown. Clinically, this proposal aims to gain molecular insights into infertilities associated with defective formation of amnion, a component of the fetal membrane, and primordial germ cells (PGC, precursors of gametes). Formation of human amnion is first seen in the pluripotent epiblast cells as implantation is initiated (within two weeks after fertilization). Thus, couples with pregnancies repeatedly accompanied by defective amnion formation would be diagnosed with infertility. Recent data indicate that, in implanting primate embryos, PGC are specified in the nascent amnion and a growing body of evidence supports the notion that a subset of the pluripotent epiblast cells form a bi- potential amnion/PGC progenitor cell population at the amnion-epiblast boundary of developing embryos. In published work using a human pluripotent stem cell (hPSC)-based Gel-3D amniogenic system, we showed that a mechanical cue initiates amnion specification by triggering a Bone Morphogenetic Protein (BMP) signaling cascade in individual cells of pluripotent cysts. Moreover, our recent single cell transcriptomic analyses of developing Gel-3D showed that, in fact, PGC-like cells (PGC-LC) are also present in Gel-3D, and that cells that are progressing to amnion and PGC-LC lineages appear to share a common progenitor labeled by Claudin 10 (CLDN10). Indeed, our preliminary results revealed the presence of CLDN10/TFAP2C/SOX17-triple positive cells at the posterior amnion-epiblast boundary of a cynomolgus macaque peri-gastrula, suggesting that this progenitor formation as well as the amnion/PGC lineage diversification from the progenitor is an important aspect of successful amniogenesis and PGC formation both in vivo and in vitro. These findings present Gel-3D as a robust, transcriptomically well-characterized and mechanosensitive model to investigate these previously unexplored aspects of amniogenesis and PGC-LC formation. Together, using the Gel-3D model, the goal of this proposal is to understand the mechanisms that triggers amniogenic BMP signaling to initiate CLDN10+ progenitor cell specification (Aim 1), as well as the mechanisms that control amnion-PGC lineage decisions in the progenitor (Aim 2). In vitro findings will be grounded using early cynomolgus macaque embryo samples. Our preliminary data lead us to hypothesize that mechanically triggered WNT/?-catenin signaling activates amniogenic BMP signaling, and, in the progenitor population, Yes-associated protein 1 (YAP1) activity determines amnion or PGC fate. Proposed studies will further expose these signaling and transcriptional machineries. Overall, the work proposed here will greatly accelerate the pace of discovery regarding critical but previously inaccessible peri-implantation events and thus will have enormous implications for understanding early processes that impact embryonic development and human fertility.

NIGMS - National Institute of General Medical Sciences

Project Summary Mobile genetic elements use strategies that closely resemble those of viruses to replicate, spread, and persist. The long interspersed element-1 (LINE-1, L1) retrotransposon is the only autonomously active retrotransposon in humans and accounts for nearly one-fifth of the genome. L1 replicates through an RNA intermediate and encodes two proteins—ORF1p and ORF2p—that assemble into a ribonucleoprotein (RNP) complex to reverse transcribe the encoding RNA into DNA and insert a new L1 copy into the genome. Despite existing among hundreds of thousands of defective relatives and competing with parasitic elements such as Alu, a small number of full-length L1s continue to replicate. This success relies on cis preference, in which L1 proteins preferentially act on the RNA that encodes them. Similarly, cis preference is observed to varying degrees in viruses. Hepatitis B virus polymerase exhibits a translation-coupled cis preference when interacting with its pregenomic RNA via the epsilon stem-loop, a crucial step in viral genome packaging and reverse transcription. RNA viruses, such as alphaviruses and flaviviruses, exhibit a related bias, with evidence of localized, co-translational coupling between viral proteins and replication templates. The central hypothesis of this proposal is that L1 cis preference is enforced co-translationally, through ribosome-linked mechanisms and/or co-assembly of RNP complexes, analogous to strategies employed by viruses. To test this, we have developed a novel RNA launch system that initiates L1 replication directly from synthetic RNA, with distinct advantages over classical DNA-based retrotransposition assays. Using this platform, we have established a quantitative barcoded assay for L1 cis preference. We will extend the system to study Alu retrotransposition and employ a retron-based surrogate system to probe the generalizability of our findings. Aim 1 will quantify L1 cis preference and determine how Alu subverts it, addressing the dynamics of competition between autonomous and parasitic elements. Aim 2 will dissect the molecular basis of ORF1p’s cis preference using in vitro translation, ribosome profiling, and deep mutational scanning. Aim 3 will define the molecular basis of ORF2p’s cis preference and test whether cis preference can be transferred to a heterologous reverse transcriptase using a bacterial retron system. The impact of this project is to resolve a long-standing mystery in retroelement biology: how cis preference is achieved. By combining innovative RNA-based tools with high-throughput discovery methods, this work will reveal general principles of viral and retroelement replication, illuminate strategies by which selfish genetic elements maintain a fitness advantage in competitive environments, and provide new entry points for biotechnology and therapeutic development.

NIAAA - National Institute on Alcohol Abuse and Alcoholism

PROJECT SUMMARY Fetal Alcohol Spectrum Disorders (FASD) represent a major public health concern, significantly contributing to intellectual disabilities with a prevalence rate of 1-5% in the United States and an estimated annual economic burden of $5 billion. Despite its substantial impact, there is currently no cure for FASD. Existing research, including our own, indicates that various brain cells and regions exhibit differential vulnerability to alcohol-induced injury, with underlying mechanisms remaining poorly understood. For instance, neurons are particularly susceptible to alcohol stress, often undergoing acute apoptosis, and those that survive exhibit impaired functionality. Our primary goal is to elucidate the brain region- and cell-specific mechanisms underlying alcohol- induced developmental neurotoxicity (AIDN) and develop cell-specific precision interventions. By addressing the following critical questions, we aim to uncover novel targets for intervention: 1) Why are neurons more vulnerable to alcohol-induced injury? What neuron-specific signaling pathways contribute to this vulnerability? What are the brain region- and cell-specific targets for neuroprotection? To achieve these objectives, we will employ cutting- edge, multidisciplinary approaches including virus-based cell-specific gene modification, spatial transcriptomics, various imaging systems (e.g., multiphoton imaging), and various behavioral tests. Our research will utilize complementary models, including human induced pluripotent stem cell-derived 3D mini brains and mouse models, to investigate the mechanisms of AIDN at molecular, cellular, tissue, and animal levels. Additionally, we will assess the neuroprotective effects of neuron-specific and mitochondria-targeted interventions in AIDN. This study aims to provide a comprehensive understanding of the mechanisms driving alcohol-induced cognitive and behavioral impairments. By mapping acute and long-term brain region- and cell-type specific gene expression profiles following developmental alcohol exposure, our research offers promising strategies for early, brain cell type-targeted precision interventions and treatments for FASD patients. Moreover, the inclusion of human stem cell-based mini brain models will enhance the translational potential of our findings, potentially offering insights applicable to other neurodevelopmental disorders.

NINDS - National Institute of Neurological Disorders and Stroke

SUMMARY Axonal degeneration of cortical projection neurons underlies several debilitating neurodegenerative disorders including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. HSPs are a large heterogeneous group of inherited diseases characterized by length-dependent degeneration of corticospinal motor neuron axons, leading to spasticity and weakness of lower limb muscles. SPG11 and SPG15, two common autosomal recessive forms of HSP, are caused by mutations in the SPG11 and ZFYVE26 that encode spatacsin and spastizin protein, respectively. Spatacsin and spastizin are mediators for autophagy lysosomal reformation that is critical for maintaining lysosome homeostasis. However, how this impairment results in axonal degeneration and how this pathway can be targeted to rescue nerve degeneration in HSP remain unknown. Using patient induced pluripotent stem cell (iPSC)-based models of SPG11 and SPG15, our previous work has identified impaired mitochondrial dynamics in these patient stem cell-derived neurons. We further found aberrant autophagy influx and reduced lysosome transport in these neurons, implying their involvement in HSP. The goal of this proposed study is to dissect the interplays between these pathological processes and to determine their roles in axonal degeneration in HSP neurons. Based on strong preliminary data, we hypothesize that perturbed spatacsin and spastizin result in autophagy lysosomal defects and impaired mitochondrial dynamics, which interact with each other to impair cytoskeleton organization and axonal transport, leading to axonal degeneration in SPG11 and SPG15. This hypothesis will be tested by pursuing the following three aims: 1) to identify the role of spatacsin and spastizin in axonal and autophagy lysosomal defects of patient cortical projection neurons; 2) to determine the interplay between autophagy lysosomal and mitochondrial defects in axonal degeneration of SPG11 and SPG15 cortical neurons; and 3) to rescue axonal degeneration by targeting autophagy lysosomal and mitochondrial defects in vitro and in vivo. By regulating autophagy lysosomal and mitochondrial pathways both genetically and pharmacologically, this study will delineate their roles in axonal degeneration in HSP. The efficacy of targeting these pathways in rescuing axonal defects will be evaluated both in vitro using iPSC models and in vivo using HSP mouse models. Thus, the combination of iPSC model, gene targeting, and HSP animal model in this study provides unique opportunities to identify novel targets and develop potential therapeutics to effectively rescue axonal degeneration in HSP.

NIGMS - National Institute of General Medical Sciences

SUMMARY Cell-cell communication is essential for coordinating complex biological processes, and extracellular vesicles (EVs) have emerged as critical mediators of intercellular signaling. This project focuses on a unique class of large EVs called midbody remnants (MBRs), which are released during cell division. MBRs represent a novel mode of intercellular communication with potential implications in various physiological and pathological contexts. The overarching goal is to elucidate the mechanisms by which MBRs facilitate the transfer of information between cells. Specific goals over the next five years are to: 1) investigate how the informational content of MBRs varies in different biological contexts by identifying and characterizing conserved and unique molecular cargo (RNAs, small RNAs, and cell surface proteins) of MBRs from different cell types, including cancer, stem, and differentiated cells, and 2) elucidating the mechanisms by which recipient cells recognize and internalize MBRs. In addition, 3) we will investigate the potential hijacking of the MBR pathway by viruses for transmission, by examining viral RNA localization, factors required for viral RNA targeting to MBRs, and the ability of virus-infected MBRs to induce infections. Lastly, we will begin to: 4) investigate the role of MBRs in neurodevelopment and neurodevelopmental disorders, like autism spectrum disorder, by profiling changes in MBR informational content during neural progenitor cell differentiation and mechanistically testing genes necessary for cell fate and proliferative function that we find altered or loss in diseased states. The research design involves isolating MBRs from diverse cell types, performing transcriptomic and cell surface proteomic analyses, functional perturbation studies, live-cell imaging, and utilizing cellular and biochemical tools. This interdisciplinary approach will provide mechanistic insights from the genome- wide scale to sub-micron resolution. The findings from this project have significant implications for public health, as they could unravel the roles of MBRs in cell proliferation, RNA signaling, and EV biology, which are crucial in cancer, stem cell biology, and diseases associated with aberrant cell division and proliferation. Furthermore, understanding MBR function may identify novel therapeutic targets and establish MBRs as potential delivery vehicles for treating various diseases.

NHLBI - National Heart Lung and Blood Institute

Project Summary/Abstract The predominant myosin heavy chain expressed in human heart, beta-MyHC, is encoded by the MYH7 gene. MYH7 variants are well described in hypertrophic cardiomyopathy and less frequently seen in dilated cardiomyopathy. A recent series of publications link variants in the 5’ end of the MYH7 gene as implicated in left ventricular noncompaction cardiomyopathy, often in the setting of a dilated ventricle with impaired function. Importantly, premature truncations as well as missense variation within the MYH7 gene has been linked to LVNC in both population studies and in individuals and families. We now generated a heterozygous premature truncation in MYH7 in human induced pluripotent stem cells (hiPSCs). When differentiated into engineered human heart tissues, we observe the heterozygous premature truncation in MYH7 produces a phenotype consistent with excess proliferation and reduced function, which are key features thought to underlie the development of LVNC in vivo. We hypothesize that truncations and missense variants identified in LVNC are associated with reduced contractility, rather than hyperdynamic MYH7 variants seen in hypertrophic cardiomyopathy. Additionally, many missense variants in MYH7 are considered variants of uncertain significance and methods such as those being used here may help adjudicate variants of risk. Through this training program under the K99 phase, Dr. Monroe will evaluate missense MYH7 variants associated with LVNC and evaluate their performance in engineered heart tissues. In his second aim, he will expand the search for LVNC-associated MYH7 variation to the population scale using linked cardiac imaging and genotype data in the in population datasets. As Dr. Monroe transitions to his independent phase, he will build from work performed earlier in his train implicating the Hippo pathway in proliferation and specification. In Aim 3, he will detail new disease relevance for the Yes-associated protein (YAP) in MYH7-associated LVNC using the models already in hand and further developed under his K99 training. Finally, in Aim 4, Dr. Monroe uses unbiased approaches to characterize human cardiomyocyte heterogeneity in healthy and LVNC engineered heart tissues in order to better delineate the range of differentiation and identify additional downstream pathways that will fuel future investigations. To promote his career development, Dr. Monroe will draw on the strengths of his mentoring committee and primary mentor which will focus on expanding his management and his own mentoring skills.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cell transplantation (HSCT) is a potentially lifesaving treatment for patients with hematological and immune disorders but poses significant risk due to prolonged immunocompromised states before donor stem cell engraftment. Successful engraftment, where hematopoietic stem and progenitor cells (HSPCs) establish themselves in the recipient's bone marrow, requires that circulating donor HSPCs transmigrate the bone marrow endothelium to reach their supportive stem cell niche. Enhancing the transendothelial migration of HSPCs offers a promising strategy to speed engraftment and boost post- transplant immune system reconstitution. The full complement of receptors and intracellular machinery within the bone marrow niche endothelial cells (ECs) that promote HSPC migration remain incompletely understood. In mammals, the transendothelial migration of HSPCs occurs deep inside opaque bones, which are largely inaccessible to imaging and experimentation in live animals. The transparent zebrafish embryo, by contrast, which has a blood and vascular system highly similar to that of humans, enables direct visualization of HSPC migration in vivo. We previously identified a conserved gene expression signature unique to the hematopoietic niche ECs that regulate HSPC transmigration. This expression program includes genes with cell adhesion and endocytosis/vesicle trafficking functions. In my preliminary studies I disrupted candidate factors from the niche EC signature, which blocked endocytosis in the niche ECs and disrupted the niche migration of HSPCs. My data suggest that a combination of specific cell adhesion and endocytosis/vesicle trafficking machinery supports the endothelial transmigration of HSPCs, a hypothesis I will test with my proposed aims. In Aim 1, I use high-resolution in vivo imaging in the zebrafish paired with human cell culture systems to determine how a candidate adhesion molecule promotes HSPC migration. In Aim 2, I will use a rapid F0 zebrafish CRISPR knock-out platform to test whether different niche EC-expressed vesicle trafficking factors are required for HSPC migration. In parallel, I will use a transcription factor reprogramming approach to assess whether induction of the niche EC signature in different human cell types can trigger the transendothelial migration of HSPCs. Together, these proposed experiments will provide key insight into how niche EC-expressed factors promote the migration of HSPCs. Results from my proposed studies could inform methods to enhance bone marrow engraftment or to direct donor HSPCs to new and/or additional niches to improve HSCT outcomes.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Craniofacial bones, unlike other bones in the skeleton, originate from both neural crest and mesoderm. Studies have indicated that bones derived from neural crest regenerate faster than those from mesoderm, but the mechanisms behind this remain poorly understood. We discovered a novel network of p75 neurotrophin receptor-positive (p75-NTR+) cells within the periosteum of neural crest-derived bones that are crucial for the rapid regeneration of these skeletal sites. My central hypothesis is that these cells represent a distinct subset of neural crest-derived stem cells that are developmentally programmed to accelerate craniofacial regeneration through two independent mechanisms: (1) direct osteolineage differentiation and (2) the establishment of a pro-regenerative immune niche. My long-term goal as a dentist-scientist is to identify endogenous mechanisms of accelerated skeletal regeneration and to translate these findings into novel therapeutics that can promote cranial and dentoalveolar bone regeneration. The proposed K99/R00 research and training is designed to help me to achieve this goal. The K99 mentored phase of the project will be carried out with Dr. Erica Scheller and Dr. Farshid Guilak (primary co-mentors) at Washington University in St. Louis. The goal of the K99 phase is to provide additional training and mentoring in three areas: 1) Lab Management, 2) Bioinformatics and in silico New Approach Methodologies (NAMs), and 3) Inflammation and Immunology. A strong Scientific Advisory Committee with members from Washington University in St. Louis, Johns Hopkins University, and the University of Pittsburg has been established with diverse expertise in bone biology, skeletal regeneration and imaging, immunology, and bioinformatics/NAMs to oversee a training plan that complements and expands my scientific background, preparing me for a transition to independence during the R00 phase as faculty at a research-focused institution. To further dissect the regenerative potential of the novel p75-NTR+ cells and their contribution to the acceleration of neural crest-derived bone healing, I am proposing two research aims in this application as follows: Aim 1. define the dual osteogenic and immunomodulatory roles of p75-NTR+ periosteal cells in craniofacial bone regeneration; and Aim 2. explore the targeting of p75-NTR+ cells as a therapeutic strategy to accelerate craniofacial regeneration. Overall, the proposed experiments aim to identify the intrinsic biological pathways that accelerate bone healing while considering the implications of these results for clinical management of skeletal disease. This research holds significant translational therapeutic potential for patients with skeletal defects and is expected to ultimately improve patient outcomes and quality of life.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Our understanding of immunity largely stems from models of infection with pathogenic microbes. However, the vast majority of microbial-immune encounters occur as a symbiotic relationship with the commensal microbiota. Recently, the contribution of commensal-specific T cells to host physiology has received significant attention. These commensal-specific responses not only control microbiota containment but also promote immune tolerance within the gastrointestinal tract. While commensal-specific CD4+ T cell responses in the lamina propria have dominated models of mucosal immune regulation, these are vastly outnumbered by CD8+ intraepithelial lymphocytes within the epithelium. How CD8+ T cell responses to gut microbiota are primed, differentiate and function under homeostasis has not been addressed. Conversely, aberrant immunity to commensal microbes has been proposed to underlie pathologies of barrier tissues, including inflammatory bowel disease (IBD), where commensal-specific T cells accumulate in blood and intestinal tissues of afflicted patients. A better understanding of the properties and functions of commensal-specific T cell responses is therefore fundamental to studies of tissue immunity in health and disease. Our long term goal is to better understand how commensal-specific T cell responses contribute to barrier tissue homeostasis, and the objective in this application is to investigate the mechanisms regulating induction of commensal-specific CD8+ T cells in homeostasis and how they become dysregulated in IBD. Our rationale for the proposed work is that uncovering these mechanisms has the potential to translate into new therapeutic approaches. Our central hypothesis is that commensal-specific CD8+ T cells develop as functionally restrained intraepithelial lymphocytes (IEL) under homeostasis, but that perturbation of local immune regulation within the intestinal epithelium, in the case of patients with ulcerative colitis, by autoantibody-mediated blockade of integrin avb6 results in aberrant CD8+ effector T cell responses in IBD. Based on strong preliminary data, we will test three specific aims: (1) Determine key antigen-presenting cells (APC) priming SFB-specific CD8⍺β+ IEL. (2) Identify how cell-intrinsic pathways drive differentiation, maintenance and restraint of SFB-specific CD8⍺β+ pIEL. (3) Determine how pathogenic KLRG1+Eomes+ CD8+ T cells arise and contribute to inflammation in murine models of ulcerative colitis Our approach is innovative as it investigates new mechanisms of immunity unique to commensal-specific CD8+ T cell responses. The proposed work is significant because it will establish new insights into the interaction and communication between commensal microbes and immune cells in the gut environment and identify potential targets for therapeutic intervention in conditions of chronic intestinal inflammation.

NIA - National Institute on Aging

Abstract Protein synthesis is a vital biological process, important for neuronal development, synaptic plasticity, and cognitive functions such as learning and memory. In contrast, dysregulated translation is a feature of many neurodegenerative disorders, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Pathogenic changes to the microtubule associated protein tau are thought to cause neurotoxicity and dysfunction in both AD and FTD in part by disrupting several molecular processes, including protein synthesis. However, the molecular mechanisms by which pathogenic tau disrupts protein synthesis remain elusive. In this application, we will determine how FTD-associated heterozygous mutations in tau impact protein synthesis in human neurons. We will use human induced pluripotent stem cell (iPSC)-derived neurons carrying FTD-associated tau mutations as a model. The iPSCs will be differentiated into neurons using Neurogenin-2, a master transcription factor capable of inducing differentiation into excitatory neurons in under two weeks. Using this platform, in the first aim we will determine the impact of FTD-associated tau mutations on translation elongation rates and will perform ribosome profiling to determine the translatome and translational efficiency associated with the FTD- associated tau mutations. In the second aim, we will determine whether the tau mutations alter tau- ribosome interactions and if they cause ribosome collisions. These studies will provide insight concerning the mechanisms by which FTD-associated mutations in tau alters protein synthesis, as well as the biology and subsequent pathobiology of tau in tauopathies such AD and FTD.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ ABSTRACT Blood vessels are the earliest organs formed during embryonic development, and the embryonic cardiovas- cular system is required for nutrient exchange and organogenesis. As early as mouse embryonic day (E) 7.5 in the extraembryonic yolk sac blood islands and embryos, mesodermal precursors undergo the first cell fate de- cision to form the endothelial cell (EC) lineage. ECs further differentiate into arterial, venous, and lymphatic subtypes that are necessary to build the vascular system. However, the clonal relationships and spatial origins of EC subtypes that form the vasculature are poorly defined, and the mechanisms of EC specification and dif- ferentiation in vivo remain incompletely understood, posing a barrier to vascular regenerative medicine. Etv2, an ETS-family transcription factor, is a master regulator of EC specification. Etv2 is transiently ex- pressed in mesoderm progenitors in the yolk sac and developing mouse embryo from E7.5 - E9.5. Mice lacking Etv2 fail to develop blood or vasculature, and Etv2 is responsible for endothelial and hematopoietic lineage specification. Conversely, forced expression of Etv2 induces EC reprogramming. We have carefully interro- gated mechanisms by which ETV2 promotes EC specification in mesoderm progenitors derived from human induced pluripotent stem cells (iPSCs), using scRNA-seq, scATAC-seq, CUT&RUN, and functional CRISPR screens. Novel observations included: (1) the strength or timing of ETV2 expression influenced arteriovenous EC differentiation; (2) in addition to stimulating EC specification, ETV2 also suppressed specification of other mesodermal lineages; and (3) ETV2 suppression of alternative fates required its recruitment of the transcrip- tional repressor REST. These observations lead to our central hypothesis that ETV2 drives EC specification and arteriovenous differentiation, with REST cooperating with ETV2 to restrict alternative lineages during em- bryonic vasculogenesis. To track cell state transitions and fate of Etv2-expressing cells during vasculogenesis, we will integrate cut- ting edge single cell, spatial transcriptomics, and barcoded lineage tracing approaches to track the progeny of Etv2-expressing progenitors. To focus our efforts, in Aim 1 we will study the initial steps of vessel formation in the yolk sac. We will perform spatial and clonal analysis of Etv2-lineage cells during early yolk sac vasculogen- esis and late vascular plexus remodeling. To determine the role of Rest in Etv2-directed EC specification, in Aim 2 we will determine the effect of Rest inactivation on vasculogenesis and Etv2-lineage diversification, un- cover the Rest-Etv2 transcriptional regulatory network, and identify Rest-regulated genes and TFs in Etv2-line- age progenitors required for EC specification. The work will produce a high-resolution phylogenetic tree of vas- cular development originating from individual Etv2+ progenitors and bring new insights into the molecular mechanisms that direct EC subtype differentiation, informing future efforts in vascular regeneration.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Sickle cell disease (SCD) is a devastating inherited hemolytic anemia that affects millions of people worldwide. Over 500,000 infants are born with SCD annually and the majority die before 5 years of age due to spleen dysfunction. In studies conducted with my K23 award, we determined that spleen damage in SCD impacts adaptive immunity. We identified an age-related decline in unswitched memory B cells (UMBC), the peripheral blood equivalent to splenic marginal zone B cells, and a corresponding increase in naïve B cells compared to children without SCD. Children with SCD/very low UMBC had >2-fold lower splenic expression of three genes important for B cell differentiation compared to SCD/low UMBC: IL21R, PF4, and CX3CR1. Expression of genes for ligands that activate B cell differentiation, DLL1 and JAG1, were significantly higher in children with SCD/very low UMBC compared to SCD/low UMBC. These data suggest that IL21R, PF4, CX3CR1, DLL1, and JAG1 have a role in the mechanism of MZB loss, B cell differentiation, and adaptive immunity in SCD. There is a critical gap in knowledge about the mechanisms of adaptive immune dysfunction in the spleen, and how to target these pathways to prevent life-threatening infections and autoimmunity in SCD. Our purpose of this limited R03 award is to prioritize genes important for B cell development in SCD spleen for future clinical and mechanistic studies. Our central hypothesis is that SCD alters expression of key genes important for B cell differentiation, leading to low UMBCs and higher naïve B cell counts. Aim 1. Develop an ex vivo system to investigate the role of IL21R, PF4, and CX3CR1 in B cell development in SCD. We will validate our findings with RNA sequencing of additional spleen samples. We will measure serum IL21, PF4, and fractalkine (the ligand for CX3CR1) by ELISA in SCD and correlate levels with B cell subsets. We will differentiate CD34+ stem cells into B cells to compare expression and activity of IL21R, PF4, and CX3CR1 in SCD- versus non-SCD-derived B cell subsets using flow cytometry and transcriptomic approaches. Aim 2. Determine the role of DLL1 and JAG1 in B cell differentiation in the spleen in SCD. We will use spatial transcriptomics to localize DLL1 and JAG1 signaling in human spleen tissue. We will use imaging flow cytometry to compare interactions between cells that express DLL1 and JAG1 and B cells in spleen tissue from patients with and without UMBC loss. We will validate the Townes SCD mouse model as a tool to examine how inflammatory stimuli influence the expression of Dll1 and Jag1 in the spleen in vivo. Impact: I expect my research will lead to significantly improved outcomes for SCD by identifying novel pathways in adaptive immunity that contribute to complications in SCD. Enhanced understanding of these pathways will yield new targets urgently needed for druggable disease modification. Within two years, I will have necessary tools and preliminary data to apply for independent funding through an R01 or similar mechanism.

NIA - National Institute on Aging

PROJECT SUMMARY/ABSTRACT This research proposal is in response to the NOT-OD-24-079 “Notice of Special Interest: Women’s Health Research” focused on health conditions that are female-specific. The ovary is the first organ to age in the human body. Ovarian aging negatively influences lifespan and a broad range of health outcomes in cardiovascular, skeletal, metabolic, immune, and neurocognitive systems in women. Despite these broad impacts, ovarian aging has received limited scientific attention. The biological mechanisms that drive ovarian aging, and how they influence broader healthspan in women, remain poorly understood. The objective of this proposal is to investigate the molecular mechanisms by which NBR2, a long non-coding RNA, contributes to the remarkably complex processes of ovarian aging. By performing genome wide association studies (GWAS) of two reproductive aging- related traits, age of natural menopause (ANM) and reproductive lifespan (RL), we found that genetic variants within a gene-rich haplotype block at the BRCA1 locus are associated with both traits. Our integrative post- GWAS analysis, combined with functional genomic studies in human ovarian cell models, identified a causal non-coding regulatory variant (rs2298862 T>C) associated with both later ANM and longer RL. Although previous ANM GWAS studies identified BRCA1 as the causal gene at this locus, our functional genomic studies experimentally validated that NBR2 at the true target gene. The variant downregulated NBR2 expression, suggesting that NBR2 is a likely driver of the reproductive longevity phenotypes. The major goal of this project is to uncover the mechanisms by which NBR2 modulates female reproductive longevity (Aim 1) and elucidate the mechanisms by which the causal regulatory variant regulates NBR2 expression in diverse ovarian cell types (Aim 2). In Aim 1, I will test the hypothesis that reduced NBR2 expression delays ovarian aging by modulating pro-longevity signaling pathways. I will generate CRISPR/Cas9-mediated knockout NBR2 cell models and perform unbiased RNA-immunoprecipitation followed by mass spectrometry to identify NBR2 interactors and downstream targets. In Aim 2, I will test the hypothesis that the rs2298862 (T>C) variant reduces NBR2 expression by altering long-range chromatin interactions, disrupting transcription factor binding, and modulating enhancer activity. I will generate multiple ovarian cell types from CRISPR-engineered human embryonic stem cells carrying the variant and assess its impact on NBR2 expression, chromatin architecture, transcription factor (TF) binding, and aging-related cellular phenotypes. By identifying pathways and regulatory mechanisms by which NBR2 and its downstream regulators influence ovarian aging, this project will provide a molecular framework for understanding human reproductive longevity and may reveal targets for preserving ovarian function and healthspan in women.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Myotonic Dystrophy Type 1 (DM1) is a progressive neuromuscular disorder affecting 1 in 8,500 individuals. Although primarily characterized by skeletal muscle dysfunction, over 80% of DM1 patients exhibit neurological manifestations, including cognitive impairment, autistic features, ADHD, depression, anxiety, sleep disturbances and excessive daytime sleepiness. DM1 is caused by a CTG repeat expansion in the 3' untranslated region of the DMPK gene. The mutant RNA transcripts containing expanded CUG repeats form nuclear RNA foci and sequester the Muscleblind-Like (MBNL) family of RNA-binding proteins, leading to their functional depletion and the dysregulation of RNA processing. Studies in skeletal muscle have revealed that characteristic DM1 symptoms, including myotonia and muscle weakness, result from mis-splicing of MBNL target genes such as the chloride channel ClC-1 and calcium channel Cav1.1. While knockout of the MBNL paralogs, MBNL1 and MBNL2, reproduces DM1-like neurological phenotypes in mice, the molecular mechanisms underlying DM1 brain pathology remain unclear, particularly whether these symptoms also stem from the dysregulation of alternative splicing or other MBNL-dependent processes such as RNA localization and stability. To investigate the neurological manifestations of DM1, I generated a novel DM1 brain mouse model, CUG960, which expresses 960 interrupted CUG repeats throughout the central nervous system (CNS). This model recapitulates key DM1 features, including nuclear RNA foci, MBNL sequestration, reduced brain weight, and behavioral abnormalities. The CUG960 mouse model is doxycycline-repressible, enabling temporal control of CUG repeat expression for rescue studies. Despite robust physiological and behavioral deficits, CUG960 mice show only modest splicing changes, suggesting additional mechanisms may contribute to the CNS pathology. The goals of this proposal are threefold. First, I will perform behavioral assays and sleep studies on the CUG960 mouse model to determine the short and long-term effects of CUG repeat RNA expression in the CNS and identify brain regions most vulnerable to CUG repeat toxicity. Second, using the CUG960 mouse model, I will identify changes in alternative splicing, gene expression, and RNA localization and determine the relative contributions of nuclear and cytoplasmic MBNL loss-of-function through rescue experiments. Third, I will use the doxycycline-repressible feature of the CUG960 mice to suppress CUG repeat expression at different timepoints to determine if the neurological DM1 symptoms are reversible and define the critical therapeutic time window. Completion of this proposal will establish the direct effects of CUG repeat RNA expression in the CNS, elucidate the molecular mechanisms driving the neurological manifestations in DM1, and determine whether and when these phenotypes can be reversed. These findings will provide crucial insights into the mechanisms underlying DM1 brain disease and inform the development of therapeutic approaches.

NIAID - National Institute of Allergy and Infectious Diseases

SUMMARY Cell therapies are a promising alternative therapy for the treatment of type 1 diabetes, and stem cell-derived insulin secreting beta cells (sBC) have demonstrated the feasibility of restoring insulin independence in clinical models. However, this strategy has limited long-term application due to the requirement of ineffective and toxic immunosuppressive drug regimens and immune rejection, which currently limits the applicable patient pool to high-risk patients. The leading commercial entities clinically testing stem cell-derived islet replacement products are pursuing macroencapsulated cell delivery methods to overcome this critical hurdle to translation. Macroencapsulation devices can blunt immune responses to the graft and confer the safety benefit of cell delivery in a single, retrievable device. However, functional clinical success of these devices has not been demonstrated to date due in part to inevitable antigen shedding resulting in indirect antigen recognition, which results in immune destruction of encapsulated cells. Thus, synergistic immunomodulatory approaches are necessary to fully immunoprotect encapsulated cell grafts, and achieve immunological tolerance in the absence of immune suppression. The fetal-maternal interface is a robust model of immune tolerance toward allogeneic tissue, where placental trophoblasts maintain tolerance by two main approaches: (1) presenting an inert surface to maternal immune cells, a strategy akin to cell encapsulation which blocks direct antigen recognition; and (2) through secretion of tolerogenic factors which induce tolerance toward fetal antigens that escape the placenta. Our preliminary data demonstrates that a macroencapsulated tolerogenic trophoblast cell therapy can evade rejection and delay rejection of bystander macroencapsulated cell grafts in a challenging xenotransplant model. In this proposal, we aim to (1) validate our preliminary studies using translatable cell sources of sBC and trophoblasts, and (2) elucidate the immunological mechanisms of trophoblast cell therapy-induced tolerance in xeno and humanized allogeneic transplantation models. This will be addressed in three Specific Aims: (1) Identify immune mechanisms of trophoblast cell therapy-induced transplant site-dependent graft tolerance, (2) Optimize and characterize tolerogenic cell therapy dose impact on tolerance induction, and (3) Untangle the contributions of antigen-specific and non-specific mechanisms of trophoblast cell therapy tolerance induction. In this work, we expect to identify the immunological mechanisms by which tolerogenic trophoblast cell therapies delay or prevent immune rejection.