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

Child development and organogenesis depend on a highly regulated interplay of inductive events, which are regulated by evolutionarily conserved cell signaling pathways. Wnt-signaling pathways are a signaling system, conserved across the animal kingdom and regulating early development by inductive events and subsequent cell growth, cell fate, and cell polarity across whole organisms and during organogenesis in the whole body. Wnt-signaling is also critical in stem cell establishment and maintenance of (almost) all tissues. At the center of these pathways are the Wnt morphogens and their receptors, the Frizzled family (Fz in Drosophila, and Fzd in mammals) of 7-pass trans-membrane proteins. The canonical Wnt-pathway also critically employs the LRP5/6 co-receptors (known as Arrow in Drosophila). While a lot is known about the architecture of the two main Wnt-pathways: the canonical Wnt/-catenin pathway and Wnt/PCP (Planar Cell Polarity) signaling, and the membrane protein complex associated activation of the canonical pathway, employing Fzd-LRP5/6 heteromeric complexes for Wnt binding, much less is known about the activation of Wnt/PCP signaling, leading to asymmetric PCP complex localization and hence cell polarity. We have strong preliminary evidence for a critical role of Arrow-LRP5/6 in Wnt/PCP signaling, which we propose to address in this application. Regulation of Wnt-signaling specificity between the Wnt/-catenin pathway and Wnt/PCP signaling remains unclear, and has so far largely been linked to the presumed specific role of Arrow-LRP5/6 in the Wnt/-catenin pathway, and differential recruitment of Dishevelled (Dsh/Dvl) proteins as the cytoplasmic mediators of Wnt- receptor interactions. As Dsh is at the node of all Wnt-signaling events and the pathways are sensitive to levels of Dsh/Dvl, we have designed and utilized a genome-wide genetic screen in Drosophila to identify novel “pathway specificity”-regulators of Wnt-signaling activation. Strikingly, this screen has revealed a critical role of Arrow-LRP5/6 in both pathways, including Wnt-PCP signaling, while it is presumed to be Wnt/-catenin pathway specific as outlined above. We are proposing here to functionally define and dissect the role of Arrow- LRP5/6 in Wnt-PCP signaling. This new function of Arrow-LRP5/6 in Wnt/PCP signaling is very unexpected and intriguing, as it challenges the pathway architecture as presented in text books, and thus it is an exciting and challenging new research avenue in the Wnt-signaling context. The specific Aims are to (1) define the phenotypic requirements of Arrow-LRP5/6 in Wnt-PCP signaling, and (2) establish the molecular mechanisms of how Arrow-LRP5/6 intersects with the well-known PCP-Core factors, including the PCP dedicated Fz receptor subset(s) and the shared effector Dsh. Our preliminary data suggest that Arrow-LRP5/6 promotes Fz- PCP signaling, acting positively upstream of Dsh in the PCP complex formation. Information acquired in this application will advance our mechanistic understanding of Arrow-LRP5/6 in the Wnt-pathways, and potentially could also lead to follow up studies in developmental disease associated contexts.

U.S. National Science Foundation

Proposals in the area of plasma physics submitted to the Division of Physics that are not governed by another solicitation (such as CAREER), should be submitted to the Division-wide solicitation: <a href="https://new.nsf.gov/funding/opportunities/division-physics-investigator-initiated-research">Division of Physics: Investigator-Initiated Research Projects</a>. The Plasma Physics program participates in multiple NSF meta-programs such as the <a href="https://new.nsf.gov/funding/opportunities/ecosystem-leading-innovation-plasma-science" target="_blank">ECosytem for Leading Innovation in Plasma Science and Engineering (ECLIPSE)</a>, <a href="https://new.nsf.gov/funding/opportunities/windows-universe-era-multi-messenger-astrophysics" target="_blank">Windows on the Universe: The Era of Multi-Messenger Astrophysics (WoU-MMA)</a>, and <a href="https://new.nsf.gov/funding/opportunities/computational-data-enabled-science-engineering-3" target="_blank">Computational and Data-enabled Science and Engineering (CDS&amp;E)</a>. Topically appropriate proposals may also be submitted to the Plasma Physics program in response to NSF Dear Colleague Letters such as <a href="https://www.nsf.gov/pubs/2022/nsf22111/nsf22111.jsp" target="_blank">Critical Aspects of Sustainability (CAS): Innovative Solutions to Sustainable Chemistry (CAS-SC)</a>. When permitted under an MOU between NSF and another funding agency or private foundation, NSF may share information from proposals submitted to this solicitation for consideration of joint funding, and may invite employees of such organizations to attend merit review panels as observers. MOUs of relevance to the Plasma Physics program presently exist with the Department of Energy/Office of Science, National Nuclear Security Administration, the Air Force Office of Scientific Research, the US-Israel Binational Science Foundation, the Czech Science Foundation, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), and the Swiss National Science Foundation. Plasma Physics is a study of matter and physical systems whose intrinsic properties are governed by collective interactions of large ensembles of free charged particles. 99.9% of the visible Universe is thought to consist of plasmas. The underlying physics of the collective behavior in plasmas has applications to space physics and astrophysics, materials science, applied mathematics, fusion science, accelerator science, and many branches of engineering. The Plasma Physics program supports research that can be categorized by several broad, sometimes overlapping, sub-areas of the discipline, including: magnetized plasmas in the laboratory, space, and astrophysical environments; high energy density plasmas; low temperature plasmas; dusty, ultra-cold, and otherwise strongly coupled plasmas; non-neutral plasmas; and intense field-matter interaction in plasmas. The focus of the Plasma Physics program is to generate an understanding of the fundamental principles governing the physical behavior of a plasma via collective interactions of large ensembles of free charged particles, as well as to improve the basic understanding of the plasma state as needed for other areas of science and engineering. Principal Investigators (PIs) are encouraged to consider including specific efforts to increase diversity of the plasma physics community and broaden participation of under-represented groups in Science, Technology, Engineering, and Mathematics (STEM) as Broader Impacts of proposed work. Development of new undergraduate and graduate plasma physics curricula, or curricula enhancement to include plasma physics topics in other courses, at institutions lacking such coursework is similarly encouraged. NSF recognizes that some research projects within this Program may require more than three years to realize demonstrable research outcomes. For such projects, PIs are encouraged to consult the above Program Director to discuss the possibility of submitting a proposal of 4- or 5-year duration. Some Plasma Physics-related activities are supported primarily by other NSF Programs. Proposals focused on the physical properties of individual or a small number of atoms or molecules, or optical physics, should be directed to the Atomic, Molecular, and Optical Physics Program within the Division of Physics. Proposals focused on understanding astrophysical systems should be directed to the Division of Astronomical Sciences. Proposals focused on understanding the Geospace environment or the Sun-Earth interactions should be directed to an appropriate program within the Geospace Section of the Division of Atmospheric and Geospace Sciences. Proposals focused on development of new materials using plasmas should be directed to an appropriate program in the Division of Materials Research. Proposals focused on plasma-assisted manufacturing should be directed to the Division of Civil, Mechanical and Manufacturing Innovation. Finally, proposals focused on use of plasmas for environmental and reaction engineering, environmental sustainability, combustion systems, or engineering of biomedical systems should be directed to an appropriate program within the Division of Chemical, Bioengineering, Environmental and Transport systems.

U.S. National Science Foundation

Proposals in the area of plasma physics submitted to the Division of Physics that are not governed by another solicitation (such as CAREER), should be submitted to the Division-wide solicitation: Division of Physics: Investigator-Initiated Research Projects. The Plasma Physics program participates in multiple NSF meta-programs such as the ECosytem for Leading Innovation in Plasma Science and Engineering (ECLIPSE), Windows on the Universe: The Era of Multi-Messenger Astrophysics (WoU-MMA), and Computational and Data-enabled Science and Engineering (CDS&amp;E). Topically appropriate proposals may also be submitted to the Plasma Physics program in response to NSF Dear Colleague Letters such as Critical Aspects of Sustainability (CAS): Innovative Solutions to Sustainable Chemistry (CAS-SC). When permitted under an MOU between NSF and another funding agency or private foundation, NSF may share information from proposals submitted to this solicitation for consideration of joint funding, and may invite employees of such organizations to attend merit review panels as observers. MOUs of relevance to the Plasma Physics program presently exist with the Department of Energy/Office of Science, National Nuclear Security Administration, the Air Force Office of Scientific Research, the US-Israel Binational Science Foundation, the Czech Science Foundation, Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), and the Swiss National Science Foundation. Plasma Physics is a study of matter and physical systems whose intrinsic properties are governed by collective interactions of large ensembles of free charged particles. 99.9% of the visible Universe is thought to consist of plasmas. The underlying physics of the collective behavior in plasmas has applications to space physics and astrophysics, materials science, applied mathematics, fusion science, accelerator science, and many branches of engineering. The Plasma Physics program supports research that can be categorized by several broad, sometimes overlapping, sub-areas of the discipline, including: magnetized plasmas in the laboratory, space, and astrophysical environments; high energy density plasmas; low temperature plasmas; dusty, ultra-cold, and otherwise strongly coupled plasmas; non-neutral plasmas; and intense field-matter interaction in plasmas. The focus of the Plasma Physics program is to generate an understanding of the fundamental principles governing the physical behavior of a plasma via collective interactions of large ensembles of free charged particles, as well as to improve the basic understanding of the plasma state as needed for other areas of science and engineering. Principal Investigators (PIs) are encouraged to consider including specific efforts to increase diversity of the plasma physics community and broaden participation of under-represented groups in Science, Technology, Engineering, and Mathematics (STEM) as Broader Impacts of proposed work. Development of new undergraduate and graduate plasma physics curricula, or curricula enhancement to include plasma physics topics in other courses, at institutions lacking such coursework is similarly encouraged. NSF recognizes that some research projects within this Program may require more than three years to realize demonstrable research outcomes. For such projects, PIs are encouraged to consult the above Program Director to discuss the possibility of submitting a proposal of 4- or 5-year duration. Some Plasma Physics-related activities are supported primarily by other NSF Programs. Proposals focused on the physical properties of individual or a small number of atoms or molecules, or optical physics, should be directed to the Atomic, Molecular, and Optical Physics Program within the Division of Physics. Proposals focused on understanding astrophysical systems should be directed to the Division of Astronomical Sciences. Proposals focused on understanding the Geospace environment or the Sun-Earth interactions should be directed to an appropriate program within the Geospace Section of the Division of Atmospheric and Geospace Sciences. Proposals focused on development of new materials using plasmas should be directed to an appropriate program in the Division of Materials Research. Proposals focused on plasma-assisted manufacturing should be directed to the Division of Civil, Mechanical and Manufacturing Innovation. Finally, proposals focused on use of plasmas for environmental and reaction engineering, environmental sustainability, combustion systems, or engineering of biomedical systems should be directed to an appropriate program within the Division of Chemical, Bioengineering, Environmental and Transport systems.

Louisa Boren STEM PTA

The Louisa Boren School Community is embarking on a project to make the playground more family friendly for use by the neighborhood. The project includes the installation of a bench to serve as a resting place, a site clean up and adding paint to 4 square courts and the area adjacent to the new bench. A community celebration at the playground, 5950 Delridge Way South West, is planned for June.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) has persistently maintained a low ~32% 5-year survival rate in spite of over three decades of medical research. With approximately 22,010 in the U.S being diagnosed with AML and 11,090 dying from the disease yearly, there remains an urgent need to develop new therapies and treatment strategies. Understanding the key protein drivers of AML and the regulatory mechanisms governing expression of these proteins has provided us with an opportunity to specifically target the disease. The most common variants of AML in both pediatric and adult patients depend upon the chromatin binding MLL protein complex. Recently developed small molecule menin-inhibitors have demonstrated remarkable clinical success by inhibiting MLL complex formation. However, close to 40% of patients treated with menin inhibitors for prolonged periods develop resistance and subsequently relapse, demonstrating an urgent need for combination therapy. Our lab has previously shown that a critical component of the MLL complex, LEDGF, is sensitive to perturbations in translation due to its short half-life. I demonstrated that LEDGF protein expression can be inhibited with the RNA helicase eIF4A1 inhibitor silvestrol, and that silvestrol has potent anti -AML properties. The following aims will test the hypothesis that eIF4A1 inhibition is an effective AML therapy in vivo, capable of circumventing menin inhibitor resistance. In Aim 1 I will generate novel mouse models of menin inhibitor resistant adult and pediatric AML to test the efficacy of eIF4A1 inhibition as an AML therapy. In Aim 2 I will identify the molecular mechanisms by which eIF4A1 contributes to LEDGF translation, exploring the role of eIF4A1 in maintaining AML cells. The long-term objectives of this project are to characterize a clinically relevant new approach to treat AML and uncover molecular mechanisms governing mRNA translation. This fellowship application is sponsored by Dr. Daisuke Nakada, PhD, an expert in hematopoietic stem cell biology and acute myeloid leukemia biology. This training plan is designed to 1) provide mentorship from experts in science and medicine; 2) develop general and field-specific scientific knowledge in stem cell, chromatin, and RNA biology; 3) grow my scientific communication skills and form professional networks; and 4) develop clinical skills and knowledge toward a pediatrician-scientist career. The clinical and scientific training environment is at Baylor College of Medicine, located in the heart of the Texas Medical Center with close ties to institutions such as Texas Children's Hospital and MD Anderson Cancer Center. This environment is ideal to foster scientific and clinical growth toward my long-term goal of becoming a physician scientist in the field of pediatric hematology-oncology.

NIGMS - National Institute of General Medical Sciences

Project Summary P-bodies are ribonucleoprotein granules that form membraneless compartments through liquid-liquid phase separation and regulate gene expression by directing mRNAs for degradation, translation, or storage. In humans, P-bodies are estimated to influence one-third of genes in the genome. Recent research highlights their involvement in stem cells, where they regulate cell cycling and cell fate decisions. Despite these advances, little is known about how P-bodies function in connective tissue or during tissue regeneration. Our recent work using in vivo proximity labeling in zebrafish identified Ddx61, a protein central to P-body formation, as critical during regeneration. Ddx61 forms condensates reminiscent of P-bodies, and its loss is associated with reduced cell proliferation and impaired tissue regeneration. Interestingly, these regenerative P-bodies form in response to injury and dissipate once regeneration is complete, suggesting they are dynamically regulated. These findings provide a strong foundation to explore the mechanisms of P-body formation and their functional significance in regeneration. In my newly established laboratory, we aim to address the mechanisms underlying P-body formation and function in the context of tissue regeneration. Specifically, we will determine the composition of regenerative P-bodies using innovative tools, including in vivo proximity labeling and super-resolution microscopy. To establish causality, we will bioengineer artificial P-bodies and test their ability to regulate mRNA fate in zebrafish. This will be one of the first comprehensive studies on the post-transcriptional regulation of gene expression by P-bodies in connective tissue and during tissue regeneration. By uncovering how regenerative P-bodies regulate cell division and differentiation, this research will advance our understanding of post-transcriptional regulation and provide a foundation for developing novel therapeutic strategies for tissue regeneration, aging, and cancer.

European Commission - HORIZON

Pragmatic clinical trials to optimise immunotherapeutic interventions for patients with refractory cancers. Call: Supporting the implementation of the Cancer Mission. Programme: HORIZON. Keywords: Anticancer therapy, Diagnostic tools (e.g. genetic, imaging), Haematology, Medical devices, Paediatrics, Pharmacogenomics, Pharmacology, pharmacogenomics, drug discovery and design, drug therapy, Radiation therapy, Stem cell therapy, Surgery. Open call from the EU Funding & Tenders Portal.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

TITLE: PRAISE (Pressure Relief Assessment Information System): A Paradigm-shifting Mobile Health Platform for Pressure Relief Adherence in Manual Wheelchair Users PROJECT SUMMARY: The proposed project aims to create a pressure relief assessment information system (PRAISE) to enhance the adherence of manual wheelchair users to Clinical Practice Guidelines (CPGs) designed to prevent pressure ulcers. The motivation of this research stems from two core challenges. First, pressure ulcers pose a serious threat to manual wheelchair users with spinal cord injuries, frequently leading to painful complications, infections, and even premature death. To reduce pressure ulcer risks, CPGs recommend that wheelchair users perform pressure relief activities (i.e., vertical pushups, lateral, and forward leans) every 15 to 30 minutes. However, research reveals that wheelchair users may not adhere to CPGs in everyday life. Second, no universally adopted tools currently exist to monitor CPG adherence, nor is the understanding of factors leading to non-adherence. As a result, the prevalence of pressure ulcers among wheelchair users with spinal cord injuries remains high. Built upon the International Classification of Functioning, Disability and Health (ICF) model, PRAISE will shift from the conventional singular focus on adherence to a holistic approach, which will cohesively integrate a user's health, personal, and environmental factors through its multidimensional design. First, PRAISE will enable users to create profiles, including demographics, wheelchair usage patterns, and medical records related to pressure ulcers. Second, this foundational data will be augmented by a spectrum of sensor data (i.e., accelerometer, heart rate, GPS, and battery life) from a smartwatch, critical for ecological momentary assessments (EMAs). Third, our novel distributed algorithm can accurately detect pressure relief activities without relying on frequent, costly internet connections. It achieves this through lightweight processing on mobile devices to capture patterns intrinsic to pressure relief activities, hence transmitting only relevant data segments to the server for fine-grained recognition. Fourth, grounded in the ICF framework, PRAISE will dynamically integrate user-specific health, personal, and environmental factors to deliver context-aware feedback and personalized guidance. Through reinforcement learning, PRAISE will continuously evolve its guidance by learning from user responses and behavior, ensuring that interventions remain effective and tailored to individual needs over time. In collaboration with a diverse advisory team, PRAISE's development will prioritize robust security, user- friendliness, advanced analytics, and customizable assessment modules. Once the advisory team completes the initial validation, a feasibility and acceptability assessment will be conducted by involving 15 manual wheelchair users for two weeks. To gain a deeper understanding of user experiences, we will employ multifaceted approaches to gather and analyze user feedback. As PRAISE strives to make pressure ulcer prevention more accessible and personalized for wheelchair users, it will help reduce health disparities, particularly for those who may not have easy access to traditional healthcare resources. Therefore, PRAISE will revolutionize care for the manual wheelchair users to achieve patient-centric, evidence-based interventions.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT A lack of proven interventions to prevent leukemia in aged populations leaves a growing demographic vulnerable to this devastating disease. While leukemic cells are subject to immune selection which influences disease progression, we lack knowledge of the stages at which T cells shape the hematopoietic stem and progenitor cell (HSPC) pool from the initiation of clonal hematopoiesis (CH) through to the progression to leukemia, which limits our ability to intervene in this process. The long-term goal of this project is to identify immuno-preventative strategies to intercept leukemogenesis at its earliest stages. The overall objective of this application is to determine the mechanisms by which, and at which stages of pre- leukemic development, HSPC clones are detected and selected by the adaptive immune system. The central hypothesis is that reduced IFNγ responsiveness enables immune evasion of CH-mutant (Dnmt3amut) HSPCs thereby promoting clonal expansion and pre-leukemic evolution. The rationale is grounded in the observation that HSPCs from humans and mice with recurrent CH driver mutations in Dnmt3a have reduced transcript and protein expression of MHC-II machinery, and reduced presentation of exogenous and endogenous antigens via MHC-II. Mechanistically, MHC-II is potently induced by IFNγ on wild-type HSPCs but to a lesser extent on Dnmt3amut HSPCs. In vitro and in vivo, we observe less activation and proliferation of CD4+ T cells by Dnmt3amut HSPCs compared to control HSPCs, supporting that Dnmt3amut HSPCs have reduced immunogenicity. The central hypothesis will be tested by pursuing two specific aims: 1) to define the stages of pre-leukemic HSPC selection that are controlled by CD4+ T cells, and 2) to evaluate decreased IFNγ response of pre-leukemic Dnmt3amut HSPCs as a mechanism of immune evasion. This research is innovative because it introduces a novel framework for understanding how adaptive immunity shapes clonal evolution of pre-leukemic HSPCs. While considerable attention has been given to genetic and cell-intrinsic drivers of CH, the role of immune surveillance—particularly adaptive immune selection—in governing HSPC clonality remains largely unexplored. Ultimately, the proposed work is significant because it will define the role of CD4⁺ T cells in HSPC clone selection during early disease phases in CH and pre-leukemia which has major therapeutic implications for immunoprevention of leukemia.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract The ultimate success of immunotherapy for brain malignancies, such as malignant glioma, will require integration of in-depth understanding of immunology with solutions for the following long-standing challenges: 1) paucity and heterogeneous expression of glioma-specific antigens; 2) on-target off-tumor toxicity and exhaustion of therapeutic T lymphocytes, such as chimeric antigen receptor (CAR) T-cells; 3) immunological privilege of the CNS and 4) immunosuppression involving tumor, neuronal, and immune cells. My laboratory has contributed to critical discoveries in these areas and integrated our findings into novel immunotherapy clinical trials for glioma patients. In the current proposal, I will enhance my research by mobilizing multiple immune mechanisms. To this end, I will collaborate with an outstanding group of investigators whose diverse expertise in multi-disciplinary areas complements my own in brain tumor immunology as the central component and apply a wide variety of resources available at UCSF and collaborators to one overarching program. I will evaluate the overarching hypothesis that the integration of novel cell-engineering and antigen-targeting approaches will allow us to develop safer and more effective immunotherapy strategies by overcoming heterogeneous expression of antigens and unique challenges in brain immunology. I will evaluate the following strategies: 1. Develop neo- junction-targeting T-cell receptor (TCR)-T cell-based immunotherapy. We will leverage our highly reliable and valuable pipeline for T-cell epitope prediction, which we established during the current funding cycle, to discover novel neoepitopes derived from tumor-specific alternative splicing events (neojunctions). 2. Develop novel cell therapies using allogeneic induced pluripotent stem cells (iPSCs) and in vivo transduction approaches. While my current NINDS R35 award allowed me to implement the first-in-human phase I study of Synthetic Notch (synNotch)-CAR T-cell therapy in patients with glioblastoma, inherent and logistical challenges associated with the use of autologous T-cells motivate us to develop these novel and alternative approaches. 3. Enhance “epitope spreading” to overcome the antigen heterogeneity. While the novel synNotch-CAR approaches are promising, one major inherent challenge is that targeting a few or several antigens by CARs or TCRs may not adequately cover the marked antigenic heterogeneity of tumors. We will enhance the effects of low-intensity pulsed ultrasound with microbubbles (LIPU/MB) to induce adaptive immune responses against heterogeneous tumor antigens. 4. Investigate the glioma-neuronal circuit-induced immune regulation. We will delineate essential mechanisms on our recent discovery of neuronal activity-driven immunosuppression as a previously unrecognized resistance mechanism of cancer immunotherapy for gliomas. These 4 strategies will be logically integrated into combination approaches. As expected per the purpose of the NINDS R35 mechanism, these strategies may involve high risks. However, based on our preliminary proof-of-principle data, we will persistently pursue our goals with long-term support from the R35 mechanism and adopt new technologies flexibly and swiftly.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Patients with obstructive vascular diseases, such as atherosclerosis or peripheral arterial disease, or acute peripheral injuries require vascular grafts to restore blood flow to areas of the body. While the use of autologous vessels is considered the gold standard of treatment, many patients lack suitable vessels due to either vascular disease, previous usage, or have a size mismatch to the injured vessel. Therefore, clinicians turn towards synthetic grafts, such as expanded polytetrafluoroethylene (ePTFE) or Dacron, for large diameter vessel reconstruction. However, these synthetic materials fail when used in clinical small-diameter vascular applications, requiring the development of novel, hemocompatible vascular grafts for these clinical needs. Previous clinical trials have investigated acellular tissue-engineered vascular grafts (TEVGs) developed using human primary smooth muscle cells seeded on biodegradable scaffolds. After robust extracellular matrix (ECM) deposition, these TEVGs were subsequently decellularized and directly investigated for vascular treatment. While promising, the acellular TEVGs lacked an endothelium, and resulted in significant occlusion and suboptimal function within patients. Therefore, developing a novel TEVG with a functional endothelium that is immunocompatible to any recipient is of great clinical need. To address this issue, we propose using human induced pluripotent stem cells (hiPSCs) to fabricate a robust TEVG lined with an endothelium that is universally accepted by any patient, mitigating allogeneic immunorejection. In this proposal, hiPSCs will be differentiated into vascular smooth muscle cells (VSMCs) and subsequently used to generate a robust TEVG in our bioreactors that is then decellularized. Of novelty, we will then endothelialize the TEVGs with hypoimmunogenic, “universal” endothelial cells (ECs) that have been previously developed in our lab by modulating human leukocyte antigens (HLA) expression. To avoid xenograft immunorejection, this proposal will develop and characterize universal pig iPSCs (piPSCs) to endothelialize the TEVGs through downregulating expression of MHC I and II molecules and upregulating expression of CD47 via CRISPR-Cas9. The aims of this grant are to (1) characterize hypoimmunogenic universal piPSC lines for vascular graft engineering and (2) to generate universal iPSC- TEVGs and investigate their hemocompatibility in a preclinical porcine carotid bypass model in vivo. By investigating the universal iPSC technology in a preclinical porcine model, future studies will investigate human universal iPSCs for vascular tissue engineering purposes, furthering our goal towards developing a universal vascular conduit accepted by any patient.

NIGMS - National Institute of General Medical Sciences

Convergent molecular evolution, especially among distantly related species, is a hallmark of adaptation, yet the drivers of such convergence (or lack thereof) are typically unknown. Variation in molecular convergence may stem from constraints on evolutionary trajectories, such as how intramolecular epistasis and broader scale interactions among genes differ across lineages. While substantial progress has been made in understanding the prevalence of epistasis for fitness-related phenotypes, particularly in microbial systems, empirical tests of the role of epistasis in convergent molecular evolution are rare, especially in metazoans. A key obstacle is the lack of tractable, highly replicated systems to investigate the extent and generality in the causes of molecular convergence. To meet this need, we have been studying a diverse group of insects which have adapted to cardenolides, a class of steroidal plant toxins that disrupts the biomedically-relevant animal protein, Na/K-ATPase. We recently documented a remarkable 30 independent origins of cardenolide-specialization in insects, spanning 350 million years of evolution (in six taxonomic orders, spanning beetles and flies to grasshoppers). Although a handful of substitutions did indeed convergently evolve in all orders, some species lack these substitutions and others have taken alternative paths. Our findings, which also show distinct patterns among groups (e.g., Coleoptera vs. Lepidoptera, each with multiple origins) suggests lineage-specific constraints of genomic background. This group of insects thus presents a treasure trove of opportunity to decipher the drivers of molecular convergence. How variable are the epistatic interactions between lineages, and do these differences drive alternative outcomes in molecular evolution? Do multiple genes coevolve, shaping patterns of convergence? For example, have ABC transporter genes involved in excretion and storage, which complement resistance to cardenolides, evolved in parallel to Na/K-ATPase substitutions? And finally, do molecular substitutions predictably track the evolution of specific toxins coevolving in host plants? This system allows for some of the strongest general tests of why adaptive phenotypic outcomes have a similar genetic basis. Beyond comparative genomics, which will reveal distinct evolutionary outcomes and genetic associations, we will integrate the power of transcriptomics, in silico models, and functional assays to directly test our hypotheses. Our five-year program is expected to reveal general rules governing when intramolecular epistasis versus broader interactions among genes drive molecular convergence.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY AND ABSTRACT Patients with inflammatory bowel disease (IBD) often require a restorative proctocolectomy and ileal pouch- anal anastomosis (IPAA) for medically refractory ulcerative colitis (UC) or colonic Crohn’s disease (CD), which involves removal of the entire colon and rectum while preserving the anal sphincter with the creation of a pouch organ by looping healthy ileum into a J-shape that serves as an internal pelvic reservoir. While an IPAA preserves continence and avoids the requirement for a permanent ileostomy, approximately 50% of patients develop pouchitis— a de novo inflammatory condition of the ileal reservoir—resulting in substantial morbidity. The pathogenesis of pouchitis remains poorly understood. Our preliminary data using ExCITE-seq, a multi- modal single-cell sequencing platform, revealed that patients who later develop pouchitis exhibit a distinct immune-epithelial signature in the pre-IPAA ileum, including enrichment of Th17 cells, epithelial remodeling, and persistent clonal T cell expansion. Additionally, stem cells derived from inflamed pouch tissue demonstrated epigenetic alterations associated with impaired viability and secretory lineage differentiation. We hypothesize that pre-existing Th17-driven immune dysregulation and aberrant epithelial responses contribute to the development of pouchitis. To test this hypothesis, in Aim 1, we will evaluate the relationship between the immune microenvironment in the pre-IPAA ileum and subsequent pouchitis. In an existing cohort of patients with longitudinal follow-up we will assess pre-IPAA ileal tissue for Th17-associated immune infiltration, epithelial apoptosis, and spatial transcriptomic profiles, and associating these findings with clinical pouch outcomes. Aim 2 will define how pouchitis-associated immune dysregulation impacts epithelial biology. We will determine the impact of Th17-mediated cytokines on epithelial cell fate and viability using organoid models derived from pre-IPAA ileum and investigate the role of the microbiome in modulating this immune-epithelial axis through integrated metagenomic analyses of pre-IPAA ileostomy stool. Collectively, this work will identify predictive immune, epithelial, and microbial biomarkers and mechanisms of pouchitis, with the potential to inform IPAA eligibility and guide preventive therapeutic strategies in pouchitis.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY Cannabis use in pregnancy is becoming increasingly common, with up to 23% of pregnant people testing positive for delta 9-tetrahydrocannabinol (Δ9-THC; the most common cannabinoid) at delivery. Prenatal exposure to cannabis has been linked to low birthweight. Our pilot data further suggests that prenatal cannabis is associated with rapid infant growth followed by higher adiposity and glucose at 5 years. While compelling, the epidemiologic data is often limited by self-report of whole cannabis use or bioanalytic testing of Δ9-THC. As such, there is a paucity of data on the potential health effects of cannabidiol (CBD). Emerging evidence suggests that one in five pregnant people use CBD products, yet we know shockingly little about its health impacts in pregnancy. Furthermore, human studies may be confounded by socioeconomic status, polysubstance use, and other lifestyle factors, which limits our ability to establish causality. Therefore, exploring the direct effects of prenatal exposure to cannabis on perinatal tissues may help to provide evidence of a causal relationship. Both Δ9-THC and CBD interact with receptors in the endocannabinoid system, as well as non-canonical metabolic receptors like peroxisome proliferator-activated receptor (PPAR)-γ. These pathways are relevant to two perinatal tissue types: placental microvascular endothelial cells (ECs) and umbilical cord tissue-derived mesenchymal stem cells (MSCs). To address these gaps in knowledge, we have designed a translational study that will combine large-scale epidemiologic investigation with in vitro experiments. We will leverage two ongoing racially and ethnically diverse Colorado-based cohorts: Healthy Start and Mile High ECHO. Our overarching goal is to assess the impact of in utero and in vitro exposure to Δ9-THC and CBD on angiogenic and adipogenic pathways in primary placenta EC and MSC cell lines derived from our participants. We will also explore the extent to which these phenotypes mediate the associations between prenatal exposure to cannabis and child adiposity and metabolic health. Finally, we will explore whether other individual-level factors (diet, tobacco) alter risk, and whether there are prenatal windows of heightened suscepticilty. Drs. Moore (contact MPI; environmental epidemiologist with established expertise on health effects of prenatal cannabis exposure) and Boyle (MPI; basic scientist with a strong focus on molecular metabolism and stem cell biology) will carry out this research. They will lead a talented, multidisciplinary team of investigators with complementary and integrated research expertise with expertise in molecular mechanisms underlying placental insufficiency (Dr. Su), cannabinoid exposure assessment (Dr. Klawitter), and community- based dissemination (Dr. Rinehart). The findings have great potential to identify biological pathways and will support a community-based dissemination plan that is designed to educate providers and empower pregnant people about the potential health risks and legal consequences of cannabis use.

NIA - National Institute on Aging

PROJECT SUMMARY The mission of our laboratory is to pursue answers to essential questions in the field vascular aging that will advance our basic understanding and translate into more effective treatments to optimize human vascular healthspan. The central thesis of this project is that endothelial cells differentiated from hiPSCs, obtained from a diverse group of healthy adults and those with vascular contributions to cognitive impairment and dementia (VCID), can be leveraged to study endothelial aging in dementia. Using a computational model to identify biosignatures that predict endothelial cell aging, we will leverage this information to probe mechanisms relevant to dementia. Our research bridges the fields of vascular biology, stem cell biology, epigenetic clocks, multi -omics, and computational modeling to close the gap in the availability of models for the study of endothelial aging in dementia. There is a tremendous opportunity to address outstanding questions in this field using the novel human induced PlurIPotent stem cell-endothELIal cell model of aging for the study of vascular coNtributIoNs to coGnitive impairment and dementia (PIPELINING) described in this application. We will (1) passage human induced pluripotent stem cells differentiated to endothelial cells (hiPSC-ECs) and identify aging endpoints modeled in vitro (mitochondrial function, senescence, and angiogenesis). (2) A computational multi-scale model will be developed to predict the aging endpoints using multi -omic biosignatures for each human donor and passage. (3) Biosignature covariates judged to be critical contributors to the PIPELINING model will be selected for further mechanistic study. Achievement of the PIPELINING model would represent a significant advance in the application of contemporary technologies (iPSCs, epigenetic clocks, -omics, computational multi-scale modeling) to VCID.

NIMH - National Institute of Mental Health

Project Summary Fragile X syndrome (FXS) stands as a prominent contributor to intellectual disability and autism spectrum disorders, stemming from mutations within the FMR1 gene. These mutations lead to severe reduction or absence of the FMRP protein. Despite extensive research, effective medical interventions for FXS remain elusive, hindered by a limited understanding of its underlying mechanisms. Biochemical investigations have consistently highlighted FMRP's role in modulating mRNA translation, with its absence correlating with increased translation levels of select FMRP- interacting mRNA targets. However, emerging evidence suggests broader dysregulation, as FXS neurons exhibit heightened overall protein synthesis, hinting at elevated translation of non-FMRP interacting mRNAs. This intriguing phenomenon underscores the need for a deeper exploration into the cellular dysfunctions characterizing FXS. This research initiative aims to unravel a novel facet of FXS pathology—nucleolar hyper-function. We propose that this hyper-function contributes to aberrant ribosome biogenesis, thus augmenting the cellular capacity for translation and driving the observed global increase in protein synthesis in FXS. Aim 1 will assess neuronal and glial nucleolar function in wild-type (WT) and Fmr1 knockout (KO) mice. Aim 2 will conduct a comparative analysis of genome-wide proteomic data encompassing nucleolar proteins in WT and Fmr1 KO samples, discerning molecular alterations integral to ribosome biogenesis and assembly. Aim 3 will assess nucleolar function in the peripheral tissue in Fmr1 KO mice, establishing the hyper-functional pathological outcome as a potential clinical biomarker. The successful execution of this exploratory R21 project promises to unveil previously unexplored cellular mechanisms underlying FXS pathology. This study will also suggest nucleolus-associated abnormalities as novel molecular/cellular measures and potential biomarkers.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary The parathyroid glands are essential endocrine organs that regulate calcium and phosphate balance through secretion of parathyroid hormone (PTH). Loss or dysfunction of parathyroid tissue—commonly occurring after thyroid or neck surgery—leads to hypoparathyroidism, a debilitating condition for which current treatments rely on chronic calcium supplementation or hormone replacement. These therapies do not achieve the precise feedback regulation of calcium levels that native parathyroid tissue provides. Stem cell-derived parathyroid cells represent a potentially curative alternative. This project seeks to generate functional parathyroid-like cells from human induced pluripotent stem cells (iPSCs) through directed differentiation informed by principles of developmental biology and organogenesis. Our approach leverages recent progress in guiding iPSCs through definitive endoderm (DE), anterior foregut endoderm (AFE), and pharyngeal endoderm (PE) stages, alongside scRNA-seq data I have collected and analyzed from developing parathyroids to identify strategies to drive specification toward a parathyroid phenotype. In parallel, we will test the inductive capacity of transcription factor modules to forward program stem cells at pluripotent, DE, AFE, and PE stages to parathyroid identity. The differentiation protocol integrates small molecule modulation of key signaling pathways, such as BMP, WNT, and SHH, augmented by transcriptional cues derived from embryonic development. To validate lineage fidelity and functional capacity, we will assess expression of parathyroid-specific markers—including GCM2, PTH, and CASR—as well as calcium-responsive PTH secretion in vitro. Single-cell RNA sequencing will map lineage trajectories and dissect genetic programs governing parathyroid fate decisions. This work is enabled by the collaborative and resource-rich environment at Yale University. The project benefits interdisciplinary supervision of both Dr. Diane S. Krause, a leader in hematopoiesis as well as iPSC to parathyroid differentiation, and Dr. Zachary D. Smith. Together, their labs offer expertise in iPSC culture and parathyroid functional assessment embryonic development, germ layer specification, and embryo manipulation, offering key insight into developmental timing and patterning. Yale's core facilities for stem cell cultivation, flow cytometry, imaging, cluster computing, and high throughput sequencing will further accelerate progress and ensure technical rigor. By bridging stem cell biology with developmental genetics, this project aims to elucidate the regulatory networks that specify parathyroid identity and establish a platform for patient-specific cell therapies. The ultimate goal is to provide an autologous source of transplantable, functional parathyroid tissue to restore calcium homeostasis in individuals with hypoparathyroidism.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary In the human brain, a large repertoire of ion channels regulates the excitability of neurons, circuits, and networks, generating complex human cognition and behavior. Most ion channels are encoded by long, multi- exon genes which undergo extensive alternative splicing. Dysregulated splicing can alter ion channel function and is implicated in disorders ranging from autism to epileptic encephalopathy. Specific splicing events in ion channels have been shown to be critical for brain development and homeostasis, such as the neonatal-to-adult splicing switch in voltage-gated sodium channels. Splice-modulating therapeutics, such as antisense oligonucleotides, have shown efficacy for multiple neurologic disorders including spinal muscular atrophy, Duchenne muscular dystrophy, and Dravet syndrome. As potential targets for both small molecules and RNA therapeutics, ion channels are particularly important candidates for the treatment of neurologic disorders. However, there has never been a systematic study of alternative splicing of ion channels to-date. This proposal applies two innovative methods to profile the ion channel transcriptome and study the functional impact of alternative splicing on neuronal physiology. Aim 1 combines long-read RNA-sequencing and transcript capture technology to comprehensively identify and annotate channel isoforms in the human cerebral cortex. Bioinformatic tools will be used to uncover ion channel isoforms which are differentially regulated during postnatal brain development. Aim 2 employs splice modulation technology to study the functional impact of alternative splicing on neuronal physiology, using the epilepsy-associated KCNMA1 gene as proof of concept. A Cas-based toolkit is developed for human stem cell-derived neurons to manipulate the splicing of KCNMA1, and whole-cell patch-clamp physiology measures the impact of splice modulation on neuronal excitability. Taken together, this proposal will generate the most comprehensive profile of ion channel isoforms to-date, uncover developmentally regulated splicing events that can be potentially targeted by RNA therapeutics, and demonstrate proof of concept for transcriptome engineering in human neurons. These skills and resources will catalyze my career as an independent researcher, which will focus on the study of alternative splicing and the development of splice-modulating therapeutics for epilepsy and other neurologic disorders.

NHLBI - National Heart Lung and Blood Institute

Project Summary Systemically administered nanoparticles (NPs) offer transformative potential for targeted drug delivery, yet their unpredictable interactions with blood components-platelets, leukocytes, erythrocytes, and plasma proteins-limit clinical translation. This project addresses a critical gap by systematically linking NP formulation parameters to blood element binding under physiologically relevant flow conditions and developing a predictive computational framework for NP-blood interactions. Aim 1 will formulate and characterize a diverse library of lipid-based NPs varying in size, charge, PEGylation, and ligand chemistry to determine how physicochemical properties influence binding to blood elements. Aim 2 will quantify NP-blood interactions in a custom-designed whole- blood flow loop to capture dynamic binding effects under shear conditions relevant to human circulation. Aim 3 will develop and validate a random forest regression model that predicts NP-blood binding profiles from formulation parameters, integrating experimental data with machine learning to guide future NP design. Innovation and Significance. This study uniquely integrates nanomaterial formulation, blood biology, and data-driven modeling to reveal the mechanistic and predictive rules governing NP–blood interactions. By generating the first experimentally validated predictive model of NP–blood binding, the project will advance rational nanomedicine design and inform safer, more effective therapeutic development. Training and Institutional Impact. Conducted at the University of New Haven, a primarily undergraduate institution, this R15 project will provide immersive research experience in nanomedicine and computational modeling for undergraduate and graduate students. Trainees will gain skills in nanoparticle synthesis, blood biology, flow cytometry, imaging, and machine learning, fostering a new generation of biomedical engineers prepared for translational research careers. Long-Term Impact. The outcomes will establish a foundational framework for designing blood- compatible nanoparticles and support future NIH R01 or collaborative studies focused on predictive nanotherapeutics. The project aligns with NIH’s mission to promote health through innovative research and STEM workforce development at undergraduate institutions.

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

PROJECT SUMMARY: The mechanisms by which stem cells orchestrate their program to become functional differentiated cells require accurate temporal regulation of specific gene expression programs. This complex network requires precise temporal regulation of transcription and degradation processes to activate specific programs in a coordinated manner. So far, most of the studies have explored the regulation of transcriptional pathways and chromatin remodeling events during the differentiation process. mRNA degradation processes may present an attractive and still poorly explored opportunity for enhancing our understanding of the differentiation process. However, the lack of technologies that can capture rapid mRNA degradation events over highly dynamic processes, such as differentiation, and the heterogeneity of the mRNA degradation machinery in composition and expression patterns during differentiation have presented major technical limitations to further exploring the role of mRNA degradation across the continuum of the differentiation program. Here I propose to explore the existence of specialized RNA degradation complexes that control the decay of specific mRNA subclasses at precise timeframes of the differentiation process. To test this, we will introduce a new platform that uses cutting-edge technologies integrated in an innovative way to interrogate the continuum of the differentiation process at an unprecedented resolution. Our programmable depletion and rescue strategy will allow us to control the expression level of each subunit of complex mRNA degradation machinery robustly and with a precise time resolution of hours. By combining this technology with a high-content imaging system, we can record phenotype changes and accurately determine the specific impact of any perturbed protein on differentiation. Additionally, the use of this platform will guide us to understand the exact gene regulatory network controlled by the machinery at the transcriptional and stability level. The conceptualization and development of this workflow have the potential to impact a broader scientific audience; due to its extremely high flexibility, it could be applied to the study of unlimited biological processes or proteins. In this essay, the application of our proposed platform has the potential to fundamentally overturn the current view of how mRNA decay is dynamically regulated, providing a definite understanding of the function of the degradation machinery on mRNAs and, at the same time, revealing the broader impact of the degradation process on differentiation.

U.S. National Science Foundation

The Promoting Research and Innovation in Methodologies for Evaluation (PRIME) program seeks to support research on evaluation with special emphasis on exploring innovative approaches for determining the impacts and usefulness of STEM education projects and programs; building on and expanding the theoretical foundations for evaluating STEM education and workforce development initiatives, including translating and adapting approaches from other fields; and growing the capacity and infrastructure of the evaluation field. Two types of proposals will be supported by the program: Exploratory Projects that include proof-of-concept and feasibility studies and more extensive Full-Scale Projects.

NCI - National Cancer Institute

Project Summary The goal of this project is to develop a strategy to effectively eradicate leukemia-initiating cells. Leukemia- initiating cells are responsible for tumor initiation and recurrence in acute myeloid leukemia (AML), making it critically important to understand and target the biology required for leukemia-initiating cell survival. LICs are characterized by their self-renewal capacity, block in differentiation, and quiescent nature making them therapy resistant. A well characterized vulnerability of leukemia-initiating cells is oxidative phosphorylation (OxPhos) a pathway responsible for energy production. Direct OxPhos inhibition has been toxic in cancer patients. Thus, the development of approaches to target processes that regulate OxPhos in leukemia-initiating cells that are dispensable in normal cells is required. Our preliminary data shows that OxPhos is regulated by a post- translational modification called protein glutathionylation in AML cells and leukemia-initiating cells but not in normal hematopoietic stem and progenitor cells (HSPCs). These data indicate that protein glutathionylation regulation may represent a mechanism for decreasing OxPhos that could be LIC/AML specific and therefore targeting protein glutathionylation may be an approach to kill LICs with a more favorable therapeutic window than other approaches. Importantly, our data suggests that depletion of mitochondrial proteins that regulate protein glutathionylation results in reduced LIC function, induction of myeloid cell differentiation and sensitizes primary human AML cells to commonly used AML therapies but does not impact HSPCs. These data further support the potential for a therapeutic window may exist to target protein glutathionylation in AML. Based on these findings, we hypothesize that the regulation of mitochondrial protein glutathionylation is essential for LIC function by regulating OxPhos. We will examine this hypothesis by determining the molecular and biological role of protein glutathionylation in regulating leukemia-initiating cells and HSPC function using primary AML specimens, patient derived xenograft (PDX) models, and normal bone marrow specimens from healthy donors. Specifically, we will quantify leukemia-initiating cell phenotypes and function upon genetic depletion of proteins that regulate glutathionylation. Further, we will interrogate the mechanism(s) by which protein glutathionylation regulates mitochondrial energy production in leukemia-initiating cells. Taken together, our studies will be the first to establish protein glutathionylation as a novel regulator of 1) leukemia-initiating cells function and 2) OxPhos in cancer.

NIBIB - National Institute of Biomedical Imaging and Bioengineering

Project Summary/Abstract Many cells are responsive to electrically conductive materials; however, to date electrical conductivity is mostly achieved through graphene or synthetic polymers. These materials have limited translational use due to a lack of biodegradability and rigid mechanical properties. To overcome these challenges, we propose the design of a recombinant engineered, conductive, injectable, and biodegradable hydrogel that has the potential to induce regeneration across a wide range of tissues. We have recently pioneered the synthesis of a fully recombinant gel that incorporates electrically-conductive protein nanowires (ePN), an engineered matrix-like protein, and the polysaccharide hyaluronic acid (HA). While the ePN provides conductivity, the engineered matrix-like protein and HA provide biochemical ligands that promote cell adhesion. The hydrogel material is crosslinked through dynamic covalent chemistry, allowing for tunable viscoelastic properties and injectability. The resulting gel supports three-dimensional cell culture and biodegrades in response to cell-secreted enzymes. As the spinal cord is an electrically conductive tissue, we will demonstrate the efficacy of our technology in a cell-based therapy for spinal cord injury (SCI). Less than 1% of SCI patients have full neurological recovery by the time of hospital discharge. We previously demonstrated with non-conductive hydrogels that intraspinal transplantation of neural progenitor cells (NPCs) can significantly improve function in a rodent SCI model, but only when they are sufficiently matured into a neuronal phenotype. We have also demonstrated that NPCs enhance their neuronal maturation in vitro when grown on conductive biomaterials that were rigid and non-biodegradable. Thus, we hypothesize that our new hydrogel will facilitate the intraspinal injection of NPCs and significantly promote their neuronal maturation, thus resulting in significant functional and histological improvements. In Aim 1, we identify the gel formulation that best promotes neuronal differentiation and maturation of human induced pluripotent stem cell-derived NPCs in vitro. Specifically, we will tune the bulk conductivity of the fabricated gels through altering the ePN concentration and amino acid sequence. Recombinant engineering of ePN allows for tunability of the electrical conductivity along a single protein wire. The cell morphology, gene expression, and protein expression of encapsulated NPCs in the gels without and with varying levels of conductivity will be quantified. In Aim 2, we will select the gel variant that provides the best in vitro results for assessment in a preclinical, rat model of cervical SCI. NPCs will be transplanted within the conductive, biodegradable gel and evaluated for functional behavior over 6 weeks. Histological outcomes include transplanted cell survival and neurite outgrowth. Controls include conductive gels without cells and non-conductive gels with cells. This study would represent the first use of conductive, biodegradable, recombinant nanowires in tissue engineering, which can have broad application in conductive tissues including brain, cardiac muscle, skeletal muscle, and skin.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary Neutropenia, defined by abnormally low neutrophil counts, compromises innate immunity and increases susceptibility to life-threatening infections. Although most cases are acquired—resulting from malignancy, chemotherapy, infections, medications, or autoimmune disease—the study of inherited forms, though rarer, offers critical insights into the core mechanisms of myelopoiesis and granulocytic differentiation. Among these, autosomal dominant, heterozygous mutations in ELANE (formerly ELA2), which encodes the neutrophil granule serine protease neutrophil elastase (NE), represent the most common cause of severe congenital neutropenia (SCN) and the primary cause of cyclic neutropenia. SCN presents at birth with lifelong neutropenia, bone marrow maturation arrest, and elevated risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In cyclic neutropenia, neutrophil counts fluctuate between zero and near-normal with a striking 21-day periodicity. Despite their clinical importance, the pathogenic mechanisms of ELANE mutations remain poorly understood, and curative treatment is currently limited to hematopoietic stem cell transplantation. Mouse models fail to recapitulate the human phenotype, highlighting the need for human systems to investigate disease biology. All known pathogenic ELANE mutations result in production of a variant NE polypeptide, which may bypass key steps of proteolytic maturation and mislocalize within developing cells. This project tests the hypothesis that ELANE mutations cause disease by disrupting the spatial or temporal control of NE activity during granulopoiesis. Using isogenic, gene-targeted human induced pluripotent stem cells (iPSCs), the proposed research will: (1) define the spatial and temporal determinants of NE pathogenicity by introducing cis-acting suppressor mutations that disrupt its processing, trafficking, and catalytic activity; (2) determine whether the NE paralogs proteinase 3 and cathepsin G function as trans-acting modifiers; and (3) test whether CD34, a critical hematopoietic surface protein with distinct properties differing between mouse and human, is an NE substrate, and whether cleavage-resistant CD34 variants can restore granulopoiesis in ELANE-mutant cells. These studies will elucidate mechanisms of protease regulation in human neutrophil development, clarify the pathogenesis of both inherited and acquired neutropenia, and identify molecular targets for potential therapeutic intervention. The proposed work aligns directly with the NIH mission to advance understanding and treatment of hematologic and immune disorders.