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NHLBI - National Heart Lung and Blood Institute

The human body produces hundreds of billions of blood cells daily, replenished by hematopoietic stem cells (HSCs) in the bone marrow. Over time, HSC clones—populations derived from a single HSC—fluctuate in size, with some clones expanding while others dwindle. Clonal dynamics have been studied by reconstructing HSC phylogenic trees from somatic mutations they have accrued using whole-genome sequencing of single-cell- derived colonies. Our group pioneered the study of clonal dynamics in myeloproliferative neoplasms (MPNs), showing that driver mutations, such as JAK2, arise decades before diagnosis and confer a fitness advantage, enabling mutant clones to dominate the population. Strikingly, similar clonal dominance is observed in aging healthy individuals, though only ~20% of expansions can be attributed to known driver mutations. Understanding why certain HSC clones expand, especially in the absence of clear genetic causes, remains a fundamental unanswered question in hematopoiesis. Clonal expansion of HSCs may result from cell-intrinsic factors, as not all HSCs are equivalent. We would like to understand how during development a heterogenous population of HSCs is generated. Extrinsic factors, such as signals from the niche or systemic inflammation, may also drive clonal expansion. However, we lack basic knowledge of the drivers of clonal dynamics in native hematopoiesis because (1) reconstructing clonal history using single-cell phylogenies is not scalable—whole-genome sequencing of colonies is invasive, slow, and costly; and (2) mouse models, while useful for perturbing clonal dynamics, fail to recapitulate human clonal dynamics. This is because, despite the fitness advantages of certain HSC clones, the short lifespan of mice does not allow sufficient time for these clones to expand and dominate the stem cell population. To resolve clonal expansions in mice, we need scalable methods to reconstruct the phylogenetic history of all HSCs, not just a subset. We propose a comprehensive research program for developing new technologies to address these challenges and uncover the drivers of HSC clonal dynamics. First, we will create a non-invasive, rapid, and cost-effective method to reconstruct HSC clonal histories using long-read bulk sequencing of methylation patterns in blood cells, reducing the cost per sample from $100,000 to $1,000 and enabling large-scale human studies. Second, we will engineer mice to record lineage and key signaling histories of HSCs directly in their own DNA by extending lineage-recording mouse models we previously developed. Phylogenetic trees of all HSCs can then be reconstructed efficiently by sequencing specific target regions instead of entire genomes. By integrating signaling activity with lineage history, we will decorate tree branches with molecular events that drive clonal expansion. These engineered mice will enable mapping the developmental origins of HSC heterogeneity and quantifying the impact of extrinsic factors on clonal dynamics. Together, these approaches will address fundamental questions in stem cell regulation and aging, improve prognosis and treatment of hematological disorders, and provide transformative tools for studying blood.

NCI - National Cancer Institute

PROJECT ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with the lowest 5-year survival rate among all solid tumors. Adoptive cell transfer (ACT) of tumor neoepitope specific CD8+ T cells has shown therapeutic promise, but current approaches rely on peptide-MHC tetramers to select high-affinity T cell receptors (TCRs), potentially overlooking functionally superior clones. Our preliminary data reveal that low- affinity TCRs—particularly those undetectable by tetramers—can mediate potent antitumor responses, suggesting that TCR bond lifetime under mechanical force, rather than affinity alone, may dictate therapeutic efficacy. To address this, I developed a novel panel of T cell receptor exchange (TRex) mice expressing PDA- specific TCRs spanning a spectrum of affinities for the same tumor neoantigen (CB101-109:H2-Db). Strikingly, the lowest-affinity TCR (TCR5.1) demonstrated enhanced tumor control compared to higher-affinity counterparts, challenging conventional TCR selection paradigms. This proposal will define how TCR biophysical properties influence CD8+ T cell fate and function in PDA through two specific aims. Aim 1, will employ a biomembrane force probe (BFP) to quantify TCR-pMHC bond lifetimes and correlate these with ACT outcomes in orthotopic PDA models, using high-resolution ultrasound and multimodal T cell profiling. In collaboration with Brian Evavold’s lab at the University of Utah, I will use BFP analysis to quantify each TCRs affinity, bond lifetime, and ideal force conditions. I will then evaluate the effector functions of each clonotype ex vivo using titrated peptide stimulation assays and assess tumor clearance in vivo through ACT in immunocompetent orthotopic PDA models. Aim 2 will dissect competitive fitness of TCRs during immunotherapy by co-transferring T cell clones into immunocompetent hosts, combining scRNAseq and spatial analyses to uncover mechanisms driving TCR-dependent persistence. By “parking” naïve TRex CD8+ T cells in mice prior to orthotopic tumor implantation, I will identify the impact of competition between TCRs of varying affinities on the anti-tumor response during immune checkpoint blockade or CD40 agonist therapies. Tracking these clonal populations through single-cell RNA sequencing and spatial analyses will allow for the identification of the molecular and cellular mechanisms that drive TCR-dependent persistence and stem-like maintenance. Together, these studies will test the central hypothesis that low-affinity, long-lived TCR interactions promote the development of durable, stem-like T cell populations that are resistant to exhaustion - a potential key to achieving sustained antitumor responses. The successful completion of this research will provide transformative insights into TCR- intrinsic determinants of therapeutic efficacy, with direct implications for the design of improved ACT protocols for PDA and other solid tumors.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Skeletal diseases contribute significantly to morbidity, yet limited therapies exist for building new bone. Understanding the molecular mechanisms driving bone formation is critical for improving skeletal health across the lifespan. Gs-GPCR signaling is a major driver of bone formation, and activating this pathway using recombinant parathyroid hormone (PTH) is an effective treatment strategy for osteoporosis, fractures, and skeletal dysplasias. The anabolic effect of Gs-GPCR activation in osteogenic lineages is well-established, but its role in non-osteogenic cells in the bone niche is poorly understood. There is compelling evidence that cells comprising the bone vasculature play an integral role in skeletal development, maintenance, and repair, yet the role of Gs-GPCR signaling in these lineages and the effect on osteogenesis has not been studied. Fibrous dysplasia of the bone (FD) is a prototypical disease of constitutive Gs-GPCR signaling that leads to expansile, fibrotic bone lesions and massive trabecular bone growth. We and others have used FD as a disease model to delineate the mechanisms by which Gs-GPCR activation in osteogenic lineages drives bone formation. The long- standing paradigm has been that Gs-GPCR signaling in osteoprogenitors drives fibrosis and aberrant osteogenesis in FD. Single-cell RNA sequencing data from human FD bone lesions shows that in addition to osteogenic lineages, endothelial and perivascular cells also harbor GNASR201H/C mutations and express an altered, fibrotic transcriptome. This unexpected finding suggests that Gs-GPCR signaling in vascular cell lineages may contribute to the dramatic FD phenotype and may regulate bone formation more broadly. The overall objective of this proposal is to elucidate the molecular mechanisms by which Gs-GPCR signaling in endothelial and perivascular cells contribute to osteogenesis and fibrosis, using FD as a disease model. Our central hypothesis is that Gs-GPCR signaling in endothelial and perivascular lineages activates critical signaling pathways downstream of Gsa, including VEGF, Wnt/ b-catenin and TGF b/BMP that promote fibrosis and osteogenesis. To test this hypothesis, we implement human induced pluripotent stem cell (hiPSC) models, primary human cells and tissue, and murine models to elucidate the role of Gs-GPCR signaling at the bone- vascular interface. Detailed knowledge of the signaling pathways activated at the bone-vascular interface will help elucidate the mechanisms by which Gs-GPCR signaling in non-skeletal lineages contribute to bone formation. This knowledge can then be leveraged to develop new therapeutics for diseases of impaired osteogenesis, including osteoporosis, fracture repair, and skeletal dysplasias.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Hirschsprung disease associated enterocolitis (HAEC) is the leading cause of death in children who lack enteric neurons in distal bowel, a birth defect called Hirschsprung disease. The etiology of HAEC is not well understood, but hypothesized disease mechanisms include altered gut microbes (“dysbiosis”), abnormal mucosal immune system and epithelial barrier defects. To date, there are no immune-targeted therapies to treat or prevent HAEC, but new treatments are needed. This proposal builds on the candidate’s preliminary data suggesting interleukin 22 (IL22) critically modulates HAEC risk and HAEC severity. The central hypothesis is that enteric nervous system (ENS) signaling induces IL22 release and facilitates IL22 epithelial responses to enhance mucosal immunity and strengthen epithelial barrier functions that prevent enterocolitis. The Piebald lethal (sl/sl) Hirschsprung disease mouse model of HAEC will be used, as survival of sl/sl mice is dramatically (> 3-fold) altered by diet (Tjaden et al, in BioRxiv and submitted) and IL22 mRNA is much higher in sl/sl fed a Protective diet that extends median survival (“late onset HAEC”). Aim 1 will define the cellular source(s) of IL22 from bowel regions of sl/sl model mice that develop early or late onset HAEC. In parallel, this aim tests the hypothesis that IL22 prevents HAEC, by using genetic and pharmacologic strategies to alter IL22 levels. Aim 2 will precisely define the role of IL22 on epithelial integrity, stem cell renewal and differentiation in organoids derived from sl/sl mice with early or late onset HAEC and from children with Hirschsprung disease with or without HAEC. Organoids facilitate studies of epithelial stem cell biology and IL22-epithelium interactions in the absence of microbes, neurons, or diffusible small molecules such as neurotransmitters. Collectively, these studies will determine cellular sources of IL22, the effect of ENS cells on IL22 secretion, the role of IL22 in enterocolitis, and the impact of Hirschsprung disease associated aganglionosis on epithelial cell biology. These studies build on the candidate’s training as a pediatric gastroenterologist, who has clinical exposure to the diagnosis and treatment of children with Hirschsprung disease and HAEC, as well as her basic science training in enteric nervous system biology. As the work proceeds, she will become an expert in mucosal immunology and epithelial biology with a focus on neuro-immune and neuro-epithelial interactions. The mentors, Dr. Robert Heuckeroth, and Dr. Kathryn Hamilton are experts in ENS biology and epithelial biology respectively. Both mentors have a strong commitment to mentorship and NIH funding track records. Experiments will be conducted at the Children’s Hospital of Philadelphia and Perelman School of Medicine at the University of Pennsylvania, a collegial, collaborative and state-of-the art institution. The professional development and training plan will position the candidate as a successful pediatrician-scientist, who is focused on the prevention and treatment of Hirschsprung associated enterocolitis. These studies should determine if IL22-based therapies would likely be successful in HAEC, and if a human clinical trial is appropriate.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Atherosclerosis-associated PAD is a leading cause of cardiovascular mortality, driven by SMC dedifferentiation into proliferative and migratory phenotypes. Despite its role in numerous vascular diseases, the mechanisms regulating SMC dedifferentiation remain unclear, limiting effective treatment development. Mechanotransduction, the process by which cells convert mechanical stimuli into biochemical signals, is altered in dedifferentiated SMCs in PAD. In this K99/R00 project, I propose to leverage (i) human induced pluripotent stem cell-derived smooth muscle cells (iPSC-SMCs), (ii) multiomics, (iii) biomaterials with tunable stiffness, and (iv) lineage tracing mouse models, to test the hypothesis that impaired mechanotransduction contributes to SMC dysfunction by translating ECM alterations into transcriptional and epigenetic changes, ultimately increasing susceptibility to atherosclerosis and PAD. In Aim 1, I will utilize soft and stiff hydrogels to explore the effects of impaired mechanotransduction mediators on iPSC-SMCs phenotype by assessing SMC function, transcriptional profile, and downstream mediators (RhoA/ROCK signaling, YAP transcription factor, αβ-integrins, FAK). In Aim 2, I will investigate how dysfunctional mechanotransduction affects SMC dynamics using an SMC-specific lineage tracing mouse model, inducing either atherosclerosis or hind limb ischemia. I will evaluate (i) plaque area (ii) blood flow restoration, (iii) SMC dedifferentiation (single nucleus RNA-seq) at different timepoints. Dysregulated targets will be prioritized by cross-integration of the mouse with the human dataset, and validation will be performed in human PAD “early” and “advanced” plaques. In Aim 3, I will identify upstream noncoding genetic regulators controlling SMC fate in response to vascular stiffness, by single nucleus ATAC-seq of human PAD and control tissues. I will conduct an arrayed CRISPRi screen targeting noncoding regions enriched in PAD compared to controls, followed by mechanistic validation. The proposed research builds upon my prior training in vascular biology and single cell analysis, while providing new opportunities for training on iPSC-SMC technology, animal models and large-scale data integration. My mentor, Dr. Joseph Wu, is a pioneer in iPSC and cardiovascular biology, and my co-mentor, Dr. Thomas Quertermous, is a renowned expert in vascular diseases and genetics, whose mentorship complements Dr. Wu’s expertise. To further strengthen my training, I have assembled an Advisory Committee comprising Dr. Chiara Giannarelli (atherosclerosis and multiomics), Dr. Ronald L. Dalman (peripheral artery disease), and Dr. Michael Snyder (large-scale functional genomics), who will provide specialized guidance and expertise across key aspects of my research. In conclusion, my path to independence is supported by rigorous training, outstanding mentorship, and exceptional research environment at Stanford. These elements will provide me with the expertise and resources needed to achieve my long-term goal of studying the genetics of mechanotransduction regulating stromal cell function in vascular diseases.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY/ ABSTRACT Cancer and chronic viral infections affect millions, with cancer being a leading cause of death. Despite significant advancements in treatment, there is a critical need to identify new therapeutic targets and enhance existing therapies. Immunotherapy, particularly immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have emerged as promising strategies; however, many patients do not respond due to CD8 T cell exhaustion, which occurs when T cells are persistently exposed to antigen and progressively lose their function. The presence of terminally exhausted (TTerm) T cells often results in poor ICB responses, and cells used for ACT often become ineffective in the tumor microenvironment (TME) due to exhaustion. Thus, discovering strategies to prevent or reverse T cell exhaustion is essential for improving immunotherapy outcomes. Epigenetic programming contributes to exhaustion progression by silencing effector-related genes through DNA methylation, which reduces T cell responsiveness to ICB. However, the upstream factors regulating this epigenetic programming in CD8 T cells remain unexplored. Among these factors is T cell receptor (TCR) signaling pathways. One such pathway activates nuclear factor of activated T cells (NFAT), known to promote exhaustion by upregulating immune checkpoints. Our preliminary studies indicate that NFAT signaling can be activated upon stimulation in all exhausted T cell (TEX) populations, suggesting this pathway is not impaired in Tex cells. Another well-known TCR pathway activates the nuclear factor-B (NF-B), which is crucial for T cell development, memory maintenance, and effector function; however, the role of NF-B signaling in the context of T cell exhaustion is largely unknown. Preliminary data suggest NF-B signaling is impaired in exhausted T cells, with its nuclear translocation as well as signaling ability decreasing through the progression of exhaustion. However, it is the most active in the progenitor population, which is associated with better ICB responses. Our preliminary data also suggest that boosting NF-B signals in chronically infected mice increases the number of progenitor exhausted cells. Because of this, we hypothesize that NF-B signals epigenetically remodels TEX cells by maintaining their stemness features, thereby increasing their ICB responsiveness, while decreasing their progression towards terminal exhaustion. To test this hypothesis, we will employ a TCR reporter system in murine CD8 T cells, followed by exhaustion modeling and epigenetic assays (Aim 1). We will also evaluate the efficacy of NF-B/NFAT signal rebalancing in treating chronic infections and cancer by adjusting these signals in cells used for ACT and in murine models receiving ICB therapy (Aim 2). Overall, this project aims to improve our understanding of NF-B signaling in TEX cells and enhance immunotherapy effectiveness. The completion of this project also aligns with my outlined training plan and will further my skills in immunotherapy research and scientific communication, taking place in a positive environment and under skilled mentorship.

NEI - National Eye Institute

PROJECT SUMMARY/ABSTRACT Candidate: Dr. James Harris, M.D., Ph.D., is completing fellowship training as a medical retina specialist in July 2026, with a career goal of becoming an independent clinician-scientist investigating the pathogenesis of age-related macular degeneration (AMD), to improve current treatments and patient outcomes. Career Development Plan: This proposal provides foundational training in the knowledge and methods required to investigate the role of intercellular interactions in retinal degeneration and the career development needed for independent research. Dr. Harris proposes to study the role of microglial-retinal pigment epithelium (RPE) interactions in geographic atrophy (GA). This tailored training plan will expand Dr. Harris’s expertise in retinal neurodegeneration, immunology, human ocular histology, induced pluripotent stem cell (iPSC) disease modeling, and in vitro assessment of RPE function. This plan is comprised of formal didactics in neuroimmunology, stem cells, bioinformatics, neurodegeneration, and responsible conduct of research, as well as structured scientific and career mentorship, seminars, faculty development workshops, presentations at national meetings, and submission of publications. Mentorship Team and Environment: The mentorship team consists of renowned scientific experts in ocular immunology, AMD, bioinformatics, and iPSCs, and retinal clinician-scientist career mentors who are ideal to advise the candidate throughout his training. The Duke Eye Center and Duke University are well-equipped with the resources to support this work. The mentorship team and institution are fully committed to preparing Dr. Harris for independence as an R01-funded scientist. Research: GA is an advanced disease state of AMD, a leading cause of legal blindness, for which more effective therapies are needed. Microglia, the resident immune cells of the nervous system, are thought to play an active role in GA, but the exact mechanisms are poorly understood. Microglia migrate to the subretinal space, gather at sites of active degeneration, upregulate genes linked to AMD by genome-wide association studies, and are protective in mouse models of outer retinal degeneration. This proposal investigates the role of microglia-RPE interaction in the pathobiology of GA. Aim 1 proposes to determine the reciprocal effects of secreted factors as mediators of microglial-RPE interaction and identify pathways that may mediate these interactions. Aim 2 proposes to elucidate the effect of APOE alleles on TREM2-mediated microglia regulation. Aim 3 investigates the role of Cyclophilin A-Basigin signaling in RPE degeneration. Doing so will elucidate key pathobiological mechanisms of AMD to inspire new therapeutic targets. This K08 will provide mentored training to develop the skills required to achieve these research and career development goals, serve as the basis for future R01 funding, and provide the PI with expertise broadly applicable for patient-oriented research.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Vascular calcification serves as the primary risk factor for predicting cardiovascular events. It involves arterial calcification, an actively regulated process mediated by cells, resembling physiological biomineralization but with impaired resorption. The vessel wall contains “calcifying vascular progenitor cells” that react to pro- calcific signals such as inflammation or infection. They then differentiate into osteoblast-like cells, which produce minerals and matrix in the vessel wall. The bulk of progenitor cells within the vasculature reside in the microanatomic progenitor niche, the tunica adventitia. Under normal conditions, these progenitors regulate vascular homeostasis and remodeling. The identity, location, function, and regulatory mechanism of adventitial progenitor cells in vascular calcification remain poorly studied. Further, uncovering the niche- specific role of adventitial progenitors in vessel calcification will guide the development of targeted therapeutic strategies. Recently, our integrated transcriptomic study on normal human blood vessel adventitia discovered a cell surface marker to typify adventitial progenitor cells. This previously undescribed marker in adventitial cell biology is Endothelial Protein C Receptor (CD201). CD201-expressing progenitor cells are spatially localized in the outer layer of the adventitia, and their expression level dictates the osteogenic potential of these cells. These recent observations raised questions regarding their role in the progression of vascular calcification and are comprehensively investigated in the present K99/R00 proposal. To achieve this, integrated spatial transcriptomics and single-cell RNA sequencing-based transcriptomic mapping of the human calcified vessel with implications of CD201+CD34+ adventitial cells in calcification will be initially performed. Additionally, reporter mice with nephrectomy-induced calcification will be also examined (Aim 1, K99 Phase). Secondly, a detailed in vitro functional characterization of FACS purified CD201High/Low cells from human calcified and healthy vessels will be performed, along with CD201 cell ablation in mouse calcification model to determine the functional role of the cell in calcification (Aim 2, K99 Phase). Lastly, the underlying signaling mechanism regulating CD201-expressing cells to be pro-calcific is investigated by CRISPR/Cas9 gene knockdowns, genetic mice calcification models, and integrated transcriptomics (Aim 3, R00 phase). Completing the proposal will greatly improve the knowledge of adventitial progenitor cell-mediated vessel calcification. The proposed research and training plan aligns with my long-term research objective. It will also significantly contribute to my scientific and career goals of establishing an independent research career in blood vessel resident stem cells and their role in vascular pathology.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Hematopoietic stem cells (HSCs) maintain blood production throughout an organism’s lifespan. Thus, HSCs must balance differentiation with self-renewal to protect stemness. To this end, HSCs are largely quiescent. Quiescence protects HSCs from genotoxic insults and functional exhaustion. Understanding the molecular mechanisms controlling HSC quiescence will yield valuable insights into mechanisms underlying hematopoietic disorders. While post-transcriptional regulation is increasingly recognized as important for hematopoietic cell fate specification, its role in HSC quiescence remains poorly understood. Preliminary data indicate that the post- transcriptional regulator DDX6 is important to maintain HSC quiescence. DDX6, an RNA helicase, orchestrates translational suppression and mRNA sequestration in cytoplasmic condensates known as P-bodies. Notably, Ddx6 knockout mice have normal mature blood cell populations but exhibit loss of HSC quiescence. Accordingly, Ddx6−⁄− HSCs exhibit increased proliferation and mitochondrial numbers, which results in diminished fitness during serial, competitive transplants. Mechanistically, initial analysis of DDX6-targeted transcripts in P-bodies revealed an enrichment for untranslated mRNAs encoding crucial regulators involved in exiting quiescence. Together, these data lead to our central hypothesis that Ddx6-mediated RNA processing is pivotal in protecting HSC quiescence and function. Aim 1 will test the hypothesis that Ddx6 is required for in situ stress hematopoiesis by challenging Ddx6−⁄− HSCs in vivo using regenerative and infectious stressors. Aim 2 will elucidate the mRNAs translationally suppressed by Ddx6 in HSCs and characterize the HSC translatome in situ both with and without Ddx6 deletion. Additionally, we will investigate the functional role of Ddx6 targeted transcripts in vivo, specifically Myc. The overall goal of this project is to elucidate a new mechanism controlling HSC function at the molecular and cellular levels and to advance strategies for treating hematologic diseases. This fellowship application is sponsored by Bruno Di Stefano, Ph.D., an expert in post-transcriptional gene regulation in stem cells, and Katherine King, M.D., Ph.D., a physician-scientist and expert in hematopoietic stem cells, who will provide close guidance throughout the fellowship period. The training plan includes strategies to 1) Learn from accomplished scientists and physician-scientists that will advise the applicant through her training goals; 2) Undergo rigorous scientific training in hematopoiesis and gene regulation; 3) Experience opportunities to improve scientific communication skills and expand professional networks; 4) Advance the applicant’s clinical training, especially in hematology. The clinical and scientific training environment at Baylor College of Medicine is within the Texas Medical Center, the largest medical research complex in the world. This environment is ideal to foster the applicant’s scientific and clinical growth toward a career as a physician-scientist investigating the role of post-transcriptional regulation in hemopoietic stem cell function and dysfunction.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Metabolism is one of the most tightly regulated cellular processes and is important for the maintenance of homeostasis; its deregulation is a hallmark for many diseases, including diabetes, cancer, and neurodegenerative diseases. The mitochondrial pyruvate carrier (MPC - comprised of the MPC1 and MPC2 proteins) is a transmembrane protein that resides in the inner mitochondria membrane and facilitates the entry of pyruvate, which is necessary in several metabolic reactions such as TCA cycle. Our lab has previously shown that low MPC expression promotes cell proliferation, and that MPC expression is negatively correlated with Wnt/β-catenin target gene expression. However, the molecular mechanism behind this correlation is not known. To test the hypothesis that Wnt/β-catenin regulates MPC expression, we performed a screen based on technology developed in the Rutter lab (TRoUT-FISH) and found that β-catenin suppresses the expression of the MPC genes. I validated this finding by showing that treatment with GSK3 inhibitors, which activate the Wnt/β- catenin pathway, caused reduced MPC1 and MPC2 mRNA and protein expression. Moreover, I examined the effects of silencing or overexpressing c-Myc (a downstream target of the Wnt/β-catenin pathway) on MPC mRNA expression using human dermal fibroblasts. I found that overexpression of c-Myc resulted in decreased MPC1 expression, whereas knockdown of c-Myc resulted in increased MPC1 expression. I hypothesize that β-catenin, through its target gene c-Myc, acts as a repressor of the MPC as part of its pro-proliferation transcriptional program. Building on my preliminary results, the objective of my K99/R00 proposal is to define the mechanisms by which β-catenin and c-Myc regulate MPC1 and MPC2 expression, and the significance of this regulation in cell metabolism and cell behavior. To test this hypothesis, I propose the following specific aims: (Aim 1) Determine the molecular mechanisms by which activation of Wnt/β-catenin represses MPC1 and MPC2 expression [K99 phase]; (Aim 2) Elucidate the role of Wnt/β-catenin in cell metabolism and cell fate [K99 phase]; and (Aim 3) Unravel the distinct roles of mitochondrial metabolism in stem cells and differentiated cells [R00 phase]. My long-term goal is to become an independent scientist at an R1 institution working at the intersection of mitochondrial metabolism and cell biology. To achieve this, I have worked with Dr. Rutter to assemble my mentoring committee which includes experts with a strong publication record in mitochondrial metabolism, cell biology/cell signaling, metabolomics/proteomics and metabolic tracing. Overall, the tools and skills learned during the mentored phase will be essential for my development as an independent scientist and for me to successfully establish a strong and differentiated research program studying the distinct roles of mitochondrial metabolism in stem cells and differentiated cells and its implication in health and disease.

Emerson Foundation

Supports STEM education, community building, and basic human needs in Emerson plant communities.

NCI - National Cancer Institute

OVERALL SUMMARY/ABSTRACT Lung cancer is the leading cause of cancer-related deaths, with more than 1.8 million deaths/year globally. It is often diagnosed at an advanced stage and is associated with poor outcomes for most patients. This revised application for the Emory Lung Cancer SPORE award includes an outstanding multi-disciplinary team of clinical oncologists, immunologists, computational scientists, drug discovery experts, and translational investigators dedicated to lung cancer research with a track-record of productive collaborations to address critical questions that will improve the outcome for patients with this lethal disease. Building on the success and findings from our current SPORE, which was first funded in 2019, our proposed program will have significant impact on: enhancing the efficacy of immunotherapy in lung cancer, using artificial intelligence (AI) to predict outcomes for stage III non-small cell lung cancer (NSCLC), and developing novel approaches to target a noncanonical mitochondrial signaling pathway that mediates lung cancer metastasis. Through strong teamwork carried out by this highly collaborative team of dedicated investigators, and building on exciting data published in leading journals by our group during the present SPORE funding period, this proposal aims to achieve substantial improvements in the management of patients with NSCLC, through three overall Specific Aims: Aim 1: To examine the novel inhibitory molecules selectively expressed by the stem-like CD8 T cells and how they orchestrate stem- like cell function and interactions with myeloid cells in NSCLC and to conduct a clinical trial to evaluate a novel immunotherapy combination approach for advanced NSCLC (Project 1); Aim 2: To utilize an innovative AI-informed systems biology approach to predict outcomes in stage III NSCLC patients, leveraging a recently completed phase III ECOG-ACRIN clinical trial (Project 2); and Aim 3: To interrogate metabolic enzyme SUCLA2 as a novel therapeutic target for NSCLC (Project 3). The Emory Lung Cancer SPORE program will be supported by three integrated cores, the Administrative Core (Core A), Pathology Core (Core B) and the Biostatistics and Biomedical Informatics Core (Core C) and will conduct Career Enhancement and Developmental Research Programs (CEP and DRP). The SPORE program will receive guidance from highly qualified External and Internal Advisory Board members and input from our patient advocates regarding its progress and direction. Our program will receive strong institutional support including modern research space, excellent shared resources, and a significant level of matching funds (totaling $2.25M). Through team-driven innovative research efforts in immunotherapies, AI-powered biomarker discovery, and targeted therapeutics, we are confident that this SPORE program, in collaboration with other NCI lung cancer SPORE sites, will have a major impact on improving the lives of patients with lung cancer.

New York State Education Department

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

NEI - National Eye Institute

PROJECT SUMMARY Neovascular Age-related Macular Degeneration (nAMD) is characterized by pathogenic choroidal neovascularization (CNV) and is a leading cause of blindness in older Americans. The current standard of care, intraocular injection of VEGF inhibitors, presents significant challenges: 25% of patients don’t respond to treatment, many patients experience vision deterioration because challenges associated with receiving frequent invasive intraocular injections lead to undertreatment, and long-term use of VEGF inhibitors may accelerate macular atrophy. This proposal aims to develop and characterize a novel VEGF degrader therapeutic that overcomes these limitations through targeted protein degradation (TPD) technology. In preliminary experiments, we identified a potent VEGF degrader (Degrader 1) which is designed to recruit VEGF to a novel lysosomal trafficking receptor for degradation. This degrader demonstrates exceptional catalytic activity, depleting a 10-fold excess of VEGF within 24 hours in stem cell-derived retinal pigment epithelial (RPE) cells. Importantly, the lysosomal trafficking receptor used has polarized expression on the apical RPE surface and thus has the potential to provide spatial specificity that may prevent the indiscriminate choroidal VEGF inhibition associated with macular atrophy. In this study, we will define Degrader 1's mode of action by: (a) confirming its dependence on the lysosomal trafficking receptor using knockdown and imaging studies; (b) defining its spatial activity on apical versus basal RPE surfaces, (c) evaluating target specificity through proteomics analysis, and (d) assessing therapeutic efficacy in the mouse laser-induced CNV model by comparing Degrader 1 to Aflibercept using fluorescein angiography, OCT, and histology as readouts. Degrader 1 represents a paradigm shift in nAMD treatment with potential advantages of increased potency at lower doses (resulting in reduced treatment frequency), spatial specificity (to prevent macular atrophy), and compatibility with AAV delivery (for long-term expression). If successful, this approach could significantly improve clinical outcomes and quality of life for nAMD patients, providing effective treatment for current non-responders while reducing injection frequency and long-term complications. The University of Wisconsin-Madison is an ideal place for successful completion of both of these aims, as it is a collaborative environment where all of the resources (both technical and intellectual) needed for this project are freely available.

NIAID - National Institute of Allergy and Infectious Diseases

Despite currently available therapeutic approaches, multiple sclerosis (MS) remains an incurable disease. Studies in MS patients and corresponding animal models show that encephalitogenic T effector cells (TEFF) promote key autoimmune responses during MS pathogenesis. Within the central nervous system (CNS) of MS patients, these autoimmune T cells infiltrate the brain and then circulate back into the peripheral immune system, where they are maintained and reactivated, making such T cells a feasible diagnostic and therapeutic target. The mechanisms responsible for the maintenance of the encephalitogenic TEFF repertoire remain unclear. Stem- like T cells (TSTEM) that continue to differentiate into TEFF were proposed to contribute to the onset and exacerbation of human MS and its animal models. However, gaps in knowledge about the molecular characteristics of these encephalitogenic T cells and technical limitations severely hamper such advances. To overcome these limitations, we have developed a novel, state-of-the-art analytical tool, Seqtometry (sequencing- to-measurement), for analyzing single-cell sequencing data. As presented in this proposal, we performed an in- depth transcriptomic analysis of encephalitogenic T cells from the CNS and peripheral blood of MS patients and successfully identified MS-linked TSTEM, which were conspicuously absent in healthy or MS-free controls. We confirmed our findings using T cells specific for myelin oligodendrocyte glycoprotein (MOG), a relevant neuronal antigen, in an animal model of MS. Our most recent studies identified CD4+ T cells programmed for differentiation into TEFF with specific transcriptomic signature profiles corresponding to the MS-linked TSTEM, highlighting the conservation of encephalitogenic TSTEM. Based on these significant findings, we hypothesize that key transcriptomic characteristics that are conserved in T cells from MS patients and relevant animal models can foretell the early disease process and offer insights into the molecular mechanisms that govern the encephalitogenic differentiation of autoimmune T cells. Our further studies revealed in encephalitogenic TSTEM specific molecular mechanisms dependent on the homeodomain-only protein (Hopx) that governs effector differentiation of these cells. We additionally hypothesize that intrinsic mechanisms of Hopx in TSTEM orchestrate key pro-encephalitogenic functions. We will test our hypotheses in three aims: In Aim 1, we will determine the trajectories of encephalitogenic differentiation, In Aim 2, we will reveal the molecular mechanisms orchestrating fate and functions of TSTEM and clarify functions of Hopx, In Aim 3, we will determine the molecular characteristics of MS-linked TSTEM. Overall, the expected results of this research would provide us with key insights into molecular mechanisms underlying differentiation of encephalitogenic T cells, also helping to usher us into developing new MS diagnostic tools.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

ABSTRACT Liver transplantation is the only curative treatment for end-stage and acute liver disease; however, the scarcity of donor organs underscores the urgent need for alternative therapies. Transplantation of human induced pluripotent stem cell (iPSC)-derived liver cells offer a promising approach to treat liver disease. The most desired cell type to transplant is the hepatocyte, the main functional cell of the liver, however, one limitation is the poor maturation of hiPSC-derived hepatocytes that prevent their clinical benefit especially in the case of acute liver diseases that require rapid replenishment of functional hepatocytes. An alternative is to transplant supportive cells that promote proliferation and function of the remaining hepatocytes. We will specifically leverage the known supportive role of liver endothelial cells in animal models for hepatocyte proliferation and function through paracrine signaling during liver repair. The goal of this proposal is therefore to evaluate the therapeutic potential of iPSC-derived endothelial cell (iEC) therapy for promoting liver regeneration in acute liver injury and elucidate the cellular and molecular mechanisms by which iECs support hepatocyte proliferation, repair and function. Using immunocompromised NSG mice, we will apply two complementary acute liver injury models: monocrotaline (MCT)-induced EC-specific damage and acetaminophen (APAP)-induced injury affecting both ECs and hepatocytes. Additionally, the master regulator of EC biology, VEGFA ligand, will be delivered via nucleoside-modified mRNA encapsulated in lipid nanoparticles (mRNA-LNP) to activate in vivo the KDR receptor on iECs, enhancing their engraftment, proliferation, and supportive function. Our preliminary data strongly support this approach: (1) We have established a protocol that efficiently generates a pure population of iECs that are able to engraft after delivery of biologically active VEGFA165 in vivo, and (2) our lab has optimized the mRNA-LNP platform for efficient and transient in vivo VEGFA165 delivery. Based on these data, our central hypothesis is that KDR activation with VEGFA mRNA-LNP in engrafted iECs will enhance their engraftment and restoration of liver function (Aim 1), through supportive endothelial factors that will promote the hepatocyte-iEC niche activation to induce hepatocyte proliferation and function, and hence to promote liver repair (Aim 2). This project will use single-cell RNA sequencing data analyses to identify and further validate critical paracrine factors secreted by iECs that mediate liver regeneration. Altogether, this study will establish the preclinical potential of iEC therapy for acute liver diseases, providing cellular and molecular mechanistic insights in this process and laying the groundwork for potential future applications in chronic liver injury. This research will be conducted at the Center for Regenerative Medicine (CReM) at Boston University, which offers cutting-edge resources for stem cell biology, advanced genomics, and in vivo disease modeling. The training plan integrates rigorous experimental work, mentorship, formal coursework, and professional development activities that will prepare the candidate for a successful academic career.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT The goal of our research program is to regenerate human hearts. We define heart regeneration as the replacement of a numerical deficit of cardiomyocytes through proliferation. This definition includes heart regeneration to replace the cardiomyocyte loss from a myocardial infarction, as well as replacing the cardiomyocyte proliferation defects in congenital heart disease, the most common birth defect. In contrast to adults, young mammals, especially neonates, have active cardiomyocyte proliferation. This points to the incredible potential for regeneration in infants. Consequently, we have directed our research to investigate the mechanisms of cardiomyocyte proliferation in neonatal mice, with the long-term goal of directing heart regeneration in pediatric patients with congenital heart disease and in adult patients after myocardial infarction. This application is for the characterization of a cardiomyocyte proliferation inhibitor, the extracellular factor, endothelin-1 (ET-1), and its receptor (ET-RA), a G protein-coupled receptor (GPCR). Although extracellular factors that stimulate cardiomyocyte proliferation have been defined, a well-characterized one being neuregulin1 (NRG1), which we have identified (Bersell et al., Cell 2009 and Polizzotti et al., Sci Translat Med 2015), the concept of inhibitors is novel. The concept of extracellular inhibition of cardiomyocyte proliferation is significant because inhibitors could dominate over pro-proliferative factors and render their administration ineffective. This is important for potential clinical application of stimulators, for example, recombinant neuregulin, and other factors, to direct cardiomyocyte proliferation in patients. The translation into the clinic may occur in the near future since administration of recombinant neuregulin is currently in clinical trials. Consequently, knowledge of the mechanisms by which inhibitors act is critical for clinical translation. Endothelin-1 receptor blockers (ET-RA), such as Ambrisentan, are in the clinic, indicating a path for translation. We propose to test the central hypothesis that endothelin-1 and its receptor, ET-RA, constitute a mechanism, whose inhibitory effect on cardiomyocyte proliferation is activated by heart injury or disease. Our preliminary results show that endothelin-1, by activating ET-RA, reduces M-phase and cytokinesis activity. These results position ET-RA as a compelling molecular target to direct cardiomyocyte proliferation. We will test the hypothesis using genetic and surgical mouse models and in induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from human patients with congenital heart disease. We will take an innovative research approach that has been validated by prior work of the investigators. This proposal combines the synergistic skills of the co-I, Dr. Honghai Liu, with whom the PI has a history of very impactful collaborations, and the co-I, Dr. Todd Evans, who will extend strengths with hiPSC. The necessary reagents are in place, and the environment is exceptional to accomplish this research at Weill Cornell Medical.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY This proposal establishes a partnership between the California State Polytechnic University, Pomona (CPP) and the University of California, Los Angeles (UCLA) to create a pipeline for a resource limited institution (RLI) serving many first-generation college students to pursue PhD programs in neuroscience. This career development program provides these students with valuable research training and exposure to an R1 research environment, addressing a critical need for highly skilled labor force in neuroscience and STEM fields. Students will begin their training at CPP, where they will engage in foundational research skills, including techniques in adeno-associated virus (AAV) applications at Cal Poly’s Armamentarium Vector Core (ArmVC). Over the course of a year (summer + academic year), they will gain hands-on experience in AAV production, purification, and capsid characterization, building skills and confidence in a familiar and supportive setting. In the 2nd summer in the program, the students will transition to UCLA to participate in intensive research experiences, applying their training to cutting-edge neuroscience projects—ideally bringing gene tools that they have prepared at the CPP ArmVC. While at UCLA, they will collaboratively use AAVs and other tools to address questions critical to PIs at UCLA, thereby benefiting UCLA faculty and projects. Upon returning to Cal Poly Pomona, students will continue their UCLA-related projects in a supportive role, ensuring continuity in their research and strengthening ties between the institutions. This may include additional AAV production, characterization of tissue samples from ongoing projects, or design and validation of novel AAVs to continue to delve deeper into a gene therapy related project. This program equips students with technical expertise, mentorship, and the experience of working in a high-level research environment, empowering them to pursue advanced degrees. By fostering a well-trained cohort of neuroscience researchers, this partnership creates a sustainable model for promoting excellence in STEM education and research.

NHLBI - National Heart Lung and Blood Institute

Project Summary / Abstract Sympathetic neurons (SNs) are critical regulators of cardiac output in response to hemodynamic stress and are known to contribute to heart development and cardiomyocyte (CM) maturation. SNs are also required for cardiac regeneration after myocardial injury in neonatal mammals but become dysregulated with adult heart injury, leading to increased risk of fatal arrhythmias. Despite documented importance, human neuro-cardiac interactions have been understudied by researchers due to the lack of high-fidelity in vitro models that both accurately recapitulate the physiology of cardiac innervation and enable mechanistic studies using cell-specific functional assessments. The Bursac Lab has previously generated highly functional, three-dimensional (3D) engineered cardiac tissues (“cardiobundles”) made from neonatal rat ventricular myocytes or human induced pluripotent stem-cell derived CMs (hiPSC-CMs). Building on these results, I have recently developed and characterized a compartmentalized 3D coculture model of human cardiobundles innervated with hiPSC-derived SNs (hiPSC- SNs). Over the 4-week coculture, this novel neuro-cardiac model supports robust axonal ingrowth into cardiobundle compartment without detriment to cardiac contractile or electrophysiological function. Importantly, CMs within cardiobundles reliably respond to stimulation of SNs by presynaptic neurotransmitters, indicating the development of functional sympathetic innervation. For the proposed project, I will utilize this human in vitro model to test two hypotheses that functional innervation of cardiobundles will: 1) promote developmental maturation of hiPSC-CMs and neurocardiac junctions and 2) stimulate the regenerative response of cardiobundles to injury, thus allowing for detailed studies of underlying mechanisms. To test these hypotheses, in Specific Aim 1 I will utilize gene and protein expression profiling, functional assays, and genetically encoded sensors of membrane voltage, calcium, and neurotransmitter release to assess the time-course of structural and functional CM maturation and robustness of formed neurocardiac junctions. In Specific Aim 2, I will apply cryoablation and CM-specific injury protocols in multicellular cardiobundle models to assess the effects of SN innervation on CM and non-CM proliferation, matrix remodeling, neurocardiac functional recovery, and molecular mechanisms underlying the resulting regenerative or pathophysiological responses. Overall, this work will establish the first tissue-engineered model of anatomically representative, highly functional innervated myocardium for in-depth studies of the human neurocardiac development and diseases of the brain-heart axis.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

ABSTRACT Rotator cuff (RC) tears are a leading cause of musculoskeletal impairment, affecting over 250,000 individuals annually in the United States. Despite surgical repair being the standard of care, failure rates remain alarmingly high, with up to 90% of cases experiencing re-tears or poor functional recovery. This failure is largely driven by irreversible muscle degeneration, including atrophy and fatty infiltration, which impair healing and compromise surgical outcomes. Current therapies primarily target the bone-tendon interface while neglecting muscle pathology, underscoring a critical need for regenerative strategies that directly address RC muscle degeneration. This proposal aims to develop a therapeutic approach leveraging lipid nanoparticles (LNPs) encapsulating WNT7a mRNA (W7a-LNP) to promote muscle regeneration and prevent degeneration following RC injury. WNT7a has been shown to increase muscle mass, enhance muscle stem cell (MuSC) expansion, and reduce fatty infiltration, but its recombinant protein form is limited by poor bioavailability and high production costs. W7a- LNP circumvents these limitations by enabling localized, sustained WNT7a production at the injury site, transforming muscle cells into `in vivo protein factories.' Our preliminary data demonstrate that W7a-LNP reduces fibro/adipogenic progenitor (FAP) adipogenesis and fatty infiltration in both in vitro and in vivo models. We will test the hypothesis that intramuscular delivery of W7a-LNP prevents and reverses RC muscle degeneration through three specific aims. Aim 1 will engineer and validate W7a-LNP as a targeted muscle regeneration platform by optimizing delivery, evaluating WNT7a expression kinetics, and assessing its effects on MuSC expansion, myofiber hypertrophy, and FAP adipogenesis in vitro and in vivo. Aim 2 will determine the efficacy of W7a-LNP in preventing RC muscle degeneration when administered at the time of injury using a clinically relevant delayed tendon repair model. Aim 3 will evaluate W7a-LNP's ability to reverse established muscle degeneration when delivered at later stages of injury, with or without mechanical loading, to assess potential synergistic effects. The impact of this research is the development of a scalable, translatable mRNA-LNP therapy that preserves and restores muscle quality in RC injuries. By addressing a critical gap in current treatment paradigms, this strategy has the potential to improve surgical outcomes, enhance functional recovery, and reduce the need for invasive salvage procedures, ultimately transforming the management of RC muscle degeneration.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY Chronic idiopathic constipation is a functional gastrointestinal disorder that affects up to 1 in 5 children. As a pediatric surgeon, often the only effective treatments I can offer are intestinal resection or diversion. Thus, I strive to better understand the mechanisms that result in this cryptic disease. The K08 program has been an ideal foundation to develop the technical and scientific skills I need to make a translational impact for my patients. Through my career development award, I have been able to explore the effect of biomechanical forces on the enteric nervous system (ENS) as a driver of persistent bowel dysfunction in Hirschsprung disease. In the last few years, we have uncovered that the chronically distended, ganglionic intestine in Hirschsprung disease has altered tissue mechanical properties. We have shown that the biomechanical forces of stretch and stiffness impact enteric neuronal phenotype and function and we have tied these changes to the mechanoreceptor, Piezo1. Taken together, our preliminary findings suggest that bowel dysfunction may be driven by mechanically- induced changes in the ENS microenvironment. The concept that biomechanical forces regulate enteric neuronal behavior and intestinal function is applicable to many gastrointestinal diseases. However, it is difficult to discern the contribution of individual biomechanical forces and the response of different cell types to biomechanical force using in vivo models alone. Thus, we seek to leverage this R03 mechanism to develop new methods and new technology to study the effect of biomechanical forces on enteric neurons and glia in vitro. This proposal lays out a two-year plan to optimize a novel engineered three-dimensional (3-D) microfluidic, co-culture system to determine the impact of biomechanical forces on enteric neurons and glia. The current proposal builds on the premise of my K08, that biomechanical forces influence ENS behavior to affect intestinal function, with two aims: (1) to test the role of biomechanical forces (stretch and shear stress) on enteric neuronal morphology, neuroglial differentiation, and neuronal function; (2) to determine how neuroglial interactions shape the ENS response to biomechanical forces. This project will leverage an established organ- on-chip platform, enteric neuronal stem cell culture, in vitro modeling, and cutting-edge cellular and molecular biology techniques to achieve these goals. Completion of these aims will further my transition to independence as an investigator and lead to a new mechanistic understanding of gastrointestinal function with the potential to improve the quality of life for millions of adult and pediatric patients.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary There are currently over 40 million people living with human immunodeficiency virus (HIV) worldwide. The gold standard treatment for HIV is antiretroviral therapy (ART) which, while capable of suppressing viremia, is unable to achieve viral clearance meaning that the therapy must be taken lifelong. Recently, cytotoxic T cells reprogrammed to express a chimeric antigen receptor (CAR) that targets the HIV envelope protein have emerged as a new treatment strategy that can potentially mediate HIV clearance through seeking out and lysing HIV infected cells. However, the cost associated with the generation of CAR T cells is prohibitively expensive due to extensive ex vivo manipulation steps performed in specialized facilities. In vivo generation of CAR T cells could substantially reduce the cost of therapy but must meet two requirements: (I) the CAR construct must be delivered specifically to T cells and (II) the CAR construct must be integrated into the genome to ensure persistence of the therapy. These two requirements are difficult to achieve with currently available in vivo gene delivery vehicles as they require the ability to target specific cells and perform off target free genome editing. Adeno-associated virus (AAV) is a leading platform for in vivo gene delivery, with a proven clinical track record, but it is constrained by broad tissue tropism and the lack of precise, off-target-free integration methods. To address these limitations, this project leverages two recent innovations from our laboratory: (1) programmable retargeting of AAV capsids through covalent linkage to antibodies or nanobodies, and (2) packaging of circular RNA (circRNA) inside AAV particles to transiently deliver gene editing tools such as Streptococcus pyogenes Cas9 (spCas9). We hypothesize that T cell-directed delivery of circRNA encoding spCas9, in combination with a homology-directed repair (HDR) template, will enable efficient and specific reprogramming of T cells into anti- HIV CAR T cells. In Aim 1, we will validate this approach by testing whether circRNA-AAV vectors encoding Cas9 and an HDR template can reprogram primary human T cells ex vivo into CAR T cells capable of targeting HIV- infected cells. In Aim 2, we will develop an induced pluripotent stem cell (iPSC) line lacking the canonical AAV receptor (AAVR), then generate T cells from this line to systematically investigate whether T cell-enriched receptors can support AAV transduction in the absence of AAVR. Identifying such alternative receptors would provide a foundation for engineering capsid-receptor interactions tailored specifically to T cells. The proposed studies are expected to (1) establish an AAV-based platform for site-specific genome integration via circRNA-encoded Cas9, (2) demonstrate efficient and safe generation of CAR T cells targeting HIV-infected cells, (3) uncover T cell-specific receptors that can mediate AAV entry, and (4) define receptor properties that enable functional transduction independent of AAVR. Together, these outcomes will advance the long-term goal of enabling in vivo generation of therapeutic CAR T cells while providing broadly applicable tools for precision AAV retargeting.

NIAID - National Institute of Allergy and Infectious Diseases

TITLE: Engineering AMR-on-a-chip to probe rejection and accommodation pathways Abstract Kidney transplantation is the preferred treatment for patients with end-stage renal disease (ESRD), offering improved quality of life, reduced long-term mortality and morbidity, and lower healthcare costs compared to dialysis. However, antibody-mediated rejection (AMR) remains a major obstacle to achieve long-term graft survival. Despite its clinical significance, investigating the pathogenesis of AMR remains challenging due to the immunological and physiological complexities of the disease in vivo, and the lack of in vitro models that faithfully replicate its pathological features. To address this unmet need, we aim to develop and validate an AMR-on-a- chip platform to recapitulate key features of AMR in vitro (Aim 1). We have developed a glomerulus-on-a-chip model using human induced pluripotent stem cell (iPSC)-derived renal endothelial cells (ECs) and podocytes. This model successfully mimics the structure and function of the glomerular filtration barrier. To induce AMR, we will incorporate immune components required to its pathogenesis, including pathological antibodies (single anti- HLA Ab, patient derived serum DSA anti-ABO Ab, and autoimmune Ab targeting podocytes), complement, and innate immune cells. Our preliminary data also demonstrate that targeting complement pathways can provide transient protection against AMR in the presence of high levels of donor-specific antibodies (DSAs), suggesting the possibility of inducing a state of accommodation. In addition, we have developed novel endopeptidases capable of selectively cleaving IgM alone or both IgG and IgM. In Aim 2, we will evaluate therapeutic strategies targeting complement and immunoglobulins using our AMR-on-a-chip platform reconstructed with the full spectrum of immune components. Specifically, we will test the efficacy of complement inhibitors (anti-C3 and anti-C5) and novel endopeptidases (IdeS, IceM and IceMG) in mitigating AMR and promoting durable protection of renal endothelial cells and podocytes upon re-exposure to pathological antibodies, a process referred to as accommodation. We hypothesize that downstream signaling triggered by HLA, ABO, or autoimmune antigens, in the absence of complement-mediated injury, may upregulate protective genes in target cells, rendering them resistant to subsequent immune challenges. This study will clarify the roles of pathological antibodies, complement, and innate immune cells in AMR; explore the possibility of antibody-independent mechanisms; and assess the local efficacy of therapeutic agents in preventing AMR in the transplant setting.

NIAID - National Institute of Allergy and Infectious Diseases

Abstract Invasive fungal infection by Aspergillus species, most commonly by Aspergillus fumigatus, remains one of the leading causes of mortality in immunocompromised patients undergoing solid organ or HSC transplantation because antifungal agents currently used in clinics are poorly effective in treating Aspergillus infection in patients with impaired immunity and neutropenia due to drug toxicities, drug-drug interactions, and the emergence of drug-resistant strains. Neutrophils provide the first line of defense against fungal infection. They effectively kill fungi by fungicidal oxidative bursts, presentation of fungal antigens to T cells, and by increasing fungal clearance by folding their hyphae and decreasing the spreading of infection with extracellular traps (NETs). However, the efficacy of neutrophil transfusions for A. fumigatus has been limited. Therefore, enhancing the antifungal properties of neutrophils is essential for advancing adoptive neutrophil transfer for treating Aspergillus infections. In this application, we aim to develop a novel therapeutic approach to effectively target A. fumigatus infections using induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) armed with anti-Aspergillus CARs. The major goal of the R21 phase is to provide proof of principle that iNeutrophils equipped with anti-Aspergillus CARs possess superior fungicidal properties. In Aim 1, we will identify the single-chain variable fragment (scFv) that is most effective in iPSC-derived neutrophils for targeting A. fumigatus. In Aim 2, we will characterize the anti-fungal potential of anti-A. fumigatus CAR-iNeutrophils in vitro. If the R21 milestones are achieved, we will advance the development of CAR-iNeutrophil therapies into the R33 phase by enhancing their antifungal potential and demonstrating their efficacy and safety in vivo. In Aim 3, we will identify the most effective CAR configuration and genetic modifications that enhance the fungicidal properties of iNeutrophils. In Aim 4, we will assess the efficacy and toxicity of CAR-iNeutrophils in vivo using zebrafish larvae and invasive pulmonary Aspergillosis mouse models. Overall, generating CAR-iNeutrophils that directly target fungal species will enable the development of a new class of antifungal therapies. These therapies will employ the adoptive transfer of readily available neutrophils with enhanced antifungal functions to treat life-threatening drug-resistant A. fumigatus infections in patients with neutropenia or dysfunctional neutrophils.