Journal of Clinical Investigation

Purpose: Neurofibromatosis type 2-related schwannomatosis (NF2-SWN) is an autosomal dominant tumor syndrome with complete penetrance and variable expressivity. Underlying patterns of disease burden and severity are largely unexplained. Methods: We comprehensively phenotyped 168 NF2-SWN patients over a mean duration of 4.5 years. We used a custom sequencing panel of NF2 and schwannomatosis genes to identify germline (n=166) and somatic variants in tumors (n=37). An optimized composite severity (CSS) score based on clinical and radiological data was created to analyze the effect of genetic variants on phenotype. Results: We found significant variable expressivity not explainable by demographic variables. Germline variants included premature termination (42%), splice-site (18%), and large deletions (16%). The CSS successfully predicted worsening clinical function in patients. Unsupervised clustering of clinical data revealed distinct phenotypic clusters that corresponded to CSS. Mosaicism, however, was not associated with CSS or any other disease severity marker. CSS was significantly associated with germline variant location along the NF2 locus. Specifically, FERM-F1 and the -helical variants were associated with increased disease severity. Within tumors, germline variants with severe effects on merlin acquired milder somatic second-hits at the NF2 locus. Conclusion: We identified a second-hit modifier to the Mendelian first-hit: severe germline variants were associated with milder somatic variants, and vice versa. This phenomenon partly explains the variable expressivity in NF2-SWN.

Genetic mitochondrial diseases (GMDs) are major challenges to human health accounting for a significant fraction of heritable neurologic diseases, myopathies, and inborn errors of metabolism. Leigh syndrome (LS) is the most common clinical presentation of GMD in pediatric patients. LS is a severe and complex disease for which effective clinical therapies are currently lacking. Preclinical therapies identified in the Ndufs4(-/-) mouse model of LS include immune-targeting interventions and chronic mild hypoxia (11% oxygen). Immune-targeting interventions include rapamycin and high-dose pexidartinib, the latter appearing to fully suppress disease. The mechanisms underlying the benefits of hypoxia remain unclear, and the relationship between hypoxia and immune interventions have not been assessed. Here, we report the immune profile of brainstem of the Ndufs4(-/-) mouse model prior to and after disease onset and the impact of pexidartinib treatment. We provide evidence that macrophages/monocytes drive pathology, consistent with recent genetic studies. We additionally find that pre-disease onset animals lack signs of inflammation, and that the elimination of leukocytes fully suppresses the molecular signature of disease. Finally, using distinct post-developmental periods of treatment, we find pexidartinib and rapamycin provide benefits which persist long beyond treatment cessation, while cessation of hypoxia results in rapid disease onset and an acceleration of disease progression. These findings are consistent with hypoxia acting upstream of immune cell activation and have major implications for the therapeutic translation of both hypoxia and immune targeting interventions. Our findings establish hypoxia-cessation as a novel method for synchronizing inflammatory disease onset in the Ndufs4(-/-) model which will be useful in future mechanistic studies.

Understanding the shared and disease-specific mechanisms that drive glomerular injury remains a major unmet need for developing more precise and effective treatments for glomerular diseases. Here, we integrate histology-guided spatial transcriptomics with trajectory inference and cell-state deconvolution to resolve glomerular injury across Membranous Nephropathy, Focal Segmental Glomerulosclerosis, and Lupus Nephritis. Herein, we show that glomerular diseases arising from distinct initiating mechanisms converge on common pathways during progressive loss of kidney function. We used spatial transcriptomics to analyze kidney biopsies from 59 individuals with glomerular diseases and controls and identified 792 glomeruli, including structurally injured glomeruli missed by transcriptomic clustering alone. Glomeruli shared interferon- and complement- associated inflammatory programs together with suppression of oxidative and lipid metabolic pathways, while retaining disease-preferential transcriptional features. Increased pseudotime correlated with lower estimated glomerular filtration rate, and cell- state deconvolution linked progression to podocyte loss and cellular remodeling. Parallel analysis of passive Heymann nephritis, a rat model of membranous nephropathy, recapitulated complement activation and localized complement activity to multiple renal cell populations extending into the periglomerular niche. These findings define a spatially resolved, cross-disease continuum of glomerular injury and identify coordinated local complement activation as a convergent feature of progressive glomerular damage.

GNB1 encephalopathy (GNB1E) is a rare neurodevelopmental disorder caused by mutations in GNB1 gene encoding the G protein subunit G{beta}1. Mechanisms linking these variants to neurological dysfunction remain unclear. We investigated the prevalent p.Ile80Thr (I80T) variant using combined clinical, cellular, and in vivo approaches. Longitudinal evaluation of a GNB1E patient revealed developmental delay, progressive peripheral spasticity, and epilepsy with Spike-Wave Activation in Sleep. Heterozygous knock-in Gnb1I80T/+ mice exhibited disease-relevant phenotypes, including impaired early development, mild adult motor and cognitive deficits and epileptiform cortical spike-and-wave discharges. Transcriptomic analysis identified 323 genes concordantly dysregulated in mouse cortex and cortical human neuronal cultures from patient-derived induced pluripotent cells. This gene set was enriched for ion-channel function, epilepsy-associated genes, and Gs/adenylyl cyclase signaling pathway. Our integrated analysis establishes the first cross-species model for GNB1E, suggests common neurological mechanisms and molecular pathways linked to GNB1E, and provides a framework for mechanistic and therapeutic studies. TeaserConserved human/mouse neurological and transcriptomic signatures in GNB1 encephalopathy.

In TLR7-driven macrophage activation syndrome (MAS), inflammatory hemophagocytes (iHPCs) differentiate from Ly6CHI monocytes, phagocytose red blood cells and promote disease, including anemia and thrombocytopenia. We demonstrate here that IRF5 is required for iHPC differentiation and MAS in TLR7-overexpressing (TLR7.1) mice. Both constitutive and myeloid-specific Irf5 deletion reduced iHPCs and improved anemia, thrombocytopenia and survival. Furthermore, therapeutic inhibition of IRF5 ameliorated MAS features and reduced splenic and circulating iHPCs. While cell-intrinsic IRF5 expression was required for iHPC differentiation, it was not required for TLR7.1 Ly6CHI monocyte differentiation and monocyte transcriptional programs. We further show that the transcriptome and chromatin landscape changed dramatically as iHPCs differentiated from TLR7.1 Ly6CHI monocytes. Many transcriptional programs gained in iHPCs were enriched in genes associated with IRF5-binding accessible chromatin regions, including those associated with NF-kB signaling, cytokine and chemokine production, and complement activation. Our data suggest that IRF5 collaborates with other transcription factor families, including NF-kB, ETS and AP1 members, to regulate iHPC gene programs. Together, our findings demonstrate that expression of IRF5 in myeloid cells is critical for MAS, for iHPC differentiation, and acts broadly across iHPC-specific gene programs in TLR7-driven inflammation.

Ribosome biogenesis defects are increasingly recognized in hematologic disorders, yet their contribution to human combined immunodeficiency (CID) remains largely unexplored. Here, we identify compound heterozygous mutations in GTF3A, encoding transcription factor IIIA (TFIIIA), in a patient with CID presenting with profound T-cell lymphopenia, diminished thymic output, and humoral failure. The patient-derived TFIIIA variants, I267S and L364Yfs*37, disrupted 5S rRNA transcription, impaired RNA-binding capacity, and compromised protein stability. Patient T and B lymphocytes exhibited intrinsic proliferative and differentiation defects, as well as increased apoptotic susceptibility in T cells, recapitulating the clinical phenotype. To model TFIIIA dosage, we generated heterozygous and progressively depleted Jurkat cell clones via sequential CRISPR editing; complete TFIIIA loss was lethal, whereas graded reduction impaired proliferation in a dose-dependent manner, fully rescued by wild-type GTF3A reconstitution. Collectively, these findings establish deficiency of GTF3A resulting in combined immunodeficiency (DoGCID) within the spectrum of ribosomopathies, highlighting the exquisite sensitivity of lymphocyte fitness to disruptions in ribosome biogenesis.

The dynamic reprogramming of the bone marrow (BM) microenvironment during metastatic progression is poorly defined. Through single-cell profiling of BM samples spanning benign conditions, high-risk bone metastatic potential localized prostate cancer (HMP) and established bone metastatic prostate cancer (PCa), we uncovered a primary tumor-to-BM signaling axis that orchestrates niche formation through neutrophil-mediated remodeling. Tumor-associated neutrophils (TANs) emerge during the HMP stage, persist throughout metastatic progression, correlate with poor patient prognosis, and exhibit direct immunosuppressive activity that contributes to a BM environment permissive to metastatic colonization. Functional and genetic studies showed that tumor-associated systemic IGF1 reprogrammed BM neutrophils through IGF1R-MAPK/ERK-KLF4 signaling, with IL-1{beta}, reactive oxygen species and nitric oxide contributing to TAN-mediated T cell suppression. Tumor-cell Igf1 perturbation, IGF1R blockade and neutrophil-specific Igf1r deletion attenuated TAN-associated BM remodeling, and reduced subsequent bone metastatic progression. In bone-metastasis models, combined IGF1R and PD-1 blockade suppressed bone-metastatic progression. Translationally, our investigator-initiated clinical trial (NCT06866548) of teprotumumab plus tislelizumab in patients with established metastatic castration-resistant prostate cancer (mCRPC) provided initial safety data and exploratory clinical observations. Together, these findings identify IGF1R-dependent neutrophil reprogramming as a functionally important component of the evolving PCa bone metastatic microenvironment and provide a rationale for therapeutic targeting of IGF1R signaling together with immune checkpoint blockade.

Systemic lupus erythematosus (SLE) features a broad array of autoantibodies against nuclear components, but the mechanisms driving this diversity are poorly understood. We investigated whether posttranslational arginine methylation creates common neo-epitopes that could unify this response. Using methylome-wide peptide arrays, we identified anti-symmetric dimethylarginine (SDMA) autoantibodies in 30-40% of SLE patients, with individualized reactivity fingerprints. A single anti-SDMA monoclonal antibody from MRL-lpr mice recognized multiple SDMA-modified antigens, and EBNA-1, an Epstein-Barr virus protein, bound SLE plasma in an SDMA-dependent manner, pointing to potential molecular mimicry. In a validation cohort of 201 patients, anti-SDMA titers correlated with SLEDAI-2K scores and inversely with C3/C4; seropositivity was overrepresented in younger patients, those with renal involvement, and anti-Sm-positive individuals. These results establish that anti-SDMA responses are common in SLE, target methylated arginine motifs across self and viral proteins, and serve as markers of active, complement-consuming disease. The patient-specific recognition of SDMA epitopes may underlie the serological heterogeneity that defines SLE.

Glucose metabolism is pivotal in regulating innate immune responses in primary human monocyte-derived macrophages (MDMs). Lipopolysaccharide (LPS) stimulation induces both inflammatory and antiviral programs; however, despite the established importance of glucose metabolism in these responses, its precise role in coordinating them remains poorly defined. Here, we identify the STAT1/NF-{kappa}B/IRF5 signaling axis as a key mediator linking glucose metabolism to inflammatory responses through the upregulation of the rate-limiting glycolytic enzyme PFKFB3. We found that LPS triggered delayed expression and activation of NF-{kappa}B p65, accompanied by increased expression of inflammatory target genes, including CD38 and CD40. Using complementary pharmacological and genetic approaches, we demonstrate that glycolysis and PFKFB3 activity are required for NF-{kappa}B p65 expression and activation. Strikingly, inhibition of PFKFB3 also suppressed LPS-induced STAT1 activation and nuclear translocation, revealing a glucose-dependent amplification loop that potentiates STAT1-mediated antiviral and NF-{kappa}B p65-mediated inflammatory responses. Collectively, these findings establish a mechanistic link between glycolytic metabolism and STAT1/IRF5- and NF-{kappa}B-dependent transcriptional programs in human MDMs responding to LPS, highlighting potential therapeutic targets for modulating innate immune responses in inflammatory disease.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common, debilitating complication of cancer therapy. Although axonal degeneration defines CIPN, the initiating cellular events remain unknown. Here, we show that CIPN is initiated by senescent peripheral fibroblasts rather than by the neuron itself. Across the mechanistically distinct chemotherapeutic agents paclitaxel and cisplatin, chemotherapy-induced senescence is unexpectedly restricted to peripheral fibroblasts rather than neurons and acts upstream of neuronal SARM1 activation. Genetic or pharmacological ablation of senescent fibroblasts prevents neuropathy and reverses established disease, demonstrating that these cells are required for both disease initiation and maintenance. Mechanistically, senescent fibroblasts drive neuropathic injury through an MK2-dependent senescence-associated secretory phenotype (SASP), and genetic or pharmacological inhibition of MK2 suppresses the SASP, preserves peripheral innervation and restores sensory function. Together, these findings redefine the cellular origin of CIPN and identify MK2-dependent fibroblast senescence as a therapeutic target.

Despite immunosuppressive regimens targeting adaptive immunity, chronic lung allograft dysfunction (CLAD) remains the major obstacle to durable lung allograft survival. Here, we identify colony-stimulating factor 1 receptor (CSF1R)-expressing interstitial macrophages as critical orchestrators of CLAD. Using lung tissue from patients with CLAD and a mouse model of mismatched lung transplantation, we show that both donor-derived tissue-resident and recipient- monocyte-derived interstitial macrophages spatially co-localize within peribronchial immune aggregates in patients with CLAD. These interstitial macrophages express distinct cytokine programs that include those implicated in the recruitment of T and B cells. Pharmacological inhibition of CSF1R after lung transplantation in mice reduced interstitial macrophage abundance and attenuated CLAD pathology. Our findings identify donor- and recipient-derived interstitial macrophages as upstream regulators of CLAD and suggest CSF1R as a therapeutic target for its prevention and treatment.

BACKGROUND: Severe influenza pneumonia with secondary bacterial infection is complicated by progressive pulmonary thrombus exacerbation, a key contributor to respiratory failure, yet anticoagulant therapies show limited efficacy and bleeding risks. Although platelet-monocyte crosstalk initiates thrombosis, whether and how it drives thrombus exacerbation via procoagulant platelets and monocyte efferocytosis remains unclear. METHODS: Clinical samples, mouse models, and isolated platelets challenged with influenza A virus followed by methicillin-resistant Staphylococcus aureus (MRSA) were analyzed. Procoagulant platelet, platelet programmed cell death and monocyte efferocytosis were assessed, and pharmacological inhibition, platelet depletion and systemic/platelet-specific Gsdmd knockout were used. Platelet proteomics and exogenous C1qa supplementation identified C1qa as a key mediator. RESULTS: We showed that elevated procoagulant platelet-driven thrombus exacerbation, rather than initial thrombus formation, was a critical driver of disease progression in influenza pneumonia with secondary bacterial infection, whereas influenza alone caused milder illness. Procoagulant platelet formation was induced via MLKL-mediated necroptosis and GSDMD-mediated pyroptosis. These platelets exacerbated pulmonary thrombosis and lung injury by inhibiting monocyte efferocytosis via complement C1qa. Platelet depletion reduced monocyte efferocytosis and worsened pneumonia, while pharmacological inhibition or platelet-specific Gsdmd knockout decreased procoagulant platelet levels, restored monocyte efferocytosis, and alleviated thrombotic and pulmonary injury. Mechanistically, C1qa impaired efferocytosis both by directly suppressing monocyte function and by reducing the proportion of reparative (M2-like) monocytes. Clinical relevance was confirmed by detection of MLKL/GSDMD-dependent procoagulant platelets and reduced efferocytosis receptor levels on monocytes in bronchoalveolar lavage fluid from severe pneumonia patients. CONCLUSIONS: Necroptosis/pyroptosis-driven procoagulant platelets exacerbate pulmonary thrombosis by suppressing monocyte efferocytosis in a C1qa-dependent manner. These findings extend platelet-monocyte crosstalk from thrombus initiation to thrombus exacerbation, identifying modulation of the interaction between procoagulant platelets and monocyte efferocytosis as a potential therapeutic strategy for thrombus-exacerbating diseases, especially in subpopulations of patients with severe pneumonia and progressive pulmonary thrombosis.

Pathogenic variants in Mitofusin2 (MFN2) cause the peripheral neuropathy Charcot-Marie-Tooth Type IIA (CMT2A), with a subset of patients presenting with myopathy. Yet how MFN2 dysfunction causes these phenotypes remains mechanistically undefined. Here, we identify a conserved pathomechanism in MFN2-linked myopathy variants, where mitochondrial DNA is released into RAB5 early endosomes, robustly activating TLR9 and cGAS-STING inflammatory signaling. We further show that MFN2 dysfunction drives two mechanistically distinct pools of extra-mitochondrial mtDNA, one in endosomes and the other present mitochondrial-derived vesicles (MDVs). These two pools of released mtDNA can be distinguished based on their size, with endosomal mtDNA forming larger puncta than mtDNA present in MDVs. Finally, we describe that pharmacological activation of the integrated stress response reduces both pools of extra-mitochondrial mtDNA. Together, our work defines a conserved cellular phenotype linking MFN2 dysfunction to myopathy and suggests a tractable therapeutic strategy for mtDNA release-associated pathologies.

Multiple sclerosis is characterized by immune-mediated demyelination and inefficient remyelination, owing to impaired differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Inflammatory cytokines within multiple sclerosis lesions inhibit OPC maturation and induce an immune-like phenotype with antigen-presenting properties, but the underlying mechanisms remain poorly defined. Here, we show that inflammation reprograms OPC lipid metabolism, linking altered metabolism to remyelination failure. In cultured rodent OPCs, interferon-{gamma} (IFN-{gamma}) induced a switch from lipid synthesis to utilization, leading to reduced intracellular fatty acid levels and increased dependence on fatty acid oxidation. Transcriptional analyses confirmed similar lipid metabolic changes in OL-lineage cells cultured from human surgical specimens or isolated from mouse models of inflammatory demyelination and human multiple sclerosis lesions. Enhancing lipid availability in OPCs through oleic acid supplementation or inhibition of fatty acid oxidation attenuated immune-like functions and increased differentiation. Pharmacologic activation of liver X receptor (LXR) transcription factors rebalanced lipid metabolism, suppressed immune-like functions, and overcame IFN-{gamma}-induced differentiation blockade in both mouse and human-derived OPCs. In an adoptive transfer-cuprizone mouse model in which inflammation directly impairs remyelination, LXR activation increased mature OL generation and augmented myelin repair. Together, these findings identify lipid metabolic remodeling as a key mechanism by which inflammation impairs OPC differentiation and highlight LXR activation as a therapeutic approach to enhance remyelination in multiple sclerosis.

Renal fibrosis represents an irreversible endpoint of chronic kidney disease. Although macrophage efferocytosis regulates the response to acute renal injury, the mechanisms by which early efferocytosis confers long-term protection against chronic fibrosis remain elusive. Here, we show that phagocyte-specific deletion of the inhibitory receptor CD300a (Cd300afl/flLyz2-Cre) mitigates obstructive renal injury and subsequent fibrosis. CD300a deficiency enhanced efferocytosis by tissue-resident macrophages during the early phase of injury, thereby suppressing the release of damage-associated molecular patterns (DAMPs) and TGF-{beta} expression. This early attenuation of the inflammatory microenvironment altered the composition of recruited neutrophils, shifting them from profibrotic SiglecF+ toward non-profibrotic SiglecF- subsets. Furthermore, loss of CD300a cell-autonomously primed neutrophils for enhanced matrix metalloproteinase-9 (MMP9) production upon injury, whereas neutrophil depletion abolished the protective effect of CD300a deficiency. Importantly, administration of a neutralizing anti-CD300a antibody to wild-type mice recapitulated these protective effects, promoting the shift toward SiglecF- neutrophils and significantly attenuating renal fibrosis. Together, our findings identify CD300a as a regulator of early macrophage efferocytosis and neutrophil-derived MMP9 production, and establish pharmacological CD300a blockade as a potential therapeutic strategy for progressive renal fibrosis.

Bispecific T cell engagers (BTCEs) have transformed the treatment of B cell malignancies, but how they remodel non-conventional T cell circuits in patients, and how they should be ideally combined with existing treatment backbones, remains unclear. Here we show that CD3-engaging BTCEs convert human FOXP3 regulatory T cells (Tregs) into an AP-1-imprinted hybrid cytotoxic state that contributes to tumor clearance. Leveraging longitudinal bone marrow and peripheral blood sampling from patients with newly diagnosed multiple myeloma (NDMM) treated with BTCE-based induction in the phase 2 (GMMG-HD10/DSMM-XX/MajesTEC-5) trial, which achieves uniformly deep, minimal residual disease (MRD)-negative remissions, we integrate single-cell RNA, ATAC and TCR sequencing with functional and metabolic profiling to define the underlying mechanism. BTCE-containing induction drives expansion of cytotoxic CD8 clones and the emergence of a Treg population characterized by GZMA, GNLY and NKG7 expression, enriched cytotoxic gene signatures, and licensed by BATF-AP-1-centered chromatin remodeling. In vitro, BTCE-exposed Tregs acquire bona fide lytic function, killing tumor cells in a target- and dose-dependent manner and undergoing metabolic rewiring towards an effector-like state while retaining canonical Treg lineage markers. Immunomodulatory drugs (IMiDs) do not initiate but quantitatively amplify and qualitatively reshape this BTCE-driven cytotoxic program, providing a mechanistic rationale for BTCE-IMiD combination regimens. Cytotoxic Treg reprogramming is conserved across BTCEs targeting BCMA, GPRC5D, and CD19 and across multiple myeloma, as well as acute lymphoblastic leukemia. These findings reveal unexpected plasticity of human Tregs under synthetic T cell engagement and establish cytotoxic Tregs as active effectors, candidate biomarkers and tractable levers for optimizing T cell-redirecting immunotherapy.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

The placenta is a transient organ that orchestrates maternal-fetal interactions essential for healthy pregnancy, yet the multicellular principles governing placental dysfunction remain poorly understood. Here, we present a multimodal single-cell atlas of the human placenta, profiling over 1.2 million cells from 68 donors using paired single-cell RNA sequencing, CITE-seq, and T cell receptor sequencing across spontaneous preterm birth, preterm and term preeclampsia, fetal growth restriction, type 1 diabetes, and healthy term and preterm pregnancies. We define 115 placental cell populations, including previously unrecognized maternal and fetal subsets, revealing unexpected cellular diversity across immune, vascular, trophoblast and stromal compartments. Cross-disease analyses demonstrate that maternal-fetal myeloid imbalance spans nearly all disease states, while trophoblasts, fetal macrophages, and fetal endothelial cells show marked, condition-specific dysregulation. We further identify conserved interferon-stimulated macrophage populations that function as constitutive homeostatic sentinels, and define candidate multicellular immune regulatory niches associated with placental T cell clonal expansion across healthy and complicated pregnancies. Together, these findings establish a comprehensive cellular framework for placental function and dysfunction that can guide precision diagnostic and therapeutic strategies across major obstetric disorders.

Loss-of-function mutations in the GATOR1 complex (NPRL2, NPRL3, and DEPDC5) are among the most common genetic causes of epilepsy. These mutations impair amino acid-dependent regulation of mTORC1 and cause hyperactive signaling, neuronal hyperexcitability, astrocytic metabolic remodeling, seizures, and early mortality. Here, we report the development of antisense oligonucleotides (ASOs) targeting RAPTOR, an mTORC1 scaffolding subunit, to correct pathological mTORC1 hyperactivity caused by defective upstream regulation. RAPTOR-targeted ASO treatment reduced RAPTOR expression in mouse and human systems and dampened mTORC1 signaling in GATOR1-deficient models. ASO-mediated RAPTOR reduction rescued neuronal hyperexcitability, corrected astrocytic mitochondrial dysfunction, and suppressed spontaneous seizures in vivo. A single treatment significantly extended longevity across neuronal and astrocytic models of GATOR1 deficiency. Collectively, these findings establish RAPTOR-targeted ASOs as a mechanism-based therapeutic approach that durably corrects cellular and circuit-level consequences of GATOR1 deficiency. By tuning RAPTOR abundance, this strategy provides a therapeutic framework for mTORC1-driven epilepsies and related diseases.

Type 2B von Willebrand disease (VWD) is characterized by missense variants in exon 28 of the von Willebrand factor (VWF) gene that encodes the VWF-A1 domain. These missense mutations result in single amino acid residue substitutions that cause activation of VWF-A1 and promote spontaneous interaction with platelet GPIba;. Importantly, enhanced VWF clearance has been shown to play a key pathogenic role in >90% patients with Type 2B VWD. Although this VWF clearance occurs via mechanisms that are independent of VWF-platelet complex formation, the biological mechanisms responsible for the increased clearance of type 2B VWD variants remain poorly understood. In this study, we investigated a series of different type 2B amino acid substitutions within the VWF-A1 domain (R1306W, R1308C, W1313C and R1379L). We demonstrate that these variants all exhibit significantly increased macrophage binding. In part, the enhanced macrophage interactions are mediated via increased binding of type 2B VWD variants to LRP1 extracellular cluster II and IV. Our findings further demonstrate that the K1405-K1408 lysine cluster in the VWF-A1 domain plays a key role in enabling enhanced LRP1 interactions for type 2B VWD variants. In addition, we show that type 2B VWF variants demonstrate significantly enhanced interaction with the macrophage MGL receptor. Finally, and importantly from a clinical perspective, we demonstrate that the increase in macrophage binding for type 2B variants is significantly attenuated in the presence of BT200. Collectively, our findings have direct translational relevance with respect to the clinical heterogeneity and treatment of type 2B VWD.