Biology subjects

Maeda, A.

Publications and source records attributed to Maeda, A..

8 recordsLinked to original sources

bioRxiv2026

Mutation-agnostic gene insertion therapy for RHO-associated autosomal dominant retinitis pigmentosa using zinc finger nucleases

PurposeAutosomal dominant retinitis pigmentosa caused by mutations in the rhodopsin gene (RHO-adRP) is among the most prevalent inherited retinal dystrophies. With nearly 100 distinct pathogenic variants identified to date, the mutational heterogeneity of RHO-adRP severely limits the clinical utility of mutation-specific therapeutic strategies. We aimed to develop a mutation-agnostic gene insertion therapy using homology-independent targeted integration (HITI) mediated by zinc finger nuclease ZF-ND1 targeting the human RHO 5'-UTR, and to validate its preclinical efficacy, safety, proof-of-concept, and proof-of-mechanism. MethodsWe developed two AAV serotype 5 (AAV5) vectors: one encoding the ZF-ND1 pair (AAV5-ZFN) and one carrying the therapeutic donor cassette (AAV5-RHO), delivered by subretinal co-injection. ZF pairs targeting the RHO 5'-UTR were arranged and refined by in vitro validation; AAV vector optimization and mechanistic verification were performed in human induced pluripotent stem cell (hiPSC)-derived retinal organoids-derived retinal organoids; longitudinal proof-of-concept efficacy and safety were assessed in a humanized RHO-T17M rat disease model by 6-month optical coherence tomography (OCT); and proof-of-mechanism was evaluated in non-human primate retina. ResultsWe identified a ZF-ND1 pair achieving cleavage efficiency comparable to the SpCas9 RNP previously validated for HITI-mediated editing in mouse retina, and optimized the ZF array composition and NLS configuration for efficient editing especially in post-mitotic photoreceptors. HITI-mediated donor integration was confirmed across multiple cell types and ZFN:donor ratios. In the humanized rat disease model, the therapeutic vector provided outer nuclear layer (ONL) preservation by 6 months, with AAV5-ZFN:AAV5-RHO ratios of 1:1 and 1:2 maintaining ONL thickness above the preservation threshold. In the non-human primate retina, the fraction of HITI-edited rod photoreceptors exceeded the 20% therapeutic correction threshold in the successfully treated individual. ConclusionsThese findings support the advancement of this therapeutic vector to first-in-human trials as a mutation-agnostic insertion therapy applicable to all patients with RHO-adRP, irrespective of the specific causative variant.

genetics↗

bioRxiv2026

RIPK3-RNASE1 axis as a potential therapeutic and clinical monitoring target in VEXAS syndrome

Vacuole, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome (VS) is a severe autoinflammatory disease driven by somatic mutations in ubiquitin-like modifier activating enzyme 1 (UBA1), for which disease activity biomarkers and therapeutic targets are needed. We conducted longitudinal deep phenotyping of patients with VS and found that ribonuclease 1 (RNASE1) expression is most strongly correlated with the disease activity score measured by the VEXAS Current Activity Form (VEXASCAF). Single-cell RNA-sequencing showed that RNASE1 was upregulated in VS monocytes. We generated human monocytic cell lines harboring UBA1 mutations (p.Met41Val, p.Met41Leu, and p.Met41Thr). These cells exhibited varying degrees of impaired ubiquitination and subsequent pathological features such as the unfolded protein response, increased pro-inflammatory cytokine production, cell death, and RNASE1 expression, mirroring the genotype-phenotype associations observed in patients with VS. Multi-omics analyses revealed that genotypes linked to greater clinical severity were enriched in pro-inflammatory, interferon, and necroptosis signatures. Notably, functional interrogation demonstrated that inhibition of receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, markedly suppressed all these pathological features, including the elevated RNASE1 expression. These results identify the RIPK3-RNASE1 axis in VS, highlighting RNASE1 as a potential biomarker and RIPK3 as an attractive therapeutic target.

immunology↗

bioRxiv2026

MDA5 multimerization on LINE RNA drives pathogenic extracellular immune complexes in autoimmunity

Autoantibodies are hallmarks of many autoimmune diseases, but their potential pathogenic roles, particularly for those targeting intracellular proteins, remain unclear. Anti-MDA5-positive dermatomyositis (anti-MDA5 DM) is characterized by autoantibodies against the intracellular protein MDA51,2, a conserved innate immune receptor that recognizes viral dsRNA by forming filaments3. Here, using four patient-derived monoclonal autoantibodies (mAbs), we reconstitute and define the molecular architecture, biogenesis, and immunological activity of pathogenic MDA5 immune complexes. Our cryo-EM analysis revealed that these mAbs bind dsRNA-scaffolded MDA5 filaments in at least two distinct binding modes, each exhibiting striking epitope convergence using germline-encoded residues. Extracellular immune complexes formed between mAbs and filamentous, but not monomeric, MDA5 potently activate multiple innate immune pathways, with the magnitude of activation determined by antibody binding mode and immune complex stoichiometry. Antibody bivalency further crosslinks MDA5 filaments into higher-order aggregates with heightened immunostimulatory activity, demonstrating an active role of autoantibodies in shaping immune complex architecture. Analysis of patient plasma reveals elevated levels of extracellular MDA5 filaments and identifies LINE retroelement-derived dsRNA as a structural scaffold. Notably, MDA5 immune complexes induce endogenous LINE dsRNA expression, likely promoting additional MDA5 filament formation and extracellular release through inflammatory cell death. These data thus support a self-amplifying inflammatory cycle as a pathogenic mechanism for anti-MDA5 DM. Collectively, our study defines a broadly applicable architectural principle, in which higher-order organization and binding modes of autoantibodies--beyond antibody affinity or nucleic acid presence alone--govern innate immune activation.

biochemistry↗

bioRxiv2025

Detecting Protein Higher-Order Structural Changes Using Kinase as a Phospho-Labeler

We developed an approach to detect protein conformational changes on a proteome-wide scale by labeling proteins with phosphate groups through in vitro kinase reactions. This structural proteomics approach combines residue-specific labeling based on substrate recognition by protein kinases with quantitative phosphoproteomics using phosphopeptide enrichment, enabling proteome-wide and site-specific profiling of protein conformational changes. By performing in vitro phosphorylation of myoglobin with and without heat denaturation and of HEK293T cell extracts with and without protease treatment, we demonstrated that differences in substrate protein structure are reflected in in vitro phosphorylation efficiency. Moreover, we successfully identified protein conformational changes upon RNA digestion in non-denatured cell extract proteome samples on a proteome-wide scale with high sensitivity by comparing phosphorylation efficiencies. This approach enables residue-specific readout of structural dynamics within the intracellular proteome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/652599v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b2c6c8org.highwire.dtl.DTLVardef@118b159org.highwire.dtl.DTLVardef@2374adorg.highwire.dtl.DTLVardef@1c14880_HPS_FORMAT_FIGEXP M_FIG C_FIG MotivationMass spectrometry (MS)-based structural analysis of proteins often involves labeling of exposed amino acid residues, followed by digestion and identification of labeled peptides by MS. However, the heterogeneity of labeled peptides and the complexity of biological samples hinder proteome-wide analysis of protein higher-order structures. To address this issue, we devised an approach that utilizes site-specific labeling with phosphate groups, taking advantage of the substrate recognition provided by protein kinases. The phosphate groups serve as enrichment handles, enabling selective extraction and highly sensitive analysis of labeled sites. HighlightsO_LIProtein labeling with phosphate groups using in vitro kinase reaction C_LIO_LIQuantitative MS-based readout of in vitro phosphorylation efficiency C_LIO_LIIn vitro phosphorylation efficiency reflects protein conformational changes C_LIO_LIProteome-wide and site-specific identification of protein conformational changes C_LI

biochemistry↗

bioRxiv2024

Integrated hepatic ferroptosis gene signature dictates pathogenic features of ferroptosis

Background & AimsFerroptosis, a distinctive form of cell death induced by iron-dependent lipid peroxidation, is implicated in various biological processes, including liver diseases. Establishing an iron overload-induced ferroptosis model and identifying hepatic gene signatures associated with ferroptosis are crucial for understanding its role in liver pathogenesis. MethodsF-box and leucine-rich repeat protein 5 (FBXL5) is a substrate-recognition component of the SCF E3 ligase complex that restricts intracellular iron levels. In this study, we used liver-specific Fbxl5-null mice to establish an iron overload-induced ferroptosis model. Transcriptome analysis identified genes involved in hepatic ferroptosis. Integrating these gene signatures with another ferroptosis model enabled the assessment of ferroptosis-related pathology in murine liver injury models and in 174 patients undergoing liver resection surgery. ResultsIron overload induced severe liver damage in liver-specific Fbxl5-null mice, characterized by elevated liver enzymes, histopathological changes, and lipid peroxidation. Transcriptome analysis revealed a distinct set of genes associated with hepatic ferroptosis response. Generating a gene signature for evaluating ferroptosis enhanced the understanding of ferroptosis-related pathologies in liver diseases. Iron overload exacerbated liver damage in murine ischemia-reperfusion injury models via ferroptosis induction. In human patients, elevated serum iron levels correlated with sustained post-operative liver damage, indicating heightened susceptibility to ferroptosis. ConclusionHere, a murine model of iron overload-induced hepatic ferroptosis was established, and a gene signature indicative of hepatic ferroptosis response in both mice and humans was identified. These findings underscore the role of ferroptosis in liver injury progression and suggest potential therapeutic targets for liver disease intervention. HIGHLIGHTSO_LILiver-specific Fbxl5 knockout mice provide an iron-induced ferroptotic injury model C_LIO_LIIntegrated gene signature of iron- and acetaminophen-induced liver injury dictates ferroptosis C_LIO_LIIron overload aggravates hepatic ischemia-reperfusion injury in mice C_LIO_LIPatients with high iron levels show delayed post-operative liver damage recovery C_LI IMPACT AND IMPLICATIONSOur study elucidated the critical role of iron in liver disease pathogenesis and ischemia-reperfusion injury (IRI). By establishing a murine model of iron overload-induced ferroptosis, we confirmed that iron overload exacerbated hepatic IRI, underscoring the importance of ferroptosis in liver damage. Additionally, the development of an integrated gene signature for hepatic ferroptosis response provides a valuable tool for evaluating ferroptosis in liver diseases. Via analysis of patient data, we also highlighted the clinical relevance of ferroptosis in post-operative liver damage, offering insights into potential therapeutic strategies targeting iron and ferroptosis to improve outcomes in patients with liver diseases.

molecular biology↗

bioRxiv2024

Genotypic identification of polyclonal plasma cells in plasma cell dyscrasias shows an aberrant single-cell phenotype with clinical implications

Multiple Myeloma (MM) is driven by clonal plasma cell (PC)-intrinsic factors and changes in the tumorigenic microenvironment (TME). To investigate if residual polyclonal PCs (pPCs) are disrupted, single-cell (sc) RNAseq and sc B-cell receptor analysis were applied in a cohort of 46 samples with PC dyscrasias and 18 healthy donors (HDs). Out of n=213,074 CD138pos PCs, 42,717 were genotypically identified as pPCs. Compared to HDs, we detected quantitative and qualitative differences in pPCs of patients showing immunoparesis, where we showed a pro-inflammatory status, driven by specific cellular interactions with TME. Finally, we derived a "hPC signature" that, once inferred in the CoMMpass dataset, was predictive of PFS and OS. Our findings show that genotypic, single-cell identification of pPCs in PC dyscrasias has relevant pathogenic and clinical implications.

cancer biology↗

bioRxiv2023

In vitro pigmentation of human iPSC-derived retinal pigment epithelium cells does not indicate their quality for cell transplantation

Retinal pigment epithelium (RPE) cells show heterogeneous level of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized in 4 clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.

cell biology↗

bioRxiv2020

Inducing Human Retinal Pigment Epithelium-like Cells from Somatic Tissue

Regenerative medicine relies on basic research to find safe and useful outcomes that are only practical when cost-effective. The human eyeball requires the retinal pigment epithelium (RPE) for support and maintenance that interfaces the neural retina and the choroid at large. Nearly 200 million people suffer from age-related macular degeneration (AMD), a blinding multifactor genetic disease among other retinal pathologies related to RPE degradation. Recently, autologous pluripotent stem cell-derived RPE cells were prohibitively expensive due to time, therefore we developed a new simplified cell reprogramming system. We stably induced RPE-like cells (iRPE) from human fibroblasts by conditional overexpression of broad plasticity and lineage-specific pioneering transcription factors. iRPE cells showed features of modern RPE benchmarks and significant in-vivo integration in transplanted chimeric hosts. Herein, we detail the iRPE system with comprehensive modern single-cell RNA (scRNA) sequencing profiling to interpret and characterize its best cells. We anticipate that our system may enable robust retinal cell induction for regenerative medicine research and affordable autologous human RPE tissue for cell therapy.

bioengineering↗