De Chiara A, Nele V, Angelillo A
… +14 more, Campani V, Campanile A, Froechlich G, Scognamiglio A, Pellino E, Greco A, Chinello C, Pagani L, Eugster R, Luciani P, Corbo C, Nicosia A, Sasso E, De Rosa G
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41736889
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Synthetic messenger RNA (mRNA) formulated in lipid nanoparticles (mRNA-LNPs) is a promising candidate for next-generation gene therapy and genetic vaccines. However, mRNA-LNP formulations require low-temperature storage...Synthetic messenger RNA (mRNA) formulated in lipid nanoparticles (mRNA-LNPs) is a promising candidate for next-generation gene therapy and genetic vaccines. However, mRNA-LNP formulations require low-temperature storage to ensure proper transport and distribution. Here, we introduce a lipid self-assembling nanoparticle (SANP) technology to address the stability challenges of mRNA-based therapeutics. SANP formulations can be prepared by simply mixing the components immediately before use, allowing mRNA vaccines to be stored and transported at 4°C without freezing, thereby enhancing their stability. SANPs loaded with mRNA (mRNA-SANPs) exhibited a sub-200 nm size, high mRNA encapsulation efficiency, colloidal stability post-assembly and in human plasma, and low hemolytic activity. Intramuscular (IM) and intravenous (IV) administration of mRNA-SANPs encoding a reporter gene in mice resulted in high levels of transgene expression, with no observed renal or hepatic toxicity and no release of pro-inflammatory cytokines. Additionally, protein fingerprint analysis of mRNA-SANPs in serum identified specific nanoparticle-protein interactions that correlated with biodistribution. Finally, mRNA-SANPs encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein elicited a significant immune response in mice following both IM and IV administration.
Chen J, Deng Q, Zhen P
… +7 more, Hu J, Wang Y, Peng J, You Z, Shang X, Zhang X, Wang T
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41736888
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Drug-drug interaction (DDI) prediction is crucial for understanding combined medication effects and preventing adverse reactions. Traditional machine learning methods rely on handcrafted features and lack generalization,...Drug-drug interaction (DDI) prediction is crucial for understanding combined medication effects and preventing adverse reactions. Traditional machine learning methods rely on handcrafted features and lack generalization, while existing deep learning approaches often fail to capture global and multi-scale drug relationships. To overcome these limitations, we propose ALG-DDI, a multi-scale feature fusion model that integrates three types of drug information: attribute (intrinsic drug structure), local correlations (with proteins and diseases), and global semantic information from the medical knowledge graph PrimeKG. We encode these using attribute masking, the idea of RGCN and GraphSAGE, and ComplEx, respectively. A transformer encoder with attention mechanism then fuses these multi-scale representations. The resulting drug pair vector is fed into a fully connected network for DDI prediction, which we also extend to DDI event prediction. Extensive evaluations on three datasets-including comparative experiments, cross-validation, retrospective analysis, and case studies-demonstrate that ALG-DDI outperforms existing state-of-the-art methods.
Karsenty CL, Betancourth D, Cao M
… +3 more, Pham QK, Park SH, Bao G
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41736887
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CRISPR-Cas9 gene editing offers the potential to transform the treatment of sickle cell disease by correcting the sickle mutation in β-globin gene (). However, in addition to alleles with homology-directed repair (HDR),...CRISPR-Cas9 gene editing offers the potential to transform the treatment of sickle cell disease by correcting the sickle mutation in β-globin gene (). However, in addition to alleles with homology-directed repair (HDR), Cas9 editing at generates a diverse spectrum of outcomes, including small insertions and deletions (indels), large deletions (LDs), and loss of allele (LOA) events, that can compromise genomic integrity and raise significant safety concerns. While new pharmacological modulators have been developed to increase the HDR rates, they may also elevate the risk of large gene modifications. To better understand the complex gene editing outcomes, we engineered a live-cell, dual-fluorescent reporter cell model enabling allele-specific monitoring of expression via GFP and blue fluorescent protein (BFP) tagging. Based on fluorescence intensities, this model can discriminate in-frame alleles, frameshift indels, LDs, and LOA, supporting high-throughput genotype-phenotype mapping. By applying HDR-enhancing agents, we further show that the reporter sensitively captures shifts in LOA outcomes that are missed by other bulk assays. This cell model provides a valuable tool for dissecting gene editing outcomes due to different DNA repair pathways and quantitatively linking editing genotypes to erythroid phenotypes and thus can be used to evaluate the safety of CRISPR/Cas9-based therapies.
Saito S, Kato S, Arai U
… +2 more, Tateishi K, Adachi N
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41736885
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DNA mismatch repair (MMR) plays a crucial role in maintaining the integrity of the human genome, and impairments in this DNA repair mechanism are responsible for more than 10% of all malignant tumors. Thus, developing a...DNA mismatch repair (MMR) plays a crucial role in maintaining the integrity of the human genome, and impairments in this DNA repair mechanism are responsible for more than 10% of all malignant tumors. Thus, developing a strategy for stage-independent killing of MMR-deficient cells is of great significance in cancer therapy. Here, we show that transient transfection of artificial DNA constructs results in efficient killing of MMR-deficient cancer cells. These constructs were designed so that an intact suicide gene is formed and expressed in the cells, depending on a functional defect in MMR that inhibits single-strand annealing-mediated DNA recombination. anticancer activity was confirmed in HCT116 xenograft tumor models. Our findings suggest that a cellular DNA recombination-dependent suicide gene can provide a novel tool to selectively kill MMR-deficient tumors, including those resistant to immunotherapy.
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41732207
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Alzheimer's disease (AD) is characterized by overlapping pathological processes, including amyloid-beta (Aβ) accumulation, tau hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation. Monogenic therapies h...Alzheimer's disease (AD) is characterized by overlapping pathological processes, including amyloid-beta (Aβ) accumulation, tau hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation. Monogenic therapies have shown limited benefits, and only in a subset of patients, as other pathological processes continue to drive disease progression. Given the multifactorial and heterogeneous nature of AD, therapeutics targeting more than one gene simultaneously represent a promising strategy to achieve broader therapeutic outcomes. This study highlights the advantages of multigene RNA-based therapeutics, which may overcome compensatory mechanisms and patient heterogeneity. Here, we report the design and functional validation of antisense oligonucleotides (ASOs) specifically engineered for simultaneous silencing of more than one AD-related gene. Using algorithm-assisted sequence design, we generated 11 bispecific gapmer ASOs from 20 candidate genes. In human and mouse cellular models, these ASOs achieved potent and sustained knockdown with picomolar to low-nanomolar IC values. Functionally, treatment led to significant reductions in Aβ42 production, up to 70%, while maintaining favorable safety and specificity profiles. Collectively, our findings establish a proof of concept for multigene silencing in AD, demonstrating that rationally designed ASOs can provide robust target suppression across key pathological pathways. This strategy introduces a new paradigm in oligonucleotide design, with the potential to deliver disease-modifying benefits for patients with AD.
Shkreta L, Delannoy A, Toutant J
… +2 more, McSwiggen J, Chabot B
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41732205
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RNA polymerase III-related disorders represent a clinically diverse spectrum of diseases. While pathogenic variants in the subunit are most commonly associated with hypomyelinating leukodystrophies, they also contribute...RNA polymerase III-related disorders represent a clinically diverse spectrum of diseases. While pathogenic variants in the subunit are most commonly associated with hypomyelinating leukodystrophies, they also contribute to other neurological or systemic deficiencies. Despite this phenotypic variability, many disease-causing variants converge mechanistically on altered RNA expression that can impair oligodendrocyte maturation and their myelination capacity. Using CRISPR-dCas13Rx, we investigated whether regions containing disease-causing missense mutations in affect RNA splicing in EcR293 and oligodendroglioma cell lines. Our analysis indicates that 20% of the interrogated exon regions of harboring disease-causing missense mutations induce significant splicing changes when targeted by an interfering guide RNA (gRNA)/dCas13Rx complex. Minigene assays confirmed that mutations residing in these regions caused splicing aberrations. We then used the CRISPR-dCas13Rx system to identify intron elements that, when targeted with gRNAs, alleviated the splicing defect caused by missense mutations in exon 14 and exon 26. Antisense oligonucleotides (ASOs) derived from these active gRNAs also improved correct splicing in EcR293 and oligodendroglioma cell lines. These findings not only highlight the therapeutic promise of gRNAs and ASOs in rescuing splicing defects caused by missense mutations but also establish CRISPR-dCas13Rx as a useful tool for identifying and correcting disease-causing splicing aberrations.
Ciancaglini M, Avanthay R, Marx AF
… +11 more, Abreu-Mota T, Finozzi D, Fixemer J, Geier F, Burri D, Wagner I, Vincenti I, Kreuzfeldt M, Merkler D, Zimmer G, Pinschewer DD
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41732204
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Replication-deficient viral vector systems hold promise for CD8 T cell-based vaccination, but the molecular mechanisms accounting for platform-specific differences in immunogenicity remain ill-defined. When comparing pro...Replication-deficient viral vector systems hold promise for CD8 T cell-based vaccination, but the molecular mechanisms accounting for platform-specific differences in immunogenicity remain ill-defined. When comparing prototypic single-cycle vaccine delivery platforms, we found that lymphocytic choriomeningitis virus (rLCMV)-based vectors, which are non-cytolytic, elicited more durable and effector-memory-differentiated CD8 T cell responses than vectors based on cytolytic vesicular stomatitis virus (rVSV). Hence, we re-engineered rVSV to be non-cytolytic (rVSVMq). This vector induced more durable and effector-differentiated CD8 T cell memory than the parental rVSV, and it afforded superior protection against challenge. Improved CD8 T cell responses of non-cytolytic rVSVMq were driven by a reinforced type I interferon (IFN-I) response and its direct sensing by vaccination-induced CD8 T cells. Many vector cargo-specific CD8 T cells in the splenic marginal zone of rVSVMq- or rLCMV-vaccinated mice were in contact with vector cargo-expressing cells that co-expressed IFN-I. In contrast, rVSV-vectored cargo-expressing contacts of specific CD8 T cells were largely IFN I-negative. Thereby, vaccination with non-cytolytic viral vectors offered an opportunity for CD8 T cells to integrate peptide-MHC (major histocompatibility complex) and IFN-I signals during priming. These mechanistic insights should help to refine vaccines aimed at eliciting durable and protective effector-memory CD8 T cell immunity.
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41726103
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This study investigated the potential of eccentric training in attenuating the deleterious effects of Marfan syndrome on skeletal muscles. Marfan syndrome and wild-type mice were divided into a trained and a control grou...This study investigated the potential of eccentric training in attenuating the deleterious effects of Marfan syndrome on skeletal muscles. Marfan syndrome and wild-type mice were divided into a trained and a control group; the former performed a downhill running protocol for 8 weeks. Morphological, molecular, and functional analyses were performed. Based on RNA sequencing analyses, the gastrocnemius and plantaris muscles were transfected with -shRNA (short hairpin RNA) and analyzed after 21 days. The muscle wet weight normalized to body weight and the percentage of myofibers with a higher cross-sectional area were lower in the muscles of Marfan syndrome mice than in wild-type mice. Marfan syndrome mice showed increased fibrosis and number of rounded myofibers, whereas eccentric training reduced these effects. Muscle-specific force was unaltered across groups. In addition, Marfan syndrome mice presented an increase in the number of CD11b myeloid cells and the mRNA levels of calcium-binding proteins and and cytokines , and in muscles, while eccentric training reversed these effects. The knockdown of induced by -shRNA transfection decreased muscle fibrosis in Marfan syndrome mice. In summary, eccentric training and knockdown of are beneficial to reducing fibrosis in muscles affected by Marfan syndrome.
He X, Zeng R, Wen S
… +9 more, Wen Z, Li H, Ai H, Gao R, Fan L, Zhou L, Shi G, Chen Y, Li S
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41717290
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Abnormal angiogenesis is the leading cause of vision loss globally, but current anti-angiogenic treatments are unsatisfactory and incompetent. Thus, novel therapies targeting angiogenesis are urgently needed. Previously,...Abnormal angiogenesis is the leading cause of vision loss globally, but current anti-angiogenic treatments are unsatisfactory and incompetent. Thus, novel therapies targeting angiogenesis are urgently needed. Previously, we revealed a positive association between cGAS-STING signaling and angiogenic factors in the mouse model with ischemic retinopathy. However, whether cGAS-STING signaling regulates retinal angiogenesis remained largely unknown. Here, we analyzed single-cell RNA sequencing databases from the epiretinal fibrovascular membranes, developing mouse retinas, and normal adult retinas from , , and . Notably, we observed spatially and temporally identical expression patterns of cGAS-STING signaling and angiogenesis. In particular, cGAS-STING signaling showed the strongest correlation with angiogenesis in retinal endothelia from mice at postnatal days 3 and 6. Endothelia-specific knockout of in mice retarded retinal vascular growth, which was due to attenuation of VEGFA-VEGFR2 signaling as suggested by bulk RNA sequencing. In human retinal vascular endothelial cells, deletion of prohibited VEGFR2 activation, down-regulated the levels of endothelial markers, and compromised endothelial proliferation and migration, which were counteracted by overexpression of . This study demonstrated an evolutionally conserved interaction between cGAS-STING signaling and VEGFA-VEGFR2 signaling in pathophysiological angiogenesis and vascular homeostasis, thus providing a novel therapeutic target for treating retinal vascular diseases.
Chatterjee P, Stevens HY, Kippner LE
… +6 more, Yeago C, Drissi H, Mautner K, Boden SD, Gibson G, Roy K
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41717289
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Knee-osteoarthritis (knee OA) is a prevalent joint disorder lacking Food and Drug Administration-approved cell therapies to halt progression. This study uses single-cell RNA sequencing to analyze bone marrow aspirate con...Knee-osteoarthritis (knee OA) is a prevalent joint disorder lacking Food and Drug Administration-approved cell therapies to halt progression. This study uses single-cell RNA sequencing to analyze bone marrow aspirate concentrate (BMAC) and stromal vascular fraction (SVF) samples in a clinical trial of autologous cell therapies. Trial site-specific variability was significant in BMAC, necessitating tailored normalization, whereas SVF was less affected, likely due to uniform subcutaneous fat sampling. Variance partitioning and tensor decomposition identified site effects in BMAC but revealed shared pathways across cell types in both tissues. Differential gene expression (DEG) analysis between responders and non-responders yielded no significant findings, although likelihood ratio test (LRT) revealed enrichment for DEG patterns linked to disease severity, potentially masked by patient heterogeneity. Key BMAC pathways included oxidative phosphorylation, unfolded protein response, and tumor necrosis factor alpha () signaling. Cell-cell communication analysis suggested enhanced human leukocyte antigen (HLA) signaling in non-responder MSCs (mesenchymal stromal cells), consistgenesent with inflammation, while responders showed more coordinated immune interactions. BMAC-MSCs promoted chondrocyte proliferation, whereas SVF-MSCs emphasized immune regulation. This study suggests that variability in therapy outcomes reflects patient heterogeneity beyond genomic factors, complicating the immediate use of genomic profiling to guide treatment. Nonetheless, as molecular pathways become better understood, integrating genomic insights into personalized strategies may become feasible.
Kliuchnikov E, Pierson D, Chou Y
… +4 more, DiLuzio W, Marx KA, Chanda A, Barsegov V
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41717288
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Elucidating the structure-function relationships of phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to limited structural data. We combined surface tension and circular dichroism (CD) spectroscop...Elucidating the structure-function relationships of phosphorodiamidate morpholino oligonucleotides (PMOs) is challenging due to limited structural data. We combined surface tension and circular dichroism (CD) spectroscopy with molecular dynamics simulations to investigate how two different PMO molecules interact with Polysorbate 80 and Polysorbate 20. In simulations of 1:1 stoichiometry complexes, we observed strong, staged, concentration-dependent PMO-surfactant interactions, with interaction energies of -60 to -80 kcal/mol for 25-mer conformers and -50 to -70 kcal/mol for 30-mer conformers. Surfactants primarily associate through surface binding but can also insert into PMO structures, preventing unfolding. Structural analysis revealed that position-dependent contacts, especially with hydrophobic surfactant tails, drive these interactions. Importantly, PMO-surfactant binding does not disrupt base pairing, base stacking, or overall chirality, consistent with CD spectra, though it slightly enlarges the tertiary structure. Surfactants reduce solvent exposure of PMO surfaces within complexes, decreasing intermolecular interactions, yet the overall PMO-surfactant complex remains more solvent-exposed. Occasionally, surfactants act in a "chaperone-like" manner, enabling refolding into more compact structures. Together, these findings highlight how surfactants stabilize PMO conformers without disrupting their essential structure. This improved understanding of PMO-surfactant interactions broadens insight into PMO physicochemical behavior and supports the rational design of RNA-mimic therapeutics.
Facal CL, Páez-Paz I, Pereyra AE
… +5 more, Gaguine C, Clerici-Delville R, Foltran R, Soiza-Reilly M, Avale ME
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41717287
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Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease, frontotemporal dementia, and progressive supranuclear palsy, characterized by the pathological accumulation of tau protein. While tau...Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease, frontotemporal dementia, and progressive supranuclear palsy, characterized by the pathological accumulation of tau protein. While tau reduction has emerged as a promising disease-modifying strategy, most preclinical studies have focused on preventive approaches, and the therapeutic potential after clinical onset remains largely unexplored. This limitation is critical, as patients are typically diagnosed after symptoms emerge. Furthermore, global tau suppression may disrupt physiological tau functions and lead to adverse effects, underscoring the need for targeted interventions. In this sense, RNA interference (RNAi)-mediated therapies using viral vectors offer high specificity, regional and cell-specific expression, and sustained target knockdown. We have engineered artificial microRNAs (Tau-miRNAs) to selectively reduce tau in vulnerable brain regions, minimizing off-target effects. Here, we tested the efficacy of these Tau-miRNAs in a tauopathy mouse model at advanced disease stages, delivering them into the prefrontal cortex after cognitive and electrophysiological deficit onset. This post-symptomatic intervention led to long-term improvements in memory, restoration of neuronal firing properties, and reduced pathological tau at synapses. Our findings highlight the potential of spatially targeted RNA-based tau-lowering strategies for late-stage intervention in tauopathies, addressing a critical unmet need in the treatment of these devastating disorders.
Du L, Billaud JN, Telwatte S
… +6 more, Kadiyala N, Dabral P, Bouzidi MS, Guatelli J, Yukl SA, Pillai SK
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41717286
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Understanding the mechanisms underlying interferon-alpha (IFNα) anti-human immunodeficiency virus type I (HIV-1) effects and HIV-1 innate immune evasion is critical to developing improved antiviral strategies. We previou...Understanding the mechanisms underlying interferon-alpha (IFNα) anti-human immunodeficiency virus type I (HIV-1) effects and HIV-1 innate immune evasion is critical to developing improved antiviral strategies. We previously reported that the microRNA (miRNA) miR-422a was the sole miRNA downregulated by IFNα treatment in people living with HIV (PLWH), and the extent of miR-422a reduction was correlated with viral load reduction. Here, we investigated the molecular basis of this relationship by examining the role of miR-422a in HIV replication and innate immune evasion. We observed that HIV-1 infection induces miR-422a expression in primary CD4+ T cells via the viral Nef accessory protein. miR-422a enhanced HIV-1 replication in CD4+ T cells by directly targeting the host factor methyl CpG binding protein 2 (MECP2). Transcriptomic analysis revealed that miR-422a depletion mimicked IFNα exposure, inducing the expression of several IFN-stimulated genes (ISGs) that restrict HIV-1 replication. Finally, we demonstrated that miR-422a overexpression or CRISPR-Cas9-mediated MECP2 knockout counteracts IFNα antiviral capacity and rescues HIV-1 replication. Together, our findings reveal that miR-422a is a key host factor induced by HIV-1 infection that supports viral replication and subverts type I IFN-mediated antiviral responses via targeting of MECP2. Virologic control in PLWH may be achieved by pharmacologic manipulation of the Nef-miR-422a-IFNα axis.
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41696513
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Nucleic acid lipid nanoparticle (LNP) technology has enabled the delivery of genetic medicines to solid tumors, opening new avenues for oncology therapeutics. Solid tumors present unique challenges to nucleic acid delive...Nucleic acid lipid nanoparticle (LNP) technology has enabled the delivery of genetic medicines to solid tumors, opening new avenues for oncology therapeutics. Solid tumors present unique challenges to nucleic acid delivery because of their complex tumor microenvironment (TME), which acts as a barrier to NP delivery. Nevertheless, nucleic acid LNPs carry diverse genetic medicine modalities that can exert anti-cancer effects. The versatility of LNPs allows both local and systemic administrations for the delivery of gene therapy payloads to solid cancer, with the additional capability to selectively target specific cell types through conjugation of targeting ligands onto the LNPs. Genetic medicines delivered by LNPs can directly affect cancer cells, such as by suppressing oncogenic drivers and cancer pathways with small non-coding RNAs, or through overexpression of toxin genes or tumor suppressors. Non-cancer components of the tumor, such as tumor-associated vasculature, can also be targeted and disrupted to inhibit the structures that support tumor growth. Alternatively, LNP delivery of genetic medicines can indirectly elicit anti-tumor effects by modifying the immune state of the tumor environment through delivery of immunomodulatory cytokines, overexpression of activatable receptors to stimulate immune cells with agonists, or antigen-binding scaffolds to direct immunity towards a cancer cell. In this review, we summarize recent advances, challenges, and prospects of LNP delivery of genetic medicines to treat solid tumors.
Kaneta H, Nakasa T, Yimiti D
… +5 more, Moriwaki D, Kawasaki R, Ogura T, Miyaki S, Adachi N
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41674725
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Ginger-derived extracellular vesicles (GDEVs) have emerged as a novel anti-inflammatory agent with advantages such as oral bioavailability, natural origin, and cost-effective large-scale production. This study evaluated...Ginger-derived extracellular vesicles (GDEVs) have emerged as a novel anti-inflammatory agent with advantages such as oral bioavailability, natural origin, and cost-effective large-scale production. This study evaluated the therapeutic potential of GDEVs in rheumatoid arthritis (RA), a chronic autoimmune disease characterized by synovial inflammation and joint destruction. We conducted both and experiments using synovial fibroblasts derived from RA patients and a collagen antibody-induced arthritis (CAIA) mouse model. , GDEVs significantly suppressed the expression of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β and downstream mediators IL-6, Cox-2, and matrix metalloproteinase 3 (MMP3) and inhibited the proliferation and migration of RA synovial fibroblasts. , oral administration of GDEVs to CAIA mice reduced arthritis severity, attenuated synovitis, preserved cartilage integrity, and suppressed osteoclast activation. GDEVs were stable against gastric digestion and were efficiently taken up by intestinal cells, supporting their oral availability. Microarray and RNA sequencing identified miR-149 as a key regulatory molecule in GDEVs, associated with the suppression of inflammation-related signaling pathways, including Ras signaling and mitogen-activated protein kinase (MAPK) cascades. These findings highlight the potential of GDEVs as an anti-inflammatory therapy for RA. Given their stability and bioavailability, the oral administration of GDEVs could be a promising non-invasive treatment for future clinical applications.