Search Results (663)

Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-α and nucleoside/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently. Full article

Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein primarily found in milk, saliva, tears, and nasal secretions. LF is well known for its antibacterial and immunomodulatory effects. However, the extraction of LF from milk is inadequate for large-scale therapeutic applications, presenting a challenge for economic mass production. Recombinant protein expression systems offer a solution to overcome this challenge and efficient production of LF. This review discusses recent progress in the translational research of LF gene transfer and biopharming, focusing on different expression systems such as bacteria, yeast, filamentous fungi, transgenic crops, and animals as well as purification methods. The optimization of expression yields, prospects for genetic engineering, and biotechnology to enhance LF production for biomedical applications are emphasized. This review systematically sourced the literature from 1987 to 2025 from leading scientific databases, including PubMed, Scopus, Web of Science, and Google Scholar. Despite ongoing debates, progress in this field indicates a viable path towards the effective use of LF in therapeutic settings. Full article

Using the fly eye as a model system, we previously demonstrated that upregulation of the fly gene mask protects against FUS- and Tau-induced photoreceptor degeneration. Building upon this finding, we investigated whether the protective role of mask is conserved in mammals. To this end, we generated a transgenic mouse line carrying Cre-inducible ANKHD1, the human homolog of mask. Utilizing the TauP301S-PS19 mouse model for Tau-related dementia, we found that expressing ANKHD1 driven by CamK2a-Cre reduced hyperphosphorylated human Tau in 6-month-old mice. Additionally, ANKHD1 expression was associated with a trend toward reduced gliosis and preservation of the presynaptic marker Synaptophysin, suggesting a protective role of ANKHD1 against TauP301S-linked neuropathology. At 9 months of age, novel object recognition (NOR) testing revealed cognitive impairment in female, but not male, PS19 mice. Notably, co-expression of ANKHD1 restored cognitive performance in the affected female mice. Together, this study highlights the novel effect of ANKHD1 in counteracting the adverse effects induced by the mutant human Tau protein. This finding underscores ANKHD1’s potential as a unique therapeutic target for tauopathies. Full article

Gene replacement using adeno-associated viral (AAV) vectors has become a major therapeutic avenue for neurodegenerative diseases (NDD). In single-gene diseases with loss-of-function mutations, the objective of gene therapy is to express therapeutic transgenes abundantly in cell populations that are implicated in the pathological phenotype. X-ALD is one of these orphan diseases. It is caused by ABCD1 gene mutations and its main clinical form is adreno-myelo-neuropathy (AMN), a disabling spinal cord axonopathy starting in middle-aged adults. Unfortunately, the main cell types involved are yet poorly identified, complicating the choice of cells to be targeted by AAV vectors. Pioneering gene therapy studies were performed in the Abcd1^-/y mouse model of AMN with AAV9 capsids carrying the ABCD1 gene. These studies tested ubiquitous or cell-specific promoters, various routes of vector injection, and different ages at intervention to either prevent or reverse the disease. The expression of one of these vectors was studied in the spinal cord of a healthy primate. In summary, gene therapy has made promising progress in the Abcd1^-/y mouse model, inaugurating gene replacement strategies in AMN patients. Because X-ALD is screened neonatally in a growing number of countries, gene therapy might be applied in the future to patients before they become overtly symptomatic. Full article

Objectives: To investigate the efficacy and underlying mechanisms of IBCar’s biological activity in breast cancer models, both in cell culture and in mice, and to compare its effects on cancer versus normal cells. Methods: The cytotoxicity of IBCar was evaluated using the MTS assay to assess metabolic activity and the clonogenic assay to determine reproductive integrity. The impact of IBCar on microtubule integrity, mitochondrial function, and multiple signaling pathways was analyzed using Western blotting, microarray analysis, and live cell imaging. The therapeutic effectiveness of orally administered IBCar was assessed in a transgenic mouse model of Luminal B breast cancer and in mice implanted with subcutaneous triple-negative breast cancer xenografts. Results: IBCar demonstrated potent cytotoxicity across a diverse panel of breast cancer cell lines, including those with mutant or wild-type TP53, and cell lines with short and long doubling times. Comparative analysis revealed distinct responses between normal and cancer cells, including differences in IBCar’s effects on the mitochondrial membrane potential, endoplasmic reticulum stress and activation of cell death pathways. In breast cancer cells, IBCar was cytotoxic at nanomolar concentrations, caused irreversible microtubule depolymerization leading to sustained mitochondrial dysfunction, endoplasmic reticulum stress, and induced apoptosis. In normal cells, protective mechanisms included reversible microtubule depolymerization and activation of pro-survival signaling via the caspase-8 and riptosome pathways. The therapeutic potential of IBCar was confirmed in mouse models of Luminal B and triple negative BC, where it exhibited strong antitumor activity without detectable toxicity. Conclusions: These findings collectively support IBCar as a promising, effective, and safe therapeutic candidate for breast cancer treatment. Full article

Proper subcellular localization is essential to exert the designated function of a protein, not only for endogenous proteins but also transgene-encoded proteins. Post-translational modification is a frequently used method to regulate the subcellular localization of a specific protein. While there are a number of tags that are widely used to direct the target protein to a specific location within a cell, these tags often fail to emulate the dynamics of protein trafficking, necessitating an alternative approach to the direct subcellular localization of transgene-encoded proteins. Here, we report the development of a new synthetic polypeptide protein tag comprised of ten amino acids, which promotes membrane localization of a target protein. This short synthetic peptide tag, named “Palmito-Tag”, induces ectopic palmitoylation on the cysteine residue within the tag, thereby promoting membrane localization of the target proteins without affecting their innate function. We show that the target proteins with the Palmito-Tag are incorporated into the membranous organelles within the cells, including the endosomes, as well as extracellular vesicles. Given the reversible nature of palmitoylation, the Palmito-Tag may allow us to shift the subcellular localization of the target protein in a context-dependent manner. With the advent of therapeutic applications of exosomes and other extracellular vesicles, we believe that the ability to reversibly modify a target protein and direct its deposition to the specific subcellular milieu will help us explore more effective venues to harness the potential of extracellular vesicle-based therapies. Full article

Background: Lipid nanoparticles (LNPs) represent one of the most effective non-viral vectors for nucleic acid delivery and have demonstrated clinical success in siRNA therapies and mRNA vaccines. While considerable research has focused on optimizing ionizable lipids and helper lipids, the impact of PEGylated lipid content on LNP-mediated mRNA delivery, especially in terms of in vitro transfection efficiency and in vivo performance, remains insufficiently understood. Methods: In this study, LNPs were formulated using a self-synthesized ionizable lipid and varying molar ratios of DMG-PEG2000. Nanoparticles were prepared via nanoprecipitation, and their physicochemical properties, mRNA encapsulation efficiency, cellular uptake, and transfection efficiency were evaluated in HeLa and DC2.4 cells. In vivo delivery efficiency and organ distribution were assessed in mice following intravenous administration. Results: The PEGylated lipid content exerted a significant influence on both the in vitro and in vivo performance of LNPs. A bell-shaped relationship between PEG content and transfection efficiency was observed: 1.5% DMG-PEG2000 yielded optimal mRNA transfection in vitro, while 5% DMG-PEG2000 resulted in the highest transgene expression in vivo. This discrepancy in optimal PEG content may be attributed to the trade-off between cellular uptake and systemic circulation: lower PEG levels enhance cellular internalization, whereas higher PEG levels improve stability and in vivo bioavailability at the expense of cellular entry. Furthermore, varying the PEG-lipid content enabled the partial modulation of organ distribution, offering a formulation-based strategy to influence biodistribution without altering the ionizable lipid structure. Conclusions: This study highlights the critical role of PEGylated lipid content in balancing nanoparticle stability, cellular uptake, and in vivo delivery performance. Our findings provide valuable mechanistic insights and suggest a straightforward formulation-based strategy to optimize LNP/mRNA systems for therapeutic applications. Full article

Despite the prevalence of fucosylated IgG in plasma, specific IgGs with low core fucosylation sporadically emerge in response to virus infections and blood cell alloantigens. This low fucosylation of IgG is implicated in the pathogenesis of SARS-CoV-2 and dengue infections. In COVID-19, the presence of IgGs with low core fucosylation (afucosylated IgGs) targeting spike protein predicts disease progression to a severe form and actively mediates this progression. This study reveals that SARS-CoV-2 infection of megakaryocytes promotes the generation of pathogenic afucosylated anti-spike IgGs, leading to outcomes, such as pulmonary vascular thrombosis, acute lung injury, and mortality in FcγRIIa-transgenic mice. Platelets from mice injected with virus-infected human megakaryocytes express significant activation biomarkers, indicating a direct link between the immune response and platelet activation. Mice injected with virus-infected human megakaryocytes demonstrate an elevated rate of thrombus formation induced by FeCl₃ (4%) and a reduction in bleeding time, emphasizing the intricate interplay of viral infection, immune response, and hemostatic complications. Treatment with inhibitors targeting FcγRIIa, serotonin, or complement anaphylatoxins of mice injected with spike-expressing MKs successfully prevents observed platelet activation, thrombus formation, and bleeding abnormalities, offering potential therapeutic strategies for managing severe outcomes associated with afucosylated IgGs in COVID-19 and related disorders. Full article

Blood–brain barrier (BBB) dysfunction is a hallmark of cerebral small vessel disease (cSVD). This study aimed to identify a mouse model that replicates BBB impairment and shares key cSVD risk factors. Transgenic db/db and LDLr^−/−.Leiden mice, both prone to obesity and hypertension, were compared to C57BL/6J controls. BBB leakage was assessed using DCE-MRI and sodium fluorescein (NaFl); cerebral blood flow (CBF) by MRI. Dyslipidemia and vascular inflammation were measured by plasma tests. Tight junction integrity, endothelial dysfunction (glucose transporter 1, GLUT-1) and neuroinflammation were evaluated with immunohistochemistry and PCR. Both transgenic models developed an obese phenotype with hyperinsulinemia, but only LDLr^−/−.Leiden mice showed human-like dyslipidemia. When fed a high-fat diet (HFD) or HFD plus cholesterol, LDLr^−/−.Leiden mice showed reduced CBF, endothelial dysfunction (lowered GLUT-1), elevated vascular inflammation (ICAM-1, VCAM-1, S-selectin), and BBB leakage, as evidenced by DCE-MRI and NaFl, together with reduced ZO-1 and claudin-5 expression. Contrastingly, db/db mice showed endothelial dysfunction without BBB leakage. Neuroinflammation (IBA-1, GFAP) was observed only in LDLr^−/−.Leiden groups, consistent with BBB disruption. These findings indicate that LDLr^−/−.Leiden mice, but not db/db mice, are a promising translational model for studying BBB dysfunction in cSVD, offering insights into disease mechanisms and a platform for therapeutic development. Full article

Background: Esophageal squamous cell carcinoma (ESCC) is a fatal malignant tumor. Several studies have demonstrated that immune checkpoint inhibitors can provide clinical benefits to patients with ESCC. However, the single-agent efficacy of these agents remains limited. Although combination therapies (e.g., radiotherapy, chemotherapy) can help to overcome immunotherapy resistance in ESCC, their severe side effects limit clinical application. This study aimed to explore new resistance mechanisms to immunotherapy in ESCC and identify novel molecular targets to overcome immunotherapy resistance. Methods: We employed immunohistochemistry staining to examine the p-FGFR1^Y654 in tumor samples obtained from 103 patients with ESCC, in addition to evaluating CD8+ T cell infiltration. In vitro expression, western blotting, CCK-8, 5-bromo-2′-deoxyuridine incorporation assays, and migration assays were used to confirm the impact of AZD4547 on p-FGFR1^Y654 expression and the proliferation and migration in ESCC cell lines. Through RNA sequencing analysis, databases such as the Cancer Genome Atlas (TCGA) and Gene Set Cancer Analysis (GSCA), and the reconstruction of transgenic mice using the humanized immune system, we validated the correlation between the expression of p-FGFR1^Y654 and CD8+ T cell infiltration. We also explored how p-FGFR1^Y654 recruits myeloid-derived suppressor cells (MDSCs) through the CXCL8–CXCR2 axis to suppress the therapeutic efficacy of immunotherapy in ESCC. Finally, the tumor-suppressive effects of AZD4547 combined with immunotherapy were confirmed in vivo in tumor-bearing mice with a humanized immune system. Results: We found that the inhibition of p-FGFR1^Y654 expression in ESCC can enhance CD8+ T cell infiltration by suppressing the CXCL8-–XCR2 recruitment of MDSCs. AZD4547, combined with immunotherapy, further promotes immunotherapeutic efficacy in ESCC. Conclusions: In conclusion, our study presents a promising model for combination therapy in ESCC immunotherapy. Full article

Astrocyte activation and gender differences play critical roles in the prognosis following stroke. Recent studies have shown that optogenetic technology can promote brain repair after stroke by activating astrocytes in male rats. However, it remains unclear whether gender differences influence the efficacy of optogenetic activation of astrocytes in regulating post-stroke brain repair and its underlying mechanisms. In this study, we activated astrocytes in the ipsilateral cortex of adult glial fibrillary acidic protein-channelrhodopsin 2-enhanced yellow fluorescent protein (GFAP-ChR2-EYFP) transgenic Sprague Dawley rats using optogenetic stimulation at 24, 36, 48, and 60 h after inducing photothrombosis stroke. Neurobehavioral tests, cresyl violet staining, RT-qPCR, Western blot, and immunofluorescence analysis were performed on both female and male rats. Our results showed that male rats exhibited significant improvements in behavioral scores and reduction in infarct size after optogenetic activation of astrocytes at three days post-stroke (p < 0.05), whereas no significant changes were observed in female rats. Additionally, in female rats, the expression of basic fibroblast growth factor (bFGF) increased after ischemic stroke and astrocytic optogenetic stimulation (p < 0.05), leading to enhanced endothelial cell proliferation compared to male rats (p < 0.05). In vitro experiments further demonstrated that the astrocyte activation was inhibited in the presence of bFGF (p < 0.05). These findings suggest that the increase in bFGF levels in females following stroke may inhibit the optogenetic activation of astrocytes, thereby attenuating the therapeutic effect of astrocyte activation on post-stroke brain repair. This study provides important insights into the gender-specific roles of astrocytes in the acute phase of ischemic stroke. Full article

Osteoarthritis (OA) is a prevalent and debilitating joint disease in older adults with a complex etiology. We investigated the role of SUMOylation, a post-translational modification, in OA pathogenesis, focusing on the mitochondrial chaperone Prohibitin (PHB1) and the cartilage homeostasis transcription factor PITX1. We hypothesized that oxidative stress-induced SUMOylation promotes PHB1 nuclear accumulation, leading to PITX1 downregulation and contributing to OA development. Analysis of cartilage specimens from 27 OA patients and 4 healthy controls revealed an increased nuclear accumulation of PHB1 in OA chondrocytes, accompanied by elevated levels of SUMO-1 and SUMO-2/3. Mechanistically, nuclear PHB1 interacted indirectly with SUMO-1 through a SUMO-interacting motif (SIM), and the deletion of this SIM prevented PHB1 nuclear trapping in OA cells. Furthermore, the SUMO-conjugating enzyme E2 (UBC9) encoded by the UBE2I gene was upregulated in knee OA cartilage, and its overexpression in vitro enhanced PHB1 nuclear accumulation. Consistently, transgenic mice overexpressing the Ube2i gene exhibited increased UBC9 in their knee cartilage, resulting in Pitx1 downregulation and the emergence of an early OA-like phenotype in articular chondrocytes. Our findings uncover a novel role for UBC9-mediated SUMOylation in primary knee and hip OA. This pathway enhances PHB1 nuclear accumulation, contributing to PITX1 repression and subsequent OA development. These results underscore the importance of SUMOylation in OA pathogenesis and suggest potential molecular targets for early diagnosis and therapeutic intervention. Full article

Among the molecules implicated in inflammation-associated tumorigenesis, Chitinase 3-like 1 (CHI3L1/YKL-40/Brp-39) has emerged as a particularly compelling target due to its multifaced roles in immune regulation, tissue remodeling, and cancer progression. Elevated CHI3L1 expression is observed in various human cancers and corresponding animal models. CHI3L1 directly promotes tumor cell proliferation and angiogenesis and also contributes to immune evasion by establishing an immunosuppressive environment in inflamed tissues. Mechanistically, CHI3L1 exerts its effects through the modulation of STAT3, MAPK, and PI3K/Akt signaling pathways and by interacting with cell surface receptors, such as IL-13Rα2 and RAGE. Studies using transgenic and knockout mouse models have revealed a strong association between CHI3L1 expression and cancer progression. In models of colon and lung cancer, CHI3L1 overexpression correlates with increased tumor size and number, whereas CHI3L1 deficiency markedly suppresses tumor formation. However, its involvement appears to be context-dependent and varies among different epithelial tumor types. These findings suggest that CHI3L1 is a potential therapeutic target and diagnostic biomarker for inflammation-associated cancers. Animal studies provide valuable insights into the immunological mechanisms of CHI3L1-mediated tumorigenesis but also highlight the need for cautious interpretation due to inherent technical limitations. Full article

This review critically examines the evolving landscape of genetic animal models for investigating cardiovascular diseases (CVDs). We analyze established models, including spontaneously hypertensive rats, Watanabe hyperlipidemic rabbits, etc., and transgenic models that have advanced our understanding of essential and secondary hypertension, atherosclerosis, and non-ischemic diseases of the heart. This review systematically evaluates the translational strengths and physiological limitations of these approaches across species barriers. Particular attention is paid to emerging technologies—AAV-mediated gene delivery, CRISPR-Cas9 editing, and chemogenetic tools—that enable unprecedented precision in manipulating cardiac-specific gene expression to study pathophysiological mechanisms. We address persistent challenges including off-target effects and transgene expression variability, while highlighting innovations in synthetic vectors and tissue-specific targeting strategies. This synthesis underscores how evolving genetic technologies are revolutionizing cardiovascular research paradigms, offering refined disease models and optimized therapeutic interventions that pave the way toward personalized medicine approaches for the world’s leading cause of mortality. Full article

Alzheimer’s disease (AD) is a chronic and complex neurodegenerative disorder characterized by progressive cognitive decline, memory loss, and irreversible impairment of brain functions. The etiology of AD is multifactorial, involving a complex interplay of genetic, environmental, and physiological factors, including the aggregation of amyloid-β (Aβ) and oxidative stress (OS). The role of OS in AD pathogenesis is of particular significance, given that an imbalance between oxidants and antioxidants promotes cellular damage, exacerbates Aβ deposition, and leads to cognitive deterioration. Despite extensive research, current therapeutic strategies have largely failed, likely due to the use of single-target drugs unable to halt the multifactorial progression of the disease. In this study, we investigated the synergistic therapeutic effect of plant-derived bioactive compounds Withanone, Apigenin, Bacoside A, Baicalin, and Thymoquinone in combination with N,N-Dimethyltryptamine (NN-DMT), a psychedelic molecule. We used a transgenic Caenorhabditis elegans model to assess the behavioral and molecular outcomes following compound exposure. Motility assays, thioflavin S staining, and survival assays under oxidative stress were employed to evaluate the treatment efficacy. The results of the behavioral and molecular analyses indicated that the combination therapy exhibited a higher efficacy than the monotherapies, leading to a significant reduction in age-related motility defects in the AD model. Furthermore, the combination treatment substantially reduced Aβ plaque burden, enhanced survival following OS insult, and demonstrated a synergistic effect in mitigating AD-related hallmarks. Taken together, these findings support the potential of combining NN-DMT with specific bioactive compounds as a promising multi-target therapeutic approach for AD. Full article