技术资料

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain,HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies,independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro,HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation,cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies,HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens,in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation,cytokine secretion,and cytotoxic activity in vitro compared to either compound alone. In conclusion,HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner,without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade. View Publication

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis,lipid metabolism,insulin sensitivity,and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications,such as pre-eclampsia and gestational diabetes. However,the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs),mimicking placental cytotrophoblasts (CTs),and their differentiation into extravillous trophoblasts (EVTs) as our models,we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation,only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity,whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries,leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally,the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta. View Publication

The hypoxic tumor microenvironment,particularly hypoxia-conditioned cancer-associated fibroblasts (CAFs),drives breast cancer (BC) progression and therapy resistance. However,the molecular mechanisms linking hypoxic CAFs to BC plasticity and chemoresistance remain elusive. Primary CAFs were isolated from high-grade BC tissues (Grade III) and characterized (α-SMA⁺/CD34⁻/pan-CK⁻),with normal fibroblasts (NFs) from reduction mammoplasty as controls. Hypoxic CAF-derived exosomal circSTAT3 stability was validated using RNase R resistance and actinomycin D assays. Exosomes were characterized via transmission electron microscopy (TEM),dynamic light scattering (DLS),and marker profiling (CD63⁺/TSG101⁺/Alix⁺,calnexin⁻). Functional effects of hypoxic CAF exosomes on TNBC cells (MDA-MB-231,SUM159) were assessed through proliferation/migration assays,stemness/epithelial-mesenchymal transition (EMT) marker analysis,and siRNA-mediated circSTAT3 knockdown. Mechanistic studies employed luciferase assays and RNA immunoprecipitation (RIP). Chemoresistance was evaluated by cisplatin half-maximal inhibitory concentration (IC₅₀). In vivo tumor growth and stemness enrichment were analyzed in xenografts. Clinical validation used BC tissues (n = 60) and plasma exosomes from BC patients (n = 40) versus healthy controls (n = 25). Hypoxic CAF-derived exosomes efficiently transferred circSTAT3 to TNBC cells,promoting proliferation,migration,EMT,and stemness marker expression. SiRNA-mediated circSTAT3 knockdown reversed these effects. Mechanistically,circSTAT3 acted as a competitive endogenous RNA (ceRNA),sponging miR-671-5p to derepress NOTCH1. Hypoxic CAF exosomes increased cisplatin IC₅₀ in TNBC cells,while circSTAT3 depletion restored chemosensitivity. In vivo,hypoxic CAF exosomes accelerated tumor growth,enriched CD44⁺/NOTCH1⁺ populations,and elevated circulating exosomal circSTAT3. Clinically,circSTAT3 was significantly upregulated in advanced BC tissues (p < 0.01) and patient plasma exosomes (p < 0.01),correlating with lymph node metastasis. This study identifies a hypoxia-driven feedforward loop wherein CAF-derived exosomal circSTAT3 promotes TNBC stemness and chemoresistance via miR-671-5p/NOTCH1 signaling. CircSTAT3 redefines stromal-tumor crosstalk as a circRNA-driven process and serves as both a circulating non-invasive biomarker and a promising therapeutic target to disrupt stromal-mediated resistance in aggressive TNBC. The online version contains supplementary material available at 10.1186/s12967-025-06794-8. View Publication

Respiratory syncytial virus (RSV) infection in the upper respiratory tract promotes disease progression and transmission,with excessive inflammation contributing to severe lower respiratory tract involvement. This study investigates the immunomodulatory effects of Lactobacillus rhamnosus D3189 on viral kinetics and innate immune responses in well-differentiated nasal epithelial cells (WD-NECs). WD-NECs from healthy adult donors (N = 8) were cultured in vitro,treated with L. rhamnosus D3189,and then infected with RSV (strain RS4) 24 hours later. Viral replication and shedding were assessed via RT-qPCR and plaque assays. Cytotoxicity and epithelial integrity were evaluated using LDH release and transepithelial electrical resistance (TEER). Inflammatory and antiviral responses were investigated using multiplex immunoassays,AlphaLISA,and ELISA. RSV infection induced robust viral replication and shedding,disrupted epithelial barrier integrity,and triggered the release of pro-inflammatory cytokines and type I/III interferons. L. rhamnosus D3189 alone did not induce cytotoxicity or inflammation. While it had no effect on viral replication,TEER,LDH release,or IFN-λ1/3 levels,D3189 significantly enhanced IFN-β production,reduced viral shedding,and attenuated RSV-induced cytokine and chemokine responses. L. rhamnosus D3189 modulates the epithelial immune response to RSV,reducing inflammation and viral shedding without compromising epithelial integrity. These findings support its potential as a novel strategy to limit RSV-associated infection and transmission. View Publication

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide,with a clinical presentation that varies between sexes. In late-stage AMD,choroidal neovascularization (CNV) triggers retinal inflammation and degeneration,processes that are exacerbated by an overactive response of retinal microglial cells. Short-chain fatty acids (SCFAs) have emerged as potential treatments for AMD due to their anti-inflammatory properties. In this study,we investigate the effects of SCFA treatment in a laser-induced CNV mouse model,focusing on sex-dependent differences in disease progression and microglial response. Our findings demonstrate distinct sex-specific patterns in the development of CNV and associated pathological hallmarks. SCFA treatment resulted in a slight increase in density of Iba1 + microglial cells in females at 3 days post-laser (3dpl),while it prevented an increase in males at 7 dpl,with both sexes showing enhanced microglial ramification. The dynamics of microglial density were likely linked to protective effects on CNV lesion,leakage size,and inflammation,which occurred earlier in females and later in males. At transcriptional level,SCFA showed mixed effects,mainly targeting inflammation resolution,mitochondrial support,and neuronal repair in a sex-dependent manner. In vitro,SCFAs reduced microglial phagocytosis of retinal debris,suggesting a potential anti-inflammatory action. This study underscores the importance of considering sex-specific responses in the development of AMD treatments,such as SCFAs,and highlights the need for personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12974-025-03508-1. View Publication

The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST,its efficiency remains low. Here,we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST -positive hPSCs from ~ 5 to ~ 43.7%,providing a robust method for stabilizing XCI in female hPSCs for diverse applications. The online version contains supplementary material available at 10.1186/s13287-025-04501-4. View Publication

Biological mechanisms are inherently dynamic,requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales,making them unsuitable for studying dynamic processes or characterizing essential genes,where chronic depletion can cause cell death. We compare five inducible protein degradation systems—dTAG,HaloPROTAC,IKZF3,and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency,basal degradation,target recovery after ligand washout,and ligand impact. This analysis identifies OsTIR1-based AID 2.0 as the most robust system. However,AID 2.0’s higher degradation efficiency comes with target-specific basal degradation and slower recovery rates. To address these limitations,we employ base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generates several gain-of-function OsTIR1 variants,including S210A,that significantly enhance the overall degron efficiency. The resulting degron system,named AID 2.1,maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout,enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts. Subject terms: Genetic engineering,CRISPR-Cas9 genome editing,Peptides View Publication

Chronic myeloid leukemia (CML) remains a therapeutic challenge,particularly in patients who develop resistance to standard tyrosine kinase inhibitors (TKIs) such as imatinib. Here,we present the first demonstration of the potent anti-leukemic activity of the histone deacetylase (HDAC) inhibitor martinostat in both TKI-sensitive and TKI-resistant CML. Structural and biochemical analyses confirmed the efficient and selective binding of martinostat to HDAC isoenzyme ligand-binding pockets,resulting in histone and tubulin hyperacetylation in both imatinib-sensitive and resistant CML cells,outperforming vorinostat,a clinically used HDAC inhibitor (HDACi). It selectively impaired CML cell proliferation and viability and induced apoptosis across various CML models,including resistant cell models and patient blasts,with minimal toxicity to healthy cells and low developmental toxicity in zebrafish. In addition to its single-agent efficacy,martinostat demonstrated enhanced anticancer effects when combined with imatinib,both in vitro and in vivo,significantly reducing tumor growth in resistant CML xenograft models. Mechanistically,mRNA-seq data showed that martinostat disrupted key survival signaling pathways and amplified apoptotic responses,contributing to its anticancer activity. These findings highlight the potential of martinostat as a selective,low-toxicity HDACi that,combined with TKIs,could provide an effective strategy to overcome drug resistance in CML and improve therapeutic outcomes. The online version contains supplementary material available at 10.1186/s13148-025-01921-0. View Publication

Secondary and tertiary lymphoid structures are a critical target of suppression in many autoimmune disorders,protein replacement therapies,and in transplantation. Although antigen-specific regulatory T cells (Tregs),such as chimeric antigen receptor (CAR) Tregs,generally persist longer and localize to target tissues more effectively than polyclonal Tregs in animal models,their numbers still progressively decline over time. A potential approach to maximize Treg activity in vivo is the expression of chemokine receptors such as CXCR5,which would enable localization of a greater number of engineered cells at sites of antigen presentation. Indeed,CXCR5 expression on follicular T helper cells and follicular Tregs enables migration toward lymph nodes,B cell zones,and tertiary lymphoid structures that appear in chronically inflamed non-lymphoid tissues. In this study,we generated human and murine CXCR5 co-expressing engineered receptor Tregs and tested them in preclinical mouse models of allo-immunity and hemophilia A,respectively. Additionally,we engineered a murine CXCR5 co-expressing clotting factor VIII (FVIII) specific T cell receptor fusion construct epsilon (FVIII TRuCe CXCR5) Treg to suppress anti-drug antibody development in a model of FVIII protein replacement therapy for hemophilia A. In vitro,anti-HLA-A2 CXCR5+ CAR-Tregs showed enhanced migratory and antigen-specific suppressive capacities compared to untransduced Tregs. When injected into an NSG mouse model of HLA-A2+ pancreatic islet transplantation,anti-HLA-A2 CXCR5+ CAR-Tregs maintained a good safety profile allowing for long-term graft survival in contrast to anti-HLA-A2 CXCR5+ conventional CAR-T (Tconv) cells that eliminated the graft. Similarly,FVIII TRuCe CXCR5 Treg demonstrated increased in vivo persistence and suppressive capacity in a murine model of hemophilia A. Collectively,our findings indicate that CXCR5 co-expression is safe and enhances in vivo localization and persistence in target tissues. This strategy can potentially promote targeted tolerance without the risk of off-target effects in multiple disease models. View Publication

CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However,its application to β 0 -thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β 0 -thalassemia/HbE patients. The editing efficiency for the BCL11A site (75–92%) was higher than for the ZBTB7A/LRF site (57–60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors ( BCL11A 26.2 ± 1.4%,ZBTB7A/LRF 27.9 ± 1.5%) and β 0 -thalassemia/HbE cells ( BCL11A 62.7 ± 0.9%,ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A -edited cells but observed at low frequencies in ZBTB7A/LRF -edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β 0 -thalassemia/HbE disease. View Publication

MED12 is a key regulator of transcription and chromatin architecture,essential for normal hematopoiesis. While its dysregulation has been implicated in hematological malignancies,the mechanisms driving its upregulation in acute myeloid leukemia (AML) remain poorly understood. We investigated MED12 expression across AML subgroups by integrating chromatin accessibility profiling,histone modification landscapes,and DNA methylation (DNAm) patterns. Functional assays using DNMT inhibition were performed to dissect the underlying regulatory mechanisms. MED12 shows subtype-specific upregulation in AML compared to hematopoietic stem and progenitor cells,independent of somatic mutations. Chromatin accessibility profiling reveals that the MED12 locus is epigenetically primed in AML blasts,with increased DNase hypersensitivity at regulatory elements. Histone modification analysis demonstrates strong H3K4me3 and H3K27ac enrichment around the transcription start site (TSS),consistent with promoter activation,while upstream and intragenic regions exhibit enhancer-associated marks (H3K4me1,H3K27ac). Notably,hypermethylation within TSS-proximal regulatory regions (TPRRs)—including promoter-overlapping and adjacent CpG islands—correlates with ectopic MED12 overexpression,challenging the canonical view of DNAm as strictly repressive. Functional studies show that DNMT inhibition via 5-azacytidine reduces MED12 expression despite promoter demethylation in cells with hypermethylated TPRRs,suggesting a noncanonical role for DNA methylation in maintaining active transcription. Furthermore,MED12 expression positively correlates with DNMT3A and DNMT3B expression,implicating these methyltransferases in sustaining its epigenetic activation. This study identifies a novel regulatory axis in which aberrant DNA methylation,rather than genetic mutation,drives MED12 upregulation in AML. Our findings suggest that TPRR hypermethylation may function noncanonically to support transcriptional activation,likely in cooperation with enhancer elements. These results underscore the importance of epigenetic mechanisms in AML and highlight enhancer-linked methylation as a potential contributor to oncogene dysregulation. Future studies should further explore the role of noncanonical methylation-mediated gene activation in AML pathogenesis and therapeutic targeting. The online version contains supplementary material available at 10.1186/s13072-025-00610-9. View Publication

This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination,homing,and mechanisms of therapy resistance. We used a gelatin-based,hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow,which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells,characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies. View Publication