技术资料

Mutational inactivation of ATRX ($\alpha$-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear,as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover,these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate,both in vitro and in vivo,that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally,we show that G4 stabilization synergizes with other DNA-damaging therapies,including ionizing radiation,in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma,while also pointing to tangible strategies for drug development. View Publication

Our understanding of the signalling pathways regulating early human development is limited,despite their fundamental biological importance. Here,we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors,along with IGF1 ligand. Consequently,we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions,we derive several pluripotent stem cell lines that express pluripotency-associated genes,retain high viability and a normal karyotype,and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos,and in both primed and na{\{i}}ve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche." View Publication

Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer,an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel,cost-effective,high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease,exemplifying the potential of our approach. View Publication

High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth,but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections,forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation,whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth,human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together,these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression. View Publication

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However,the effects of antigen copy number,spacing and affinity,as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here,we display the clinical vaccine immunogen eOD-GT8,an engineered outer domain of the HIV-1 glycoprotein-120,on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to {\~{}}25-30 nm monotonically increases B-cell receptor activation. Moreover,scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses. View Publication

Although the association between the microbiome and IBD and liver diseases is known,the cause and effect remain elusive. By connecting human microphysiological systems of the gut,liver,and circulating Treg and Th17 cells,we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity,depending on the involvement of effector CD4 T cells. Using multiomics,we found SCFAs increased production of ketone bodies,glycolysis,and lipogenesis,while markedly reducing innate immune activation of the UC gut. However,during acute T cell-mediated inflammation,SCFAs exacerbated CD4+ T cell-effector function,partially through metabolic reprograming,leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity,metabolism,and tissue homeostasis. View Publication

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast,lung,colon and prostate. Using CR,we have established matched normal and tumor cultures,GUMC-29 and GUMC-30 respectively,from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly,only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice,demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry,both normal and tumor CR cells expressed basal,luminal,and stem cell markers,with the majority of the normal and tumor CR cells expressing prostate basal cell markers,CD44 and Trop2,as well as luminal marker,CD13,suggesting a transit-amplifying phenotype. Consistent with this phenotype,real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5,KRT14 and p63),and low levels of luminal markers. When the CR tumor cells were injected into SCID mice,the expression of luminal markers (AR,NKX3.1) increased significantly,while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor,but undergo differentiation once injected into SCID mice. Genomic analyses,including SNP and INDEL,identified genes mutated in tumor cells,including components of apoptosis,cell attachment,and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. View Publication

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here,we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development,from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells,whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements,normalized T cell development,and corrected the NK cell-like phenotype. In conclusion,we succeeded in generating an iPSC-based RAG2-SCID model,which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks. View Publication

Human-induced Pluripotent Stem Cell (hiPSC)-derived models have advanced the study of neurodegenerative diseases,including Parkinson's disease (PD). While age is the strongest risk factor for these disorders,hiPSC-derived models represent rejuvenated neurons. We developed hiPSC-derived Aged dopaminergic and cholinergic neurons to model PD and related synucleinopathies. Our new method induces aging through a `semi-natural' process,by passaging multiple times at the Neural Precursor Cell stage,prior to final differentiation. Characterization of isogenic hiPSC-derived neurons using heterochromatin and nuclear envelope markers,as well as DNA damage and global DNA methylation,validated our age-inducing method. Next,we compared neurons derived from a patient with SNCA-triplication (SNCA-Tri) and a Control. The SNCA-Tri neurons displayed exacerbated nuclear aging,showing advanced aging signatures already at the Juvenile stage. Noteworthy,the Aged SNCA-Tri neurons showed more $\alpha$-synuclein aggregates per cell versus the Juvenile. We suggest a link between the effects of aging and SNCA overexpression on neuronal nuclear architecture. View Publication

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here,we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1{\&}2),a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS,for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1{\&}2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1{\&}2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore,PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway,and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1{\&}2 reversed arthritis in mice,providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies. View Publication

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication