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Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here,we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system,when optimized in human U87 cells,provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs,targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs,the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette,demonstrating the feasibility of cassette exchange. Moreover,as assessed by measuring γ-H2AX expression levels,genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency,flexibility in transgene exchange and low genome toxicity,our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells. View Publication

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here,we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels,we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis,phospholipid composition,and inner leaflet surface charge,thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together,our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration. The biomechanical mechanisms enabling the invasive growth of brain tumors remain opaque. Here,Junqueira Alves et al. reveal that the guidance receptor Plexin-B2 controls membrane tension,facilitating confined migration of brain tumor cells. View Publication

IntroductionInnate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. MethodsTo gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection,we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression,transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. ResultsWe found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover,all ILC subsets displayed a significantly higher frequency of CD69-expressing cells,indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation,activation and homeostasis. In addition,differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. DiscussionOverall,this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection. View Publication

AbstractT cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection,including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT,but current sampling methods require a healthcare provider and are expensive,limiting the ability to study these populations longitudinally. To address these challenges,we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive,self-applied,and low in cost. To demonstrate reproducibility,we sampled the cervicovaginal fluid of healthy,reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells,and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live,CD45+ leukocytes,as well as distinct populations of T cells and B cells. Within the T cell compartment,activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches,including identification of both tissue-resident and migratory populations. In addition,the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies. View Publication

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success,but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion,hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet,the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable,multiplexed modulation of gene expression with greater safety. Here,we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes,PDCD1 and LAG3,both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion,limiting functional assessment,gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore,transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features. Graphical abstract Epigenome editing is used to engineer CAR T cells targeting prostate cancer by stably silencing the PDCD1 and LAG3 genes,which encode key inhibitory checkpoint receptors. This DNA break-free approach enhances safety by avoiding genomic damage and holds promise as a next-generation strategy for safer,more durable cancer immunotherapy. View Publication

Acute myeloid leukaemia (AML) is a disease of the haematopoietic stem cells(HSCs) that is characterised by the uncontrolled proliferation and impaired differentiation of normal haematopoietic stem/progenitor cells. Several pathways that control the proliferation and differentiation of HSCs are impaired in AML. Activation of the Wnt/beta-catenin signalling pathway has been shown in AML and beta-catenin,which is thought to be the key element of this pathway,has been frequently highlighted. The present study was designed to determine beta-catenin expression levels and beta-catenin-related genes in AML. In this study,beta-catenin gene expression levels were determined in 19 AML patients and 3 controls by qRT-PCR. Transcriptome analysis was performed on AML grouped according to beta-catenin expression levels. Differentially expressed genes(DEGs) were investigated in detail using the Database for Annotation Visualisation and Integrated Discovery(DAVID),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),STRING online tools. The transcriptome profiles of our AML samples showed different molecular signature profiles according to their beta-catenin levels(high-low). A total of 20 genes have been identified as hub genes. Among these,TTK,HJURP,KIF14,BTF3,RPL17 and RSL1D1 were found to be associated with beta-catenin and poor survival in AML. Furthermore,for the first time in our study,the ELOV6 gene,which is the most highly up-regulated gene in human AML samples,was correlated with a poor prognosis via high beta-catenin levels. It is suggested that the identification of beta-catenin-related gene profiles in AML may help to select new therapeutic targets for the treatment of AML. View Publication

The intestinal epithelial repair after injury is coordinated by intestinal stem cells (ISCs). Fucosylation catalyzed by fucosyltransferase 2 (FUT2) of the intestinal epithelium is beneficial to mucosal healing but poorly defined is the influence on ISCs. The dextran sulfate sodium (DSS) and lipopolysaccharide (LPS) model were used to assess the role of FUT2 on ISCs after injury. The apoptosis,function,and stemness of ISCs were analyzed using intestinal organoids from WT and Fut2?ISC (ISC-specific Fut2 knockout) mice incubated with LPS and fucose. N-glycoproteomics,UEA-1 chromatography,and site-directed mutagenesis were monitored to dissect the regulatory mechanism,identify the target fucosylated protein and the corresponding modification site. Fucose could alleviate intestinal epithelial damage via upregulating FUT2 and ?-1,2-fucosylation of ISCs. Oxidative stress,mitochondrial dysfunction,and cell apoptosis were impeded by fucose. Meanwhile,fucose sustained the growth and proliferation capacity of intestinal organoids treated with LPS. Contrarily,FUT2 depletion in ISCs aggravated the epithelial damage and disrupted the growth and proliferation capacity of ISCs via escalating LPS-induced endoplasmic reticulum (ER) stress and initiating the IRE1/TRAF2/ASK1/JNK branch of unfolded protein response (UPR). Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine (Asn) 862 mediated by FUT2 was identified to facilitate ISCs survival and self-renewal,and improve ISCs resistance to ER stress and inflammatory injury. Our study highlights a fucosylation-dependent protective mechanism of ISCs against inflammation,which may provide a fascinating strategy for treating intestinal injury disorders. View Publication

Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity,we engineered induced pluripotent stem cell-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity,non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity. In addition,we introduced a membrane-bound IL-15/IL-15R fusion protein to promote in vivo persistence. These engineered cells,termed iDuo NK cells,displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models,iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore,iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19,features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represent a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies. View Publication

The airway epithelium contains ionocytes,a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR),a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high ({\~{}}3{\%}) ionocyte abundance in ex vivo nasal samples,with no difference between CF and control individuals. In bronchi,ionocytes instead appeared very rarely as previously reported,thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture,which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure,we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions,thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern. View Publication

Management of chronic kidney disease (CKD) remains a major challenge due limited therapeutic options to reverse fibrosis,which is a critical feature in CKD. Partial epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) is a key driver of fibrosis,and has become an important focus for kidney protection strategies. Blood platelets,a major source of circulating transforming growth factor beta (TGF-β),are implicated in pathogenesis of CKD,but their involvement in EMT and kidney fibrosis remains uncertain. Methods: We used two mouse models of renal fibrosis—diabetic kidney disease (DKD) and unilateral ureter obstruction (UUO)—to examine the connection between platelets,partial EMT,and fibrosis. Platelet inhibition or depletion was performed to assess EMT,cell cycle arrest,and fibrosis. In vitro,platelets were applied to TECs and kidney organoids. To determine the role of TGF-β signaling,we used TGF-βRI inhibitor. Expression of EMT,and fibrosis markers,as well as TGF-β1 signaling,were analyzed using western blot,reverse transcription quantitative PCR (RT-qPCR),enzyme-linked immunosorbent assay (ELISA),and immunostaining. Results: In both animal models,platelet inhibition or depletion resulted in reduced expression of cell cycle arrest marker p21,partial EMT and fibrosis. In vitro,activated platelets stimulated cell cycle arrest,EMT,and fibrosis in TECs and kidney organoids. Chronically injured TECs experience cell-cycle arrest which promote a paracrine EMT program in TECs,jointly leading to fibrosis. This platelet-mediated effect on cell cycle arrest and EMT was driven by TGF-β1 signaling,as selective inhibition of the TGF-β receptor rescued these dysfunctional phenotypes. Conclusions: Our study demonstrates that platelets activate the TGF-β1 pathway,leading to cell cycle arrest,EMT and renal fibrosis. These findings suggest that antiplatelet therapies may have potential renoprotective effects by protecting tubular homeostasis,attenuating partial EMT and fibrosis. View Publication

Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease,and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive,most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND),which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines,and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was,then,used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines,as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND,to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First,the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second,the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed,along with the relevance of these two different strategies for disease modeling. View Publication

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication