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Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet,evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. In this study,scRNAseq on organoids reveals the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A,progenitor proliferation and structures are disrupted while organoid composition shifts away from proliferative cell types. We observe impacts on non-neuronal cells,including choroid plexus enrichment. Furthermore,EMX1+ cortical progenitors are negatively impacted; potentially disrupting corticogenesis and delaying excitatory neuron maturation. This work reveals impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential therapeutic targets. Human cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A. View Publication

SummaryTumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here,we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly,ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies. Graphical abstract Highlights•Tumor-derived factors upregulate PARP11 in the tumor-infiltrating Treg cells•PARP11 supports the immunosuppressive properties of Treg cells•Pharmacologic inhibition of PARP11 inactivates intratumoral Treg cells•PARP11 inhibitor augments the efficacy of immunotherapies Basavaraja et al. demonstrate that induction of PARP11 in the intratumoral regulatory T (Treg) cells is required for their regulatory functions and contributes to the immunosuppressive tumor microenvironment. The selective inhibitor of PARP11 ITK7 inactivates tumor Treg cells and improves the efficacy of immunotherapies against tumors. View Publication

AbstractBackgroundNatural killer (NK) cells’ unique ability to kill transformed cells expressing stress ligands or lacking major histocompatibility complexes (MHC) has prompted their development for immunotherapy. However,NK cells have demonstrated only moderate responses against cancer in clinical trials.MethodsAdvanced genome engineering may thus be used to unlock their full potential. Multiplex genome editing with CRISPR/Cas9 base editors (BEs) has been used to enhance T cell function and has already entered clinical trials but has not been reported in human NK cells. Here,we report the first application of BE in primary NK cells to achieve both loss-of-function and gain-of-function mutations.ResultsWe observed highly efficient single and multiplex base editing,resulting in significantly enhanced NK cell function in vitro and in vivo. Next,we combined multiplex BE with non-viral TcBuster transposon-based integration to generate interleukin-15 armored CD19 chimeric antigen receptor (CAR)-NK cells with significantly improved functionality in a highly suppressive model of Burkitt’s lymphoma both in vitro and in vivo.ConclusionsThe use of concomitant non-viral transposon engineering with multiplex base editing thus represents a highly versatile and efficient platform to generate CAR-NK products for cell-based immunotherapy and affords the flexibility to tailor multiple gene edits to maximize the effectiveness of the therapy for the cancer type being treated. View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Apigenin (API),a traditionally sourced flavonoid,is recognized for its anti-neoplastic properties. Despite well-documented effects on tumorigenesis,the detailed therapeutic impact on breast cancer stem cells (BCSCs) and the associated molecular mechanisms are yet to be clarified. The objective of this study is to elucidate the therapeutic effects of API on BCSCs and to uncover its molecular mechanisms through network pharmacology and experimental validation. Interactions of API with candidate targets were examined through target screening,enrichment analysis,construction of protein-protein interaction networks,and molecular docking. MCF-7-derived BCSCs were utilized as a model system to investigate and substantiate the anti-BCSC effects of API and the underlying mechanism. Molecular docking studies have shown that API and TP53 exhibit favorable binding affinity. Compared with the negative control group,API effectively suppressed the expression of BCSC-related proteins such as ALDH1A1,NANOG,EpCAM,and MYC,downregulated p-PI3K and p-AKT,and upregulated p53. This study demonstrates that API can play an anti-BCSC role by regulating the PI3K/AKT/p53 pathway in BCSCs of MCF-7 cells,highlighting its potential as a therapeutic agent for targeting BCSCs. View Publication

To produce pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and culture and differentiate them into vascular organoids,producing a disease model for cerebral small vessel disease (CSVD). (1) PMBCs from patients clinically diagnosed with CADASIL ( NOTCH3 p.R141C) were induced to differentiate into pluripotent stem cells (iPSCs); the quality and differentiation ability of the iPSCs were determined. (2) CADASIL-derived iPSCs and control iPSCs were cultured and differentiated into vascular organoids. The differences in the morphological structure of the two differentiated groups of vascular organoids were observed,and both were identified. (1) No mycoplasma infections were detected in the iPSCs prepared from the PBMCs of patients with CADASIL. The short tandem repeat (STR) identification verified that the iPSCs originated from the patient,and the karyotype was normal. Flow cytometry and immunofluorescence detection revealed that the iPSCs expressed SSEA4,OCT4,and NANOG stem proteins. Tri-germ differentiation testing confirmed that the iPSCs expressed the endoderm markers SOX17 and FOXA2,the mesoderm markers Brachyury and α-SMA,and the ectoderm markers Pax6 and β-III Tubulin. (2) CADASIL-derived iPSCs and control iPSCs were induced to differentiate and produce endothelial networks and vascular networks,ultimately forming vascular organoids. Compared with control vascular organoids,CADASIL vascular organoids exhibited lower growth density,earlier blood vessel sprouting,longer and thinner vascular filaments,and smaller final vascular organoids. The vascular organoids from the two sources expressed the endothelial cell marker CD31,the vascular smooth muscle marker α-SMA,and the pericyte marker PDGFR-β. Reprogramming technology can be used to induce PBMCs to become iPSCs,and a CSVD disease model can be successfully constructed by culturing and differentiating the iPSCs into CADASIL vascular organoids. The NOTCH3 p.R141C mutation suppresses the vascular differentiation process in CADASIL. 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

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. View Publication

Outcomes of 159 young patients with inherited metabolic disorders (IMDs) undergoing transplantation with partially HLA-mismatched unrelated donor umbilical cord blood were studied to investigate the impact of graft and patient characteristics on engraftment,overall survival (OS),and graft-versus-host disease (GVHD). Patients received myeloablative chemotherapy (busulfan,cyclophosphamide,ATG) and cyclosporine-based GVHD prophylaxis. Infused cell doses were high (7.57 x 10(7)/kg) because of the patients' young age (median,1.5 years) and small size (median,12 kg). Median follow-up was 4.2 years (range,1-11 years). The cumulative incidences of neutrophil and platelet engraftment were 87.1% (95% confidence interval [CI],81.8%-92.4%) and 71.0% (95% CI,63.7%-78.3%). A total of 97% achieved high (textgreater 90%) donor chimerism. Serum enzyme normalized in 97% of patients with diseases for which testings exist. Grade III/IV acute GVHD occurred in 10.3% (95% CI,5.4%-15.2%) of patients. Extensive chronic GVHD occurred in 10.8% (95% CI,5.7%-15.9%) of patients by 1 year. OS at 1 and 5 years was 71.8% (95% CI,64.7%-78.9%) and 58.2% (95% CI,49.7%-66.6%) in all patients and 84.5% (95% CI,77.0%-92.0%) and 75.7% (95% CI,66.1%-85.3%) in patients with high (80-100) performance score. In multivariate analysis,favorable factors for OS were high pretransplantation performance status,matched donor/recipient ethnicity,and higher infused colony forming units. View Publication

We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003,8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%),with 3 complete responses,2 partial responses,and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect. View Publication