67 research outputs found
27. Aberrant Expression of the Stem Cell microRNA-126 Induces B Cell Malignancy
MicroRNAs are essential regulators of normal and malignant hematopoiesis. miRNAs are relevant for gene therapy, since they can be exploited to fine-tune the expression profile of vector constructs or to alter viral tropism (GentnerN Chiriaco et al, 2014; Escobar et al, 2014) and described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell malignancies. LV insertion site (IS) analysis revealed that all tumors were monoclonal. We then tracked back leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and leukemic clone suggests stem or multipotent progenitor cell as origin for most tumors. Importantly, we show that miR-126 is the direct cause of genesis and maintenance of leukemia, since leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and established symptomatic leukemia completely regresses when miR-126 is switched off by doxy through induction of apoptosis. Transcriptional profiling indicated that miR-126 regulates multiple genes in p53 pathway both in murine blasts and in normal human CD34+ cells. Previous work suggested expression of miR-126 in acute lymphoblastic leukemia (ALL) and germinal center lymphoma. To further establish the relevance of miR-126 in human disease, we measured miR-126 expression in blasts from 16 adult patients with ALL. miR-126 was highly expressed in most studied ALL cases (Phil+: n=11, Phil-: n=5), at similar levels as CD34+ cells. We then down-regulated miR-126 in primary blasts from human B-ALL patients (n=5), and we observed increased apoptosis and impaired engraftment in xenograft models after primary and secondary transplantation (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL. In conclusion, we present a novel spontaneous mouse model for high grade B cell malignancies which are addicted to miR-126 expression, provide insight into the dynamic process of leukemogenesis by clonal IS tracking and unveil key tumor signaling pathways controlled by miR-126. Down-regulation of miR-126 could be exploited as therapeutic strategy in ALL, since it would deplete leukemic cells while expanding normal HSC, two ways to restore normal hematopoieis
MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation
The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system
現地観測に基づく河川流の乱流特性に関する研究
博士(工学)神戸大
IMMU-01. TEM-GBM: AN OPEN-LABEL, PHASE I/IIA DOSE-ESCALATION STUDY EVALUATING THE SAFETY AND EFFICACY OF GENETICALLY MODIFIED TIE-2 EXPRESSING MONOCYTES TO DELIVER IFN-A WITHIN GLIOBLASTOMA TUMOR MICROENVIRONMENT
Abstract
Temferon is a macrophage-based treatment relying on ex-vivo transduction of autologous HSPCs to express immune-payloads within the TME. Temferon targets the immune-modulatory molecule IFN-a, to a subset of tumor infiltrating macrophages known as Tie-2 expressing macrophages (TEMs) due to the Tie2 promoter and a post-transcriptional regulation layer represented by miRNA-126 target sequences. As of 31st May 2021, 15-patients received Temferon (D+0) with follow-up of 3 – 693 days. After conditioning neutrophil and platelet engraftment occurred at D+13 and D+13.5, respectively. Temferon-derived differentiated cells, as determined be the number of vector copy per genome, were found within 14 days post treatment and persisted albeit at lower levels up to 18-months. Very low concentrations of IFN-a in the plasma (8.7 pg/ml-D+30) and in the CSF (1.6 pg/ml-D+30) were detected, suggesting tight regulation of transgene expression. Five-deaths occurred at D+322, +340, +402, +478 and +646 due to PD, and one at D+60 due to complications following the conditioning regimen. Eight-patients had progressive disease (range: D-11 to +239) as expected for this tumor type. SAEs include GGT elevation (possibly related to Temferon) and infections, venous thromboembolism, brain abscess, hemiparesis, seizures, anemia and general physical condition deterioration, compatible with ASCT, concomitant medications and PD. Four-patients underwent 2ndsurgery. Recurrent tumors had gene-marked cells and increased expression of ISGs compared to first surgery, indicative of local IFNa release by TEMs. In one patient, a stable lesion had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased ISGs than in the progressing lesion, detected in the same patient. Tumor-associated clones expanded in the periphery. TME characterization by scRNA and TCR-sequencing is ongoing. To date, Temferon is well tolerated, with no DLTs identified. The results provide initial evidence of Temferon potential to activate the immune system of GBM patients, as predicted by preclinical studies
miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells
SummaryTo investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance
Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis
Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis
A Double-Switch Vector System Positively Regulates Transgene Expression by Endogenous microRNA Expression (miR-ON Vector)
International audienceTo better understand and exploit microRNA (miR) regulation, a more precise characterization of miR expression patterns within a tissue or a lineage during development, differentiation, and homeostasis is needed. We previously showed that lentiviral vectors (LV) can be made responsive to miR to stringently control transgene expression as well as to report miR activity "live" and at the single-cell level. Although very useful, this approach reports miR activity by transgene suppression, hampering the direct identification and selection of miR-expressing cells. Here, we describe a strategy to couple transgene expression to the activity of the miR of interest. To this aim, we generated LV encoding two in-series OFF switches: a transcriptional repressor tagged with miR target sequences and a reporter cassette under the control of the repressor. Reporter expression is ON only when the miR is active and represses translation of the transcriptional repressor. We successfully applied this design to different types of repressors, multiple gene encoding vectors and delivered the system either by two separate or a self-contained vector. We demonstrated its performance by live monitoring of two miRs in different stages of human primary hematopoietic stem/progenitor cell differentiation in vivo. Further applications of this approach include imaging of rare miR-expressing cells and positive regulation of a therapeutic or selector gene in target cells identified by the expression of selected miRs
Engineered tumor-infiltrating macrophages as gene delivery vehicles for interferon-α activates immunity and inhibits breast cancer progression
An immunosuppressive tumor microenvironment is a cancer hallmark and a major impediment to successful immunotherapy. We engineered hematopoietic progenitors to target expression of an interferon-α (IFNα) transgene specifically to their monocytic progeny, including tumor-infiltrating macrophages. Mice chimeric for these IFNα-expressing macrophages showed activation of innate and adaptive immune cells against breast cancer and inhibited disease progression
Regulated and Multiple miRNA and siRNA Delivery Into Primary Cells by a Lentiviral Platform
RNA interference (RNAi) has tremendous potential for investigating gene function and developing new therapies. However, the design and validation of proficient vehicles for stable and safe microRNA (miR) and small interfering RNA (siRNA) delivery into relevant target cells remains an active area of investigation. Here, we developed a lentiviral platform to efficiently coexpress one or more natural/artificial miR together with a gene of interest from constitutive or regulated polymerase-II (Pol-II) promoters. By swapping the stem–loop (sl) sequence of a selected primary transcript (pri-miR) with that of other miR or replacing the stem with an siRNA of choice, we consistently obtained robust expression of the chimeric/artificial miR in several cell types. We validated our platform transducing a panel of engineered cells stably expressing sensitive reporters for miR activity and on a natural target. This approach allowed us to quantitatively assess at steady state the target suppression activity and expression level of each delivered miR and to compare it to those of endogenous miR. Exogenous/artificial miR reached the concentration and activity typical of highly expressed natural miR without perturbing endogenous miR maturation or regulation. Finally, we demonstrate the robust performance of the platform reversing the anergic/suppressive phenotype of human primary regulatory T cells (Treg) by knocking-down their master gene Forkhead Transcription Factor P3 (FOXP3)
- …