[Show abstract][Hide abstract] ABSTRACT: To 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.
[Show abstract][Hide abstract] ABSTRACT: Globoid cell leukodystrophy (GLD) is a demyelinating lysosomal storage disease due to the deficiency of the galactocerebrosidase (GALC) enzyme. The favorable outcome of hematopoietic stem and progenitor cell (HSPC)-based approaches in GLD and other similar diseases suggests HSPC gene therapy as a promising therapeutic option for patients. The path to clinical development of this strategy was hampered by a selective toxicity of the overexpressed GALC in the HSPC compartment. Here, we presented the optimization of a lentiviral vector (LV) in which miR-126 regulation was coupled to codon optimization of the human GALC cDNA to obtain a selective and enhanced enzymatic activity only upon transduced HSPCs differentiation. The safety of human GALC overexpression driven by this LV was extensively demonstrated in vitro and in vivo on human HSPCs from healthy donors. No perturbation in the content of proapoptotic sphingolipids, gene expression profile, and capability of engraftment and mutlilineage differentiation in chimeric mice was observed. The therapeutic potential of this LV was then assessed in a severe GLD murine model that benefited from transplantation of corrected HSPCs with longer survival and ameliorated phenotype as compared to untreated siblings. This construct has thus been selected as a candidate for clinical translation.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Although allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) can accomplish apparent leukemia eradication in most patients, residual tumor cells can persist over time and eventually outgrow, resulting in clinical relapse. The genetic landscape of relapsing leukemia is often markedly different from its counterpart at diagnosis, due to clonal evolution (Ding et al, Nature, 2012) and selection of treatment-resistant variants (Vago et al, N Engl J Med, 2009). A potential solution to treat, or even prevent, relapse is to identify and specifically target mutations occurring very early in the leukemic transformation process, and thus putative hallmarks of cancer progenitors. Next-Generation Sequencing (NGS) provides the opportunity to track disease subclones during the clinical history of patients, pinpoint founder alterations and identify the mechanisms by which leukemia evades elimination.METHODS: In the present study, we combined immunogenetic analyses and NGS to detail the complex
[Show abstract][Hide abstract] ABSTRACT: Regulated transgene expression may improve safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked Chronic Granulomatous Disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34+ HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.Molecular Therapy (2014); doi:10.1038/mt.2014.87.
[Show abstract][Hide abstract] ABSTRACT: Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: New neurons, originating from the subventricular zone, are continuously integrating into neuronal circuitry in the olfactory bulb (OB). Using a transgenic sensor mouse, we found that adult-born OB interneurons express microRNA-125 (miR-125), whereas the pre-existing developmentally generated OB interneurons represent a unique population of cells in the adult brain, without miR-125 activity. Stable inhibition of miR-125 in newborn OB neurons resulted in enhanced dendritic morphogenesis, as well as in increased synaptic activation in response to odour sensory stimuli. These data demonstrate that miR-125 controls functional synaptic integration of adult-born OB interneurons. Our results also suggest that absence of an otherwise broadly expressed miRNA is a novel mechanism with which to achieve neuronal subtype specification.
[Show abstract][Hide abstract] ABSTRACT: The immunosuppressive tumor microenvironment represents a major hurdle to cancer therapy. We developed a gene transfer strategy into hematopoietic stem cells (HSCs) to target transgene expression to tumor-infiltrating monocytes/macrophages. Using a combination of transcriptional and microRNA-mediated control, we achieved selective expression of an interferon-α (IFN-α) transgene in differentiated monocytes of human hematochimeric mice. We show that IFN-α transgene expression does not impair engraftment and long-term multilineage repopulation of NSG (NOD/LtSz-scidIL2Rγ(null)) mice by transplanted human HSCs. By providing a source of human cytokines in the mice, we improved the functional reconstitution of human myeloid, natural killer, and T cell lineages, and achieved enhanced immune-mediated clearance of transplanted human breast tumors when hematopoiesis was engineered for tumor-targeted IFN-α expression. By applying our strategy to mouse breast cancer models, we achieved inhibition of tumor progression and experimental metastases in an autologous setting, likely through enhanced generation of effector T cells and their recruitment to the neoplastic tissues. By forcing IFN-α expression in tumor-infiltrating macrophages, we blunted their innate protumoral activity and reprogrammed the tumor microenvironment toward more effective dendritic cell activation and immune effector cell cytotoxicity. Overall, our studies validate the feasibility, safety, and therapeutic potential of a new cancer gene therapy strategy, and open the way to test this approach as adjuvant therapy in advanced breast cancer patients.
Full-text · Article · Jan 2014 · Science translational medicine
[Show abstract][Hide abstract] ABSTRACT: Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy of cancer. Selecting a target expressed in multiple-tumor types and required for tumor growth would widen disease indications and prevent immune escape due to the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematological and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human AML and MM cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM, while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and IL-7/IL-15 was required for antitumor efficacy in vivo. Finally, co-expressing a suicide gene enabled fast and efficient pharmacological ablation of CD44v6-targeted T cells and complete rescue from hyper-acute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.
[Show abstract][Hide abstract] ABSTRACT: Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.
No preview · Article · Jul 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Several microRNAs (miRNAs) that are either specifically enriched or highly expressed in neurons and glia have been described, but the identification of miRNAs modulating neural stem cell (NSC) biology remains elusive. In this study, we exploited high throughput miRNA expression profiling to identify candidate miRNAs enriched in NSC/early progenitors derived from the murine subventricular zone (SVZ). Then, we used lentiviral miRNA sensor vectors (LV.miRT) to monitor the activity of shortlisted miRNAs with cellular and temporal resolution during NSC differentiation, taking advantage of in vitro and in vivo models that recapitulate physiological neurogenesis and gliogenesis and using known neuronal- and glial-specific miRNAs as reference. The LV.miRT platform allowed us monitoring endogenous miRNA activity in low represented cell populations within a bulk culture or within the complexity of CNS tissue, with high sensitivity and specificity. In this way we validated and extended previous results on the neuronal-specific miR-124 and the astroglial-specific miR-23a. Importantly, we describe for the first time a cell type- and differentiation stage-specific modulation of miR-93 and miR-125b in SVZ-derived NSC cultures and in the SVZ neurogenic niche in vivo, suggesting key roles of these miRNAs in regulating NSC function.