Tumor Hypoxia Blocks Wnt Processing and Secretion through the Induction of Endoplasmic Reticulum Stress▿

Poorly formed tumor blood vessels lead to regions of microenvironmental stress due to depletion of oxygen and glucose and accumulation of waste products (acidosis). These conditions contribute to tumor progression and correlate with poor patient prognosis. Here we show that the microenvironmental stresses found in the solid tumor are able to inhibit the canonical Wnt/β-catenin signaling pathway. However, tumor cells harboring common β-catenin pathway mutations, such as loss of adenomatous polyposis coli, are insensitive to this novel hypoxic effect. The underlying mechanism responsible is hypoxia-induced endoplasmic reticulum (ER) stress that inhibits normal Wnt protein processing and secretion. ER stress causes dissociation between GRP78/BiP and Wnt, an interaction essential for its correct posttranslational processing. Microenvironmental stress can therefore block autocrine and paracrine signaling of the Wnt/β-catenin pathway and negatively affect tumor growth. This study provides a general paradigm relating oxygen status to ER function and growth factor signaling

Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias

Episomal vectors based on S/MAR and the beta-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient gamma-globin activation in haematopoietic cells

We report the development of episomal vectors for the specific gamma-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the beta-globin Replicator, the DNA replication-Initiation Region from the beta-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV-Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine beta-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34(+) cells, with transfection efficiencies of 46.3 +/- 5.2%, 23.0 +/- 2.1% and 24.2 +/- 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of gamma-globin mRNA by 3.3 +/- 0.13, of gamma-globin protein by 6.75 +/- 3.25 and HbF protein by 2 +/- 0.2 fold, while the vector remained episomal and non integrated. In murine beta-YAC BMCs the vector mediated the activation of the silent human gamma-globin gene and in CD34+ cells, increased gamma-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of beta-globin Replicator, transferred into CD34+ cells, produced CD34(+) eGFP(+) cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 +/- 0.13 fold increased gamma-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors