Hypoxia specific gene expression system by transcirptional and post-translational regulation using the erythropoietin enhancer and the oxygen-dependent degradation domain

Abstract

Background Gene therapy with angiogenic factors such as vascular endothelial growth factor is a promising strategy for treatment of ischemic diseases. However, unregulated and sustained expression of angiogenic factor may induce pathological angiogenesis. Therefore, the angiogenic factor expression should be tightly regulated. Most hypoxia specific expression systems have been developed to induce gene expression under hypoxia. However, gene expression by the basal promoter activity under normoxia should also be reduced to avoid pathological angiogenesis. In this study, a hypoxia specific expression system was developed with an oxygen-dependent domain (ODD) to reduce gene expression under normoxia and the erythropoietin (Epo) enhancer to induce gene expression under hypoxia. Method pSV-Luc-ODD and pEpo-SV-Luc-ODD were constructed with ODD and the Epo enhancer. For transfection assays, Neuro2A and HEK293 cells were used. Transgene expression was measured by luciferase assay. Results Luciferase-ODD produced by pSV-Luc-ODD is rapidly degraded under normoxia. Therefore, luciferase activity in the pSV-Luc-ODD transfected cells was much lower under normoxia than hypoxia. The luciferase mRNA levels under hypoxia and normoxia were not significantly different, suggesting that the reduction of activity under normoxia is due to post-translational degradation. To increase gene expression under hypoxia, pEpo-SV-Luc-ODD was constructed. In a transfection assay, pEpo-SV-Luc-ODD showed over 100 times higher gene expression under hypoxia than normoxia. Under normoxia, gene expression by pEpo-SV-Luc-ODD was 100 times lower than that by pSV-Luc, suggesting that the gene expression by the basal promoter activity can be overcome by this approach. Conclusion This hypoxia specific expression system reduced gene expression under normoxia, minimizing the effect of the basal promoter activity. In addition, the Epo enhancer increased gene expression under hypoxia, increasing therapeutic efficiency in a target tissue. Therefore, the hypoxia specific expression system with ODD and the Epo enhancer will be useful and safe for angiogenic factor gene therapy.