Transcriptional and post-translational regulation system for hypoxia specific gene therapy

Abstract

Therapeutic angiogenesis is a promising strategy for the treatment of ischemic diseases. However, unregulated and sustained expression of an angiogenic factor may induce pathological angiogenesis and tumor growth. Therefore, the angiogenic factor expression must be tightly regulated for safe ischemic disease gene therapy. Most hypoxia specific expression systems have been developed to induce gene expression under hypoxia. However, leaky expression by the basal promoter activity under normoxia should also be reduced to avoid pathological angiogenesis. In this study, a hypoxia specific expression plasmid was developed with an oxygen-dependent degradation (ODD) domain for rapid degradation of a target protein under normoxia and the erythropoietin (Epo) enhancer for more hypoxia specific gene expression under hypoxia. pSV-Luc-ODD was constructed with the Hif-1 ODD domain. In vitro transfection assay showed that luciferase activity in the pSV-Luc-ODD transfected cells was much lower under normoxia than hypoxia. However, the luciferase mRNA levels under hypoxia and normoxia were not significantly different. The results suggesting that the reduction of luciferase activity under normoxia is not due to pre-translational events, but to post-translational degradation. This post-translational regulation system was combined with a transcriptional induction system of the Epo enhancer for more hypoxia specific gene expression. pEpo-SV-Luc-ODD was constructed with the ODD domain and the Epo enhancer. pSV-Luc was used as a control. In a transfection assay, pEpo-SV-Luc-ODD showed over 1,000 times higher gene expression under hypoxia in Neuro2A cells, compared to normoxia. However, pSV-Luc did not induce luciferase expression under hypoxia. Reoxygenation studies after hypoxia incubation showed that gene expression was decreased in response to increased oxygen concentration. Therefore, the hypoxia specific expression system with the ODD domain and the Epo enhancer will be useful and safe for gene therapy of ischemic diseases.