Transcriptional and post-translational regulatory system for hypoxia specific gene expression using the erythropoietin enhancer and the oxygen-dependent degradation domain

Abstract

Gene therapy with angiogenic factors is a promising strategy for the treatment of ischemic diseases. However, unregulated expression of an angiogenic factor may induce pathological angiogenesis. In this study, a hypoxia specific gene expression plasmid, pSV-Luc-ODD, was constructed with the oxygen-dependent degradation (ODD) domain for rapid degradation of a target protein under normoxia. In the transfection assay, luciferase activity in the pSV-Luc-ODD transfected cells was much lower under normoxia than that under hypoxia. However, the luciferase mRNA levels under hypoxia and normoxia were not significantly different. Therefore, decrease of luciferase activity under normoxia is not due to pre-translational events such as change of transcription rate or mRNA stability, but to post-translational degradation. For more hypoxia specific gene expression, pEpo-SV-Luc-ODD was constructed with the erythropoietin (Epo) enhancer and the ODD domain. pEpo-SV-Luc-ODD showed more than 1000 times increase of gene expression under hypoxia in Neuro2A cells, compared to normoxia. In addition, reoxygenation studies after hypoxia incubation showed that gene expression was decreased in response to increased oxygen concentration. This highly hypoxia specific gene expression system will be useful for development of targeting gene therapy for ischemic diseases.