Enhanced hypoxia specific expression activity of the Epo enhancer and SV40 promoter by co-transfection of the HIF-1α gene

Abstract

Gene therapy with the vascular endothelial growth factor (VEGF) gene is a potential treatment for ischemic diseases. However, the unregulated VEGF expression had deleterious effect. The tight regulation of the gene expression is one of the requirements for VEGF gene therapy. Therefore, many efforts have been made to develop ischemic specific gene expression system. For this purpose, several hypoxia responsive systems have been developed. Our group developed the erythropoietin (Epo) enhancer-SV40 promoter system for a hypoxia-inducible gene expression. In this system, hypoxia inducible gene expressions are mediated by endogenous hypoxia inducible factor 1 (HIF-1 ). In normal tissues, HIF-1 is rapidly degraded via ubiquitin-dependent proteolysis pathway. However, in ischemic tissue, HIF-1 is stabilized and induces the activity of the Epo enhancer-SV40 promoter. In the present study, we developed a hypoxia specific gene expression system, in which the promoter activity of the Epo enhancer-SV40 promoter was further enhanced without compromise of the specificity by the co-transfection of the HIF-1 gene. The HIF-1 cDNA was amplified from the total RNA of 293 cells by RT-PCR using specific primers. pSV-HIF1 was constructed by the insertion of the HIF-1a cDNA into pSI. In the transfection assay for optimization of the ratio between pEpo-SV-Luc and pSV-HIF1 , the luciferase expression of each sample was not significantly different. Therefore, a 1:1 ratio was used for the following experiments. The co-transfection of pSV-HIF1 and pEpo-SV-Luc increased the promoter activity of the Epo enhancer and SV40 promoter system without compromise of hypoxia specificity, showing higher gene expression than pEpo-SV-Luc only. This result was confirmed with the VEGF expression plasmid, pEpo-SV-VEGF. In ELISA, the co-transfection of pSV-HIF1 and pEpo-SV-VEGF increased the VEGF expression under hypoxia condition without compromise of specificity. In addition, pSV-HIF1a may induce the endogenous hypoxia responsive angiogenic genes, which will be beneficial for therapeutic angiogenesis. Indeed, the co-trasfection of pSV-HIF1 and pEpo-SV-Luc increased the expression of the endogenous growth factor, Angiopoietin-1. In conclusion, the co-transfection of pSV-HIF1 and pEpo-SV-VEGF may be useful for gene therapy for ischemic disease.