Augmentation of erythropoietin enhancer-mediated hypoxia-inducible gene expression by co-transfection of a plasmid encoding hypoxia-inducible factor 1α for ischemic tissue targeting gene therapy

Abstract

Therapeutic angiogenesis with gene encoding vascular endothelial growth factor (VEGF) is a potential treatment for ischemic diseases. However, VEGF expression should be tightly regulated to avoid side effects such as tumor growth. Previously, our group developed the erythropoietin (Epo) enhancer–SV40 promoter system for hypoxia-specific gene expression. In the present study, the activity of the Epo enhancer–SV40 promoter system was further enhanced without significant decrease in its specificity by co-transfection of the hypoxia-inducible factor 1α (HIF1α) gene. pSV-HIF1α was constructed by the insertion of the HIF1α cDNA into pSI. At a 1:1 ratio, co-transfection of pSV-HIF1α and pEpo-SV-Luc increased the promoter activity of the Epo enhancer–SV40 promoter system, showing at least three times higher gene expression under hypoxia as compared with the pEpo-SV-Luc single-plasmid transfection. Furthermore, co-transfection showed significant hypoxia specificity. Also, co-transfection of pEpo-SV-VEGF with pSV-HIF1α showed the enhanced VEGF expression without loss of hypoxia specificity, as compared with pEpo-SV-VEGF single-plasmid transfection. Furthermore, pSV-HIF1α induced the endogenous hypoxia-responsive genes such as angiopoietin-1, which would be beneficial for therapeutic angiogenesis. Therefore, with hypoxia specificity and higher gene expression, co-transfection of pSV-HIF1α and pEpo-SV-VEGF may be useful for ischemia targeting gene therapy.