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

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 1alpha (HIF1alpha) gene. pSV-HIF1alpha was constructed by the insertion of the HIF1alpha cDNA into pSI. At a 1:1 ratio, co-transfection of pSV-HIF1alpha 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-HIF1alpha showed the enhanced VEGF expression without loss of hypoxia specificity, as compared with pEpo-SV-VEGF single-plasmid transfection. Furthermore, pSV-HIF1alpha 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-HIF1alpha and pEpo-SV-VEGF may be useful for ischemia targeting gene therapy.