193. Hypoxia-Inducible VEGF Gene Therapy Using the RTP801 Promoter

Abstract

Background; The angiogenic gene therapy using VEGF is a new potential treatment for ischemic disease. Gene expression in ischemic tissue must be locally enhanced and regulated to be safe and effective. Previously, it was reported that the RTP801 promoter was strongly induced under hypoxic conditions. This strong induction of the RTP801 promoter suggests its applicability to the regulation of VEGF gene in the VEGF gene therapy. In this study, we evaluated the role of the RTP801-VEGF plasmid as a therapeutic plasmid in vitro and in vivo study. Method; To identify the cis-regulatory element, the RTP801 promoter was analyzed by various methods. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter upstream of the VEGF cDNA. pEpo-SV-VEGF and pSV-VEGF were used as positive controls and WSLP only injection without plasmid was used as a negative control the in vivo model. In vitro cell transfection study, polyethylenimine (PEI, 25,000 Da) was used as a gene carrier and 293T cells were used. In an in vivo VEGF expression study, we used water-soluble lipopolymer (WSLP) as a gene carrier. The circumflex artery of a New Zealand white rabbit was ligated with a 6-0 Prolene suture and WSLP-plasmid complex (150 ug/750 ul) was injected into the infarcted myocardium using a 30-guage needle. The hearts were harvested 4 days after the injections and homogenized in lysis buffer. The expression level of the VEGF gene was measured by ELISA. Result; In luciferase assay, the region between -495 and -445 was responsible for the hypoxia-induced transcription. In this region, there was a potential Sp1 binding element. The mutation of the Sp1 site reduced the hypoxia-induced transcription of the RTP801 promoter. In addition, co-transfection with antisense Sp1 oligonucleotide decreased the promoter activity. These results suggest that the hypoxia induction of the RTP801 promoter is mediated by Sp1. The pRTP801-VEGF had stronger VEGF expression than pEpo-SV-VEGF, which contained the Epo (erythropoietin) enhancer and the SV 40 promoter system. In addition, the VEGF expression by pRTP801-VEGF was induced under hypoxic condition. In the animal study, the expression level of VEGF by pRTP801-VEGF in the ischemic myocardium was 5.7 times higher than controls (p < 0.001) and 2.5 times higher than the pSV-VEGF (p < 0.01) and 2 times higher than the pEpo-SV-VEGF (p < 0.05). Conclusion; Therefore pRTP801-VEGF will be useful for the VEGF gene therapy for ischemic heart disease with strong induction under hypoxic condition.