1052. A Versatile Chimeric Enhancer Vector That Is Highly Inducible by Hypoxia and Metals

Abstract

To develop a potent hypoxia-inducible vector, we evaluated the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient trasfection assays, combination of three copies of HRE (3xHRE) with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3xHRE (E-M-H), a hypoxia induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus (HCMV) promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. The E-M-H was also tested for induction by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+turned out to be potent inducers whereas hydrogen peroxide worked as a moderate inducer. Gel shift assay together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1|[alpha]| (HIF-1|[alpha]|), metal transcription factor-1 (MTF-1), and Egr-1 may be associated with the high inducibility of E-M-H enhancer. In vivo evaluation of the E-M-H vector in ischemic calf muscle revealed that luciferase expression was significantly higher in E-M-H group than in control, H, or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.