Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers.
One of the current challenges in gene therapy is to construct a vector that will target specific tissues. Targeting expression to endothelium is of particular interest in the treatment of several pathologies. We have shown previously that defined regions of the E-selectin and KDR promoters confer endothelial cell specific expression following retroviral delivery. However, the levels of expression were low. In an attempt to increase expression but to preserve the tissue specificity we have examined hypoxic and cytokine-inducible enhancer elements in combination with the KDR and E-selectin promoters. Both enhancers should be active in the tumour environment, boosting expression and giving additional specificity of gene expression in the tumour endothelium. The hypoxia response element (HRE) of the murine phosphoglycerate kinase-1 (PGK-1) promoter was used as a hypoxic enhancer and the tandem-binding site for NFKB from the murine vascular cell adhesion molecule-1 (VCAM-1) promoter as a cytokine-inducible enhancer. The HRE conferred hypoxia inducibility to the KDR and E-selectin promoters. Endothelial specificity of expression was retained with the KDR but not the E-selectin promoter. The NFKB-binding site conferred responsiveness to TNF-alpha to the KDR promoter, however the level of induction was less than that achieved with the HRE. Retrovirus combining both enhancer elements transferred inducibility by hypoxia and TNF-alpha, and reached the highest expression levels upon stimulation. These results confirm that heterologous enhancer elements may operate on a single endothelial cell specific promoter. These findings make the use of inducible enhancers a promising strategy for increasing tissue specific gene expression.
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Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, UK.
Pugh
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Bicknell
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D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D019040:E-Selectin / Q000235:genetics; D004727:Endothelium / Q000166:cytology* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D051379:Mice; D016328:NF-kappa B / Q000235:genetics; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D017978:Receptors, Growth Factor / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor; D014162:Transfection; D014407:Tumor Cells, Cultured
D019040:E-Selectin; D016328:NF-kappa B; D017978:Receptors, Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor