Abstract
We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.
Highlights
The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can selectively and efficiently deliver genes to target organs with minimal toxicity (Niidome and Huang, 2002)
That a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region
A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon
Summary
The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can selectively and efficiently deliver genes to target organs with minimal toxicity (Niidome and Huang, 2002). Development of efficient endothelial cell selective vector systems will be invaluable to cancer gene therapy as well as other vascular diseases (Martin and Murray, 2000). A few studies have reported construction of endothelial cell specific viral vectors using sequences from von-Willebrand factor, intracellular adhesion molecule 2, vascular endothelial growth factor receptor, or E-selectin (Modlich et al, 2000; He et al, 2001). To develop highly efficient and endothelial cell specific expression plasmids, we chose to use the preproendothelin-1 (ppET-1) gene promoter because it's regulation and expression in endothelial cells are well studied We show that our newly constructed vector based on ppET-1 gene promoter and 5' untranslated region drives high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxiamimic conditions due to a natural hypoxia responsive element within the promoter region. Our results show that the vector could be a potentially useful tool for vascular targeting of vascular disease and cancer gene therapy
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