Endothelial cell-specific expression of tumor necrosis factor-alpha from the KDR or E-selectin promoters following retroviral delivery.
The tumor vasculature offers a target for anti-cancer gene therapy which has the advantages both of good accessibility to systemically delivered therapy and comparative homogeneity across solid tumor types. We aimed to develop retroviruses carrying endothelial-specific promoters for the delivery of genes to proliferating endothelial cells in vitro and to tumor endothelial cells in vivo. This paper reports the generation of such retroviral vectors and the level of expression of murine tumor necrosis factor-alpha (mTNF-alpha) cDNA following infection into endothelial cells and stromal fibroblasts. Retroviral vectors carrying mTNF-alpha have been generated whose expression is controlled either by the retroviral long terminal repeat or by 5' proximal promoter sequences from the endothelial-specific kinase insert domain receptor (KDR)/VEGF receptor and E-Selectin promoters within the context of a self-inactivating (SIN) vector backbone. Both KDR and E-Selectin have been shown to be upregulated on tumor endothelium. A putative polyadenylation sequence AAATAAA within the E-Selectin promoter was mutated to permit faithful transmission of retroviral vectors carrying this promoter. We demonstrate a 10- to 11-fold increase in mTNF-alpha expression from promoter elements within sEND endothelioma cells as compared to NIH-3T3 fibroblasts. Suggestions for further improvements in vector design are discussed.
Jaggar
R T
RT
Imperial Cancer Research Fund Molecular Oncology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK.
Chan
H Y
HY
Harris
A L
AL
Bicknell
R
R
D016475:3T3 Cells; D000818:Animals; D019040:E-Selectin / Q000235:genetics*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D017978:Receptors, Growth Factor / Q000235:genetics*; D040262:Receptors, Vascular Endothelial Growth Factor; D012190:Retroviridae; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D019040:E-Selectin; D017978:Receptors, Growth Factor; D014409:Tumor Necrosis Factor-alpha; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor