Development and characterization of a synthetic promoter for selective expression in proliferating endothelial cells.
BACKGROUND
Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues.


METHODS
Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter.


RESULTS
The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues.


CONCLUSIONS
These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors.
Berlex, Richmond, CA, USA.
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P
Szymanski
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Anwer
S M
SM
Sullivan
D000818:Animals; D002417:Cattle; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D016232:Endothelins / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection; D019737:Transplants
C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D016232:Endothelins; D009687:Nuclear Proteins