Development of a hypoxia-responsive vector for tumor-specific gene therapy.
We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. As vascular endothelial growth factor (VEGF) is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with five copies of hypoxia-responsive elements (HREs) derived from the 5'-untranslated region (UTR) of the human VEGF showed excellent transcriptional activation at low oxygen tension relevant to tumor hypoxia. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. In addition, we also investigated whether the 3' UTR of the VEGF gene would confer increased post-transcriptional mRNA stability under hypoxic conditions. However, despite increases in the hypoxic/aerobic ratio of luciferase activity, gene expression with 3' UTR was lower due to mRNA destabilization by AU-rich elements (AREs). Thus, we found no benefit from the inclusion of the 3' UTR in our vectors. Of all the vectors tested, the combination of 5HRE and a CMV minimal promoter exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter. We propose that this vector would be useful for tumor selective gene therapy.
Shibata
T
T
Mayer Cancer Biology Research Laboratory, Department of Radiation Oncology, Stanford University School of Medicine, CA 94305-5468, USA.
Giaccia
A J
AJ
Brown
J M
JM
D020121:5' Untranslated Regions / Q000235:genetics; D015687:Cell Hypoxia; D003587:Cytomegalovirus / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D016228:Endothelial Growth Factors / Q000235:genetics*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases / Q000235:genetics; D008222:Lymphokines / Q000235:genetics*; D009369:Neoplasms / Q000378:metabolism / Q000628:therapy*; D009687:Nuclear Proteins / Q000235:genetics; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D016228:Endothelial Growth Factors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008222:Lymphokines; D009687:Nuclear Proteins; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D008156:Luciferases