Development of Chimeric Gene Regulators for Cancer-specific Gene Therapy with both Transcriptional and Translational Targeting

Cancer gene therapy has been of great challenge in achieving maximal high levels of specificity and more rational efficiency in target cancer cell. We herein developed a novel approach for cancer-specific gene therapy using both transcriptional and translational targeting regulation. We integrated the tumor-specific gene promoter of hTERT, the 5'UTR of bFGF-2, the enhancer of woodchuck hepatitis virus post-transcriptional regulatory element (WRE), and/or the 3'UTR of the human EGFR into two major chimeric gene regulators. We found that chimeric gene regulator I (hTERT_5'UTR center dot center dot center dot WRE_BGHpolyA) enhanced the specificity of expression in hepatocellular carcinoma (HCC) cells up to 300% in total due to increases at both the transcriptional and translational levels but only 120-200% enhancement at the transcriptional level and 120-180% enhancement at the translational level. In addition, chimeric gene regulator II (hTERT_5'UTR center dot center dot center dot WRE_3'UTR_BGHpolyA) improved the specificity to 550% and also highly strengthened the stability of the mRNA. In vitro cytotoxicity assays demonstrated that HCC cell growth was inhibited by HSV-1 TK expression under the control of both chimeric regulators, with a relative cell viability of similar to 80% for 2 days and similar to 85% for 4 days after transfection, respectively. These observations represent a new approach for highly tumor-specific gene expression and also provide insights into application to cancer gene therapy.