Study On Targeting Transcriptional Vector Driven By Human Telomerase Gene Promoter To Restrict To Tumors In Vitro

One of the major challenges encountered in gene therapy is appropriate spatial and temporal control of the expression of the therapeutic gene. This is especially true in the numerous cancer gene therapy regimens where extratumoral expression of the transgene can result in the destruction of normal tissue and lead to undesirable toxicity. Gene transfer vectors will dramatically increase the safety and effectiveness of the therapeutic products to target tumors. Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in immortalized cells and >85% of human cancers but is quiescent in most normal somatic cells. As the RNA and catalytic components of telomerase are sufficient to reconstitute telomerase activity in vitro, transcription initiation at the hTERT promoter appears to bethe limiting step in the production of active telomerase. Therefore, the use of hTERT promoter-driven vector system could restrict the expression of therapeutic products to telomerase-positive tumors. In this study, we constructed the expression vector of report gene afforded by the hTERT promoter and investigated its effect on tumor cells and normal cells. One promoter fragment was generated that comprise 2000 basepairs of sequences upstream of the hTERT initiation codon, and constructed retrovirus vector carrying the hTERTp/egfp or hTERTp/p53 or hTERTp/tk cassette retains the tumor-specific expression imparted by the hTERT promoter. In transfection experiments, the hTERT promoter driven EGFP report gene were transfected by Lipofectamine-mediated gene transfer. Forty-eight hours after transfection, the EGFP component could be detected in telomerase-positive tumor cells using a fluorescent microscope with a FITC filter set, and not found in telomerase-negative normal cells. Transient transfection with the hTERT-promoter/P53 construct induced apoptosis in telomerase-positive tumor cells of wide range by flow cytometry analysis. In contrast, normal fibroblast cells without telomerase didnot undergo apoptosis following the hTERT-promoter/P53 transfer. The hTERT promoter driven thymidine kinase gene conferred ganciclovir sensitivity to all tumor and immortal cell lines tested, whereas normal somatic cells retained largely unaffected. These results indicated that by restricting the interest gene to tumor cells, the hTERT promoter allows the tumoricidal effect of the interest gene to be exerted without detrimental consequences on healthy tissue and vital organs. Thus, the application of the hTERT promoter-system is expected to represent a promising approach for the cancer gene therapy of a wide range of human malignancies.