Construction Of Transcriptional Targeting Replication-defective Recombinant Adenoviruses Containing FCY₁ Driven By Modified HTERT Core Promoter

The strategy of enzyme-prodrug has been widely used in experimental research of ovarian cancer gene therapy. However, the strategy itself cannot achieve specificity for ovarian career cells, and will still result in side effect to normal cells. In recent years, transcriptional targeting strategy has become one of the focus of tumor gene therapy. Quite a few studies have combined these two strategies, and have shown, to some extent, selective cytotoxicity to cancer cells. However, the activities of the promoters employed in transcriptional targeting strategy have been detected , though very low , in many other tissues therefore limit the clinical application of the combined strategy.In this study, what we've tried to construct is a promoter sequence efficiently and specificially activated in ovarian cancer cells, by using which to control the transcription of prodrug-converting enzyme gene. To achieve tissue-specificity and tumor-specificity, we designed and constructed the tandem sequences of estrogen responsive element(ERE) and hypoxia-responsive element(HRE) to doubly modify hTERT core promoter as cis-acting elements, based on the facts of estrogenenvironment of ovary, hypoxia micro-enviroment of tumor and relationship between telomerase and tumors. Then, we constructed replication-defective recombinant advenovirus containing FCYj gene driven by modified promoters to estimate the activity of the promoters and cytotoxicity to ovarian cancer cells, so that we could determine whether the modified promoter is suitable for clinical application.1. Modification of hTERT core promoterTandem sequences of estrogen response element and hypoxia-responsive element were constructed and subcloned to the upstream of hTERT core promoter. Thus the promoter sequence with specificity for ovarian carcinoma was obtained.2. Construction of shuttle plasmidThe promoter sequence and the FCYj gene fragment were subcloned into shuttle plasmid.3. Construction of recombinant adenovirusesThe shuttle plasmid and rescue plasmid were used to co-transfect the HEK 293 cells. Infected cells typically remain intact but round up and may detach from the plate, and these changes are collectively referred to as the cytopathic effect (CPE), which appears within 7 to 10 days after the co-transfection.4. Identification of recombinant adenovirusesThe DNA was extracted from the virus culture medium and identified by PCR using FCYi specific primers. The results show that, by PCR, we have obtained the122bp targeted fragment from the viruses.5. Amplification of adenoviruses and determination of the recombinantadenoviral titersAfter the amplification, the recombinant adenoviral titers were determined by end-point dilution assay. Titer of the recombinant adenoviruses is 6.2X 107pfu / ml.In conclusion, we successfully constructed recombinant replication defective adenoviruses with FCY1 gene driven by hTERT core promoter modified by tandem sequences of estrogen response element and hypoxia-responsive element, which may enable us to further evaluate the use of ERE, HRE and hTERT core promoter in transcriptional targeting gene therapy of ovarian carcinoma.