An Experimental Research On Gene Therapy Of Osteosarcoma By Targeted Transcription Of FCY1 Gene

With the progress of gene therapy, the results of researches using strategies of transcriptional targeting and gene delivered enzyme-prodrug are exciting. It has been reported that the recombinant adenovirus, which contains the osteocalcin promoter that drives the expression of herpes simplex virus thymidine kinase, can selectively be cytotoxic to the osteosarcoma and its lung metastasis. However, the activity of osteocalcin promoter has been detected though very low in many non-osteogenic tissues therefore limit its clinical application.More and more research results demonstrate that the reactivation of telomerase plays an important role in cancer development and progression. And the experiments in which hTERT promoter is used as a transcriptional targeting promoter driving therapeutic genes had been proved to be successful in treating several types of malignancies. In order to further improving the selectivity and the specificity of the activity of hTERT promoter and restricting the expression of transgene to the tumor tissues, we designed and constructed several tandem sequences of hypoxia-responsive element and OSE2 based on the facts that hypoxiais a common feature of solid tumors and osteosarcoma is osteogenic.3The tandem sequences were confirmed by sequencing and then were used to modify the hTERT core promoter.Two groups of replication-defective recombinant advenoviruses containing FCYl or luciferase gene driven by modified promoters were constructed in order to estimate the activity of the promoters and the cytotoxicity to osteosarcoma cells, so that we can determine whether the modified promoter are suitable for clinical application in the future.1. Co-transfection the HEK 293 cells and obtaining the recombinant adenovirusesThe shuttle plasmids and the rescue plasmid are 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. After the co-transfection, cytopathic effect appeared within 7 to 10 days.2. Evaluating recombinant adenoviruses by detection the virus DNA The DNA were extracted from the recombinant virus culture mediumand detected by PCR using FCYl or luciferase specific primers. The results show that all the eight viruses of the FCYl group can amplify the 122bp targeted fragment and all the eight viruses of the luciferase group can amplify the 312bp targeted fragment.3. Determining the recombinant adenoviral titersAfter the amplification, the recombinant adenoviral titers were determined by plaque assay and end-point dilution assay. Though the titers determined by the two methods are different, they confirmed that we obtained recombinant adenoviruses with high titers.44. Evaluating the recombinat adenoviruses by detecting the virus mRNAA telomerase positive cell line A549 was infected by the two groups of adenoviruses. Fouty-eight hours later, total RNA were isolated from the infected cells to perform RT-PCR using FCY1 specific primers. Primers specific for human 0 -actin serves as internal control. The results show that all the samples infected with recombinant adenoviruses can amplify the 122bp targeted fragment as well as the 185bp?-actin targeted fragment at the same time.In conclusion, we have successfully constructed two groups of recombinant replication defective adenoviruses with FCY1 or luciferase gene driven by hTERT core promoter modified by tandem sequences of hypoxia-responsive element and OSE2. These two groups of viruses can enable us further evaluate whether the modified hTERT core promoter can be used in clinical gene therapy of osteosarcoma in the future.