| The concept of virotherapy, an approach to the treatment of cancer with viruses, was inspired in the early 19th by the observation of occasional tumor regressions in cancer patients suffering from virus infections or receiving vaccinations. Many different viruses were subsequently tested in clinical trials as lytic agents of tumor cells, but a low efficacy/toxicity ratio led to their abandonment. Following the development of virology, molecular biology and oncology, virus has been genetically modified with the aim of making tumor-specific replication virus. Without the replicative potential in normal cells, tumor-specific replication virus will selectively replicate in tumor cells, lyse them and then cause a further infection in adjacent tumor cells. Currently there have been more than 10 kinds of replication-selective viruses being tested in clinical trials. Among them, ONYX-015 is the most effective one, which efficiently replicates in tumor cells with a^ W ?1*. * 'Hi ,'ii K K-fc ?ifiL vfJM ?ft * -il> - 9 -deletion of a gene encoding a p53 protein, E1B-55KD, would be selective for tumors that had already lost p53 function. p53 function is lost in the majority of human cancers. Now phase HI clinical test of ONYX-015 has been carried out. The published data about Chemotherapy-naive head and neck cancer patients treated with combination chemotherapy and ONYX-015 showed that 7 patients among 10 got significant rumor regression (<50%). Up to date, this is the most effective biological therapy. However, single agent responses of ONYX-015 across all studies were rare. Considering the severe side effect of chemotherapy, it is obvious that neither ONYX-015 alone nor the combination of ONYX-015 with chemotherapy is ideal, and hence to find a more effective anti-tumor strategy based on tumor-specific replication becomes urgent and meaningful.Cytokine therapy is another important biological method to cure carcinoma. But the half-life of cytokine is generally very short and the high dose, repeating injection are needed and cause severe side effect. Cytokine gene therapy, an approach to transfer cytokine or cytokine receptor gene into patients, has been studied broadly and made great progress recently. But the curative effect of cytokine gene therapy is still- 10-not very ideal due to two main obstacles lying in this method, one is the vector lack of selectivity, the other is low efficiency of gene transfer during gene therapy.In this study, combined the virus of virotherapy and cytokine gene therapy, we have established a tumor-specific replication-competent adenovirus vector system that carry interferon- Y gene in order to get a system which a deletion of ElB55KD.The properties and anti-tumor effects of this vector have been studied.1. Constructions of the tumor-specific replication-competent adenovirus-mediated transfer of mIFN- Y .First, the vector pCA13 was digested at the site of 764bp and 1214bp by the endonuclease BamH I and EcoR I ,then obtained 6460bp fragment. Second, linked the fragment with the mIFN- Y gene, a 466bp fragment, named as pCA13-mIFN-Y .Third, pCA13-mIFN-Y , an El region deficient adenovirus vector, was digested by the restriction endonuclease Bgl II , and the obtained fragment containing CMV promoter, mIFN- Y gene and SV40 poly-A signal was inserted in the Bgl II restriction site of the vector pXC-delElb. Fourth, the needed recombination were selected by the method of PCR. Fifth, the plasmidspXC-delElb-mlFN- y were transfected into the 293 cell plant together with the plasmid pBGHE3 containing the right arm sequence of adenovirus type 5.The virus plague was sublimated three times, and adenovirus DNA was amplified by the method of PCR. After the sequence was verify, the vector was named as CNHK200-mlFN- y .And the virus was propagated in 293 cells, the amount of propagated viruses was titrated by TCID50 method.2. Constructions of the tumor replication-defected adenovirus -mediated...
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