| Background:Virotherapy, an approach to treat cancers with viruses, was inspired early in 19th by the observation of occasional tumor regressions in cancer patients suffering from virus infections or receiving vaccinations. Over last century, a wide varity of wild-type viruses have been used in the treatment of cancer patients, but the approach was temporarily abandoned due to the toxicity of viruses. With the development of virology, molecular biology and oncology, to improve the safety and antitumoral capacity of viruses through genetically engineering them became possible. This reburned the study of virotherapy, while the discovery of the replication-selective viruses further expanded it. Up to date, at least ten different engineering viral species have been or will soon be in clinical trials, including conditionally replicative adenovirus (CRAD), herpes simplex virus, vaccinia virus, reovirus and Newcastle disease virus. Tumor-specific replicative viruses are the viruses that do not replicate in normal cells, but selectively replicate in tumor cells, kill them, and subsequently infect adjacent tumor cells until all tumor cells are killed. ONYX-015 is the most successful tumor-specific replicative adenovirus used for cancer therapy. With the deletion of the E1B-55KD gene region, which is responsible for binding with p53, ONYX-015 therefore only targets cancer cells with genetic defect of p53 function. ONYX-015 is being in a phase III clinical trial. Studies showed that treating chemotherapy-naive head and neck cancer patients with acombination of chemotherapy and ONYX-015, 7 among 10 of them obtained significant tumor regression. This is the most effective biological therapy by now. However, single-agent antitumor activity with ONYX-015 was minimal (<20%) despite repeated daily injections.The gene of p53 is one of the most important tumor suppressors, which closely correlate with the occurrence, development, and the prognosis of patients suffering from cancer. The function of p53 pathway is lost in most human tumors through diverse mechanisms; the wide type p53 transduction is then an important method to cure carcinoma. But the currently used vector systems are not satisfying because of their low transfer rate. Considering that tumor-specific replicative adenovirus can selectively replicate in tumor cells, constructing a gene-viral vector, i.e., inserting the anti-tumor gene such as p53 into the genome of the tumor-specific replicative virus may be able to combine the advantages of the gene therapy and virus therapy and hence a good stratage to overcome the above obstacle.Herein, we generated a replication-selective adenovirus CNHK600-p53, which carried the anti-tumor gene p53, deleted the E1A-CR2 gene region that is responsible for binding/inactivating pRB family members, retained the adenovirus E3 gene region that is responsible for the escapase of adenovirus from the immune response of host cells and for its lysis on host cells, improved the tumor selectivity of the adenovirus by controlling the expression of adenovirus E1A gene by the hTERT promoter and that of E1B gene by the hypoxia response promoter. This vector possesed all the advantages of gene therapy and virus therapy, increased the expression of p53, thus displayed efficient tumor-selective killing capacity both in vitro and in vivo.Methods and Results:1.Construction of replication-selective adenovirus CNHK600-p53.The vector pClonl7 was digested by the endonuclease EcoR I and Nhe I , then joined the bigger fragment with the p53 gene which was 1179bp, named as pClonl7-p53. Digestion of the vector with Age I ^i Not I at the site of 403bp and 2872bp led to one 2469bp fragment containing CMV promotor, the gene of p53 and SV40 poly-A. Insertion of this fragment into the CNHK600produced a new plasmid CNHK600-p53. The plasmid CNHK600-p53 was co-transfected with ppE3 into 293 cells to produce the new recombinant adenovirus CNHK600-p53 by homologous recombination. CNHK600-p53 adenovirus was propagated in 293 cells and purified by cesium chloride gradient centrifugation. The viral titer was 1.99 X 1010 pfu/ml determined by TCID50 method. 2.Selective replication and oncolysis of CNHK600-p53 in vitro.Viral replication experiments were performed to evaluate the selective replication ability of CNHK600-p53 in tumor cells. In tested tumor cell lines, replication of CNHK600-p53 is satisfying especially in lung cancer cells NCI-H1299 where the replication times of the virus for 96h was as high as 40625. On the contrary, CNHK600-p53 hardly replicate in normal cell lines, indicating the low toxicity of CNHK-p53 in normal cells. MTT assay was performed to determine cell viability at various viral MOIs. Results showed that the MOI of CNHK600-p53 that killed 90% of Hep3B cells was 0.06 ± 0.02, while Ad-p53 was 542.33 + 71.84.3.Antitumor efficacy of CNHK600-p53 lung cancer xenografts in BALB/C mice.
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