AIM: To develop a conditionally replicative gene-viral vector system called CNHK500-p53, which contains dual promoters within the E1 region, and combines the advantages of oncolytic virus and gene therapies for hepatocellular carcinoma (HCC).METHODS: CNHK500-p53 was constructed by using human telomerase reverse transcriptase (hTERT)promoter to drive adenovirus E1a gene and hypoxia response element (HRE) promoter to drive adenovirus E1b gene. p53 gene expressing cassette was inserted into the genome of replicative virus. Viral replication experiments, cytopathic effect (CPE) and methyl thiazolyl tetrazolium (MTT) assay were performed to test the selective replication and oncolytic efficacy of CNHK500-p53.RESULTS: Immunohistochemistry verified that infection with CNHK500-p53 was associated with selective replication of adenovirus and production of p53 protein in telomerase-positive and hypoxia-inducible factordependent HCC cells. p53 protein secreted from HepG2,infected with CNHK500-p53 was significantly higher than that infected with nonreplicative adenovirus Ad-p53 in vitro (388 ± 34.6 μg/L vS 76.3 + 13.17 μg/L).Viral replication experiments showed that replication of CNHK500-p53 and CNHK500 or WtAd5, was much stronger than that of Ad-p53 in tested HCC cell lines. CPE and MTT assay indicated that CNHK500-p53 selectively replicated in and killed HCC cells while leaving normal cells unaffected.CONCLUSION: A more efficient gene-viral system is developed by combining selective oncolysis with exogenous expression of p53 against HCC cells.