Construction of a highly hepatoma specific adenoviral vector system carrying NIS gene activated by AFP enhancer

1548 Objectives: Cancer specific killing can be achieved by therapeutic gene activated by a cell specific promotor. Degree of over-expression by enhancer element is a major component for specific cancer killing effect. This study was performed to construct a recombinant adenoviral vector carrying highly efficient hepatoma specific AFP enhancer element with therapeutic sodium iodide symporter (NIS) gene for targeting AFP producing hepatocellular carcinoma. Methods: AFP enhancer, that -4.1~-3.3kb upstream to the human AFP gene, was selected by PCR technique with genomic DNA template. The selected AFP enhancer fragment was fused to pGL3-promoter vector with luciferase gene. Hepatoma cell specific expression of AFP ehhancer was calculated with measured luciferase activity using luminometer in hepatoma (HepG2, Hep3B, SNU423, SNU354)and non-hepatoma cell lines(293 : human embryonic kidney cell), which were transfected with the plasmid with/without AFP enhancer by lipofectamine and plus reagent assistance. The constructed AFP enhancer with NIS gene replaced cytomegalovirus promoter region in pShuttle and pAdTrack, and a recombinant adenovirus vector carrying NIS gene activated by AFP enhancer was constructed with the AdEasy system. hepatoma and non-hepatoma cell lines were transfected with the adenovirus. Expression of the NIS gene at mRNA level was elucidated by RT-PCR. Results: A 0.8 kb AFP enhancer was successfully synthesized. NIS gene with AFP enhancer were directly inserted into pShuttle and pAdTrack. These insertion of the target gene to shuttle vectors were confirmed by restriction enzyme digestion and DNA sequencing technique. Hepatoma cell lines with NIS gene with AFP enhancer has higher luciferase activity than that of non hepatoma cell lines (Table 1). A recombinant adenoviral vector system carrying NIS gene activated by AFP enhancer was also successfully constructed. Hepatoma cell lines transfected by the adenoviral system showed marked over-expression of the NIS gene, but non hepatoma cell line transfected by the adenovirus showed trivial expression of the gene. Conclusions: We constructed a highly efficient and hepatoma specific AFP enhancer, and also constructed NIS gene activated by the specific promoter system. This adenoviral system can be used as a potent and specific therapy for the AFP producing carcinomas.