Selection of tumour specific promoters for adenoviral gene therapy of cholangiocarcinoma

Abstract

The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector. mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes. mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines. The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.