Promoter elements for gene therapy of regenerating hepatic tissue.

We have identified and characterized two transcriptional modules derived from the promoters of liver genes whose expression is specific for proliferating hepatocytes: A) An enhancer module from the HNF1β promoter. HNF1α and β are two trans-acting factors that play a central role in the expression of liver-specific genes. We reported that, during mouse and rat development, HNF1β expression precedes that of HNF1α and is inducible by retinoic acid treatment. Moreover HNF1β (but not HNF1α) is expressed in undifferentiated hepatocytes. A combined strategy of deletions and site-specific mutagenesis allowed us to identify the cis-acting elements of the HNF1β promoter. In our transient expression assay, the -435/+33 region of the promoter is sufficient to reproduce the expression pattern of the endogenous gene. This region can be further subdivided into a tissue-specific enhancer and a proximal promoter element. The transcriptional activity of the enhancer element, as measured by co-transfection assays and band-shift experiments, depends on the binding of two transcriptional activators: the liver-specific HNF-4 factor (-255/-235), and the RARα/RXRα heterodimeric protein, that recognize a RARE element partially overlapping with the HNF-4 binding site. B) The proximal element of the alpha-foetoprotein promoter. The alpha-fetoprotein (AFP) gene is expressed in the prenatal life in the yolk sac and in the foetal liver. At birth, the AFP gene is "shut down" at the transcriptional level, and it can be re-expressed in the adult liver only in pathologic conditions, like hepatic parenchyma regeneration or liver tumours. Therefore, the AFP gene must contain cis acting regulatory elements able to activate transcription only in proliferating hepatocytes. We have isolated and individually analysed the key elements of the AFP transcriptional regulation located in the proximal promoter region, where two partially overlapping AP1 and GRE elements dictate the activation or repression of the gene, respectively. Our results suggest that, while two adjacent HNF1α/β binding sites restrict the AFP promoter activity to liver cell, changes in the GR/AP1 ratio can account for the AFP reactivation in the regenerating hepatocyte. We have constructed a series of artificial promoters by assembling different combinations of these transcriptional modules. The most efficient combinations have been used to construct SV40-derived episomal vectors, that we plan to use for gene therapy of proliferating hepatocytes. These experiments are currently in progress.