Targeted and regulable expression of transgenes in hepatic stellate cells using an adenoviral Cre-loxP system

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Hepatic stellate cells (HSCs) are responsible for extracellular matrix synthesis accumulation, leading to liver fibrosis. The aim of this project was to develop a regulable adenoviral transduction system in order to modulate HSC activation and ultimately to either abrogate their generation of extracellular matrix or promote apoptosis. For this purpose, we constructed two recombinant adenoviral systems ; one expressing the Cre gene under the control of specific promoters and the other containing a potent expression unit that was activated by Cre recombinase-mediated recombination to remove an upstream LoxP-flanked ‘stuffier' sequence, thereby transcribing the downstream transgene of interest (Cre-loxP system). This Cre-loxP system was analyzed as a potential method to achieve the regulated expression of specific genes in HSCs. When promoter for the collagen 1A2 gene drove Cre recombinase expression in primary quiescent rat HSC, expression of reporter gene was observed. However, in activated HSC, the collagen promoter effectively drove Cre recombinase activity, as assessed by the expression. In contrast, the expression was barely observed when the collagen promoter was expressed in hepatocytes. HSC-specific expression of Smad7 significantly reduced the expression of type I collagen in culture and decreased fibrosis in two liver fibrosis models. These results demonstrate the potential utility of transcriptionally controlled gene therapy using a Cre/loxP system to ameliorate hepatic fibrosis in vivo.