Targeted and regulable expression of transgenes in hepatic stellate cells and myofibroblasts in culture and in vivo using an adenoviral Cre/loxP system to antagonise hepatic fibrosis.
BACKGROUND
Activated hepatic stellate cells (HSCs) are an attractive target for antifibrotic therapy based on their key role in extracellular matrix accumulation during liver injury.


OBJECTIVE
: To develop a system for regulable and cell-specific gene expression in HSCs to enable targeted delivery of therapeutic genes.


METHODS
Two types of recombinant adenoviral vectors were constructed, one expressing the Cre gene under the surveillance of specific promoters and the other containing a potent expression unit that was activated by Cre recombinase-mediated recombination to remove an upstream lox-flanked "stuffer" sequence, thereby amplifying the expression of downstream transgene of interest while maintaining specificity.


RESULTS
When the promoter of the collagen 1A2 gene drove Cre recombinase expression in primary quiescent rat HSC, modest green fluorescence protein (GFP) expression was observed. However, in activated HSC, the collagen promoter effectively drove Cre recombinase activity, as assessed by the increased expression of GFP. In contrast, GFP expression was barely observed when the collagen promoter was expressed in hepatocytes. HSC-specific expression of Smad7 considerably reduced the expression of type I collagen in culture and decreased fibrosis in two liver fibrosis models. Finally, to achieve targeted clearance of activated HSC in culture and in vivo, thymidine kinase was selectively expressed under the control of the collagen promoter, which conferred cell-specific killing by ganciclovir leading to reduced fibrosis.


CONCLUSIONS
Our results show the potential utility of transcriptionally controlled gene therapy using a Cre/loxP system to ameliorate hepatic fibrosis in vivo.
Kinoshita
Kohji
K
First Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan.
Iimuro
Yuji
Y
Fujimoto
Jiro
J
Inagaki
Yutaka
Y
Namikawa
Kazuhiko
K
Kiyama
Hiroshi
H
Nakajima
Yuji
Y
Otogawa
Kohji
K
Kawada
Norifumi
N
Friedman
Scott L
SL
Ikeda
Kazuo
K
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D022781:Hepatocytes / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D008106:Liver Cirrhosis, Experimental / Q000378:metabolism / Q000628:therapy*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D051906:Smad7 Protein / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism; D019076:Transgenes
D051906:Smad7 Protein; C497808:Smad7 protein, rat; D013937:Thymidine Kinase