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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
  1. Kohji Kinoshita1,
  2. Yuji Iimuro1,
  3. Jiro Fujimoto1,
  4. Yutaka Inagaki2,
  5. Kazuhiko Namikawa3,
  6. Hiroshi Kiyama3,
  7. Yuji Nakajima4,
  8. Kohji Otogawa5,
  9. Norifumi Kawada5,
  10. Scott L Friedman6,
  11. Kazuo Ikeda4
  1. 1First Department of Surgery, Hyogo College of Medicine, Nishinomiya, Japan
  2. 2Liver Fibrosis Research Unit, Department of Community Health, Tokai University School of Medicine, Isehara, Japan
  3. 3Department of Anatomy & Neurobiology, Graduate School of Medicine, Osaka City University, Osaka, Japan
  4. 4Department of Anatomy, Graduate School of Medicine, Osaka City University, Osaka, Japan
  5. 5Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan
  6. 6Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, USA
  1. Correspondence to:
    Dr K Ikeda
    Department of Anatomy, Graduate School of Medicine, Osaka City University, 1-4-3, Asahimachi, Abeno, Osaka, 545-8585, Japan;ikeda{at}med.osaka-cu.ac.jp

Abstract

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.

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

Method: 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.

Conclusion: Our results show the potential utility of transcriptionally controlled gene therapy using a Cre/loxP system to ameliorate hepatic fibrosis in vivo.

  • BDL, bile duct ligation
  • CAG, cytomegalovirus enhancer, chicken β-actin promoter and a part of 3′ untranslated region of rabbit β-globin
  • COL, collagen
  • GAPDH, glyceraldehyde-3-phosphate dehydrogenase
  • GCV, ganciclovir
  • GFAP, glial fibrillar acidic protein
  • GFP, green fluorescent protein
  • HSC, hepatic stellate cell
  • LCM, Laser capture microdissection
  • LNL, loxP-neo-loxP
  • MOI, multiplicity of infection
  • PDGF, platelet-derived growth factor
  • PFU, plaque-forming unit
  • RT-PCR, reverse transcription polymerase chain reaction
  • αSMA, smooth muscle-α actin
  • TAA, thioacetamide
  • TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling

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Footnotes

  • Published Online First 6 September 2006

  • Funding: This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan (C16590150 to KI), and the NIH (DK56601 to SLF), as well as the Feld Fibrosis Program (to SLF).

  • Competing interests: None.

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