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Thromb Haemost 2003; 90(03): 398-405
DOI: 10.1160/TH03-01-0010
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Regulation of human factor IX expression using doxycycline-inducible gene expression system

Mahmoud A. Srour   *
1   Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
,
Henry Fechner   *
2   Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
,
Xiaomin Wang
2   Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
,
Ulrike Siemetzki
2   Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
,
Thilo Albert
1   Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
,
Johannes Oldenburg
3   Institute of Transfusion Medicine and Immune Hematology of the German Red Cross Blood Donor Service Baden Württemberg–Hessen, Frankfurt, Germany
,
Peter Hanfland
1   Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
,
Wolfgang Poller
2   Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
,
Hans-Hermann Brackmann
1   Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
,
Rainer Schwaab
1   Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
› Author AffiliationsFinancial support: This study was supported by a research grant from Aventis, Germany. Additionally, MAS was supported by a scholarship from the German Academic Exchange Service.
Further Information

Publication History

Received 08 January 2003

Accepted after resubmission 09 June 2003

Publication Date:
05 December 2017 (online)

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Summary

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovec-tors: a response vector expressing hFIX (Ad5. TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5. CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5. TRE.hFIX in all cell lines in combination with Ad5. CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5. TRE.hFIX/Ad5. CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5. TRE.hFIX/Ad5. CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5. CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5. CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.

Keywords

Human factor IX - Tet-On-system - adenovirus - doxycycline - human alpha1-antitrypsin promoter

* Both authors contributed equally to this work


 

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