PDF herunterladen
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
› InstitutsangabenFinancial 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.
Weitere Informationen

Publikationsverlauf

Received 08. Januar 2003

Accepted after resubmission 09. Juni 2003

Publikationsdatum:
05. Dezember 2017 (online)

Auch verfügbar auf
    Lizenzen und Reprints

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


 

  • References

  • 1 Gossan M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 9: 5547-51.
    PubMedSuche in Google Scholar
  • 2 Gossan M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. Transcriptional activation by tetracycline in mammalian cells. Science 1995; 268: 1766-9.
    CrossrefPubMedSuche in Google Scholar
  • 3 No D, Yao TP, Evans RM. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 1996; 93: 3346-51.
    CrossrefPubMedSuche in Google Scholar
  • 4 Wang Y, Xu J, Pierson T, O’Malley BW, Tsai SY. Positive and negative regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. Gene Ther 1997; 4: 432-41.
    CrossrefPubMedSuche in Google Scholar
  • 5 Burcin MM, Schiedner G, Kochanek S, Tsai SY, O’Malley BW. Adenovirus-mediated regulatabale target gene expression in vitro . Proc Natl Acad Sci USA 1999; 96: 355-60.
    CrossrefPubMedSuche in Google Scholar
  • 6 Rivera VM, Clackson T, Natesan S. et al. A humanized system for pharmacologic control of gene expression. Nature Med 1996; 2: 1028-32.
    CrossrefPubMedSuche in Google Scholar
  • 7 Kung SH, Hagstrom JN, Cass D. et al. Human factor IX corrects the bleeding diathesis of mice with hemophilia B. Blood 1998; 91: 784-90.
    PubMedSuche in Google Scholar
  • 8 Wang L, Takabe K, Bidlingmaier SM, Ill CR, Verma IM. Sustained correction of bleeding disorder in hemophilia B mice b gene therapy. Proc Natl Acad Sci USA 1999; 96: 3906-10.
    CrossrefPubMedSuche in Google Scholar
  • 9 White GC. Gene therapy in hemophilia: clinical trials update. Thromb Haemost 2001; 86: 172-7.
    Thieme ConnectPubMedSuche in Google Scholar
  • 10 Kay MA, Manno CS, Ragni MV. et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nature Genet 2000; 24: 257-61.
    CrossrefPubMedSuche in Google Scholar
  • 11 Roth DA, Tawa NE, O’Brien JM, Treco DA, Selden RF. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A. N Engl J Med 2001; 344: 1735-42.
    CrossrefPubMedSuche in Google Scholar
  • 12 Smith TAG, Mehaffey MG, Kayda DB. et al. Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nature Genet 1993; 5: 397-402.
    CrossrefPubMedSuche in Google Scholar
  • 13 Kay MA, Landen CN, Rothenberg SR. et al. In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs. Proc Natl Acad Sci USA 1994; 91: 2353-7.
    CrossrefPubMedSuche in Google Scholar
  • 14 Dai Y, Schwarz EM, Gu D, Zhang WW, Sarvetnick N, Verma IM. Cellular and humoral immune response to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci USA 1995; 92: 1401-5.
    CrossrefPubMedSuche in Google Scholar
  • 15 Poller W, Schneider-Rasp S, Lieber U. et al. Stabilization of transgene expression by incorporation of E3 region genes int an adenoviral factor IX vector and by treatment by transient anti-CD4 treatment of the host. Gene Ther 1996; 3: 521-32.
    PubMedSuche in Google Scholar
  • 16 Walter J, You Q, Hagstrom JN, Sands M, High KA. Successful expression of human factor IX following repeat administration of an adenoviral vector in mice. Proc Natl Acad Sci USA 1996; 93: 3056-61.
    CrossrefPubMedSuche in Google Scholar
  • 17 Herzog RW, Hagstrom JM, Kung S-H. et al. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci USA 1997; 94: 5804-9.
    CrossrefPubMedSuche in Google Scholar
  • 18 Nakai H, Herzog RW, Hagstrom JN. et al. Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. Blood 1998; 91: 4600-7.
    PubMedSuche in Google Scholar
  • 19 Herzog RW, Yang EY, Couto LB. et al. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nature Med 1999; 5: 56-63.
    CrossrefPubMedSuche in Google Scholar
  • 20 Hagstrom JN, Couto LB, Scallan C. et al. Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/promoter. Blood 2000; 95: 2536-42.
    PubMedSuche in Google Scholar
  • 21 Louis DS, Verma IM. An alternative approach to somatic cell gene therapy. Proc Natl Acad Sci USA 1988; 85: 3150-4.
    CrossrefPubMedSuche in Google Scholar
  • 22 Yao S-H, Wilson JM, Nabel EG, Kurachi S, Hachiya HL, Kurachi K. Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B. Proc Natl Acad Sci USA 1991; 88: 8101-5.
    CrossrefPubMedSuche in Google Scholar
  • 23 Kay MA, Rothenberg S, Landen CN. et al. In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs. Science 1993; 262: 117-9.
    CrossrefPubMedSuche in Google Scholar
  • 24 Miao CH, Thompson AR, Loeb K, Ye X. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther 2001; 3: 947-57.
    CrossrefPubMedSuche in Google Scholar
  • 25 Fields PA, Armstrong E, Hagstrom JN. et al. Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice. Gene Ther 2001; 8: 354-61.
    CrossrefPubMedSuche in Google Scholar
  • 26 Van Linthout S, Collen D, De Geest B. Effects of promoters and enhancers on expression, transgene DNA persistence, and hepatotoxicity after adenoviral gene transfer of human apolipoprotein A-I. Hum Gene Ther 2002; 13: 829-40.
    CrossrefPubMedSuche in Google Scholar
  • 27 Van Hylckama Vlieg A, van der Linden IM, Bertina RM, Rossendaal FR. High levels of factor IX increase the risk of venous thrombosis. Blood 2000; 95: 3678-82.
    PubMedSuche in Google Scholar
  • 28 Hu SX, Ji W, Zhou Y, Logothetis C, Xu HJ. Development of an adenovirus vector with tetracycline rgulatable human tumor necrosis factor alpha gene expression. Cancer Res 1997; 57: 3339-43.
    PubMedSuche in Google Scholar
  • 29 Nakagawa S, Massie B, Hawley RG. Tetracycline regulatable adenovirus vectors: pharmacologic properties and clinical potential. Europ J Pharm Sci 2001; 13: 53-60.
    CrossrefPubMedSuche in Google Scholar
  • 30 Corti O, Sabate O, Horellou P. et al. A single adenovirus vector mediates doxycycline controlled expression of tyrosine hydroxylase in brain grafts of human neural progenitors. Nat Biotechnol 1999; 17: 349-54.
    CrossrefPubMedSuche in Google Scholar
  • 31 Corti O, Sanchez-Capelo A, Colin P, Hanoun N, Hamon M, Mallet J. Long-term doxycycline controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat model of Parkinson’s disease. Proc Natl Acad Sci USA 1999; 96: 12120-5.
    CrossrefPubMedSuche in Google Scholar
  • 32 Bohl D, Salvetti A, Moullier P, Heard JM. Control of erythropoietin delivery in mice after intramuscular injection of adeno-associated vector. Blood 1998; 92: 1512-7.
    PubMedSuche in Google Scholar
  • 33 Rendahl KG, Quiroz D, Lander M. et al. Tightly regulated long-term erythropoietin expression in vivo using Tet-inducible recombinant adeno-associated viral vectors. Hum Gene Ther 2002; 13: 335-42.
    CrossrefPubMedSuche in Google Scholar
  • 34 Bohl D, Heard JM. Modulation of erythropoietin delivery from engineered muscles in mice. Hum Gene Ther 1997; 8: 195-204.
    CrossrefPubMedSuche in Google Scholar
  • 35 Bohl D, Naffakh N, Heard JM. Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nature Med 1997; 3: 299-305.
    CrossrefPubMedSuche in Google Scholar
  • 36 Serguera C, Bohl D, Rolland E, Prevost P, Heard JM. Control of erythropoietin by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells. Hum Gene Ther 1999; 10: 375-83.
    CrossrefPubMedSuche in Google Scholar
  • 37 Verhoef K, Marzio G, Hillen W, Bujard H, Berkhout B. Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat. J Virol 2001; 75: 979-87.
    CrossrefPubMedSuche in Google Scholar
  • 38 Lamartina S, Roscil G, Rinaudo CD. et al. Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activator. Hum Gene Ther 2002; 13: 199-210.
    CrossrefPubMedSuche in Google Scholar
  • 39 Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W. Exploring the sequence space for tetracycline-dependent transactivators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 2000; 97: 7963-8.
    CrossrefPubMedSuche in Google Scholar
  • 40 Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-9.
    CrossrefPubMedSuche in Google Scholar
  • 41 Hafenrichter DG, Wu X, Rettinger SD. et al. Evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes. Blood 1994; 84: 3394-404.
    PubMedSuche in Google Scholar
  • 42 Fechner H, Wang X, Wang H. et al. Transcomplementation of vector replication versus coxsackie adenovirus receptor over-expression to improve transgene expression in poorly permissive cancer cells. Gene Ther 2000; 7: 1954-68.
    CrossrefPubMedSuche in Google Scholar
  • 43 Hacket NR, Crystal RG. Adenovirus vectors for gene therapy. In: Gene therapy: therapeutic mechanisms and strategies. Templeton NS, Lasic DD. eds Marcel Dekker Inc; 2000: 17-40.
    Suche in Google Scholar
  • 44 Miao CH, Oashi K, Patijn GA. et al. Inclusion of the hepatic locus control region, an intron and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro . Mol Ther 2000; 1: 522-32.
    CrossrefPubMedSuche in Google Scholar
  • 45 Shen RF, Clift SM, DeMayo JL, Sifers RN, Finegold MJ, Woo SLC. Tissue-specific regulation of human alpha1-antitrypsin gene expression in transgeneic mice. DNA 1989; 8: 101-8.
    CrossrefPubMedSuche in Google Scholar
  • 46 Kriegler M. Gene transfer and expression: a laboratory manual. New York: W.H; Freeman and company 1991
    Suche in Google Scholar
  • 47 Yao S-N, Farjo A, Rossler BJ, Davidson BL, Kurachi K. Adenovirus-mediated transfer of human factor IX gene in immunodeficient and normal mice: evidence for prolonged and activity of transgene in liver. Viral Immunol 1996; 9: 141-53.
    CrossrefPubMedSuche in Google Scholar
  • 48 Haack A, Poller W, Schneider-Rasp S. et al. Highly sensitive and species-specific assay for quantification of human transgene expression levels. Haemophilia 1999; 5: 334-9.
    CrossrefPubMedSuche in Google Scholar