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Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

Gene Therapy volume 13, pages 117–126 (2006)Cite this article

Abstract

Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.

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References

  1. Sultan Y . Prevalence of inhibitors in a population of 3435 hemophilia patients in France. French Hemophilia Study Group. Thromb Haemost 1992; 67: 600–602.

    Article  CAS  Google Scholar 

  2. Kamiya T, Nagao T, Yoshioka A . A retrospective study on the development of inhibitors in Japanese hemophiliacs (second report, 1994 study). Research Group of Blood Products for Hemophilia Inhibitor. Rinsho Ketsueki 1998; 39: 402–404.

    CAS  PubMed  Google Scholar 

  3. Ljung R, Petrini P, Tengborn L, Sjorin E . Haemophilia B mutations in Sweden: a population-based study of mutational heterogeneity. Br J Haematol 2001; 113: 81–86.

    Article  CAS  Google Scholar 

  4. Ghosh K, Shetty S, Kulkarni B, Nair S, Pawar A, Khare A et al. Development of inhibitors in patients with haemophilia from India. Haemophilia 2001; 7: 273–278.

    Article  CAS  Google Scholar 

  5. Knobe KE, Sjorin E, Tengborn LI, Petrini P, Ljung RC . Inhibitors in the Swedish population with severe haemophilia A and B: a 20-year survey. Acta Paediatr 2002; 91: 910–914.

    Article  CAS  Google Scholar 

  6. Warrier I, Ewenstein BM, Koerper MA, Shapiro A, Key N, DiMichele D et al. Factor IX inhibitors and anaphylaxis in hemophilia B. J Pediatr Hematol Oncol 1997; 19: 23–27.

    Article  CAS  Google Scholar 

  7. Tengborn L, Hansson S, Fasth A, Lubeck PO, Berg A, Ljung R . Anaphylactoid reactions and nephrotic syndrome – a considerable risk during factor IX treatment in patients with haemophilia B and inhibitors: a report on the outcome in two brothers. Haemophilia 1998; 4: 854–859.

    Article  CAS  Google Scholar 

  8. Evans GL, Morgan RA . Genetic induction of immune tolerance to human clotting factor VIII in a mouse model for hemophilia A. Proc Natl Acad Sci USA 1998; 95: 5734–5739.

    Article  CAS  Google Scholar 

  9. Kootstra NA, Matsumura R, Verma IM . Efficient production of human FVIII in hemophilic mice using lentiviral vectors. Mol Ther 2003; 7: 623–631.

    Article  CAS  Google Scholar 

  10. Moayeri M, Ramezani A, Morgan RA, Hawley TS, Hawley RG . Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells. Mol Ther 2004; 10: 892–902.

    Article  CAS  Google Scholar 

  11. Bagley J, Tian C, Sachs DH, Iacomini J . T cells mediate resistance to genetically modified bone marrow in lethally irradiated recipients. Transplantation 2002; 74: 1454–1460.

    Article  CAS  Google Scholar 

  12. Bagley J, Tian C, Sachs DH, Iacomini J . Induction of T-cell tolerance to an MHC class I alloantigen by gene therapy. Blood 2002; 99: 4394–4399.

    Article  CAS  Google Scholar 

  13. Fraser CC, Sykes M, Lee RS, Sachs DH, LeGuern C . Specific unresponsiveness to a retrovirally-transferred class I antigen is controlled through the helper pathway. J Immunol 1995; 154: 1587–1595.

    CAS  PubMed  Google Scholar 

  14. Tian C, Bagley J, Kaye J, Iacomini J . Induction of T cell tolerance to a protein expressed in the cytoplasm through retroviral-mediated gene transfer. J Gene Med 2003; 5: 359–365.

    Article  CAS  Google Scholar 

  15. Demaison C, Parsley K, Brouns G, Scherr M, Battmer K, Kinnon C et al. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency (correction of immunodeficiency) virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 2002; 13: 803–813.

    Article  CAS  Google Scholar 

  16. Guenechea G, Gan OI, Dorrell C, Dick JE . Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001; 2: 75–82.

    Article  CAS  Google Scholar 

  17. Waddington SN, Nivsarkar MS, Mistry AR, Buckley SM, Kemball-Cook G, Mosley KL et al. Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy. Blood 2004; 104: 2714–2721.

    Article  CAS  Google Scholar 

  18. Siapati EK, Bigger BW, Miskin J, Chipchase D, Parsley KL, Mitrophanous K et al. Comparison of HIV- and EIAV-based vectors on their efficiency to transduce murine and human hematopoietic stem cells. Mol Ther 2005; 12: 537–546.

    Article  CAS  Google Scholar 

  19. Ailles L, Schmidt M, Santoni de Sio FR, Glimm H, Cavalieri S, Bruno S et al. Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells. Mol Ther 2002; 6: 615–626.

    Article  CAS  Google Scholar 

  20. Cui YZ, Hisha H, Yang GX, Fan TX, Jin T, Li Q et al. Optimal protocol for total body irradiation for allogeneic bone marrow transplantation in mice. Bone Marrow Transplant 2002; 30: 843–849.

    Article  Google Scholar 

  21. Osawa M, Nakamura K, Nishi N, Takahasi N, Tokuomoto Y, Inoue H et al. In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/−) hemopoietic stem cells. J Immunol 1996; 156: 3207–3214.

    CAS  PubMed  Google Scholar 

  22. Puig T, Kadar E, Limon A, Cancelas JA, Eixarch H, Luquin L et al. Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene. Gene Therapy 2002; 9: 1472–1479.

    Article  CAS  Google Scholar 

  23. Rao SS, Peters SO, Crittenden RB, Stewart FM, Ramshaw HS, Quesenberry PJ . Stem cell transplantation in the normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment. Exp Hematol 1997; 25: 114–121.

    CAS  PubMed  Google Scholar 

  24. Fields PA, Armstrong E, Hagstrom JN, Arruda VR, Murphy ML, Farrell JP et al. Intravenous administration of an E1/E3-deleted adenoviral vector induces tolerance to factor IX in C57BL/6 mice. Gene Therapy 2001; 8: 354–361.

    Article  CAS  Google Scholar 

  25. McVicar DW, Winkler-Pickett R, Taylor LS, Makrigiannis A, Bennett M, Anderson SK et al. Aberrant DAP12 signaling in the 129 strain of mice: implications for the analysis of gene-targeted mice. J Immunol 2002; 169: 1721–1728.

    Article  CAS  Google Scholar 

  26. Mingozzi F, Liu YL, Dobrzynski E, Kaufhold A, Liu JH, Wang Y et al. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J Clin Invest 2003; 111: 1347–1356.

    Article  CAS  Google Scholar 

  27. Wong W, Billing JS, Stranford SA, Hyde K, Fry J, Morris PJ et al. Retroviral transfer of donor MHC class I or MHC class II genes into recipient bone marrow cells can induce operational tolerance to alloantigens in vivo. Hum Gene Ther 2003; 14: 577–590.

    Article  CAS  Google Scholar 

  28. Fry JW, Morris PJ, Wood KJ . Adenoviral transfer of a single donor-specific MHC class I gene to recipient bone marrow cells can induce specific immunological unresponsiveness in vivo. Gene Therapy 2002; 9: 220–226.

    Article  CAS  Google Scholar 

  29. Schumacher IK, Newberg MH, Jackson JD, Hammel JM, Rubocki RJ, Engelhard VH et al. Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen. Transplantation 1996; 62: 831–836.

    Article  CAS  Google Scholar 

  30. Wong W, Wood KJ . Transplantation tolerance by donor MHC gene transfer. Curr Gene Ther 2004; 4: 329–336.

    Article  CAS  Google Scholar 

  31. Andersson G, Denaro M, Johnson K, Morgan P, Sullivan A, Houser S et al. Engraftment of retroviral EGFP-transduced bone marrow in mice prevents rejection of EGFP-transgenic skin grafts. Mol Ther 2003; 8: 385–391.

    Article  CAS  Google Scholar 

  32. Sykes M, Sachs DH . Mixed chimerism. Philos Trans R Soc London B 2001; 356: 707–726.

    Article  CAS  Google Scholar 

  33. Li Y, Li XC, Zheng XX, Wells AD, Turka LA, Strom TB . Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat Med 1999; 5: 1298–1302.

    Article  CAS  Google Scholar 

  34. Wekerle T, Kurtz J, Bigenzahn S, Takeuchi Y, Sykes M . Mechanisms of transplant tolerance induction using costimulatory blockade. Curr Opin Immunol 2002; 14: 592–600.

    Article  CAS  Google Scholar 

  35. Mapara MY, Pelot M, Zhao G, Swenson K, Pearson D, Sykes M . Induction of stable long-term mixed hematopoietic chimerism following nonmyeloablative conditioning with T cell-depleting antibodies, cyclophosphamide, and thymic irradiation leads to donor-specific in vitro and in vivo tolerance. Biol Blood Marrow Transplant 2001; 7: 646–655.

    Article  CAS  Google Scholar 

  36. Joffre O, Gorsse N, Romagnoli P, Hudrisier D, van Meerwijk JP . Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes. Blood 2004; 103: 4216–4221.

    Article  CAS  Google Scholar 

  37. Bowman JE, Reese JS, Lingas KT, Gerson SL . Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients. Mol Ther 2003; 8: 42–50.

    Article  CAS  Google Scholar 

  38. Tsui LV, Kelly M, Zayek N, Rojas V, Ho K, Ge Y et al. Production of human clotting Factor IX without toxicity in mice after vascular delivery of a lentiviral vector. Nat Biotechnol 2002; 20: 53–57.

    Article  CAS  Google Scholar 

  39. Follenzi A, Battaglia M, Lombardo A, Annoni A, Roncarolo MG, Naldini L . Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice. Blood 2004; 103: 3700–3709.

    Article  CAS  Google Scholar 

  40. Wang L, Zoppe M, Hackeng TM, Griffin JH, Lee KF, Verma IM . A factor IX-deficient mouse model for hemophilia B gene therapy. Proc Natl Acad Sci USA 1997; 94: 11563–11566.

    Article  CAS  Google Scholar 

  41. Wang LJ, Chen YM, George D, Smets F, Sokal EM, Bremer EG et al. Engraftment assessment in human and mouse liver tissue after sex-mismatched liver cell transplantation by real-time quantitative PCR for Y chromosome sequences. Liver Transplant 2002; 8: 822–828.

    Article  Google Scholar 

  42. Waddington SN, Buckley SM, Nivsarkar M, Jezzard S, Schneider H, Dahse T et al. In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor. Blood 2003; 101: 1359–1366.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Professor Inder Verma for the kind gift of the factor IX knockout mouse. This work was supported by the Wellcome Trust and the Katharine Dormandy Trust.

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Author notes

  1. B W Bigger and E K Siapati: These authors have contributed equally to this work.

Authors and Affiliations

  1. Gene Therapy Research Group, Faculty of Medicine, Imperial College London, South Kensington, London, UK

    B W Bigger, S N Waddington, M S Nivsarkar, L Jacobs, R Perrett, M V Holder, C Coutelle & M Themis

  2. Haematopoietic Stem Cell Laboratory, Cancer Research UK, London Research Unit, London, UK

    E K Siapati, C Ridler & D Bonnet

  3. Molecular Immunology Unit, Institute of Child Health, London, UK

    A Mistry, R R Ali & A J Thrasher

  4. Haemostasis Research, MRC Clinical Sciences Centre, Imperial College, Hammersmith Campus, London, UK

    G Kemball-Cook

  5. Department of Medicine, Imperial College, St Mary's Hospital, London, UK

    S J Forbes

  6. Histopathology Unit, Cancer Research UK, London Research Unit, London, UK

    N Wright

  7. Department of Histopathology, Imperial College, Hammersmith Campus, London, UK

    M Alison

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  1. B W Bigger
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Correspondence to B W Bigger.

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Bigger, B., Siapati, E., Mistry, A. et al. Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX. Gene Ther 13, 117–126 (2006). https://doi.org/10.1038/sj.gt.3302638

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  • DOI: https://doi.org/10.1038/sj.gt.3302638

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