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Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction

Gene Therapy volume 10, pages 1917–1925 (2003)Cite this article

Abstract

Autologous transplantation of gene-modified hematopoietic stem cells may provide a therapeutic strategy for several monogeneic disorders. In previous studies, retroviral gene transfer of coagulation factor VIII (FVIII) into FVIII−/− mouse bone marrow (BM) cells did not result in detectable plasma FVIII levels. However, specific immune tolerance was achieved against neo-antigenic FVIII. Here, we used lentiviral vectors to study the ability of various hematopoietic cell types to synthesize and secrete recombinant FVIII. Several myeloid, monocytic and megakaryocytic cell lines (K-562, TF-1, Monomac-1, Mutz-3, Meg-01) expressed FVIII at 2–12 mU/104 cells. In contrast, two lymphatic cell lines, BV-173 and Molt-4, were less-efficiently transduced and did not express detectable FVIII. Similarly, peripheral blood-derived primary monocytes were transduced efficiently and expressed up to 20 mU/104 cells, whereas primary lymphocytes did not express FVIII. Although human and canine CD34+ cells were transduced efficiently, the cells expressed very low levels of FVIII (up to 0.8 mU/104 cells). Following xenotransplantation of transduced CD34+ into NOD/SCID mice, ELISA failed to detect FVIII in the plasma of engrafted mice. However, NOD/SCID repopulating cell (SRC)-derived human monocytes isolated from BM of these mice secreted functional recombinant FVIII after culture ex vivo. Again, SRC-derived human lymphocytes did not secrete FVIII. Therefore, certain hematopoietic cell types are able to synthesize and secrete functional recombinant FVIII. Our results show for the first time that transplantation of transduced CD34+ progenitors may give rise to differentiated hematopoietic cells secreting a nonhematopoietic recombinant protein.

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References

  1. High KA . Gene transfer as an approach to treating hemophilia. Circ Res 2001; 88: 137–144.

    Article  CAS  Google Scholar 

  2. Chuah MK, Collen D, VandenDriessche T . Gene therapy for hemophilia. J Gene Med 2001; 3: 3–20.

    Article  CAS  Google Scholar 

  3. White II GC . Gene therapy in hemophilia: clinical trials update. Thromb Haemost 2001; 86: 172–177.

    Article  CAS  Google Scholar 

  4. Connelly S et al. Complete short-term correction of canine hemophilia A by in vivo gene therapy. Blood 1996; 88: 3846–3853.

    CAS  Google Scholar 

  5. Connelly S et al. Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy. Blood 1998; 91: 3273–3281.

    CAS  Google Scholar 

  6. Lipshutz GS et al. Short-term correction of factor VIII deficiency in a murine model of hemophilia A after delivery of adenovirus murine factor VIII in utero. Proc Natl Acad Sci USA 1999; 96: 13324–13329.

    Article  CAS  Google Scholar 

  7. Balagué C et al. Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 2000; 95: 820–828.

    PubMed  Google Scholar 

  8. Sarkar R et al. Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum Gene Ther 2000; 11: 881–894.

    Article  CAS  Google Scholar 

  9. Bristol JA et al. Adenovirus-mediated factor VIII gene expression results in attenuated anti-factor VIII-specific immunity in hemophilia A mice compared with factor VIII protein infusion. Hum Gene Ther 2001; 12: 1651–1661.

    Article  CAS  Google Scholar 

  10. Gallo-Penn AM et al. Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs. Blood 2001; 97: 107–113.

    Article  CAS  Google Scholar 

  11. Chuah MK et al. Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors. Blood 2002; 101: 1734–1743.

    Article  Google Scholar 

  12. Burton M et al. Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein. Proc Natl Acad Sci USA 1999; 96: 12725–12730.

    Article  CAS  Google Scholar 

  13. Chao H, Mao L, Bruce AT, Walsh CE . Sustained expression of human factor VIII in mice using a parvovirus-based vector. Blood 2000; 95: 1594–1599.

    CAS  Google Scholar 

  14. Chao H, Walsh CE . Induction of tolerance to human factor VIII in mice. Blood 2001; 97: 3311–3312.

    Article  CAS  Google Scholar 

  15. Chao H et al. Expression of human factor VIII by splicing between dimerized AAV vectors. Mol Ther 2002; 5: 716–722.

    Article  CAS  Google Scholar 

  16. Andrews JL et al. Evaluation of the duration of human factor VIII expression in nonhuman primates after systemic delivery of an adenoviral vector. Hum Gene Ther 2002; 13: 1331–1336.

    Article  CAS  Google Scholar 

  17. VandenDriessche T et al. Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc Natl Acad Sci USA 1999; 96: 10379–10384.

    Article  CAS  Google Scholar 

  18. Park F, Ohashi K, Kay MA . Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver. Blood 2000; 96: 1173–1176.

    CAS  Google Scholar 

  19. Stein CS et al. In vivo treatment of hemophilia A and mucopolysaccharidosis type VII using nonprimate lentiviral vectors. Mol Ther 2001; 3: 850–856.

    Article  CAS  Google Scholar 

  20. VandenDriessche T et al. Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo. Blood 2002; 100: 813–822.

    Article  CAS  Google Scholar 

  21. Hoeben RC et al. Toward gene therapy for hemophilia A: long-term persistence of factor VIII-secreting fibroblasts after transplantation into immunodeficient mice. Hum Gene Ther 1993; 4: 179–186.

    Article  CAS  Google Scholar 

  22. Dwarki VJ et al. Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice. Proc Natl Acad Sci USA 1995; 92: 1023–1027.

    Article  CAS  Google Scholar 

  23. Roth DA et al. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A. N Engl J Med 2001; 344: 1735–1742.

    Article  CAS  Google Scholar 

  24. Chuah MK et al. Bone marrow stromal cells as targets for gene therapy of hemophilia A. Hum Gene Ther 1998; 9: 353–365.

    Article  CAS  Google Scholar 

  25. Chuah MK et al. Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum Gene Ther 2000; 11: 729–738.

    Article  CAS  Google Scholar 

  26. Hoeben RC et al. Toward gene therapy in haemophilia A: retrovirus-mediated transfer of a factor VIII gene into murine haematopoietic progenitor cells. Thromb Haemost 1992; 67: 341–345.

    Article  CAS  Google Scholar 

  27. 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 

  28. Hollestelle MJ et al. Tissue distribution of factor VIII gene expression in vivo – a closer look. Thromb Haemost 2001; 86: 855–861.

    Article  CAS  Google Scholar 

  29. Uchida N et al. HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc Natl Acad Sci USA 1998; 95: 11939–11944.

    Article  CAS  Google Scholar 

  30. Miyoshi H et al. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283: 682–686.

    Article  CAS  Google Scholar 

  31. Evans JT, Kelly PF, O'Neill E, Garcia JV . Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors. Hum Gene Ther 1999; 10: 1479–1489.

    Article  CAS  Google Scholar 

  32. Case SS et al. Stable transduction of quiescent CD34(+)CD38(−) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci USA 1999; 96: 2988–2993.

    Article  CAS  Google Scholar 

  33. Scherr M et al. Lentiviral gene transfer into peripheral blood-derived CD34+ NOD/SCID-repopulating cells. Blood 2002; 99: 709–712.

    Article  CAS  Google Scholar 

  34. Zennou V et al. HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 2000; 101: 173–185.

    Article  CAS  Google Scholar 

  35. Sirven A et al. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood 2000; 96: 4103–4110.

    CAS  Google Scholar 

  36. Zennou V et al. The HIV-1 DNA flap stimulates HIV vector-mediated cell transduction in the brain. Nat Biotechnol 2001; 19: 446–450.

    Article  CAS  Google Scholar 

  37. Park F, Kay MA . Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo. Mol Ther 2001; 4: 164–173.

    Article  CAS  Google Scholar 

  38. Chinnasamy D et al. Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins. Blood 2000; 96: 1309–1316.

    CAS  Google Scholar 

  39. Matzinger P . Tolerance, danger, and the extended family. Annu Rev Immunol 1994; 12: 991–1045.

    Article  CAS  Google Scholar 

  40. Brown BD, Lillicrap D . Dangerous liaisons: the role of ‘danger’ signals in the immune response to gene therapy. Blood 2002; 100: 1133–1140.

    Article  CAS  Google Scholar 

  41. Heim DA et al. Introduction of a xenogeneic gene via hematopoietic stem cells leads to specific tolerance in a rhesus monkey model. Mol Ther 2000; 1: 533–544.

    Article  CAS  Google Scholar 

  42. Kang E et al. In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting. Hum Gene Ther 2001; 12: 1663–1672.

    Article  CAS  Google Scholar 

  43. Cameron C et al. The canine factor VIII cDNA and 5′ flanking sequence. Thromb Haemost 1998; 79: 317–322.

    Article  CAS  Google Scholar 

  44. Naldini L et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267.

    Article  CAS  Google Scholar 

  45. Naldini L et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93: 11382–11388.

    Article  CAS  Google Scholar 

  46. Zufferey R et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 1997; 15: 871–875.

    Article  CAS  Google Scholar 

  47. Scherr M et al. Quantitative determination of lentiviral vector particle numbers by real-time PCR. Biotechniques 2001; 31: 520–526.

    Article  CAS  Google Scholar 

  48. Eder M et al. Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation. Leukemia 1999; 13: 1383–1389.

    Article  CAS  Google Scholar 

  49. Schiedlmeier B et al. Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice. Blood 2000; 95: 1237–1248.

    CAS  Google Scholar 

Download references

Acknowledgements

The authors acknowledge D Trono (Geneva, Switzerland) for providing the lentiviral plasmid system, and D Lillicrap (Kingston, Canada) for providing B-domain deleted canine factor VIII cDNA. M Ballmaier and G Cleves are acknowledged for help with cell sorting. Supported in part by a grant of the HW and J Hector Stiftung (to MS and ME), by a grant of Hannover Medical School (HilF to AT), and by Cytonet GmbH & Co., Hannover.

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Authors and Affiliations

  1. Department of Hematology and Oncology, Hannover Medical School, Hannover, Germany

    A Tiede, M Eder, M von Depka, K Battmer, S Luther, A Ganser & M Scherr

  2. Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, Washington, USA

    H-P Kiem

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  1. A Tiede
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  2. M Eder
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  8. M Scherr
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Tiede, A., Eder, M., von Depka, M. et al. Recombinant factor VIII expression in hematopoietic cells following lentiviral transduction. Gene Ther 10, 1917–1925 (2003). https://doi.org/10.1038/sj.gt.3302093

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