Gene Therapy Protocols pp 203-212

Part of the Methods in Molecular Biology™ book series (MIMB, volume 433)

Retroviral Modification of Mesenchymal Stem Cells for Gene Therapy of Hemophilia

  • Christopher B. Doering

Summary

Mesenchymal stem cells (MSCs) are a promising target for the delivery of secreted proteins due to their ease of isolation, expansion, and genetic modification. The bleeding disorder hemophilia A results from the deficiency of a secreted blood clotting factor termed factor VIII (fVIII). Hemophilia A could be cured by gene-transfer-based procedures targeting virtually any cell type, including MSCs. Here, we describe methods for retroviral modification of MSCs incorporating a high-expression porcine (HEP)-fVIII transgene and a murine model of hemophilia A. MSCs were isolated from bone marrow of hemophilia A mice, expanded, and transduced ex vivo. Genetically modified MSCs secreted high levels of HEP-fVIII into the conditioned medium. HEP-fVIII was purified from the conditioned medium and demonstrated to have a specific activity, relative electrophoretic mobility, and proteolytic activation pattern similar to HEP-fVIII produced by other commercial cell lines. Collectively, these data support the concept that MSCs can be utilized as a cellular vehicle for successful gene-transfer-based therapy of hemophilia A and other disorders resulting from the deficiency of a secreted protein.

Keywords

Mesenchymal stem cells marrow-derived stromal cells gene therapy retroviral vector murine stem cell virus hemophilia A porcine factor VIII 

References

  1. 1.
    Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–4 (1997).CrossRefPubMedGoogle Scholar
  2. 2.
    Devine, S.M. Mesenchymal stem cells: will they have a role in the clinic? J Cell Biochem Suppl 38, 73–9 (2002).CrossRefPubMedGoogle Scholar
  3. 3.
    Friedenstein, A.J., Chailakhjan, R.K. and Lalykina, K.S. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 3, 393–403 (1970).PubMedGoogle Scholar
  4. 4.
    Friedenstein, A.J., Gorskaja, J.F. and Kulagina, N.N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 4, 267–74 (1976).PubMedGoogle Scholar
  5. 5.
    Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I. and Frolova, G.P. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6, 230–47 (1968).CrossRefPubMedGoogle Scholar
  6. 6.
    Pittenger, M.F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–7 (1999).CrossRefPubMedGoogle Scholar
  7. 7.
    Meirelles Lda, S. and Nardi, N.B. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 123, 702–11 (2003).CrossRefPubMedGoogle Scholar
  8. 8.
    Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41–9 (2002).CrossRefPubMedGoogle Scholar
  9. 9.
    Peister, A., Mellad, J.A., Larson, B.L., Hall, B.M., Gibson, L.F. and Prockop, D.J. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103, 1662–8 (2004).CrossRefPubMedGoogle Scholar
  10. 10.
    Horwitz, E.M. et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 5, 309–13 (1999).CrossRefPubMedGoogle Scholar
  11. 11.
    Horwitz, E.M. et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 97, 1227–31 (2001).CrossRefPubMedGoogle Scholar
  12. 12.
    Ding, L., Lu, S., Batchu, R., Iii, R.S. and Munshi, N. Bone marrow stromal cells as a vehicle for gene transfer. Gene Ther 6, 1611–6 (1999).CrossRefPubMedGoogle Scholar
  13. 13.
    Gao, J., Dennis, J.E., Muzic, R.F., Lundberg, M. and Caplan, A.I. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 169, 12–20 (2001).CrossRefPubMedGoogle Scholar
  14. 14.
    Sato, Y. et al. Human mesenchymal stem cells xenografted directly to rat liver differentiated into human hepatocytes without fusion. Blood 106, 756–63 (2005).CrossRefPubMedGoogle Scholar
  15. 15.
    Eliopoulos, N., Al-Khaldi, A., Crosato, M., Lachapelle, K. and Galipeau, J. A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice. Gene Ther 10, 478–89 (2003).CrossRefPubMedGoogle Scholar
  16. 16.
    Eliopoulos, N., Gagnon, R.F., Francois, M. and Galipeau, J. Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure. J Am Soc Nephrol 17, 1576–84 (2006).CrossRefPubMedGoogle Scholar
  17. 17.
    Eliopoulos, N., Lejeune, L., Martineau, D. and Galipeau, J. Human-compatible collagen matrix for prolonged and reversible systemic delivery of erythropoietin in mice from gene-modified marrow stromal cells. Mol Ther 10, 741–8 (2004).CrossRefPubMedGoogle Scholar
  18. 18.
    Gangadharan, B., Parker, E.T., Ide, L.M., Spencer, h.T. and Doering, C.B. High-level expression of porcine factor VIII from genetically modified bone marrow-derived stem cells. Blood 107, 3859–64 (2006).CrossRefPubMedGoogle Scholar
  19. 19.
    Bi, L., Lawler, A.M., Antonarakis, S.E., High, K.A., Gearhart, J.D. and Kazazian, h.H. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet 10, 119–21 (1995).CrossRefPubMedGoogle Scholar
  20. 20.
    Doering, C.B., Healey, J.F., Parker, E.T., Barrow, R.T. and Lollar, P. High-level expression of recombinant porcine coagulation factor VIII. J Biol Chem 277, 38345–9 (2002).CrossRefPubMedGoogle Scholar
  21. 21.
    Miura, M. et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 24, 1095–103 (2006).CrossRefPubMedGoogle Scholar
  22. 22.
    Rubio, D., Garcia-Castro, J., Martín, M.C., de la Fuente, R., Cigudosa, J.C., Lloyd, A.C. and Bernad, A. Spontaneous human adult stem cell transformation. Cancer Res 65, 3035–9 (2005).PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Christopher B. Doering
    • 1
  1. 1.Aflac Cancer Center and Blood Disorders Service, Department of PediatricsEmory UniversityGA

Personalised recommendations