Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin

Gene Therapy volume 12, pages 1099–1108 (2005)Cite this article

Abstract

Gene therapy for Duchenne muscular dystrophy (DMD) will require sustained expression of therapeutic dystrophins in striated muscles. Lentiviral vectors have a relatively large transgene carrying capacity and can integrate into nondividing cells. We therefore explored the use of lentiviral vectors for transferring genes into mouse skeletal muscle cells. These vectors successfully transferred a minidystrophin expression cassette into mdx muscles, and minidystrophin expression persisted and prevented subsequent muscle fiber degeneration for at least 6 months. However, only low to moderate levels of skeletal muscle transduction could be obtained by intramuscular injection of the highest currently available lentiviral doses. Using cultured cells, the lentiviral vectors effectively transduced proliferating and terminally differentiated muscle cells, indicating that cell cycling is not essential for transduction of myogenic cells. We further showed that lentiviral vectors efficiently transduced both primary myoblasts and multipotent adult progenitor cells (MAPCs) in vitro, and the cells persistently expressed transgenes without any obvious toxicity. When mdx primary myoblasts were genetically modified with minidystrophin vectors and transplanted into mdx skeletal muscles, significant numbers of dystrophin-expressing myofibers formed. Finally, we showed that a short, highly active CK6 regulatory cassette directed muscle-specific activity in the context of the lentiviral vectors. The ability of lentiviral vectors to transduce myogenic progenitors using a minidystrophin cassette regulated by a muscle-specific promoter suggests that this system could be useful for ex vivo gene therapy of muscular dystrophy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Emery AEH (ed). The Muscular Dystrophies. Oxford University Press Inc.: New York, Oxford, 2001.

    Book  Google Scholar 

  2. Gregorevic P, Chamberlain JS . Gene therapy for muscular dystrophy – a review of promising progress. Expert Opin Biol Ther 2003; 3: 803–814.

    CAS  PubMed  Google Scholar 

  3. Cox GA et al. Overexpression of dystrophin in transgenic mdx mice eliminates dystrophic symptoms without toxicity. Nature 1993; 364: 725–729.

    Article  CAS  PubMed  Google Scholar 

  4. Harper SQ et al. Modular flexibility of dystrophin: implications for gene therapy of Duchenne muscular dystrophy. Nat Med 2002; 8: 253–261.

    Article  CAS  PubMed  Google Scholar 

  5. DelloRusso C et al. Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin. Proc Natl Acad Sci USA 2002; 99: 12979–12984.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gregorevic P et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat Med 2004; 10: 828–834.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Vincent-Lacaze N et al. Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle. J Virol 1999; 73: 1949–1955.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Schnepp BC et al. Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J Virol 2003; 77: 3495–3504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 1996; 70: 8098–8108.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Snyder RO et al. Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum Gene Ther 1997; 8: 1891–1900.

    Article  CAS  PubMed  Google Scholar 

  11. Harui A, Suzuki S, Kochanek S, Mitani K . Frequency and stability of chromosomal integration of adenovirus vectors. J Virol 1999; 73: 6141–6146.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Kafri T et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 1997; 17: 314–317.

    Article  CAS  PubMed  Google Scholar 

  13. Seppen J, Barry SC, Harder B, Osborne WR . Lentivirus administration to rat muscle provides efficient sustained expression of erythropoietin. Blood 2001; 98: 594–596.

    Article  CAS  PubMed  Google Scholar 

  14. MacKenzie TC et al. Efficient transduction of liver and muscle after in utero injection of lentiviral vectors with different pseudotypes. Mol Ther 2002; 6: 349–358.

    Article  CAS  PubMed  Google Scholar 

  15. Kobinger GP et al. Correction of the dystrophic phenotype by in vivo targeting of muscle progenitor cells. Hum Gene Ther 2003; 14: 1441–1449.

    Article  CAS  PubMed  Google Scholar 

  16. Bischoff R . The satellite cell and muscle regeneration. In: Andrew G, Engel CF-A (eds). Myology. McGraw-Hill Inc., 1994: pp 97–118.

    Google Scholar 

  17. Gussoni E et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999; 401: 390–394.

    CAS  PubMed  Google Scholar 

  18. Jankowski RJ, Haluszczak C, Trucco M, Huard J . Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells. Hum Gene Ther 2001; 12: 619–628.

    Article  CAS  PubMed  Google Scholar 

  19. Jiang Y et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002; 30: 896–904.

    Article  CAS  PubMed  Google Scholar 

  20. Bujold M et al. Autotransplantation in mdx mice of mdx myoblasts genetically corrected by an HSV-1 amplicon vector. Cell Transplant 2002; 11: 759–767.

    Article  PubMed  Google Scholar 

  21. Floyd Jr SS et al. Ex vivo gene transfer using adenovirus-mediated full-length dystrophin delivery to dystrophic muscles. Gene Therapy 1998; 5: 19–30.

    Article  CAS  PubMed  Google Scholar 

  22. Moisset PA, Gagnon Y, Karpati G, Tremblay JP . Expression of human dystrophin following the transplantation of genetically modified mdx myoblasts. Gene Therapy 1998; 5: 1340–1346.

    Article  CAS  PubMed  Google Scholar 

  23. Hartigan-O'Connor D et al. Immune evasion by muscle-specific gene expression in dystrophic muscle. Mol Ther 2001; 4: 525–533.

    Article  CAS  PubMed  Google Scholar 

  24. Cordier L et al. Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies. Hum Gene Ther 2001; 12: 205–215.

    Article  CAS  PubMed  Google Scholar 

  25. Hauser MA et al. Analysis of muscle creatine kinase regulatory elements in recombinant adenoviral vectors. Mol Ther 2000; 2: 16–25.

    Article  CAS  PubMed  Google Scholar 

  26. Neville C et al. Skeletal muscle cultures. Methods Cell Biol 1997; 52: 85–116.

    Article  CAS  PubMed  Google Scholar 

  27. Clegg CH, Linkhart TA, Olwin BB, Hauschka SD . Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol 1987; 105: 949–956.

    Article  CAS  PubMed  Google Scholar 

  28. Chamberlain JS, Jaynes JB, Hauschka SD . Regulation of creatine kinase induction in differentiating mouse myoblasts. Mol Cell Biol 1985; 5: 484–492.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jaynes JB et al. Transcriptional regulation of the muscle creatine kinase gene and regulated expression in transfected mouse myoblasts. Mol Cell Biol 1986; 6: 2855–2864.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Naldini L . Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol 1998; 9: 457–463.

    Article  CAS  PubMed  Google Scholar 

  31. Nguyen TH et al. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes. Mol Ther 2002; 6: 199–209.

    Article  CAS  PubMed  Google Scholar 

  32. Bonci D et al. ‘Advanced’ generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo. Gene Therapy 2003; 10: 630–636.

    Article  CAS  PubMed  Google Scholar 

  33. 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  PubMed  PubMed Central  Google Scholar 

  34. Chen W et al. Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiated mice. Stem Cells 2000; 18: 352–359.

    Article  CAS  PubMed  Google Scholar 

  35. Montanaro F et al. Skeletal muscle engraftment potential of adult mouse skin side population cells. Proc Natl Acad Sci USA 2003; 100: 9336–9341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bachrach E et al. Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells. Proc Natl Acad Sci USA 2004; 101: 3581–3586.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Acsadi G et al. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity. Hum Mol Genet 1994; 3: 579–584.

    Article  CAS  PubMed  Google Scholar 

  38. Nalbantoglu J, Pari G, Karpati G, Holland PC . Expression of the primary coxsackie and adenovirus receptor is downregulated during skeletal muscle maturation and limits the efficacy of adenovirus-mediated gene delivery to muscle cells. Hum Gene Ther 1999; 10: 1009–1019.

    Article  CAS  PubMed  Google Scholar 

  39. Bilbao R et al. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors. Gene Therapy 2003; 10: 1821–1829.

    Article  CAS  PubMed  Google Scholar 

  40. Guibinga GH, Miyanohara A, Esko JD, Friedmann T . Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells. Mol Ther 2002; 5: 538–546.

    Article  CAS  PubMed  Google Scholar 

  41. Cao B, Mytinger JR, Huard J . Adenovirus mediated gene transfer to skeletal muscle. Microsc Res Technol 2002; 58: 45–51.

    Article  CAS  Google Scholar 

  42. Flint SJ, Enquist LW, Krug RM, Racaniello VR, Skalka AM . Principles of Virology, Molecular Biology, Pathogenesis, and Control. ASM press: Washington DC, 2000.

    Google Scholar 

  43. Scherr M et al. Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography. Gene Therapy 2002; 9: 1708–1714.

    Article  CAS  PubMed  Google Scholar 

  44. Follenzi A, Naldini L . Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 2002; 346: 454–465.

    Article  CAS  PubMed  Google Scholar 

  45. Hartigan-O'Connor D, Barjot C, Salvatori G, Chamberlain JS . Generation and growth of gutted adenoviral vectors. Methods Enzymol 2002; 346: 224–246.

    Article  CAS  PubMed  Google Scholar 

  46. Potter M et al. Streamlined large-scale production of recombinant adeno-associated virus (rAAV) vectors. Methods Enzymol 2002; 346: 413–430.

    Article  CAS  PubMed  Google Scholar 

  47. Blankinship MJ et al. Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6. Mol Ther 2004; 10: 671–678.

    Article  CAS  PubMed  Google Scholar 

  48. Barry SC et al. Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression. Hum Gene Ther 2001; 12: 1103–1108.

    Article  CAS  PubMed  Google Scholar 

  49. Hawley RG, Lieu FH, Fong AZ, Hawley TS . Versatile retroviral vectors for potential use in gene therapy. Gene Therapy 1994; 1: 136–138.

    CAS  PubMed  Google Scholar 

  50. Miller JB, Crow MT, Stockdale FE . Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures. J Cell Biol 1985; 101: 1643–1650.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Luigi Naldini and William R Osborne for providing lentiviral vector backbones, and James Allen, Paul Gregorevic, and Jay Han for critical reading of this paper. These studies were supported by grants from the National Institutes of Health (AR44533, NS46788, AR18860, and HL64387) and the Muscular Dystrophy Association (USA) (to JSC and SDH). SL was supported by a Research Development Grant from the Muscular Dystrophy Association (USA).

Author information

Authors and Affiliations

  1. Department of Neurology, University of Washington School of Medicine, Seattle, WA, USA

    S Li, E Kimura, B M Fall, M Reyes & J S Chamberlain

  2. Senator Paul D Wellstone Muscular Dystrophy Cooperative Research Center, University of Washington School of Medicine, Seattle, WA, USA

    S Li, E Kimura, B M Fall, M Reyes, J C Angello, R Welikson, S D Hauschka & J S Chamberlain

  3. Department of Biochemistry, University of Washington School of Medicine, Seattle, WA, USA

    J C Angello, R Welikson, S D Hauschka & J S Chamberlain

  4. Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA

    J S Chamberlain

Authors
  1. S Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E Kimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. B M Fall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J C Angello
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R Welikson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S D Hauschka
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. J S Chamberlain
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, S., Kimura, E., Fall, B. et al. Stable transduction of myogenic cells with lentiviral vectors expressing a minidystrophin. Gene Ther 12, 1099–1108 (2005). https://doi.org/10.1038/sj.gt.3302505

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302505

Keywords

This article is cited by

Search

Advanced search

Quick links