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:

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

Gene Therapy volume 10, pages 795–802 (2003)Cite this article

Abstract

Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3′untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Harper PS . Myotonic Dystrophy, 3rd edn. W.B. Saunder: London, UK, 1989, pp 293–322.

    Google Scholar 

  2. Brooke JD et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. Cell 1992; 68: 799–808.

    Article  Google Scholar 

  3. Fu YH et al. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 1992; 255: 1256–1258.

    Article  CAS  Google Scholar 

  4. Mahadevan M et al. Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. Science 1992; 255: 1253–1255.

    Article  CAS  Google Scholar 

  5. Fu YH et al. Decreased expression of myotonin–protein kinase mRNA and protein in adult form of myotonic dystrophy. Science 1993; 260: 235–238.

    Article  CAS  Google Scholar 

  6. Novelli G et al. Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle. Biochem Mol Biol Int 1993; 29: 291–297.

    CAS  PubMed  Google Scholar 

  7. Hofmann-Radvanyi H et al. Myotonic dystrophy: absence of CTG enlarged transcript in congenital forms, and low expression of the normal allele. Hum Mol Genet 1993; 2: 1263–1267.

    Article  CAS  Google Scholar 

  8. Boucher CA et al. A novel homeodomain-encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG)n repeat. Hum Mol Genet 1995; 4: 1919–1925.

    Article  CAS  Google Scholar 

  9. Jansen G et al. Structural organization and development expression pattern of the DMR-N9 gene immediately upstream of the myotonic dystrophy locus. Hum Mol Genet 1995; 4: 843–852.

    Article  CAS  Google Scholar 

  10. Davis MB et al. Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci 1997; 94: 7388–7393.

    Article  CAS  Google Scholar 

  11. Timchenko LT et al. Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucl Acid Res 1996; 24: 4407–4414.

    Article  CAS  Google Scholar 

  12. Roberts R et al. Altered phosphorylation and intracellular distribution of a (CUG)(n) triplet repeat RNA binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice. Proc Natl Acad Sci 1997; 94: 13221–13226.

    Article  CAS  Google Scholar 

  13. Philips AV, Timchenko LT, Cooper TA . Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science 1998; 280: 737–741.

    Article  CAS  Google Scholar 

  14. Timchenko NA et al. RNA CUG repeats sequester CUGBP1 and alter protein levels and activity of CUGBP1. J Biol Chem 2001; 276: 7820–7826.

    Article  CAS  Google Scholar 

  15. Tiscornia G, Mahadevan MS . Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios. Mol Cell 2000; 5: 959–967.

    Article  CAS  Google Scholar 

  16. Miller JW et al. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J 2000; 19: 4439–4448.

    Article  CAS  Google Scholar 

  17. Mankodi A et al. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science 2000; 289: 1769–1773.

    Article  CAS  Google Scholar 

  18. Weiss B, Davidkova G, Zhou LW . Antisense RNA gene therapy for studying and moduling biological processes. Cell Mol Life Sci 1999; 55: 334–358.

    Article  CAS  Google Scholar 

  19. Weiss B . Antisense Oligonucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents. CRC Press: Boca Raton, FL, 1997.

    Google Scholar 

  20. Birikh KR, Heaton PA, Eckstein F . The structure, function and application of the hammerhead ribozyme. Eur J Biochem 1997; 245: 1–16.

    Article  CAS  Google Scholar 

  21. Caplen NJ et al. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001; 98: 9742–9747.

    Article  CAS  Google Scholar 

  22. Bass BL, Weintraub H . An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 1988; 55: 1089–1098.

    Article  CAS  Google Scholar 

  23. Scadden AD, Smith CW . Specific cleavage of hyper-edited dsRNAs. EMBO J 2001; 20: 4243–4252.

    Article  CAS  Google Scholar 

  24. Deschenes I et al. Increase in the proliferative capacity of human myoblasts by using the T antigen under the vimentin promoter control. Muscle & Nerve 1997; 20: 437–445.

    Article  CAS  Google Scholar 

  25. Furling D, Lemieux D, Taneja K, Puymirat J . Decreased levels of myotonic dystrophy protein kinase (DMPK) and delayed differentiation in human myotonic dystrophy myoblasts. Neuromuscul Disord 2001; 11: 728–735.

    Article  CAS  Google Scholar 

  26. Furling D, Marette A, Puymirat J . Insulin-like growth factor-I circumvents defective insulin action in human myotonic dystrophy skeletal muscle cells. Endocrinology 1999; 140: 4244–4250.

    Article  CAS  Google Scholar 

  27. Samuel CE . Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: phosphorylation of protein synthesis initiation factor elF-2 and ribosome-associated protein P1. Virology 1979; 93: 281–285.

    Article  CAS  Google Scholar 

  28. Samuel CE . Protein–nucleic acid interactions and cellular responses to interferon. Methods 1998; 15: 161–165.

    Article  CAS  Google Scholar 

  29. Zhou A, Hassel BA, Silverman RH . Expression cloning of 2-5A-dependent RNase: a uniquely regulated mediator of interferon action. Cell 1993; 72: 753–765.

    Article  CAS  Google Scholar 

  30. Silverman RH . 2-5A dependent RNase L: a regulated endoribonuclease in the interferon system. In: D'Alessio G, Riodan JF (eds). Ribonucleases: Structure and Functions. Academic Press: New York, NY, 1997; pp 515–551.

    Chapter  Google Scholar 

  31. Davis MB et al. Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci 1997; 94: 7388–7393.

    Article  CAS  Google Scholar 

  32. Furling D et al. Defective satellite cells in congenital myotonic dystrophy. Hum Mol Genet 2001; 10: 2079–2087.

    Article  CAS  Google Scholar 

  33. Timchenko NA et al. Molecular basis for impaired muscle differentiation in myotonic dystrophy. Mol Cell Biol 2001; 2: 6927–6938.

    Article  Google Scholar 

  34. Savkur RS, Philips AV, Cooper TA . Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 2001; 29: 40–47.

    Article  CAS  Google Scholar 

  35. Denizot F, Lang R . Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89: 271–277.

    Article  CAS  Google Scholar 

  36. Sarabia V et al. Glucose transport in human skeletal muscle cells in culture. Stimulation by insulin and metformin. J Clin Invest 1992; 90: 1386–1395.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by MDA (USA) grants and by grants from the Institutes of Health Research of Canada (IRSC), National Institutes of Health (AR01D44387 and AG16392) and the French Myopathy Association (AFM). We thank Dr Mahadevan for PUC18 5′UTR DMPK plasmid.

Author information

Authors and Affiliations

  1. Unit of Human Genetics, CHU Laval Research Center, Quebec, Canada

    D Furling, G Doucet, M-A Langlois, E Belanger, L Cossette & J Puymirat

  2. CNRS UMR 7000, Faculté de Médecine Pitie-Salpetriere, Université Paris VI, France

    D Furling

  3. Department of Medicine, Cardiovascular Sciences Section, Baylor College of Medicine, Houston, TX, USA

    L Timchenko

Authors
  1. D Furling
    View author publications

    Search author on:PubMed Google Scholar

  2. G Doucet
    View author publications

    Search author on:PubMed Google Scholar

  3. M-A Langlois
    View author publications

    Search author on:PubMed Google Scholar

  4. L Timchenko
    View author publications

    Search author on:PubMed Google Scholar

  5. E Belanger
    View author publications

    Search author on:PubMed Google Scholar

  6. L Cossette
    View author publications

    Search author on:PubMed Google Scholar

  7. J Puymirat
    View author publications

    Search author on:PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Furling, D., Doucet, G., Langlois, MA. et al. Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions. Gene Ther 10, 795–802 (2003). https://doi.org/10.1038/sj.gt.3301955

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/sj.gt.3301955

Keywords

This article is cited by

Search

Advanced search

Quick links