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Engineering Exon-Skipping Vectors Expressing U7 snRNA Constructs for Duchenne Muscular Dystrophy Gene Therapy

  • Aurélie Goyenvalle
  • Kay E. DaviesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 709)

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. In most cases, the open-reading frame is disrupted which results in the absence of a functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and has recently been shown to correct the reading frame and restore dystrophin expression in vitro and in vivo. Specific exon skipping can be achieved using synthetic oligonucleotides or viral ­vectors encoding modified snRNAs, by masking important splicing sites.

We have recently demonstrated that enhanced exon skipping can be induced by a U7 snRNA carrying binding sites for the heterogeneous ribonucleoprotein A1. In DMD patient cells, bifunctional U7 snRNAs harboring silencer motifs induce complete skipping of exon 51 and thus restore dystrophin expression to near wild-type levels. Furthermore, we have confirmed the efficacy of these constructs in vivo in transgenic mice carrying the entire human DMD locus after intramuscular injection of AAV vectors encoding the bifunctional U7 snRNA. These new constructs are very promising for the optimization of therapeutic exon skipping for DMD, but also offer powerful and versatile tools to modulate pre-mRNA splicing in a wide range of applications. Here, we outline the design of these U7 snRNA constructs to achieve efficient exon skipping of the dystrophin gene. We also describe methods to evaluate the efficiency of such U7 snRNA constructs in vitro in DMD patient cells and in vivo in the transgenic hDMD mouse model, using lentiviral and recombinant adeno-associated viral vectors, respectively.

Key words

AAV vector Duchenne muscular dystrophy Lentiviral vector Exon skipping U7 snRNA Gene therapy Antisense Exonic splicing silencer 

Notes

Acknowledgments

We would like to thank Vincent Mouly (Institut de Myologie, Paris) for providing the immortalized myoblasts used in this study. We also thank Annemieke Aarstma-Rus and Johan T. den Dunnen (Leiden University Medical Center, the Netherlands) for providing the transgenic human DMD mice. This work was supported by Action Duchenne, the Association Monegasque contre les myopathies, and Duchenne Parent Project de France. A.G. was supported by an EMBO long-term postdoctoral fellowship.

References

  1. 1.
    Koenig, M., Beggs, A. H., Moyer, M., Scherpf, S., Heindrich, K., Bettecken, T., Meng, G., Muller, C. R., Lindlof, M., Kaariainen, H., and et al. (1989) The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 45, 498–506.PubMedGoogle Scholar
  2. 2.
    Monaco, A. P., Bertelson, C. J., Liechti-Gallati, S., Moser, H., and Kunkel, L. M. (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2, 90–95.PubMedCrossRefGoogle Scholar
  3. 3.
    Aartsma-Rus, A., Janson, A. A., Heemskerk, J. A., De Winter, C. L., Van Ommen, G. J., and Van Deutekom, J. C. (2006) Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides. Ann NY Acad Sci 1082, 74–76.PubMedCrossRefGoogle Scholar
  4. 4.
    Mann, C. J., Honeyman, K., Cheng, A. J., Ly, T., Lloyd, F., Fletcher, S., Morgan, J. E., Partridge, T. A., and Wilton, S. D. (2001) Antisense-induced exon skipping and synthesis of dystrophin in the mdx mouse. Proc Natl Acad Sci USA 98, 42–47.PubMedCrossRefGoogle Scholar
  5. 5.
    De Angelis, F. G., Sthandier, O., Berarducci, B., Toso, S., Galluzzi, G., Ricci, E., Cossu, G., and Bozzoni, I. (2002) Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48–50 DMD cells. Proc Natl Acad Sci USA 99, 9456–9461.PubMedCrossRefGoogle Scholar
  6. 6.
    Brun, C., Suter, D., Pauli, C., Dunant, P., Lochmuller, H., Burgunder, J. M., Schumperli, D., and Weis, J. (2003) U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping. Cell Mol Life Sci 60, 557–566.PubMedCrossRefGoogle Scholar
  7. 7.
    Schumperli, D., and Pillai, R. S. (2004) The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein. Cell Mol Life Sci 61, 2560–2570.PubMedCrossRefGoogle Scholar
  8. 8.
    Goyenvalle, A., Vulin, A., Fougerousse, F., Leturcq, F., Kaplan, J. C., Garcia, L., and Danos, O. (2004) Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306, 1796–1799.PubMedCrossRefGoogle Scholar
  9. 9.
    Dunckley, M. G., Manoharan, M., Villiet, P., Eperon, I. C., and Dickson, G. (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet 7, 1083–1090.PubMedCrossRefGoogle Scholar
  10. 10.
    Mann, C. J., Honeyman, K., McClorey, G., Fletcher, S., and Wilton, S. D. (2002) Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy. J Gene Med 4, 644–654.PubMedCrossRefGoogle Scholar
  11. 11.
    Aartsma-Rus, A., De Winter, C. L., Janson, A. A., Kaman, W. E., Van Ommen, G. J., Den Dunnen, J. T., and Van Deutekom, J. C. (2005) Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites. Oligonucleotides 15, 284–297.PubMedCrossRefGoogle Scholar
  12. 12.
    Fairbrother, W. G., Yeh, R. F., Sharp, P. A., and Burge, C. B. (2002) Predictive identification of exonic splicing enhancers in human genes. Science 297, 1007–1013.PubMedCrossRefGoogle Scholar
  13. 13.
    Cartegni, L., and Krainer, A. R. (2003) Correction of disease-associated exon skipping by synthetic exon-specific activators. Nat Struct Biol 10, 120–125.PubMedCrossRefGoogle Scholar
  14. 14.
    Zhang, X. H., and Chasin, L. A. (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18, 1241–1250.PubMedCrossRefGoogle Scholar
  15. 15.
    Aartsma-Rus, A., Bremmer-Bout, M., Janson, A. A., den Dunnen, J. T., van Ommen, G. J., and van Deutekom, J. C. (2002) Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscul Disord 12 Suppl 1, S71–S7.PubMedCrossRefGoogle Scholar
  16. 16.
    Wilton, S. D., Fall, A. M., Harding, P. L., McClorey, G., Coleman, C., and Fletcher, S. (2007) Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript. Mol Ther 15, 1288–1296.PubMedCrossRefGoogle Scholar
  17. 17.
    Popplewell, L. J., Trollet, C., Dickson, G., and Graham, I. R. (2009) Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene. Mol Ther 17, 554–561.PubMedCrossRefGoogle Scholar
  18. 18.
    Goyenvalle, A., Babbs, A., van Ommen, G. J., Garcia, L., and Davies, K. E. (2009) Enhanced exon-skipping induced by U7 snRNA carrying a splicing silencer sequence: Promising tool for DMD therapy. Mol Ther 17, 1234–1240.PubMedCrossRefGoogle Scholar
  19. 19.
    Bremmer-Bout, M., Aartsma-Rus, A., de Meijer, E. J., Kaman, W. E., Janson, A. A., Vossen, R. H., van Ommen, G. J., den Dunnen, J. T., and van Deutekom, J. C. (2004) Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther 10, 232–240.PubMedCrossRefGoogle Scholar
  20. 20.
    t Hoen, P. A., de Meijer, E. J., Boer, J. M., Vossen, R. H., Turk, R., Maatman, R. G., Davies, K. E., van Ommen, G. J., van Deutekom, J. C., and den Dunnen, J. T. (2008) Generation and characterization of transgenic mice with the full-length human DMD gene. J Biol Chem 283, 5899–5907.Google Scholar
  21. 21.
    Suter, D., Tomasini, R., Reber, U., Gorman, L., Kole, R., and Schumperli, D. (1999) Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations. Hum Mol Genet 8, 2415–2423.PubMedCrossRefGoogle Scholar
  22. 22.
    Burd, C. G., and Dreyfuss, G. (1994) RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. Embo J 13, 1197–1204.PubMedGoogle Scholar
  23. 23.
    van Deutekom, J. C., Bremmer-Bout, M., Janson, A. A., Ginjaar, I. B., Baas, F., den Dunnen, J. T., and van Ommen, G. J. (2001) Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet 10, 1547–1554.PubMedCrossRefGoogle Scholar
  24. 24.
    Maquat, L. E. (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA 1, 453–465.PubMedGoogle Scholar
  25. 25.
    Aartsma-Rus, A., Janson, A. A., Kaman, W. E., Bremmer-Bout, M., den Dunnen, J. T., Baas, F., van Ommen, G. J., and van Deutekom, J. C. (2003) Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 12, 907–914.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.MRC Functional Genomics Unit, Department of Physiology, Anatomy, and GeneticsUniversity of OxfordOxfordUK

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