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Exon Skipping pp 325-347 | Cite as

Antisense Genes to Induce Exon Inclusion

  • Rachel Nlend Nlend
  • Daniel Schümperli
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 867)

Abstract

Many inherited diseases are associated with changed splicing patterns, and alternative splicing influences several biological processes as well as the replication of certain viral pathogens. For this reason, there is a broad interest in modulating individual splicing events for therapeutic purposes. Based on the small nuclear RNA (snRNA) U7, we have developed expression vectors for short antisense RNAs that accumulate in the cell nucleus where splicing occurs and that can very specifically modulate the splicing of individual exons. More specifically, in the context of the fatal neuromuscular disorder Spinal Muscular Atrophy (SMA), we have shown that U7 snRNA constructs can restore the inclusion of exon 7 in the SMN2 gene and thereby alleviate or even fully cure disease symptoms in a severe mouse model for SMA. Here we describe more generally procedures to produce U7 constructs to induce exon inclusion and to test their efficiency in cell culture experiments at the level of RNA as well as protein. The analytical methods comprise reverse transcription (RT-)PCR to detect the splicing changes, quantitative real-time RT-PCR to measure U7 snRNA expression levels and western blot and immunofluorescence methods to detect a restoration of protein expression. Additionally, we indicate how U7 cassettes can be introduced into gene transfer vectors for in vivo experiments in animal models or to transduce cell systems that are not readily amenable to DNA transfection.

Key words

U7 small nuclear RNA Antisense Exon inclusion Gene therapy Alternative splicing reporter minigene Lentivirus AAV RT-PCR Western-blot 

Notes

Acknowledgements

This work was supported by the Kanton Bern as well as by grants of the AFM (Association Française contre les Myopathies), EURASNET (European Network of Excellence on Alternative Splicing), and the Swiss National Science Foundation (grant 3100A0-120064) to D. Schümperli. R.Nlend Nlend was partly supported by a postdoctoral fellowship from SMA Europe.

References

  1. 1.
    Wirth B (2000) An update of the mutation spectrum of the survival motor neuron gene (SMN1) in autosomal recessive spinal muscular atrophy (SMA). Hum Mutat 15:228–237PubMedCrossRefGoogle Scholar
  2. 2.
    Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA 96:6307–6311PubMedCrossRefGoogle Scholar
  3. 3.
    Lim SR, Hertel KJ (2001) Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3′ splice site pairing. J Biol Chem 276:45476–45483PubMedCrossRefGoogle Scholar
  4. 4.
    Singh NK, Singh NN, Androphy EJ, Singh RN (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol Cell Biol 26:1333–1346PubMedCrossRefGoogle Scholar
  5. 5.
    Hua Y, Vickers TA, Okunola HL, Bennett CF, Krainer AR (2008) Antisense Masking of an hnRNP A1/A2 Intronic Splicing Silencer Corrects SMN2 Splicing in Transgenic Mice. Am J Hum Genet 82:834–848PubMedCrossRefGoogle Scholar
  6. 6.
    Cartegni L, Krainer AR (2003) Correction of disease-associated exon skipping by synthetic exon-specific activators. Nat Struct Biol 10:120–125PubMedCrossRefGoogle Scholar
  7. 7.
    Skordis LA, Dunckley MG, Yue B, Eperon IC, Muntoni F (2003) Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci USA 100:4114–4119PubMedCrossRefGoogle Scholar
  8. 8.
    Baughan TD, Dickson A, Osman EY, Lorson CL (2009) Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy. Hum Mol Genet 18:1600–1611PubMedCrossRefGoogle Scholar
  9. 9.
    Asparuhova M, Kole R, Schümperli D (2004) Antisense derivatives of U7 and other small nuclear RNAs as tools to modify pre-mRNA splicing patterns. Gene Ther Regul 2:321–349CrossRefGoogle Scholar
  10. 10.
    Schümperli D, Pillai RS (2004) The special Sm core structure of the U7 snRNP: far-reaching significance of a small nuclear ribonucleoprotein. Cell Mol Life Sci 60:2560–2570CrossRefGoogle Scholar
  11. 11.
    Marquis J, Meyer K, Angehrn L, Kämpfer SS, Rothen-Rutishauser B, Schümperli D (2007) Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. Mol Ther 15:1479–1486PubMedCrossRefGoogle Scholar
  12. 12.
    Meyer K, Marquis J, Trüb J, Nlend Nlend R, Verp S, Ruepp MD et al (2009) Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation. Hum Mol Genet 18:546–555PubMedCrossRefGoogle Scholar
  13. 13.
    Müller B, Schümperli D (1997) The U7 snRNP and the hairpin binding protein: key players in histone mRNA metabolism. Semin Cell Dev Biol 8:567–576CrossRefGoogle Scholar
  14. 14.
    Grimm C, Stefanovic B, Schümperli D (1993) The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J 12:1229–1238PubMedGoogle Scholar
  15. 15.
    Suter D, Tomasini R, Reber U, Gorman L, Kole R, Schümperli D (1999) Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations. Hum Mol Genet 8:2415–2423PubMedCrossRefGoogle Scholar
  16. 16.
    Zhang ML, Lorson CL, Androphy EJ, Zhou J (2001) An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA. Gene Ther 8:1532–1538PubMedCrossRefGoogle Scholar
  17. 17.
    Meister G, Bühler D, Laggerbauer B, Zobawa M, Lottspeich F, Fischer U (2000) Characterization of a nuclear 20S complex ­containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins. Hum Mol Genet 9:1977–1986PubMedCrossRefGoogle Scholar
  18. 18.
    Madocsai C, Lim SR, Geib T, Lam BJ, Hertel KJ (2005) Correction of SMN2 pre-mRNA splicing by antisense U7 small nuclear RNAs. Mol Ther 12:1013–1022PubMedCrossRefGoogle Scholar
  19. 19.
    Goyenvalle A, Babbs A, van Ommen GJ, Garcia L, Davies KE (2009) Enhanced exon-skipping induced by U7 snRNA carrying a splicing silencer sequence: promising tool for DMD therapy. Mol Ther 17:1234–1240PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Institute of Cell BiologyUniversity of BernBernSwitzerland

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