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Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells

Gene Therapy volume 11, pages 1391–1398 (2004)Cite this article

Abstract

As small molecule drugs for Duchenne muscular dystrophy (DMD), antisense oligonucleotides (AONs) have been shown to restore the disrupted reading frame of DMD transcripts by inducing specific exon skipping. This allows the synthesis of largely functional Becker muscular dystrophy (BMD)-like dystrophins and potential conversion of severe DMD into milder BMD phenotypes. Thus far we have used 2′-O-methyl phosphorothioate (2OMePS) AONs. Here, we assessed the skipping efficiencies of different AON analogs containing morpholino-phosphorodiamidate, locked nucleic acid (LNA) or peptide nucleic acid (PNA) backbones. In contrast to PNAs and morpholinos, LNAs have not yet been tested as splice modulators. Compared to the most effective 2OMePS AON directed at exon 46, the LNA induced higher skipping levels in myotubes from a human control (85 versus 20%) and an exon 45 deletion DMD patient (98 versus 75%). The morpholino-induced skipping levels were only 5–6%, whereas the PNA appeared to be ineffective. Further comparative analysis of LNA and 2OMePS AONs containing up to three mismatches revealed that LNAs, while inducing higher skipping efficiencies, show much less sequence specificity. This limitation increases the risk of adverse effects elsewhere in the human genome. Awaiting further improvements in oligochemistry, we thus consider 2OMePS AONs currently the most favorable compounds, at least for targeted DMD exon 46 skipping.

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References

  1. Braasch DA, Corey DR . Novel antisense and peptide nucleic acid strategies for controlling gene expression. Biochemistry 2002; 41: 4503–4510.

    Article  CAS  PubMed  Google Scholar 

  2. Suwanmanee T et al. Repair of a splicing defect in erythroid cells from patients with beta-thalassemia/HbE disorder. Mol Ther 2002; 6: 718–726.

    Article  CAS  PubMed  Google Scholar 

  3. Friedman KJ et al. Correction of aberrant splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by antisense oligonucleotides. J Biol Chem 1999; 274: 36193–36199.

    Article  CAS  PubMed  Google Scholar 

  4. Mercatante DR, Sazani P, Kole R . Modification of alternative splicing by antisense oligonucleotides as a potential chemotherapy for cancer and other diseases. Curr Cancer Drug Targets 2001; 1: 211–230.

    Article  CAS  PubMed  Google Scholar 

  5. Cartegni L, Krainer AR . Correction of disease-associated exon skipping by synthetic exon-specific activators. Nat Struct Biol 2003; 10: 120–125.

    Article  CAS  PubMed  Google Scholar 

  6. Hoffman EP, Brown Jr RH, Kunkel LM . Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987; 51: 919–928.

    Article  CAS  PubMed  Google Scholar 

  7. Hoffman EP et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med 1988; 318: 1363–1368.

    Article  CAS  PubMed  Google Scholar 

  8. Koenig M et al. The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion. Am J Hum Genet 1989; 45: 498–506.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Ervasti JM et al. Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature 1990; 345: 315–319.

    Article  CAS  PubMed  Google Scholar 

  10. Di Blasi C et al. Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers. Acta Neuropathol (Berl) 1996; 92: 369–377.

    Article  CAS  Google Scholar 

  11. Koenig M, Monaco AP, Kunkel LM . The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 1988; 53: 219–226.

    Article  CAS  PubMed  Google Scholar 

  12. Dunckley MG et al. Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet 1998; 7: 1083–1090.

    Article  CAS  PubMed  Google Scholar 

  13. Wilton SD et al. Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides. Neuromuscular Disord 1999; 9: 330–338.

    Article  CAS  Google Scholar 

  14. De Angelis FG et al. 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 2002; 99: 9456–9461.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Takeshima Y et al. Oligonucleotides against a splicing enhancer sequence led to dystrophin production in muscle cells from a Duchenne muscular dystrophy patient. Brain Dev 2001; 23: 788–790.

    Article  CAS  PubMed  Google Scholar 

  16. van Deutekom JC et al. Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet 2001; 10: 1547–1554.

    Article  CAS  PubMed  Google Scholar 

  17. Aartsma-Rus A et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 2003; 12: 907–914.

    Article  CAS  PubMed  Google Scholar 

  18. Aartsma-Rus A et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74: 83–92; Epub 2003 Dec 2016.

    Article  CAS  PubMed  Google Scholar 

  19. Lu QL et al. Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 6: 6.

    Google Scholar 

  20. Agrawal S . Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides. Biochim Biophys Acta 1999; 1489: 53–68.

    Article  CAS  PubMed  Google Scholar 

  21. Manoharan M . Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug Dev 2002; 12: 103–128.

    Article  CAS  PubMed  Google Scholar 

  22. Ekker SC, Larson JD . Morphant technology in model developmental systems. Genesis 2001; 30: 89–93.

    Article  CAS  PubMed  Google Scholar 

  23. Summerton J . Morpholino antisense oligomers: the case for an RNase H-independent structural type. Biochim Biophys Acta 1999; 1489: 141–158.

    Article  CAS  PubMed  Google Scholar 

  24. Morcos PA . Achieving efficient delivery of morpholino oligos in cultured cells. Genesis 2001; 30: 94–102.

    Article  CAS  PubMed  Google Scholar 

  25. Schmajuk G, Sierakowska H, Kole R . Antisense oligonucleotides with different backbones. Modification of splicing pathways and efficacy of uptake. J Biol Chem 1999; 274: 21783–21789.

    Article  CAS  PubMed  Google Scholar 

  26. Nasevicius A, Ekker SC . Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 2000; 26: 216–220.

    Article  CAS  PubMed  Google Scholar 

  27. Sazani P et al. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat Biotechnol 2002; 20: 1228–1233.

    Article  CAS  PubMed  Google Scholar 

  28. Gebski BL et al. Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 2003; 12: 1801–1811.

    Article  CAS  PubMed  Google Scholar 

  29. Braasch DA, Liu Y, Corey DR . Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucleic Acids Res 2002; 30: 5160–5167.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Leumann CJ . DNA analogues: from supramolecular principles to biological properties. Bioorg Med Chem 2002; 10: 841–854.

    Article  CAS  PubMed  Google Scholar 

  31. Fluiter K et al. In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides. Nucleic Acids Res 2003; 31: 953–962.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Larsen HJ, Bentin T, Nielsen PE . Antisense properties of peptide nucleic acid. Biochim Biophys Acta 1999; 1489: 159–166.

    Article  CAS  PubMed  Google Scholar 

  33. Mologni L et al. Additive antisense effects of different PNAs on the in vitro translation of the PML/RARalpha gene. Nucleic Acids Res 1998; 26: 1934–1938.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ray A, Norden B . Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future. FASEB J 2000; 14: 1041–1060.

    Article  CAS  PubMed  Google Scholar 

  35. Sazani P et al. Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic Acids Res 2001; 29: 3965–3974.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Karras JG et al. Deletion of individual exons and induction of soluble murine interleukin-5 receptor-alpha chain expression through antisense oligonucleotide-mediated redirection of pre-mRNA splicing. Mol Pharmacol 2000; 58: 380–387.

    Article  CAS  PubMed  Google Scholar 

  37. Aartsma-Rus A et al. Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscular Disord 2002; 12: S71.

    Article  Google Scholar 

  38. Arzumanov A et al. Inhibition of HIV-1 Tat-dependent trans activation by steric block chimeric 2′-O-methyl/LNA oligoribonucleotides. Biochemistry 2001; 40: 14645–14654.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was financially supported by the Duchenne Parent Project (The Netherlands), the Princess Beatrix Fund (The Netherlands) and the MDA (USA).

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Authors and Affiliations

  1. Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg, AL Leiden, The Netherlands

    A Aartsma-Rus, W E Kaman, M Bremmer-Bout, A A M Janson, J T den Dunnen, G-J B van Ommen & J C T van Deutekom

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  1. A Aartsma-Rus
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  2. W E Kaman
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Aartsma-Rus, A., Kaman, W., Bremmer-Bout, M. et al. Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle cells. Gene Ther 11, 1391–1398 (2004). https://doi.org/10.1038/sj.gt.3302313

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  • DOI: https://doi.org/10.1038/sj.gt.3302313

Keywords

  • antisense
  • exon skipping
  • 2′-O-methyl phosphorothioate
  • LNA
  • morpholino
  • dystrophin

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