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Exon Skipping Therapy Using Phosphorodiamidate Morpholino Oligomers in the mdx52 Mouse Model of Duchenne Muscular Dystrophy

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Duchenne Muscular Dystrophy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1687))

Abstract

Exon skipping therapy using synthetic DNA-like molecules called antisense oligonucleotides (ASOs) is a promising therapeutic candidate for overcoming the dystrophin mutation that causes Duchenne muscular dystrophy (DMD). This treatment involves splicing out the frame-disrupting segment of the dystrophin mRNA, which restores the reading frame and produces a truncated yet functional dystrophin protein. Phosphorodiamidate morpholino oligomer (PMO) is the safest ASO for patients among ASOs and has recently been approved under the accelerated approval pathway by the U.S. Food and Drug Administration (FDA) as the first drug for DMD. Here, we describe the methodology and protocol of PMO transfection and evaluation of the exon skipping efficacy in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting. The mdx52 mouse model offers advantages over the mdx mouse, a spontaneous DMD model with a nonsense mutation in exon 23, in terms of the deletion in a hotspot of deletion mutations in DMD patients, the analysis of caveolae and also Dp140 and Dp260, shorter dystrophin isoforms.

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References

  1. Shimizu-Motohashi Y, Miyatake S, Komaki H, Takeda S, Aoki Y (2016) Recent advances in innovative therapeutic approaches for Duchenne muscular dystrophy: from discovery to clinical trials. Am J Transl Res 8(6):2471–2489

    PubMed  PubMed Central  Google Scholar 

  2. Aoki Y, Nakamura A, Yokota T, Saito T, Okazawa H, Nagata T, Takeda S (2010) In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther 18(11):1995–2005. doi:10.1038/mt.2010.186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Aoki Y, Yokota T, Nagata T, Nakamura A, Tanihata J, Saito T, Duguez SM, Nagaraju K, Hoffman EP, Partridge T, Takeda S (2012) Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery. Proc Natl Acad Sci U S A 109(34):13763–13768. doi:10.1073/pnas.1204638109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kole R, Krainer AR, Altman S (2012) RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov 11(2):125–140. doi:10.1038/nrd3625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Arora V, Devi GR, Iversen PL (2004) Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 5(5):431–439

    Article  CAS  PubMed  Google Scholar 

  6. Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J, Jadoon A, Bou-Gharios G, Partridge T (2005) Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci U S A 102(1):198–203. doi:10.1073/pnas.0406700102

    Article  CAS  PubMed  Google Scholar 

  7. Yokota T, Lu QL, Partridge T, Kobayashi M, Nakamura A, Takeda S, Hoffman E (2009) Efficacy of systemic morpholino exon-skipping in Duchenne dystrophy dogs. Ann Neurol 65(6):667–676. doi:10.1002/ana.21627

    Article  PubMed  Google Scholar 

  8. Guncay A, Yokota T (2015) Antisense oligonucleotide drugs for Duchenne muscular dystrophy: how far have we come and what does the future hold? Future Med Chem 7(13):1631–1635. doi:10.4155/fmc.15.116

    Article  CAS  PubMed  Google Scholar 

  9. Touznik A, Lee JJ, Yokota T (2014) New developments in exon skipping and splice modulation therapies for neuromuscular diseases. Expert Opin Biol Ther 14(6):809–819. doi:10.1517/14712598.2014.896335

    Article  CAS  PubMed  Google Scholar 

  10. Dowling JJ (2016) Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line. Nat Rev Neurol 12(12):675–676. doi:10.1038/nrneurol.2016.180

    Article  CAS  PubMed  Google Scholar 

  11. Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science (New York, NY) 244(4912):1578–1580

    Article  CAS  Google Scholar 

  12. Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT (2006) Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 34(2):135–144. doi:10.1002/mus.20586

    Article  CAS  PubMed  Google Scholar 

  13. Aartsma-Rus A, Fokkema I, Verschuuren J, Ginjaar I, van Deutekom J, van Ommen GJ, den Dunnen JT (2009) Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. Hum Mutat 30(3):293–299. doi:10.1002/humu.20918

    Article  PubMed  Google Scholar 

  14. Esposito G, Schiattarella GG, Perrino C, Cattaneo F, Pironti G, Franzone A, Gargiulo G, Magliulo F, Serino F, Carotenuto G, Sannino A, Ilardi F, Scudiero F, Brevetti L, Oliveti M, Giugliano G, Del Giudice C, Ciccarelli M, Renzone G, Scaloni A, Zambrano N, Trimarco B (2015) Dermcidin: a skeletal muscle myokine modulating cardiomyocyte survival and infarct size after coronary artery ligation. Cardiovasc Res. doi:10.1093/cvr/cvv173

  15. Araki E, Nakamura K, Nakao K, Kameya S, Kobayashi O, Nonaka I, Kobayashi T, Katsuki M (1997) Targeted disruption of exon 52 in the mouse dystrophin gene induced muscle degeneration similar to that observed in Duchenne muscular dystrophy. Biochem Biophys Res Commun 238(2):492–497. doi:10.1006/bbrc.1997.7328

    Article  CAS  PubMed  Google Scholar 

  16. Aoki Y, Nagata T, Yokota T, Nakamura A, Wood MJ, Partridge T, Takeda S (2013) Highly efficient in vivo delivery of PMO into regenerating myotubes and rescue in laminin-alpha2 chain-null congenital muscular dystrophy mice. Hum Mol Genet 22(24):4914–4928. doi:10.1093/hmg/ddt341

    Article  CAS  PubMed  Google Scholar 

  17. Kameya S, Araki E, Katsuki M, Mizota A, Adachi E, Nakahara K, Nonaka I, Sakuragi S, Takeda S, Nabeshima Y (1997) Dp260 disrupted mice revealed prolonged implicit time of the b-wave in ERG and loss of accumulation of beta-dystroglycan in the outer plexiform layer of the retina. Hum Mol Genet 6(13):2195–2203

    Article  CAS  PubMed  Google Scholar 

  18. Prior TW, Bartolo C, Pearl DK, Papp AC, Snyder PJ, Sedra MS, Burghes AH, Mendell JR (1995) Spectrum of small mutations in the dystrophin coding region. Am J Hum Genet 57(1):22–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Helderman-van den Enden AT, Straathof CS, Aartsma-Rus A, den Dunnen JT, Verbist BM, Bakker E, Verschuuren JJ, Ginjaar HB (2010) Becker muscular dystrophy patients with deletions around exon 51; a promising outlook for exon skipping therapy in Duchenne patients. Neuromuscul Disord 20(4):251–254. doi:10.1016/j.nmd.2010.01.013

    Article  CAS  PubMed  Google Scholar 

  20. van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A, Bremmer-Bout M, den Dunnen JT, Koop K, van der Kooi AJ, Goemans NM, de Kimpe SJ, Ekhart PF, Venneker EH, Platenburg GJ, Verschuuren JJ, van Ommen GJ (2007) Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med 357(26):2677–2686. doi:10.1056/NEJMoa073108

    Article  PubMed  Google Scholar 

  21. Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, Guglieri M, Ashton E, Abbs S, Nihoyannopoulos P, Garralda ME, Rutherford M, McCulley C, Popplewell L, Graham IR, Dickson G, Wood MJ, Wells DJ, Wilton SD, Kole R, Straub V, Bushby K, Sewry C, Morgan JE, Muntoni F (2009) Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol 8(10):918–928. doi:10.1016/s1474-4422(09)70211-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nakamura A, Shiba N, Miyazaki D, Nishizawa H, Inaba Y, Fueki N, Maruyama R, Echigoya Y, Yokota T (2017) Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy. J Hum Genet 62(4):459–463. doi:10.1038/jhg.2016.152

    Article  CAS  PubMed  Google Scholar 

  23. Shibuya S, Wakayama Y, Murahashi M, Kojima H, Oniki H, Matsuzaki T, Nonaka I (2001) Muscle plasma membrane changes in dystrophin gene exon 52 knockout mouse. Pathol Res Pract 197(6):441–447. doi:10.1078/0344-0338-00058

    Article  CAS  PubMed  Google Scholar 

  24. Hagiwara Y, Fujita M, Imamura M, Noguchi S, Sasaoka T (2006) Caveolin-3 deficiency decreases the gene expression level of osteopontin in mdx mouse skeletal muscle. Acta Myol 25(2):53–61

    CAS  PubMed  Google Scholar 

  25. Lehto T, Castillo Alvarez A, Gauck S, Gait MJ, Coursindel T, Wood MJ, Lebleu B, Boisguerin P (2014) Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells. Nucleic Acids Res 42(5):3207–3217. doi:10.1093/nar/gkt1220

    Article  CAS  PubMed  Google Scholar 

  26. Gervasio OL, Whitehead NP, Yeung EW, Phillips WD, Allen DG (2008) TRPC1 binds to caveolin-3 and is regulated by Src kinase - role in Duchenne muscular dystrophy. J Cell Sci 121(Pt 13):2246–2255. doi:10.1242/jcs.032003

    Article  CAS  PubMed  Google Scholar 

  27. Echigoya Y, Lee J, Rodrigues M, Nagata T, Tanihata J, Nozohourmehrabad A, Panesar D, Miskew B, Aoki Y, Yokota T (2013) Mutation types and aging differently affect revertant fiber expansion in dystrophic mdx and mdx52 mice. PLoS One 8(7):e69194. doi:10.1371/journal.pone.0069194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. t Hoen PA, de Meijer EJ, Boer JM, Vossen RH, Turk R, Maatman RG, Davies KE, van Ommen GJ, van Deutekom JC, den Dunnen JT (2008) Generation and characterization of transgenic mice with the full-length human DMD gene. J Biol Chem 283(9):5899–5907. doi:10.1074/jbc.M709410200

    Article  CAS  Google Scholar 

  29. Kudoh H, Ikeda H, Kakitani M, Ueda A, Hayasaka M, Tomizuka K, Hanaoka K (2005) A new model mouse for Duchenne muscular dystrophy produced by 2.4 Mb deletion of dystrophin gene using Cre-loxP recombination system. Biochem Biophys Res Commun 328(2):507–516. doi:10.1016/j.bbrc.2004.12.191

    Article  CAS  PubMed  Google Scholar 

  30. Aartsma-Rus A, Krieg AM (2017) FDA approves Eteplirsen for Duchenne muscular dystrophy: the next chapter in the Eteplirsen saga. Nucleic Acid Ther 27(1):1–3. doi:10.1089/nat.2016.0657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. (2016) Railroading at the FDA. Nat Biotechnol 34(11):1078. doi:10.1038/nbt.3733

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Acknowledgements

This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Research Activity Start-up (grant to Y.A., number 15H06883) and the Japan Agency for Medical Research and Development (AMED) (16ek0109154h0002 and 16am0301021h0002). We thank Dr. Rika Maruyama for scientific advice and Dr. Motoya Katsuki for the production of mdx52 mice.

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Authors and Affiliations

  1. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo, 187-8551, Japan

    Shouta Miyatake, Yoshitaka Mizobe, Hotake Takizawa, Yuko Hara, Shin’ichi Takeda & Yoshitsugu Aoki

  2. Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada, T6G 2H7

    Toshifumi Yokota

Authors
  1. Shouta Miyatake
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  2. Yoshitaka Mizobe
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  3. Hotake Takizawa
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  4. Yuko Hara
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  5. Toshifumi Yokota
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  6. Shin’ichi Takeda
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  7. Yoshitsugu Aoki
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Correspondence to Yoshitsugu Aoki .

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Editors and Affiliations

  1. Istituto di Anatomia Umana e Biologia Cellulare, Università Cattolica del Sacro Cuore, Roma, Italy

    Camilla Bernardini

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Miyatake, S. et al. (2018). Exon Skipping Therapy Using Phosphorodiamidate Morpholino Oligomers in the mdx52 Mouse Model of Duchenne Muscular Dystrophy. In: Bernardini, C. (eds) Duchenne Muscular Dystrophy. Methods in Molecular Biology, vol 1687. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7374-3_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7374-3_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7373-6

  • Online ISBN: 978-1-4939-7374-3

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