Abstract
Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (AONs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Multiple exon skipping utilizing a cocktail of AONs can theoretically treat 80–90% of patients with Duchenne muscular dystrophy (DMD). The success of multiple exon skipping by the systemic delivery of a cocktail of AONs called phosphorodiamidate morpholino oligomers (PMOs) in a DMD dog model has made a significant impact on the development of therapeutics for DMD, leading to clinical trials of PMO-based drugs. Here, we describe the systemic delivery of a cocktail of PMOs to skip multiple exons in dystrophic dogs and the evaluation of the efficacies and toxicity in vivo.
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References
Arora V, Devi GR, Iversen PL (2004) Neutrally charged phosphorodiamidate morpholino antisense oligomers: uptake, efficacy and pharmacokinetics. Curr Pharm Biotechnol 5(5):431–439
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
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
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
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
Mendell JR, Rodino-Klapac LR, Sahenk Z, Roush K, Bird L, Lowes LP, Alfano L, Gomez AM, Lewis S, Kota J, Malik V, Shontz K, Walker CM, Flanigan KM, Corridore M, Kean JR, Allen HD, Shilling C, Melia KR, Sazani P, Saoud JB, Kaye EM, Eteplirsen Study G (2013) Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann Neurol 74(5):637–647. doi:10.1002/ana.23982
Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51(6):919–928
Flanigan KM (2014) Duchenne and Becker muscular dystrophies. Neurol Clin 32(3):671–688. doi:10.1016/j.ncl.2014.05.002viii
Yu X, Bao B, Echigoya Y, Yokota T (2015) Dystrophin-deficient large animal models: translational research and exon skipping. Am J Transl Res 7(8):1314–1331
Rodrigues M, Echigoya Y, Fukada S, Yokota T (2016) Current translational research and murine models for Duchenne muscular dystrophy. J Neuromuscul Dis 3(1):29–48
Shimatsu Y, Katagiri K, Furuta T, Nakura M, Tanioka Y, Yuasa K, Tomohiro M, Kornegay JN, Nonaka I, Takeda S (2003) Canine X-linked muscular dystrophy in Japan (CXMDJ). Exp Anim 52(2):93–97
Shimatsu Y, Yoshimura M, Yuasa K, Urasawa N, Tomohiro M, Nakura M, Tanigawa M, Nakamura A, Takeda S (2005) Major clinical and histopathological characteristics of canine X-linked muscular dystrophy in Japan, CXMDJ. Acta Myol 24(2):145–154
Urasawa N, Wada MR, Machida N, Yuasa K, Shimatsu Y, Wakao Y, Yuasa S, Sano T, Nonaka I, Nakamura A, Takeda S (2008) Selective vacuolar degeneration in dystrophin-deficient canine Purkinje fibers despite preservation of dystrophin-associated proteins with overexpression of Dp71. Circulation 117(19):2437–2448
Yugeta N, Urasawa N, Fujii Y, Yoshimura M, Yuasa K, Wada MR, Nakura M, Shimatsu Y, Tomohiro M, Takahashi A, Machida N, Wakao Y, Nakamura A, Takeda S (2006) Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ): electrocardiographic, echocardiographic, and morphologic studies. BMC Cardiovasc Disord 6:47. doi:10.1186/1471-2261-6-47
Sharp NJ, Kornegay JN, Van Camp SD, Herbstreith MH, Secore SL, Kettle S, Hung WY, Constantinou CD, Dykstra MJ, Roses AD et al (1992) An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 13(1):115–121
Aoki Y, Yokota T, Wood MJ (2013) Development of multiexon skipping antisense oligonucleotide therapy for Duchenne muscular dystrophy. Biomed Res Int 2013:402369. doi:10.1155/2013/402369
Echigoya Y, Yokota T (2014) Skipping multiple exons of dystrophin transcripts using cocktail antisense oligonucleotides. Nucleic Acid Ther 24(1):57–68. doi:10.1089/nat.2013.0451
Nakamura A, Yoshida K, Fukushima K, Ueda H, Urasawa N, Koyama J, Yazaki Y, Yazaki M, Sakai T, Haruta S, Takeda S, Ikeda S (2008) Follow-up of three patients with a large in-frame deletion of exons 45–55 in the Duchenne muscular dystrophy (DMD) gene. J Clin Neurosci 15(7):757–763
Echigoya Y, Aoki Y, Miskew B, Panesar D, Touznik A, Nagata T, Tanihata J, Nakamura A, Nagaraju K, Yokota T (2015) Long-term efficacy of systemic multiexon skipping targeting dystrophin exons 45–55 with a cocktail of vivo-morpholinos in mdx52 mice. Mol Ther Nucleic Acids 4:e225. doi:10.1038/mtna.2014.76
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
Acknowledgements
This work was supported by The Friends of Garrett Cumming Research Chair Fund, HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), Canadian Institutes of Health Research (CIHR), Jesse’s Journey—The Foundation for Gene and Cell Therapy, the University of Alberta Faculty of Medicine and Dentistry, Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowships for Research Abroad, and the Women and Children’s Health Research Institute (WCHRI).
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Maruyama, R., Echigoya, Y., Caluseriu, O., Aoki, Y., Takeda, S., Yokota, T. (2017). Systemic Delivery of Morpholinos to Skip Multiple Exons in a Dog Model of Duchenne Muscular Dystrophy. In: Moulton, H., Moulton, J. (eds) Morpholino Oligomers. Methods in Molecular Biology, vol 1565. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6817-6_17
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DOI: https://doi.org/10.1007/978-1-4939-6817-6_17
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