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Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting

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Mouse Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2224))

Abstract

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.

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References

  1. Duchenne (1867) The pathology of paralysis with muscular degeneration (paralysie myosclerotique), or paralysis with apparent hypertrophy. Br Med J 2(363):541–542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Nowak KJ, Davies KE (2004) Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment. EMBO Rep 5(9):872–876. https://doi.org/10.1038/sj.embor.7400221

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Koenig M, Hoffman EP, Bertelson CJ et al (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50(3):509–517

    Article  CAS  PubMed  Google Scholar 

  4. Manzur AY, Muntoni F (2009) Diagnosis and new treatments in muscular dystrophies. J Neurol Neurosurg Psychiatry 80(7):706–714. https://doi.org/10.1136/jnnp.2008.158329

    Article  CAS  PubMed  Google Scholar 

  5. Yiu EM, Kornberg AJ (2015) Duchenne muscular dystrophy. J Paediatr Child Health 51(8):759–764. https://doi.org/10.1111/jpc.12868

    Article  PubMed  Google Scholar 

  6. Nichols B, Takeda S, Yokota T (2015) Nonmechanical roles of dystrophin and associated proteins in exercise, neuromuscular junctions, and brains. Brain Sci 5(3):275–298. https://doi.org/10.3390/brainsci5030275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  8. Nudel U, Zuk D, Einat P et al (1989) Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature 337(6202):76–78. https://doi.org/10.1038/337076a0

    Article  CAS  PubMed  Google Scholar 

  9. Sato K, Yokota T, Ichioka S et al (2008) Vasodilation of intramuscular arterioles under shear stress in dystrophin-deficient skeletal muscle is impaired through decreased nNOS expression. Acta Myol 27:30–36

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kobayashi YM, Rader EP, Crawford RW et al (2008) Sarcolemma-localized nNOS is required to maintain activity after mild exercise. Nature 456(7221):511–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Petrof BJ (2002) Molecular pathophysiology of myofiber injury in deficiencies of the dystrophin-glycoprotein complex. Am J Phys Med Rehabil 81(11 Suppl):S162–S174. https://doi.org/10.1097/01.PHM.0000029775.54830.80

    Article  PubMed  Google Scholar 

  12. Vita G, Vita GL, Musumeci O et al (2019) Genetic neuromuscular disorders: living the era of a therapeutic revolution. Part 2: diseases of motor neuron and skeletal muscle. Neurol Sci 40(4):671–681. https://doi.org/10.1007/s10072-019-03764-z

    Article  PubMed  Google Scholar 

  13. Matsuo M, Masumura T, Nishio H et al (1991) Exon skipping during splicing of dystrophin mRNA precursor due to an intraexon deletion in the dystrophin gene of Duchenne muscular dystrophy kobe. J Clin Invest 87(6):2127–2131. https://doi.org/10.1172/JCI115244

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hua Y, Krainer AR (2012) Antisense-mediated exon inclusion. Methods Mol Biol 867:307–323. https://doi.org/10.1007/978-1-61779-767-5_20

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Rodrigues M, Yokota T (2018) An overview of recent advances and clinical applications of exon skipping and splice modulation for muscular dystrophy and various genetic diseases. Methods Mol Biol 1828:31–55. https://doi.org/10.1007/978-1-4939-8651-4_2

    Article  CAS  PubMed  Google Scholar 

  16. Wood M, Yin H, McClorey G (2007) Modulating the expression of disease genes with RNA-based therapy. PLoS Genet 3(6):e109. https://doi.org/10.1371/journal.pgen.0030109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kole R, Krieg AM (2015) Exon skipping therapy for Duchenne muscular dystrophy. Adv Drug Deliv Rev 87:104–107. https://doi.org/10.1016/j.addr.2015.05.008

    Article  CAS  PubMed  Google Scholar 

  18. Dunckley MGMM, Villiet P, Eperon IC, Dickson G (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Hum Mol Genet 7(7):1083–1090

    Article  CAS  PubMed  Google Scholar 

  19. Love DR, Byth BC, Tinsley JM et al (1993) Dystrophin and dystrophin-related proteins: a review of protein and RNA studies. Neuromuscul Disord 3(1):5–21

    Article  CAS  PubMed  Google Scholar 

  20. Nakamura A, Shiba N, Miyazaki D et al (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. https://doi.org/10.1038/jhg.2016.152

    Article  CAS  PubMed  Google Scholar 

  21. Nakamura A, Fueki N, Shiba N et al (2016) Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy. J Hum Genet 61(7):663–667. https://doi.org/10.1038/jhg.2016.28

    Article  CAS  PubMed  Google Scholar 

  22. Wein N, Vulin A, Findlay AR et al (2017) Efficient skipping of single exon duplications in DMD patient-derived cell lines using an antisense oligonucleotide approach. J Neuromuscul Dis 4(3):199–207. https://doi.org/10.3233/JND-170233

    Article  PubMed  Google Scholar 

  23. Lim KRQ, Echigoya Y, Nagata T et al (2019) Efficacy of multi-exon skipping treatment in duchenne muscular dystrophy dog model neonates. Mol Ther 27(1):76–86. https://doi.org/10.1016/j.ymthe.2018.10.011

    Article  CAS  PubMed  Google Scholar 

  24. Lu QL, Rabinowitz A, Chen YC et al (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. https://doi.org/10.1073/pnas.0406700102

    Article  CAS  PubMed  Google Scholar 

  25. Aoki Y, Nakamura A, Yokota T et al (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. https://doi.org/10.1038/mt.2010.186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shimo T, Hosoki K, Nakatsuji Y et al (2018) A novel human muscle cell model of Duchenne muscular dystrophy created by CRISPR/Cas9. J Hum Genet 63(3):365–375

    Article  CAS  PubMed  Google Scholar 

  27. Aartsma-Rus A, Bremmer-Bout M, Janson AA et al (2002) Targeted exon skipping as a potential gene correction therapy for Duchenne muscular dystrophy. Neuromuscul Disord 12(Suppl 1):S71–S77

    Article  PubMed  Google Scholar 

  28. Miyatake S, Mizobe Y, Takizawa H et al (2018) Exon Skipping therapy using phosphorodiamidate morpholino oligomers in the mdx52 mouse model of Duchenne muscular dystrophy. Methods Mol Biol 1687:123–141. https://doi.org/10.1007/978-1-4939-7374-3_9

    Article  CAS  PubMed  Google Scholar 

  29. Fletcher S, Bellgard MI, Price L et al (2017) Translational development of splice-modifying antisense oligomers. Expert Opin Biol Ther 17(1):15–30. https://doi.org/10.1080/14712598.2017.1250880

    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. https://doi.org/10.1089/nat.2016.0657

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Stein CA (2016) Eteplirsen approved for Duchenne muscular dystrophy: the FDA faces a difficult choice. Mol Ther 24(11):1884–1885. https://doi.org/10.1038/mt.2016.188

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Michelson D, Ciafaloni E, Ashwal S et al (2018) Evidence in focus: Nusinersen use in spinal muscular atrophy: Report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology. Neurology 91(20):923–933. https://doi.org/10.1212/WNL.0000000000006502

    Article  PubMed  Google Scholar 

  33. Lim KR, Maruyama R, Yokota T (2017) Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Des Devel Ther 11:533–545. https://doi.org/10.2147/DDDT.S97635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Aartsma-Rus A, van Ommen GJ (2009) Less is more: therapeutic exon skipping for Duchenne muscular dystrophy. Lancet Neurol 8(10):873–875. https://doi.org/10.1016/S1474-4422(09)70229-7

    Article  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Yokota T, Pistilli E, Duddy W et al (2007) Potential of oligonucleotide-mediated exon-skipping therapy for Duchenne muscular dystrophy. Expert Opin Biol Ther 7(6):831–842. https://doi.org/10.1517/14712598.7.6.831

    Article  CAS  PubMed  Google Scholar 

  37. Echigoya Y, Lim KRQ, Trieu N et al (2017) Quantitative antisense screening and optimization for exon 51 skipping in Duchenne muscular dystrophy. Mol Ther 25(11):2561–2572. https://doi.org/10.1016/j.ymthe.2017.07.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Anwar S, Yokota T (2020) Golodirsen for Duchenne muscular dystrophy. Drugs Today 56:491–504. https://doi.org/10.1016/j.omtn.2018.09.017

  39. Roshmi RR, Yokota T (2019) Viltolarsen for the treatment of Duchenne muscular dystrophy. Drugs Today 55(10):627–639. https://doi.org/10.3390/jpm9010001

  40. Aslesh T, Maruyama R, Yokota T (2018) Skipping multiple exons to treat DMD-promises and challenges. Biomedicine 6(1):1. https://doi.org/10.3390/biomedicines6010001

    Article  CAS  Google Scholar 

  41. Touznik A, Maruyama R, Hosoki K et al (2017) LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts. Sci Rep 7(1):3672. https://doi.org/10.1038/s41598-017-03850-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Maruyama R, Touznik A, Yokota T (2018) Evaluation of exon inclusion induced by splice switching antisense oligonucleotides in SMA patients fibroblasts. J Vis Exp 135:57530

    Google Scholar 

  43. Echigoya Y, Aoki Y, Miskew B et al (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. https://doi.org/10.1038/mtna.2014.76

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gait MJ, Arzumanov AA, McClorey G et al (2019) Cell-penetrating peptide conjugates of steric blocking oligonucleotides as therapeutics for neuromuscular diseases from a historical perspective to current prospects of treatment. Nucleic Acid Ther 29(1):1–12. https://doi.org/10.1089/nat.2018.0747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hammond SM, Hazell G, Shabanpoor F et al (2016) Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy. Proc Natl Acad Sci U S A 113(39):10962–10967. https://doi.org/10.1073/pnas.1605731113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Betts C, Saleh AF, Arzumanov AA et al (2012) Pip6-PMO, a new generation of peptide-oligonucleotide conjugates with improved cardiac exon skipping activity for DMD treatment. Mol Ther Nucleic Acids 1:e38. https://doi.org/10.1038/mtna.2012.30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Yin H, Saleh AF, Betts C et al (2011) Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice. Mol Ther 19(7):1295–1303. https://doi.org/10.1038/mt.2011.79

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hammond SM, Wood MJ (2010) PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy. Curr Opin Mol Ther 12(4):478–486

    CAS  PubMed  Google Scholar 

  49. Echigoya Y, Nakamura A, Nagata T et al (2017) Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy. Proc Natl Acad Sci U S A 114(16):4213–4218. https://doi.org/10.1073/pnas.1613203114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Singh NK, Singh NN, Androphy EJ et al (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(4):1333–1346. https://doi.org/10.1128/MCB.26.4.1333-1346.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Melo D, Maruyama R, Yokota T (2018) Systemic injection of peptide-PMOs into humanized DMD mice and evaluation by RT-PCR and ELISA. Methods Mol Biol 1828:263–273. https://doi.org/10.1007/978-1-4939-8651-4_16

    Article  CAS  PubMed  Google Scholar 

  52. Bremmer-Bout M, Aartsma-Rus A, de Meijer EJ et al (2004) Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of human-specific antisense oligonucleotides. Mol Ther 10(2):232–240. https://doi.org/10.1016/j.ymthe.2004.05.031

    Article  CAS  PubMed  Google Scholar 

  53. Kudoh H, Ikeda H, Kakitani M et al (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. https://doi.org/10.1016/j.bbrc.2004.12.191

    Article  CAS  PubMed  Google Scholar 

  54. Sicinski P, Geng Y, Ryder-Cook AS et al (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244(4912):1578–1580

    Article  CAS  PubMed  Google Scholar 

  55. Anderson LV, Davison K (1999) Multiplex Western blotting system for the analysis of muscular dystrophy proteins. Am J Pathol 154(4):1017–1022. https://doi.org/10.1016/S0002-9440(10)65354-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Anthony K, Arechavala-Gomeza V, Taylor LE et al (2014) Dystrophin quantification: Biological and translational research implications. Neurology 83(22):2062–2069. https://doi.org/10.1212/WNL.0000000000001025

    Article  PubMed  PubMed Central  Google Scholar 

  57. Miskew Nichols B, Aoki Y, Kuraoka M et al (2016) Multi-exon skipping using cocktail antisense oligonucleotides in the canine X-linked muscular dystrophy. J Vis Exp 111:53776. https://doi.org/10.3791/53776

    Article  CAS  Google Scholar 

  58. Echigoya Y, Mouly V, Garcia L et al (2015) In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy. PLoS One 10(3):e0120058. https://doi.org/10.1371/journal.pone.0120058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Maruyama R, Aoki Y, Takeda S et al (2018) In vivo evaluation of multiple exon skipping with peptide-PMOs in cardiac and skeletal muscles in Dystrophic dogs. Methods Mol Biol 1828:365–379. https://doi.org/10.1007/978-1-4939-8651-4_23

    Article  CAS  PubMed  Google Scholar 

  60. Lim KRQ, Yokota T (2018) Quantitative evaluation of exon skipping in immortalized muscle cells in vitro. Methods Mol Biol 1828:127–139. https://doi.org/10.1007/978-1-4939-8651-4_7

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Faculty of Medicine and Dentistry, Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Rika Maruyama & Toshifumi Yokota

  2. The Friends of Garrett Cumming Research & Muscular Dystrophy Canada HM Toupin Neurological Science Research Chair, Edmonton, AB, Canada

    Toshifumi Yokota

Authors
  1. Rika Maruyama
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  2. Toshifumi Yokota
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Correspondence to Toshifumi Yokota .

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Editors and Affiliations

  1. Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA

    Shree Ram Singh

  2. Department of Surgery, University of California, AntiCancer, Inc., San Diego, CA, USA

    Robert M. Hoffman

  3. Department of Biology, University of Dayton, Dayton, OH, USA

    Amit Singh

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Maruyama, R., Yokota, T. (2021). Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive Detection of Dystrophin Using Western Blotting. In: Singh, S.R., Hoffman, R.M., Singh, A. (eds) Mouse Genetics . Methods in Molecular Biology, vol 2224. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1008-4_15

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  • DOI: https://doi.org/10.1007/978-1-0716-1008-4_15

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