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Morpholino-Mediated Exons 28–29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay

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Muscular Dystrophy Therapeutics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2587))

Abstract

Dysferlinopathies are a group of disabling muscular dystrophies  that includes limb girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy, and distal myopathy with anterior tibial onset (DMAT) as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for many patients with dysferlinopathies. It was reported that exons 28–29 of DYSF are dispensable for dysferlin functions. Here, we present a method for multiexon skipping of DYSF exons 28–29 using a cocktail of two phosphorodiamidate morpholino oligomers (PMOs) on cells derived from a dystrophinopathy patient. Also, we describe assays to characterize the multiexon skipped dysferlin at several levels by using one-step RT-PCR, immunoblotting, and a membrane wounding assay.

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Acknowledgments

This work was supported by the University of Alberta Faculty of Medicine and Dentistry, 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 Women and Children’s Health Research Institute (WCHRI), and Alberta Innovates Health Solutions (AIHS). S.A. is supported by scholarships from the Maternal and Child Health (MatCH) Program and the Alberta Innovates Graduate Student Scholarship (AIGSS).

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Authors and Affiliations

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

    Saeed Anwar & Toshifumi Yokota

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

    Toshifumi Yokota

Authors
  1. Saeed Anwar
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  2. Toshifumi Yokota
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Correspondence to Toshifumi Yokota .

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Editors and Affiliations

  1. Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Rika Maruyama

  2. Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Toshifumi Yokota

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Anwar, S., Yokota, T. (2023). Morpholino-Mediated Exons 28–29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT-PCR, Immunoblotting, and Membrane Wounding Assay. In: Maruyama, R., Yokota, T. (eds) Muscular Dystrophy Therapeutics. Methods in Molecular Biology, vol 2587. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2772-3_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2772-3_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2771-6

  • Online ISBN: 978-1-0716-2772-3

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