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

  • Shouta Miyatake
  • Yoshitaka Mizobe
  • Hotake Takizawa
  • Yuko Hara
  • Toshifumi Yokota
  • Shin’ichi Takeda
  • Yoshitsugu Aoki
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Phosphorodiamidate morpholino oligomer (PMO) mdx mdx52 Duchenne/becker muscular dystrophies (DMD/BMD) Exon skipping Dystrophin 

Notes

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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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Shouta Miyatake
    • 1
  • Yoshitaka Mizobe
    • 1
  • Hotake Takizawa
    • 1
  • Yuko Hara
    • 1
  • Toshifumi Yokota
    • 2
  • Shin’ichi Takeda
    • 1
  • Yoshitsugu Aoki
    • 1
  1. 1.Department of Molecular TherapyNational Institute of Neuroscience, National Center of Neurology and PsychiatryTokyoJapan
  2. 2.Department of Medical GeneticsUniversity of Alberta Faculty of Medicine and DentistryEdmontonCanada

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