Exons 45–55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene

Yusuke Echigoya, KRQ Lim, Yoshitsugu Aoki, Shin'ichi Takeda, Vincent Mouly, William Duddy, Toshifumi Yokota

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45–55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45–55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45–55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45–55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

LanguageEnglish
Pages2005-2017
Number of pages13
JournalMolecular Therapy
Volume27
Issue number11
Early online date25 Jul 2019
DOIs
Publication statusPublished - 6 Nov 2019

Fingerprint

Morpholinos
Duchenne Muscular Dystrophy
Exons
Dystrophin
Mutation
Genes
Frameshift Mutation
Muscle Cells
Western Blotting

Keywords

  • Duchenne muscular dystrophyDMDBecker muscular dystrophyBMDdystrophinexon skippingantisense oligonucleotidephosphorodiamidate morpholino oligomerPMOexons 45–55humanized mouse
  • Becker muscular dystrophy
  • humanized mouse
  • PMO
  • antisense oligonucleotide
  • exon skipping
  • Duchenne muscular dystrophy
  • phosphorodiamidate morpholino oligomer
  • BMD
  • DMD
  • exons 45–55
  • dystrophin

Cite this

Echigoya, Yusuke ; Lim, KRQ ; Aoki, Yoshitsugu ; Takeda, Shin'ichi ; Mouly, Vincent ; Duddy, William ; Yokota, Toshifumi. / Exons 45–55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene. In: Molecular Therapy. 2019 ; Vol. 27, No. 11. pp. 2005-2017.
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abstract = "Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45–55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45–55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45–55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45–55 skipping and treat over 65{\%} of DMD patients carrying out-of-frame or in-frame deletions.",
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Exons 45–55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene. / Echigoya, Yusuke; Lim, KRQ; Aoki, Yoshitsugu; Takeda, Shin'ichi; Mouly, Vincent; Duddy, William; Yokota, Toshifumi.

In: Molecular Therapy, Vol. 27, No. 11, 06.11.2019, p. 2005-2017.

Research output: Contribution to journalArticle

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