Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery

Yoshitsugu Aoki, Toshifumi Yokota, Tetsuya Nagata, Akinori Nakamura, Jun Tanihata, Takashi Saito, Stephanie Duguez, Kanneboyina Nagaraju, Eric P Hoffman, Terence Partridge, Shin'ichi Takeda

Research output: Contribution to journalArticle

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Abstract

Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.
LanguageEnglish
Pages13763-13768
JournalPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume109
Issue number34
DOIs
Publication statusAccepted/In press - 28 Jun 2012

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Exons
Dystrophin
Morpholinos
Duchenne Muscular Dystrophy
Skeletal Muscle Fibers
Mutation
Muscular Dystrophies
Sequence Deletion
Intramuscular Injections
Muscle Strength
Skeletal Muscle
Animal Models
Phenotype
Injections
Genes
Proteins

Keywords

  • personalized medicine nucleic acid therapy molecular therapy oligonucleotides gene therapy

Cite this

Aoki, Yoshitsugu ; Yokota, Toshifumi ; Nagata, Tetsuya ; Nakamura, Akinori ; Tanihata, Jun ; Saito, Takashi ; Duguez, Stephanie ; Nagaraju, Kanneboyina ; Hoffman, Eric P ; Partridge, Terence ; Takeda, Shin'ichi. / Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery. In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2012 ; Vol. 109, No. 34. pp. 13763-13768.
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abstract = "Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60{\%} of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15{\%} of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.",
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Aoki, Y, Yokota, T, Nagata, T, Nakamura, A, Tanihata, J, Saito, T, Duguez, S, Nagaraju, K, Hoffman, EP, Partridge, T & Takeda, S 2012, 'Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery', PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 109, no. 34, pp. 13763-13768. https://doi.org/10.1073/pnas.1204638109

Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery. / Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi.

In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol. 109, No. 34, 28.06.2012, p. 13763-13768.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery

AU - Aoki, Yoshitsugu

AU - Yokota, Toshifumi

AU - Nagata, Tetsuya

AU - Nakamura, Akinori

AU - Tanihata, Jun

AU - Saito, Takashi

AU - Duguez, Stephanie

AU - Nagaraju, Kanneboyina

AU - Hoffman, Eric P

AU - Partridge, Terence

AU - Takeda, Shin'ichi

PY - 2012/6/28

Y1 - 2012/6/28

N2 - Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.

AB - Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model.

KW - personalized medicine nucleic acid therapy molecular therapy oligonucleotides gene therapy

U2 - 10.1073/pnas.1204638109

DO - 10.1073/pnas.1204638109

M3 - Article

VL - 109

SP - 13763

EP - 13768

JO - Proceedings of the National Academy of Sciences

T2 - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

SN - 0027-8424

IS - 34

ER -