Efficacy of systemic morpholino exon-skipping in duchenne dystrophy dogs

Authors

  • Toshifumi Yokota PhD,

    1. Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC
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  • Qi-long Lu MD, PhD,

    1. McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/Amyotrophic Lateral Sclerosis Center, Carolinas Medical Center, Charlotte, NC
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  • Terence Partridge PhD,

    1. Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC
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  • Masanori Kobayashi DVM,

    1. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan
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  • Akinori Nakamura MD, PhD,

    1. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan
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  • Shińichi Takeda MD, PhD,

    Corresponding author
    1. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan
    • Department of Molecular Therapy National Institute of Neuroscience, National Center of Neurology and Psychiatry 4-1-1 Ogawa-higashi, Kodaiva, Tokyo, Japan
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  • Eric Hoffman PhD

    Corresponding author
    1. Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC
    • Research Center for Genetic Medicine, Children's National Medical Center, Department of Integrative Systems Biology, George Washington University School of Medicine, 111 Michigan Avenue NW, Washington, DC 20010
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  • Potential conflict of interest: Nothing to report.

Abstract

Objective

Duchenne muscular dystrophy (DMD) is caused by the inability to produce dystrophin protein at the myofiber membrane. A method to rescue dystrophin production by antisense oligonucleotides, termed exon-skipping, has been reported for the mdx mouse and in four DMD patients by local intramuscular injection. We sought to test efficacy and toxicity of intravenous oligonucleotide (morpholino)-induced exon skipping in the DMD dog model.

Methods

We tested a series of antisense drugs singly and as cocktails, both in primary cell culture, and two in vivo delivery methods (intramuscular injection and systemic intravenous injection). The efficiency and efficacy of multiexon skipping (exons 6–9) were tested at the messenger RNA, protein, histological, and clinical levels.

Results

Weekly or biweekly systemic intravenous injections with a three-morpholino cocktail over the course of 5 to 22 weeks induced therapeutic levels of dystrophin expression throughout the body, with an average of about 26% normal levels. This was accompanied by reduced inflammatory signals examined by magnetic resonance imaging and histology, improved or stabilized timed running tests, and clinical symptoms. Blood tests indicated no evidence of toxicity.

Interpretation

This is the first report of widespread rescue of dystrophin expression to therapeutic levels in the dog model of DMD. This study also provides a proof of concept for systemic multiexon-skipping therapy. Use of cocktails of morpholino, as shown here, allows broader application of this approach to a greater proportion of DMD patients (90%) and also offers the prospect of selecting deletions that optimize the functionality of the dystrophin protein. Ann Neurol 2009

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