In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide

  • An Erratum to this article was published on 01 November 2000

Abstract

In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription–polymerase chain reaction (RT-PCR) analysis of exons 5–10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)–PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.

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Figure 1: Chimeric oligonucleotide used for GRMD mutation repair.
Figure 2: RT-PCR of total RNA from canine skeletal muscle.
Figure 3: Quantitative RT-PCR of cDNA from canine skeletal muscle.
Figure 4: In situ RT-PCR of canine muscle.
Figure 5: Epitope mapping of dystrophin antibodies and western blotting.
Figure 6: Immunofluorescence localization of dystrophin in canine muscle.
Figure 7: Genomic PCR analysis of mutation repair.

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Acknowledgements

The authors wish to thank Walter Bradley (University of Miami School of Medicine) for initial critical reading of this manuscript. The authors also wish to acknowledge the following for support of this research: Association Francais contre les Myopathies, AFM (R.J.B.), Muscular Dystrophy Group of Great Britain and Northern Ireland (G.E.M.) for support of antibody production, and the Parents Project for Muscular Dystrophy (J.N.K.), and MDA (J.N.K.) for their support of the canine animal colony. One of the authors (R.J.B.) is a member of the Scientific Advisory Board of Kimeragen Pharmaceuticals, Inc. (Newtown, PA) and acknowledges some support for this research from that company. Finally, the authors wish to acknowledge sustaining support for this research from the Diabetes Research Institute Foundation (Hollywood, FL).

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Correspondence to Richard J. Bartlett.

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Bartlett, R., Stockinger, S., Denis, M. et al. In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotechnol 18, 615–622 (2000). https://doi.org/10.1038/76448

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