Abstract
Duchenne muscular dystrophy (DMD) is caused by the mutations in the DMD gene resulting in no dystrophin production. Skipping DMD exons using phosphorodiamidate morpholino oligomers (PMOs) is an emerging treatment strategy that can restore the reading frame of the mutated gene and produce truncated but functional dystrophin protein. To date, four PMOs, including eteplirsen, casimersen, viltolarsen, and golodirsen, have been conditionally approved by the FDA for the treatment of DMD. Since degeneration of muscle fibers and irreversible fibrosis occur from childhood, the earlier treatment is preferred. The canine X-linked muscular dystrophy in Japan (CXMDj), a dog model of DMD, produces no dystrophin and exhibits a severe phenotype similar to human patients from early childhood. As such, CXMDj, which harbors a splice site mutation in intron 6, is a useful model for examining the long-term effects of early PMO treatment. In this chapter, we describe the systemic delivery of a cocktail of four PMOs that can successfully induce multiple exon skipping (exons 6–9) in neonatal dystrophic dogs. We also describe the procedures to evaluate the efficacy and toxicity, including clinical grading of dystrophic dogs, ELISA-based quantification of PMOs, histology, RT-PCR, and western blotting.
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Acknowledgments
This work was supported by the Friends of Garrett Cumming Research Chair Fund, HM Toupin Neurological Science Research Chair Fund, Muscular Dystrophy Canada, Alberta Innovates, the University of Alberta Faculty of Medicine and Dentistry, and the Women and Children’s Health Research Institute (WCHRI). We also would like to thank Kenji Rowel Q Lim for his assistance.
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Shah, M.N.A., Yokota, T. (2023). Restoring Dystrophin Expression by Skipping Exons 6 and 8 in Neonatal Dystrophic Dogs. In: Maruyama, R., Yokota, T. (eds) Muscular Dystrophy Therapeutics. Methods in Molecular Biology, vol 2587. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2772-3_6
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DOI: https://doi.org/10.1007/978-1-0716-2772-3_6
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