Abstract
Antisense oligonucleotide induced exon skipping emerges as a promising therapeutic strategy for patients suffering from a devastating muscle disorder Duchenne muscular dystrophy (DMD). Systemic administration of antisense phosphorodiamidate morpholino oligomers (PMOs) targeting exons 6 and 8 in dystrophin mRNA of the canine X-linked muscular dystrophy model in Japan (CXMDJ) that lacks exon 7, restored dystrophin expression throughout skeletal muscle and ameliorated skeletal muscle pathology and function. However, the antisense PMO regime used in CXMDJ could not be considered for a direct application to DMD patients so far, because this type of mutation is quite rare. We have identified a DMD patient with an exon 7 deletion; and tried a direct translation of the antisense PMOs used in dog models to the DMD patient’s cells. We converted fibroblasts obtained from CXMDJ dogs and from the DMD patient to myotubes by MyoD transduction using fluorescence-activated cell sorting (FACS). We subsequently designed antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 and administered them as a cocktail to the in vitro generated dog or human myotubes. In both cases, we observed comparable skipping efficacy of exons 6 and 8 and restoration of dystrophin protein. The accompanying skipping of exon 9, which does not alter the reading frame, varied according to the cell origin. The antisense PMOs originally administered to the CXMDJ dog model were capable of inducing multi-exon skipping of the dystrophin gene on the FACS-aided MyoD-transduced fibroblasts derived from an exon 7-deleted DMD patient. These data support the suitability of dog as a laboratory model for DMD because the similarity of dystrophin sequences allowed a successful translation of the dog’s PMOs to DMD patients cells.
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Acknowledgments
This work was supported by Intramural Research Grant (22-5) for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry (NCNP); Health and Labour Sciences Research Grants for Translation Research (H21-Translational Research-011); Health and Labour Sciences Research Grants for Translation Research (H21-Clinical Research-015); Comprehensive Research on Disability Health and Welfare (H23-Neuromuscular Disease-005) from the Ministry of Health, Labour, and Welfare of Japan; Foundation to Eradicate Duchenne; US Department of Defense (W81XWH-09-1-0599); the National Institutes of Health (1P50AR060836, 5T32AR056993, U54HD071601, R24HD050846, and K26OD011171); Muscular Dystrophy Association; University of Alberta; The Friends of Garrett Cumming Research; HM Toupin Neurological Science Research; and Muscular Dystrophy Canada.
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Nakamura, A., Aoki, Y., Tsoumpra, M., Yokota, T., Takeda, S. (2018). In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient. In: Yokota, T., Maruyama, R. (eds) Exon Skipping and Inclusion Therapies. Methods in Molecular Biology, vol 1828. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8651-4_9
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DOI: https://doi.org/10.1007/978-1-4939-8651-4_9
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