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In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient.
Methods Mol Biol. 2018; 1828:151-163.MM

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.

Authors+Show Affiliations

Third Department of Medicine, Shinshu University School of Medicine, Matsumoto, Japan. anakamu@shinshu-u.ac.jp. Department of Neurology, Matsumoto Medical Center, National Hospital Organization, Matsumoto, Japan. anakamu@shinshu-u.ac.jp.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, AB, Canada.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Japan.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

30171540

Citation

Nakamura, Akinori, et al. "In Vitro Multiexon Skipping By Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient." Methods in Molecular Biology (Clifton, N.J.), vol. 1828, 2018, pp. 151-163.
Nakamura A, Aoki Y, Tsoumpra M, et al. In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient. Methods Mol Biol. 2018;1828:151-163.
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. Methods in Molecular Biology (Clifton, N.J.), 1828, 151-163. https://doi.org/10.1007/978-1-4939-8651-4_9
Nakamura A, et al. In Vitro Multiexon Skipping By Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient. Methods Mol Biol. 2018;1828:151-163. PubMed PMID: 30171540.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient. AU - Nakamura,Akinori, AU - Aoki,Yoshitsugu, AU - Tsoumpra,Maria, AU - Yokota,Toshifumi, AU - Takeda,Shin'ichi, PY - 2018/9/2/entrez PY - 2018/9/2/pubmed PY - 2019/4/2/medline KW - Antisense oligonucleotide KW - Canine X-linked muscular dystrophy in Japan (CXMDJ) KW - Duchenne/Becker muscular dystrophies (DMD/BMD) KW - Dystrophin KW - Exon skipping KW - Multiexon skipping KW - Phosphorodiamidate morpholino oligomers (PMOs) SP - 151 EP - 163 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 1828 N2 - 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. SN - 1940-6029 UR - https://www.unboundmedicine.com/medline/citation/30171540/In_Vitro_Multiexon_Skipping_by_Antisense_PMOs_in_Dystrophic_Dog_and_Exon_7_Deleted_DMD_Patient_ L2 - https://dx.doi.org/10.1007/978-1-4939-8651-4_9 DB - PRIME DP - Unbound Medicine ER -