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Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.
Mol Ther. 2017 11 01; 25(11):2561-2572.MT

Abstract

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.

Authors+Show Affiliations

Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Center for Genetic Medicine Research, Children's National Medical Center, 111 Michigan Avenue Northwest, Washington, DC 20010, USA.Center for Genetic Medicine Research, Children's National Medical Center, 111 Michigan Avenue Northwest, Washington, DC 20010, USA.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.UPMC-Sorbonne Universités-University Paris 6, UPMC/INSERM UMRS974, CNRS FRE 3617, Myology Centre for Research, Paris Cedex 13 75651, France.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan.Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, SUNY-Binghamton University, Binghamton, NY 13902-6000, USA.UPMC-Sorbonne Universités-University Paris 6, UPMC/INSERM UMRS974, CNRS FRE 3617, Myology Centre for Research, Paris Cedex 13 75651, France.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada.Northern Ireland Centre for Stratified Medicine, Altnagelvin Hospital Campus, Ulster University, Londonderry BT47 6SB, UK.Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada; Muscular Dystrophy Canada Research Chair, Edmonton, AB T6G 2H7, Canada. Electronic address: toshifum@ualberta.ca.

Pub Type(s)

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

Language

eng

PubMed ID

28865998

Citation

Echigoya, Yusuke, et al. "Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy." Molecular Therapy : the Journal of the American Society of Gene Therapy, vol. 25, no. 11, 2017, pp. 2561-2572.
Echigoya Y, Lim KRQ, Trieu N, et al. Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy. Mol Ther. 2017;25(11):2561-2572.
Echigoya, Y., Lim, K. R. Q., Trieu, N., Bao, B., Miskew Nichols, B., Vila, M. C., Novak, J. S., Hara, Y., Lee, J., Touznik, A., Mamchaoui, K., Aoki, Y., Takeda, S., Nagaraju, K., Mouly, V., Maruyama, R., Duddy, W., & Yokota, T. (2017). Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy. Molecular Therapy : the Journal of the American Society of Gene Therapy, 25(11), 2561-2572. https://doi.org/10.1016/j.ymthe.2017.07.014
Echigoya Y, et al. Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy. Mol Ther. 2017 11 1;25(11):2561-2572. PubMed PMID: 28865998.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy. AU - Echigoya,Yusuke, AU - Lim,Kenji Rowel Q, AU - Trieu,Nhu, AU - Bao,Bo, AU - Miskew Nichols,Bailey, AU - Vila,Maria Candida, AU - Novak,James S, AU - Hara,Yuko, AU - Lee,Joshua, AU - Touznik,Aleksander, AU - Mamchaoui,Kamel, AU - Aoki,Yoshitsugu, AU - Takeda,Shin'ichi, AU - Nagaraju,Kanneboyina, AU - Mouly,Vincent, AU - Maruyama,Rika, AU - Duddy,William, AU - Yokota,Toshifumi, Y1 - 2017/07/28/ PY - 2017/04/19/received PY - 2017/07/21/revised PY - 2017/07/25/accepted PY - 2017/9/4/pubmed PY - 2018/6/15/medline PY - 2017/9/4/entrez KW - BMD KW - Becker muscular dystrophy KW - Duchenne muscular dystrophy KW - Exondys 51 KW - antisense morpholino KW - clinical trial candidate screening KW - drisapersen KW - eteplirsen KW - exon skipping KW - hDMD/Dmd-null mice KW - machine learning KW - mdx52 mice SP - 2561 EP - 2572 JF - Molecular therapy : the journal of the American Society of Gene Therapy JO - Mol Ther VL - 25 IS - 11 N2 - Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. SN - 1525-0024 UR - https://www.unboundmedicine.com/medline/citation/28865998/Quantitative_Antisense_Screening_and_Optimization_for_Exon_51_Skipping_in_Duchenne_Muscular_Dystrophy_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1525-0016(17)30355-6 DB - PRIME DP - Unbound Medicine ER -