Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression

Joshua Lee, Takashi Saito, William Duddy, Shin'ichi Takeda, Toshifumi Yokota

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45–55 skipping in dystrophin is potentially applicable to approximately 47% of DMD patients because many mutations occur within this “mutation hotspot.” In addition, patients naturally harboring a dystrophin exons 45–55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45–55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45–55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.
LanguageEnglish
Title of host publicationMethods in Molecular Biology
Subtitle of host publicationMethods and Protocols
EditorsToshifumi Yokota, Rika Maruyama
Chapter8
Pages141-150
Number of pages10
ISBN (Electronic)978-1-4939-8651-4
Publication statusPublished - 3 Aug 2018

Fingerprint

Dystrophin
Duchenne Muscular Dystrophy
Skeletal Muscle Fibers
Exons
Fibroblasts
Sequence Deletion
Antisense Oligonucleotides
Phenotype
Myogenic Regulatory Factors
Research Personnel
In Vitro Techniques
Therapeutics
Muscles
Mutation
Inborn Genetic Diseases
RNA Precursors
Traction
Muscle Cells
Open Reading Frames
Biopsy

Cite this

Lee, J., Saito, T., Duddy, W., Takeda, S., & Yokota, T. (2018). Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. In T. Yokota, & R. Maruyama (Eds.), Methods in Molecular Biology: Methods and Protocols (pp. 141-150)
Lee, Joshua ; Saito, Takashi ; Duddy, William ; Takeda, Shin'ichi ; Yokota, Toshifumi. / Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. Methods in Molecular Biology: Methods and Protocols. editor / Toshifumi Yokota ; Rika Maruyama. 2018. pp. 141-150
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abstract = "Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach for the treatment of various genetic diseases and a therapy which has gained significant traction in recent years following FDA approval of new antisense-based drugs. Exon skipping for Duchenne muscular dystrophy (DMD) works by modulating dystrophin pre-mRNA splicing, preventing incorporation of frame-disrupting exons into the final mRNA product while maintaining the open reading frame, to produce a shortened-yet-functional protein as seen in milder Becker muscular dystrophy (BMD) patients. Exons 45–55 skipping in dystrophin is potentially applicable to approximately 47{\%} of DMD patients because many mutations occur within this “mutation hotspot.” In addition, patients naturally harboring a dystrophin exons 45–55 in-frame deletion mutation have an asymptomatic or exceptionally mild phenotype compared to shorter in-frame deletion mutations in this region. As such, exons 45–55 skipping could transform the DMD phenotype into an asymptomatic or very mild BMD phenotype and rescue nearly a half of DMD patients. In addition, this strategy is potentially applicable to some BMD patients as well, who have in-frame deletion mutations in this region. As the degree of exon skipping correlates with therapeutic outcomes, reliable measurements of exon skipping efficiencies are essential to the development of novel antisense-mediated exon skipping therapeutics. In the case of DMD, researchers have often relied upon human muscle fibers obtained from muscle biopsies for testing; however, this method is highly invasive and patient myofibers can display limited proliferative ability. To overcome these challenges, researchers can generate myofibers from patient fibroblast cells by transducing the cells with a viral vector containing MyoD, a myogenic regulatory factor. Here, we describe a methodology for assessing dystrophin exons 45–55 multiple skipping efficiency using antisense oligonucleotides in human muscle cells derived from DMD patient fibroblast cells.",
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Lee, J, Saito, T, Duddy, W, Takeda, S & Yokota, T 2018, Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. in T Yokota & R Maruyama (eds), Methods in Molecular Biology: Methods and Protocols. pp. 141-150.

Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. / Lee, Joshua; Saito, Takashi; Duddy, William; Takeda, Shin'ichi; Yokota, Toshifumi.

Methods in Molecular Biology: Methods and Protocols. ed. / Toshifumi Yokota; Rika Maruyama. 2018. p. 141-150.

Research output: Chapter in Book/Report/Conference proceedingChapter

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M3 - Chapter

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Lee J, Saito T, Duddy W, Takeda S, Yokota T. Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45–55 Skipping Accompanied by Rescue of Dystrophin Expression. In Yokota T, Maruyama R, editors, Methods in Molecular Biology: Methods and Protocols. 2018. p. 141-150