Restoration of Dystrophin Protein Expression by Exon Skipping Utilizing CRISPR-Cas9 in Myoblasts Derived from DMD Patient iPS Cells
| Autor: | Ifuku Masataka, Akitsu Hotta, Kumiko Iwabuchi, Masami Tanaka, Mandy S. Y. Lung |
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| Rok vydání: | 2018 |
| Předmět: |
musculoskeletal diseases
0301 basic medicine congenital hereditary and neonatal diseases and abnormalities Duchenne muscular dystrophy Biology medicine.disease Exon skipping Cell biology 03 medical and health sciences Exon genomic DNA 030104 developmental biology Genome editing RNA splicing medicine biology.protein Dystrophin Gene |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493986507 |
| DOI: | 10.1007/978-1-4939-8651-4_12 |
| Popis: | Duchenne muscular dystrophy (DMD) is a congenital X-linked disease caused by mutations in the gene encoding the dystrophin protein, which is required for myofiber integrity. Exon skipping therapy is an emerging strategy for restoring the open reading frame of the dystrophin gene to produce functional protein in DMD patients by skipping single or multiple exons. Although antisense oligonucleotides are able to target pre-mRNA for exon skipping, their half-lives are short and any therapeutic benefit is transient. In contrast, genome editing by DNA nucleases, such as the CRISPR-Cas9 system, could offer permanent correction by targeting genomic DNA. Our laboratory previously reported that disrupting the splicing acceptor site in exon 45 by plasmid delivery of the CRISPR-Cas9 system in iPS cells, derived from a DMD patient lacking exon 44, successfully restored dystrophin protein expression in differentiated myoblasts. Herein, we describe an optimized methodology to prepare myoblasts differentiated from iPS cells by mRNA transfection of the CRISPR-Cas9 system to skip exon 45 in myoblasts, and evaluate the restored dystrophin by RT-PCR and Western blotting. |
| Databáze: | OpenAIRE |
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