title: Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.
abstract: Duchenne muscular dystrophy (DMD) is a severe muscle-degenerative disease caused by a mutation in the dystrophin gene. Genetic correction of patient-derived induced pluripotent stem cells (iPSCs) by TALENs or CRISPR-Cas9 holds promise for DMD gene therapy; however, the safety of such nuclease treatment must be determined. Using a unique k-mer database, we systematically identified a unique target region that reduces off-target sites. To restore the dystrophin protein, we performed three correction methods (exon skipping, frameshifting, and exon knockin) in DMD-patient-derived iPSCs, and found that exon knockin was the most effective approach. We further investigated the genomic integrity by karyotyping, copy number variation array, and exome sequencing to identify clones with a minimal mutation load. Finally, we differentiated the corrected iPSCs toward skeletal muscle cells and successfully detected the expression of full-length dystrophin protein. These results provide an important framework for developing iPSC-based gene therapy for genetic disorders using programmable nucleases.
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Hongmei Lisa
authors.initials: HL
authors.lastname: Li
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Fujimoto
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Sasakawa
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Saya
authors.initials: S
authors.lastname: Shirai
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Tokiko
authors.initials: T
authors.lastname: Ohkame
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Sakuma
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Amano
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Sakurai
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan; Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
authors.forename: Shinya
authors.initials: S
authors.lastname: Yamanaka
authors.affiliation: Center for iPS Cell Research and Application, Kyoto University, Kyoto 606-8507, Japan; iCeMS, Kyoto University, Kyoto 606-8501, Japan; PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan. Electronic address: akitsu.hotta@cira.kyoto-u.ac.jp.
authors.forename: Akitsu
authors.initials: A
authors.lastname: Hotta
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D056915:DNA Copy Number Variations; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D023061:Gene Order; D018390:Gene Targeting; D056426:Genetic Loci; D015316:Genetic Therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D059785:Karyotype; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D016254:Mutagenesis, Insertional; D009154:Mutation; D016364:Reading Frames; D017384:Sequence Deletion
chemical_list: D016189:Dystrophin