title: Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.
abstract: Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Ousterout
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: Ami M
authors.initials: AM
authors.lastname: Kabadi
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: Pratiksha I
authors.initials: PI
authors.lastname: Thakore
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Perez-Pinera
authors.affiliation: Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Brown
authors.affiliation: Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Majoros
authors.affiliation: 1] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA [2] Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Reddy
authors.affiliation: 1] Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA [2] Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA [3] Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
mesh_terms: D000818:Animals; D001483:Base Sequence; D016189:Dystrophin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D018274:Electroporation; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005091:Exons; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008969:Molecular Sequence Data; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D032446:Myoblasts / Q000378:metabolism / Q000473:pathology; D016366:Open Reading Frames; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D017393:RNA Editing; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: C484258:DMD protein, human; D016189:Dystrophin; D012333:RNA, Messenger; D004720:Endonucleases