title: Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.
abstract: Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.
authors.affiliation: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom.
authors.forename: Linda
authors.initials: L
authors.lastname: Popplewell
authors.forename: Taeyoung
authors.initials: T
authors.lastname: Koo
authors.forename: Xavier
authors.initials: X
authors.lastname: Leclerc
authors.forename: Aymeric
authors.initials: A
authors.lastname: Duclert
authors.forename: Kamel
authors.initials: K
authors.lastname: Mamchaoui
authors.forename: Agnés
authors.initials: A
authors.lastname: Gouble
authors.forename: Vincent
authors.initials: V
authors.lastname: Mouly
authors.forename: Thomas
authors.initials: T
authors.lastname: Voit
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
authors.forename: Frédéric
authors.initials: F
authors.lastname: Cédrone
authors.forename: Olga
authors.initials: O
authors.lastname: Isman
authors.forename: Rafael J
authors.initials: RJ
authors.lastname: Yáñez-Muñoz
authors.forename: George
authors.initials: G
authors.lastname: Dickson
mesh_terms: D015153:Blotting, Western; D004260:DNA Repair / Q000235:genetics; D003851:Deoxyribonucleases / Q000378:metabolism; D005091:Exons / Q000235:genetics; D055879:Gene Knock-In Techniques / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D016086:Lentivirus; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000628:therapy*; D009154:Mutation / Q000235:genetics*; D032446:Myoblasts / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D052416:Targeted Gene Repair / Q000379:methods*
chemical_list: D016376:Oligonucleotides, Antisense; D003851:Deoxyribonucleases