title: Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.
abstract: Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Ignazio
authors.initials: I
authors.lastname: Maggio
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands Facoltà di Scienze Matematiche Fisiche e Naturali, Universitá di Roma Tor Vergata, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Stefanucci
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Josephine M
authors.initials: JM
authors.lastname: Janssen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Center for Research in Myology, UMRS 974 UPMC-INSERM, FRE 3617 CNRS, Paris, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Mouly
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, Einthovenweg 20, 2333 ZC Leiden, The Netherlands M.Goncalves@lumc.nl.
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000483:Alleles; D001483:Base Sequence; D015153:Blotting, Western; D064113:CRISPR-Cas Systems; D002460:Cell Line; D059766:DNA End-Joining Repair; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D009154:Mutation; D032446:Myoblasts / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D016189:Dystrophin; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases