title: In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide.
abstract: In the canine model of Duchenne muscular dystrophy in golden retrievers (GRMD), a point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA, and the consequent frameshift causes early termination of translation. We have designed a DNA and RNA chimeric oligonucleotide to induce host cell mismatch repair mechanisms and correct the chromosomal mutation to wild type. Direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and necropsy samples, demonstrated in vivo repair of the GRMD mutation that was sustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclusion with time. An isolated exon 7-specific dystrophin antibody confirmed synthesis of normal-sized dystrophin product and positive localization to the sarcolemma. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism (RFLP)-PCR and sequencing the PCR product. This work provides evidence for the long-term repair of a specific dystrophin point mutation in muscle of a live animal using a chimeric oligonucleotide.
authors.affiliation: Department of Veterinary Medicine and Surgery, Dalton Cardiovascular Research Center, College of Veterinary Medicine, Columbia, MO 65202, USA. richard_bartlett@nih.gov
authors.forename: R J
authors.initials: RJ
authors.lastname: Bartlett
authors.forename: S
authors.initials: S
authors.lastname: Stockinger
authors.forename: M M
authors.initials: MM
authors.lastname: Denis
authors.forename: W T
authors.initials: WT
authors.lastname: Bartlett
authors.forename: L
authors.initials: L
authors.lastname: Inverardi
authors.forename: T T
authors.initials: TT
authors.lastname: Le
authors.forename: N
authors.initials: N
authors.lastname: thi Man
authors.forename: G E
authors.initials: GE
authors.lastname: Morris
authors.forename: D J
authors.initials: DJ
authors.lastname: Bogan
authors.forename: J
authors.initials: J
authors.lastname: Metcalf-Bogan
authors.forename: J N
authors.initials: JN
authors.lastname: Kornegay
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004195:Disease Models, Animal; D004285:Dogs; D016189:Dystrophin / Q000235:genetics*; D018604:Epitope Mapping; D005091:Exons; D016368:Frameshift Mutation; D007150:Immunohistochemistry; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000628:therapy; D009841:Oligonucleotides / Q000627:therapeutic use*; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D012313:RNA / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012508:Sarcolemma; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
chemical_list: D016189:Dystrophin; D009841:Oligonucleotides; D012313:RNA; D004247:DNA