In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide

In the canine model of Duchenne muscular dystrophy in golden retrievers (GR MD), 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 chi meric oligonucleotide to induce host cell mismatch repair mechanisms and co rrect the chromosomal mutation to wild type. Direct skeletal muscle injecti on of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog, and subsequent analysis of biopsy and ne cropsy samples, demonstrated in vivo repair of the GRMD mutation that was s ustained for 48 weeks. Reverse transcription-polymerase chain reaction (RT- PCR) analysis of exons 5-10 demonstrated increasing levels of exon 7 inclus ion with time. An isolated exon 7-specific dystrophin antibody confirmed sy nthesis of normal-sized dystrophin product and positive localization to the sarcolemma, Chromosomal repair in muscle tissue was confirmed by restricti on fragment length polymorphism (RFLP)-PCR and sequencing the PCR product, This work provides evidence for the long-term repair of a specific dystroph in point mutation in muscle of a live animal using a chimeric oligonucleoti de.