Human Molecular Genetics Advance Access originally published online on November 24, 2004
Human Molecular Genetics 2005 14(2):221-233; doi:10.1093/hmg/ddi020
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Human Molecular Genetics, Vol. 14, No. 2 © Oxford University Press 2005; all rights reserved
Strand bias in oligonucleotide-mediated dystrophin gene editing
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA, 2MRIC Biochemistry Group, North East Wales Institute, Mold Road, Wrexham LL11 2AW, UK and 3GRECC, VA Palo Alto Health Care System, Palo Alto, CA, USA
* To whom correspondence should be addressed at: Department of Neurology and Neurological Sciences, Stanford University Medical Center, Room A343, Stanford, CA 94305-5235, USA. Tel: +1 6508583976; Fax: +1 6508583935; Email: rando{at}stanford.edu
Received August 13, 2004; Revised October 13, 2004; Accepted November 9, 2004
Defects in the dystrophin gene cause the severe degenerative muscle disorder, Duchenne muscular dystrophy (DMD). Among the gene therapy approaches to DMD under investigation, a gene editing approach using oligonucleotide vectors has yielded encouraging results. Here, we extend our studies of gene editing with self-pairing, chimeric RNA/DNA oligonucleotides (RDOs) to the use of oligodeoxynucleotides (ODNs) to correct point mutations in the dystrophin gene. The ODN vectors offer many advantages over the RDO vectors, and we compare the targeting efficiencies in the mdx5cv mouse model of DMD. We found that ODNs targeted to either the transcribed or the non-transcribed strand of the dystrophin gene were capable of inducing gene repair, with efficiencies comparable to that seen with RDO vectors. Oligonucleotide-mediated repair was demonstrated at the genomic, mRNA and protein levels in muscle cells both in vitro and in vivo, and the correction was stable over time. Interestingly, there was a strand bias observed with the ODNs, with more efficient correction of the non-transcribed strand even though the dystrophin gene is not transcribed in proliferating myoblasts. This finding demonstrates that strand bias of ODN-mediated gene repair is likely to be due to the specific sequence of the target gene in addition to any effects of transcription. A better understanding of how the efficiency of gene editing relates to the target sequence will offer the opportunity for rational oligonucleotide design for further development of this elegant approach to gene therapy for DMD and other genetic diseases.
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