Strand bias in oligonucleotide-mediated dystrophin gene editing.

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 mdx(5cv) 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.