P07 The feasibility of exon skipping to restore the reading frame in DMD patients with duplications

Duchenne muscular dystrophy (DMD) is caused by mutations in the
dystrophin gene, leading to disruption of its reading frame. Exon
skipping trials, using antisense oligonucleotides (AONs) in DMD
boys with out-of-frame deletions (targeting exon 51) have shown
promising results. AON could theoretically be beneficial for patients
with out-of-frame duplications (15% of all DMD mutations).
However, the application of AONs to duplicated patients faces
unique challenges. Firstly, it is not known what is the genomic
configuration of the duplicated regions is (tandem or not); secondly
AONs not only recognise duplicated exons, but also normal exons,
leading to complex patterns of skipping. Furthermore, double
skipping to remove duplicated regions could be problematic in
patients with large duplications. Finally efficient skipping of whole
duplicated exons may lead to produce unstable proteins.
We firstly characterised the genomic configuration of the
duplications, using both transcription studies from skeletal muscle
or muscle cell cultures and Comparative Genomic Hybridization
(CGH) arrays.
Secondly we evaluated the splicing patterns in patients with
duplications to assess the feasibility of exon skipping strategies
to restore the disrupted reading frame in cultured myoblasts.
Our results show that there was a good correlation between CGH
array result and transcription studies except in one case. In cultures
of selected duplicated patients, AONs induced multiple skipping of
duplicated exons, and this was accompanied by the production of
dystrophin protein as detected by western blotting, suggesting that
this approach could be viable in at least some of the DMD with
duplications.