Skipping of Duplicated Dystrophin Exons: In Vitro Induction and Assessment

Duplications of one or more dystrophin exons that disrupt the reading frame account for about 15% of all Duchenne cases, and like the more common genomic deletions, most pathogenic duplications of single or multiple dystrophin exons are also amenable to targeted exon skipping. However, additional considerations must be taken into account: (1) skipping of all duplicated exons, and, flanking exons as necessary, will frequently be required to restore the reading frame and generate an in-frame Becker muscular dystrophy-like mRNA, (2) the phosphorodiamidate morpholino oligomer chemistry is more effective than the 2′-O-methyl modified oligonucleotides at inducing multiple exon skipping, and (2) the apparent efficiency of exon skipping can be confounded by the choice of RT-PCR system. Standard RT-PCR systems can preferentially amplify the shorter amplicons, implying more efficient exon skipping than may actually be induced. Unless high fidelity RT-PCR systems are used, strand slippage during annealing/elongation steps will generate normal length transcripts that are artifacts of the amplification.