Screening for antisense modulation of dystrophin pre-mRNA splicing

Most gene therapy approaches to genetic disorders aim to compensate loss-of-function by introducing recombinant cDNA-based minigenes into diseased tissues. The current report represents an ongoing series of studies designed to correct genetic mutations at the post-transcriptional level. This strategy modifies the binding of components of the spliceosome by high affinity hybridisation of small complementary (antisense) RNA oligonucleotides to specific pre-mRNA sequences. These, so-called ‘splicomer’ reagents are chemically modified to impart bio-stability, and are designed to cause skipping of mutant frame-shifting exon sequences leading to restoration of the reading frame and an internally deleted but partially functional gene product. For instance, Duchenne muscular dystrophy is generally caused by frame-shift mutations in the dystrophin gene, whereas in-frame deletions of up to 50% of the central portion of the gene cause Becker muscular dystrophy, a much milder myopathy, which in some cases can remain asymptomatic to old age. In the mdx mouse model of Duchenne muscular dystrophy, a mutation in exon 23 of the dystrophin gene creates a stop codon and leads to a dystrophin-deficient myopathy in striated muscle. In previous studies, we have demonstrated that forced skipping of this mutant exon by treatment of mdx muscle cells with splicomer oligonucleotides can generate in-frame dystrophin transcripts and restore dystrophin expression. Here, we report the results of an optimisation of splicomer sequence design by the use of both high-throughput arrays and biological screens. This has resulted in specific and, importantly, exclusive skipping of the targeted exon in greater than 60% of dystrophin mRNA, leading to the de novo synthesis and localisation of dystrophin protein in cultured mdx muscle cells.