The activity of synthetic antisense oligonucleotides (splicomers) designed to block pre-mRNA splicing at specific exons has been demonstrated in a number of model systems, including constitutively spliced exons in mouse dystrophin RNA. Splicomer reagents directed to Duchenne muscular dystrophy (DMD) RNAs might thus circumvent nonsense or frame-shifting mutations, leading to therapeutic expression of partially functional dystrophin, as occurs in the milder, allelic (Becker) form of the disease (BMD).
Functional and hybridisation array screens have been used to select optimised splicomers directed to exon 23 of dystrophin mRNA which carries a nonsense mutation in the mdx mouse. Splicomers were transfected into cultured primary muscle cells, and dystrophin mRNA assessed for exon exclusion. Splicomers were also administered to the muscles of mdx mice.
Oligonucleotide array analyses with dystrophin pre-mRNA probes revealed strong and highly specific hybridisation patterns spanning the exon 23/intron 23 boundary, indicating an open secondary structure conformation in this region of the RNA. Functional screening of splicomer arrays by direct analysis of exon 23 RNA splicing in mdx muscle cultures identified a subset of biologically active reagents which target sequence elements associated with the 5' splice site region of dystrophin intron 23; splicomer-mediated exclusion of exon 23 was specific and dose-responsive up to a level exceeding 50% of dystrophin mRNA, and Western blotting demonstrated de novo expression of dystrophin protein at 2-5% of wild-type levels. Direct intramuscular administration of optimised splicomer reagents in vivo resulted in the reappearance of sarcolemmal dystrophin immunoreactivity in > 30% of muscle fibres in the mdx mouse
These results suggest that correctly designed splicomers may have direct therapeutic value in vivo, not only for DMD, but also for a range of other genetic disorders.