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.
Dickson
G
G
Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway--University of London, Surrey, TW20 0EX, UK. g.dickson@rhul.ac.uk
Hill
V
V
Graham
I R
IR
D000818:Animals; D016189:Dystrophin / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx; D009137:Muscular Dystrophy, Animal / Q000628:therapy; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012322:RNA Precursors; D012326:RNA Splicing
D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors