title: Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays.
abstract: BACKGROUND
  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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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
  
  
  CONCLUSIONS
  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.
authors.affiliation: Centre for Biomedical Science, School of Biological Sciences, Royal Holloway-University of London, Egham TW20 0EX, UK. igraham@rhul.ac.uk
authors.forename: Ian R
authors.initials: IR
authors.lastname: Graham
authors.forename: Vanessa J
authors.initials: VJ
authors.lastname: Hill
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.forename: Gopal B
authors.initials: GB
authors.lastname: Inamati
authors.forename: George
authors.initials: G
authors.lastname: Dickson
mesh_terms: D000483:Alleles; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016189:Dystrophin / Q000096:biosynthesis / Q000235:genetics*; D005091:Exons; D016368:Frameshift Mutation; D015316:Genetic Therapy; D007150:Immunohistochemistry; D007438:Introns; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000628:therapy; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D016189:Dystrophin; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012313:RNA; D004247:DNA