title: Phosphorothioate modification of chimeric 2´-O-methyl RNA/ethylene-bridged nucleic acid oligonucleotides increases dystrophin exon 45 skipping capability and reduces cytotoxicity.
abstract: BACKGROUND
  Antisense oligonucleotide (AO)-mediated exon skipping is the most promising way to express internally deleted dystrophin in Duchenne muscular dystrophy (DMD), by correcting the reading frame of dystrophin mRNA. An antisense chimeric oligonucleotide consisting of 2´-O-methyl RNA and ethylene-bridged nucleic acid (ENA), targeting exon 45 of the dystrophin gene, AO85, has been shown to induce exon 45 skipping efficiently. Since phosphorothioate (PS)-modification of AO85 has never been explored, we produced a PS-modified AO85 (AO88) and examined its exon skipping capability and cytotoxicity.
  
  
  METHODS
  Exon 45 skipping activity was examined in primary muscle cells established from a DMD patient carrying a deletion of dystrophin exon 44. Cytotoxicity was assessed by MTT assay.
  
  
  RESULTS
  AO88 induced dystrophin exon 45 skipping from 50 nM. More than 90% of products lacked exon 45 at 400 nM. AO88 showed significantly higher exon skipping activity than AO85. The EC50 of AO88 was 94.8 nM, while EC50 of AO85 was 66.7 nM. Cytotoxicity was lower for AO88 than for AO85.
  
  
  CONCLUSIONS
  the PS-modified RNA/ENA chimera displayed stronger exon skipping activity and lower cytotoxicity than the phosphodiester-RNA/ENA chimera. AO88 has better potential for clinical use than AO85.
authors: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180 Japan.
authors: Rusdy Ghazali
authors: RG
authors: Malueka
authors: Department of Pediatrics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Ery Kus
authors: EK
authors: Dwianingsih
authors: Department of Pediatrics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Mariko
authors: M
authors: Yagi
authors: Department of Pediatrics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Tomoko
authors: T
authors: Lee
authors: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180 Japan.
authors: Atsushi
authors: A
authors: Nishida
authors: Department of Pediatrics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Kazumoto
authors: K
authors: Iijima
authors: Department of Pediatrics, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017 Japan.
authors: Yasuhiro
authors: Y
authors: Takeshima
authors: Department of Medical Rehabilitation, Faculty of Rehabilitation, Kobegakuin University, 518 Arise, Ikawadani-cho, Nishi-ku, Kobe 651-2180 Japan.
authors: Masafumi
authors: M
authors: Matsuo
mesh_terms: D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016189:Dystrophin / Q000235:genetics*; D005030:Ethylenes; D005091:Exons; D006801:Humans; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D012313:RNA
chemical_list: D016189:Dystrophin; D005030:Ethylenes; D016376:Oligonucleotides, Antisense; D054735:Phosphorothioate Oligonucleotides; D012313:RNA; C036216:ethylene
keywords: Antisense oligonucleotide; Cytotoxicity; Exon skipping; Phosphorothioate; RNA/ENA chimera