title: Antisense oligonucleotide induced dystrophin exon 45 skipping at a low half-maximal effective concentration in a cell-free splicing system.
abstract: Antisense oligonucleotides (AOs) can facilitate the expression of internally deleted dystrophin in dystrophin-deficient Duchenne muscular dystrophy (DMD) by correcting the reading frame of the pre-mRNA with AO-mediated exon skipping. An antisense 18-mer 2'-O-methyl RNA/ethylene-bridged nucleic acid chimera AO targeting exon 45 of the dystrophin gene, AO85, can induce exon 45 skipping efficiently in cultured cells. AO85 is expected to facilitate dystrophin expression in 8%-9% of all DMD patients. Here, we examined the kinetics of AO85-mediated exon 45 skipping in a cell-free splicing system. In vitro transcribed pre-mRNAs containing dystrophin exon 45 and part of its flanking introns within a hybrid minigene were incubated with HeLa cell nuclear extract, and the resultant mRNAs were amplified by semiquantitative reverse transcriptase-polymerase chain reaction. Time-course analysis revealed that the splicing process fitted well to first order kinetics. Addition of AO85 produced an extra spliced product, deleting exon 45 (Δexon 45), indicating AO85-mediated exon 45 skipping. Production of Δexon 45 increased linearly with increasing concentrations of AO85, reaching a maximum of nearly 80% of the transcripts. The half-maximal effective concentration (EC(50)) of AO85 was 58.0 nM. The percentage of Δexon 45 among the transcripts decreased inversely with the pre-mRNA concentration; Lineweaver-Burk plotting revealed a competitive fashion of AO85 action. The low EC(50) indicates high potential of AO85 for clinical application.
authors.affiliation: Department of Pediatrics, Graduate School of Medicine, Kobe University, Japan.
authors.forename: Rusdy Ghazali
authors.initials: RG
authors.lastname: Malueka
authors.forename: Mariko
authors.initials: M
authors.lastname: Yagi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Awano
authors.forename: Tomoko
authors.initials: T
authors.lastname: Lee
authors.forename: Ery Kus
authors.initials: EK
authors.lastname: Dwianingsih
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishida
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeshima
authors.forename: Masafumi
authors.initials: M
authors.lastname: Matsuo
mesh_terms: D001667:Binding, Competitive; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D016189:Dystrophin / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D015870:Gene Expression / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects*; D012266:Ribose / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D016189:Dystrophin; C527155:ENA nucleotide; D009711:Nucleotides; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D012266:Ribose