T.P.20 Antisense oligonucleotide induced dystrophin exon 45 skipping at a low EC50 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.