P.11.13 Phosphorothioate modification increases capability of dystrophin exon 45 skipping and reduces cytotoxicity of RNA/ENA chimera

Antisense oligonucleotides (AOs)-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) against exon 45 of the dystrophin gene, AO85, has been shown efficient in inducing exon 45 skipping. Phosphorothioate (PS)-modification is the most widely performed chemical modification of AO. Here, we introduced PS-modification to AO85 (AO88) and examined its exon skipping capability and cytotoxicity. The activity to induce exon 45 skipping was examined using primary muscle cells established from a DMD patient with a deletion of dystrophin exon 44. AO88 and AO85 were added to the culture medium and resulting dystrophin mRNAs were semiquantitated by a micro capillary electrophoresis of RT-PCR amplified products. AO88 was found to show activity to induce dystrophin exon 45 skipping from 50nM. More than 90% of splicing products lacked exon 45 at 400 nM. Remarkably, AO88 showed significantly higher exon skipping activity than AO85. Cytotoxicity assessed by the MTT Cell Proliferation Kit was lower in AO88 than AO85. In conclusion, PS-modification of RNA/ENA chimera was shown to render stronger exon skipping activity and lower cytotoxicity than PO-RNA/ENA chimera. It was assumed that AO88 has higher potential for clinical use than AO85.