Intraperitoneal administration of phosphorothioate antisense oligodeoxynucleotide against splicing enhancer sequence induced exon skipping in dystrophin mRNA expressed in mdx skeletal muscle.

Antisense oligodeoxynucleotide against the splicing enhancer sequence (SES) in exon 19 of the dystrophin gene have been shown to induce exon 19 skipping and promote the expression of internally deleted dystrophin by correcting the translational reading frame in the cultured Duchenne muscular dystrophy (DMD) myocytes with the deletion of exon 20. Transfection of the antisense oligodeoxynucleotide, therefore, has been proposed as a promising means for therapeutic modification of dystrophin mRNA of DMD, a fatal disorder caused by defects in the dystrophin gene. A systemic delivery method targeting the large number of diseased muscles remains to be established for clinical application of antisense oligodeoxynucleotide. In this study, we investigated capability of oligodeoxynucleotide transfer into the skeletal muscles of mdx mouse, a mouse model of DMD. Thirty-one mer phosphorothioate oligodeoxynucleotide complementary to the SES of dystrophin exon 19 was intraperitoneally administered to mdx mice without any carrier. Histochemical study disclosed that fluorescence-labeled oligodeoxynucleotide appeared in the nuclei of femoral skeletal muscle cell at the second day after injection of 20 mg/kg BW oligodeoxynucleotide, and still visible at 14th day. Reverse transcription (RT)-PCR analysis of dystrophin transcript in these cells disclosed that a proportion of it showed skipping of exon 19 from second to seventh day after injection. These results showed that the intraperitoneally administered oligodeoxynucleotide could be transfected to nucleus of mdx skeletal muscle without any carrier and was able to induce exon skipping in vivo.