Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides

The mdx mouse, which carries a nonsense mutation in exon 23 of the dystroph in gene, has been used as an animal model of Duchenne muscular dystrophy to evaluate cell or gene replacement therapies. Despite the mdx mutation, whi ch should preclude the synthesis of a functional dystrophin protein, rare, naturally occurring dystrophin-positive fibres have been observed in mdx mu scle tissue. These dystrophin-positive fibres are thought to have arisen fr om an exon-skipping mechanism, either somatic mutations or alternative spli cing. increasing the frequency of these fibres may offer another therapeuti c approach to reduce the severity of Duchenne muscular dystrophy. Antisense oligonucleotides have been shown to block aberrant splicing in the human b eta-globin gene. We wished to use a similar approach to re-direct normal pr ocessing of the dystrophin pre-mRNA and induce specific exon skipping. Anti sense 2'-O-methyl-oligoribonucleotides, directed to the 3' and 5' splice si tes of introns 22 and 23, respectively in the mdx pre-mRNA, were used to tr ansfect myoblast cultures. The 5' antisense oligonucleotide appeared to eff iciently displace factors normally involved in the removal of intron 23 so that exon 23 was also removed during the splicing of thr dystrophin pre-mRN A. Approximately 50% of the dystrophin gene mRNAs were missing this exon 6 h after transfection of primary mdr myotubes, with all transcripts showing skipping of exon 23 after 24 h. Deletion of exon 23 does not disrupt the re ading frame and should allow the synthesis of a shorter but presumably func tional Becker-like dystrophin. Molecular intervention at dystrophin pre-mRN A splicing has the potential to reduce the severity of a Duchenne mutation to the milder Becker phenotype. (C) 1999 Elsevier Science B.V. All rights r eserved.