Improved antisense oligonucleotide induced exon skipping in the mdx mouse model of muscular dystrophy

Background Duchenne muscular dystrophy (DMD) is a fatal genetic. disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx Mouse model of DMD. Methods H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR. and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies. Results We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro. Conclusions These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique. Copyright (C) 2002 John Wiley Sons, Ltd.