Duchenne & Becker muscular dystrophies (DMDIBMD) are allelic disorders caused by mutations in a 2.5Mb gene on the Xchromosome( 1). Over two-thirds of mutations are deletions(2). causing a shift in the reading frame or an in-frame loss of part of the dystrophin molecule associated with DMD or BMD forms of the disease respectively(3). The remaining disease-causing mutations are probably due to point mutations, or defects in transcriptional regulation(4). While muscle fibres of DMD patients and animal models of the disease are generally characterised by a lack of detectable dystrophin at the sarcolemma(5), rare dystrophin-positive “revertant” myofibres are encountered in muscle biopsies(6,7). Proposed mechanisms include reinitiation of translation at downstream methionine codons, frame-restoring mutations, and exon skipping to frame-restoring exons in a proportion of dystrophin mRNAs(8). Evidence of exon skipping has been found by reverse transcriptase-mediated PCR (RT-PCR) analysis of patients’ muscle tissue(9,lO) but it is unclear whether these transcripts can improve muscle function. It is clear from BMD patients, however, that in-frame gene products are normally at least semi-functional and lead to milder muscle pathology(1 l), so it has been proposed that the severe muscle disease experienced by DMD patients may be alleviated by increasing the proportion of in-frame transcripts arising in their myofibre nuclei( 12). This study aims to induce exon-skipping across DMD mutations during pre-mRNA splicing in order to force production of framerestored mRNA transcripts. Initial work seeks to analyse revertant fibres in the mdr mouse model by RT-PCR of muscle transcripts, followed by PCR-mediated construction of a recombinant premRNA template in order to study splicing using in vitro transcription assays( 13). Transcription of the template is driven by a “7 promoter and incorporates exon 23 (mutated in the mdx mouse) and flanking intron sequences within the context of a partial P-globin genome. The 5’ region of mouse dystrophin intron 23 has now been cloned and sequenced using the vectorette PCR technique for inclusion in the template DNA. Sequence-specific antisense 2’-O-methyl oligoribonucleotides targeted to splicing signals will be added to these assays to try to induce exonskipping at high frequency(l4). Parallel studies will focus on the effective delivery of oligonucleotides to the nuclei of cultured muscle cells from normal and dystrophic animals/patients. Methods to be tested include various liposomelnucleic acid formulations( 15, 16) and oligonucleotides tagged with nucleartargeting peptides(l7). Ultimately, these studies will be drawn together in order to induce exon skipping by the introduction of antisense oligonucleotides into cultured cells. Carefully-controlled analyses will involve RT-PCR of total and poly(A)+ RNA from in vitro assays, cultured cells and tissues and Northern blotting( 19) as well as immunocytochemistry using exon-specific monoclonal antibodies(20). Direct sequencing of PCR products will be performed to examine the precise nature of mRNAs in both experimental and control preparations(2 1). REFERENCES: