MODIFICATION OF SPLICING IN THE DYSTROPHIN GENE IN CULTURED MDX MUSCLE-CELLS BY ANTISENSE OLIGORIBONUCLEOTIDES

Deletions and point mutations in the gene encoding the cytoskeletal pr otein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Decker muscul ar dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively, Frameshift mutations tend to result i n a lack of dystrophin at the sarcolemma, destabilization of the membr ane and degeneration of skeletal muscle, The mdx mouse is a valuable a nimal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy, This report represents a novel approach to correct dystrophin deficiency at the post-transcri ptional level by transfection of muscle cells with antisense RNA. Esse ntially, 2'-O-methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after tran sfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22, Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relativel y mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.