Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48 -50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5 and 3 splice sites are coexpressed in the same cell. D uchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1 in every 3,500 males. It is characterized by the absence of the cytoskeletal dystrophin (427-kDa protein) that in turn produces a severe and progressive muscle deterioration. Most of the DMD mutations consist in deletions and point mutations in the 2.5-Mb dystrophin gene that introduce stop codons and consequently premature translation termination. A milder myopathy is the Becker muscular dystrophy; in this case, deletions inside the gene produce in frame mRNAs and consequently shorter but semifunctional dystrophin proteins (1). A third of DMD cases are the results of a de novo mutation (2, 3), therefore the disease can never be eliminated through genetic screening and counseling. For this reason, many efforts are now being devoted to the development of a treatment for this disorder, and several strategies have been designed that might provide an insight into finding a cure. One of these strategies involves the transplantation of normal myoblasts into the muscle tissues that lack this protein (4, 5), whereas the other strategy tries to restore correct expression of the dystrophin through a gene therapy approach. In this direction, several groups have tried to deliver full-length or mini cDNA copies of dystrophin into cells with the mutated gene (6-8). Even if this approach is very promising, several problems still remain to be solved, such as size capacity and transducing activity of the vector and immune response to the ''therapeutic'' gene (9).Another powerful approach of gene therapy is based on the fact that internal in-frame deletions in the protein produce only mild myopathic symptoms; therefore, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore ...