Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Delta 48-50 DMD cells.
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.
De Angelis
Fernanda Gabriella
FG
Institute Pasteur Fondazione Cenci-Bolognetti, Department of Genetics and Molecular Biology, University La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy.
Sthandier
Olga
O
Berarducci
Barbara
B
Toso
Silvia
S
Galluzzi
Giuliana
G
Ricci
Enzo
E
Cossu
Giulio
G
Bozzoni
Irene
I
D000818:Animals; D016189:Dystrophin / Q000096:biosynthesis* / Q000235:genetics*; D005091:Exons; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D009865:Oocytes / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000494:pharmacology; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology; D012342:RNA, Small Nuclear / Q000235:genetics* / Q000494:pharmacology; D017384:Sequence Deletion; D014161:Transduction, Genetic; D014982:Xenopus laevis
D016189:Dystrophin; D012322:RNA Precursors; D016372:RNA, Antisense; D012342:RNA, Small Nuclear; C039919:RNA, recombinant; D012313:RNA