Human Molecular Genetics, Vol 8, 2415-2423, Copyright © 1999 by Oxford University Press
D Suter, R Tomasini, U Reber, L Gorman, R Kole and D Sch#mperli
We have used three beta-thalassemic mutations, IVS2-654, -705 and -745,
that create aberrant 5' splice sites (5' ss) and activate a common cryptic
3' ss further upstream in intron 2 of the human beta-globin gene to
optimize a generally applicable exon-skipping strategy using antisense
derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7
snRNA gene carrying an antisense sequence against the cryptic 3' ss into
cultured cells expressing the mutant beta-globin genes, restored correct
beta-globin mRNA splicing for all three mutations, but the efficiency was
much weaker for IVS2-654 than for the other mutations. The length of
antisense sequence influenced the efficiency with an optimum of
approximately 24 nucleotides. Combining two antisense sequences directed
against different target sites in intron 2, either on separate antisense
RNAs or, even better, on a single U7 snRNA, significantly enhanced the
efficiency of splicing correction. One double-target U7 RNA was expressed
on stable transformation resulting in permanent and efficient suppression
of the IVS2-654 mutation and production of beta-globin. These results
suggest that forcing the aberrant exon into a looped secondary structure
may strongly promote its exclusion from the mRNA and that this approach may
be used generally to induce exon skipping.
ARTICLES
Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations
Abteilung f#r Entwicklungsbiologie, Zoologisches Institut der Universit#t Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland,
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