Journal Article

Double-Target Antisense U7 snRNAs Promote Efficient Skipping of an Aberrant Exon in Three Human β-Thalassemic Mutations

Daniel Suter, Reto Tomasini, Ueli Reber, Linda Gorman, Ryszard Kole and Daniel Schümperli

in Human Molecular Genetics

Volume 8, issue 13, pages 2415-2423
Published in print December 1999 | ISSN: 0964-6906
Published online December 1999 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/8.13.2415
Double-Target Antisense U7 snRNAs Promote Efficient Skipping of an Aberrant Exon in Three Human β-Thalassemic Mutations

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We have used three β-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 β-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 β-globin genes, restored correct β-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 ∼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 β-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.

Journal Article.  6271 words.  Illustrated.

Subjects: Genetics and Genomics

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