Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human β-thalassemic mutations

Abstract

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.