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

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, tha t 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 carryin g an antisense sequence against the cryptic 3' ss into cultured cells expre ssing the mutant beta-globin genes, restored correct beta-globin mRNA splic ing for all three mutations, but the efficiency was much weaker for IVS2-65 4 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of similar to 24 nucleotides, Combining two antisense sequences directed against different target sites in intron 2, e ither on separate antisense RNAs or, even better, on a single U7 snRNA, sig nificantly enhanced the efficiency of splicing correction. One double-targe t U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globi n, These results suggest that forcing the aberrant exon into a looped secon dary structure may strongly promote its exclusion from the mRNA and that th is approach may be used generally to induce exon skipping.