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

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 skippin