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

Abstract

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.

Repair of pre-mRNA splicing

“…4A and inset). As was the case for AONs, such modified U7 snRNAs were initially almost exclusively designed to induce exon skipping in genes involved in human diseases such as β-thalassemia, [134][135][136][137] Duchenne muscular dystrophy, [138][139][140] or HIV/AIDS. [141][142][143] Focusing on SMA, a number of different U snRNA-based approaches have been used with variable success (Fig.…”

Evolutionary conservation of the U7 small nuclear ribonucleoprotein inDrosophila melanogaster

“…Drosophila U7 snRNA, histone H3 pre-mRNA, and ␤-actin premRNA were amplified by RT-PCR using the rTth kit (Perkin Elmer) and ␣-32P-dCTP, essentially as described (Suter et al 1999). The primers and conditions used are listed in Table 1.…”

Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

“…8 Here we used a newly designed, more efficient, U7 snRNA, named U7-Do3-Ex1, that targets a first sequence within the aberrant exon and a second sequence downstream of it. This construct is characterized in Supplementary Figure 2 online.…”

“…24 Conditions for cell culture, DNA transfections, lentiviral vector production in 293-T cells, titration on HeLa cells and transduction have been described. 8,14 Modifications used here were that HS2 cells were kept under constant selection pressure with 500 mg ml À1 G418 (Invitrogen, Carlsbad, CA, USA) and that their media contained 10% tetracycline-free foetal calf serum (BioConcept, Allschwil, Switzerland). Doxycyline (Sigma, St Louis, MO, USA) was kept at À20 1C as a 2 mg ml À1 stock solution, freshly diluted at the time of the experiment and changed twice a week for experiments requiring long-term use of the drug.…”

“…Our group has developed such an antisense expression system relying on the use of a modified U7 snRNA (U7 Sm OPT) (reviewed in Asparuhova et al 6 ). This system has been successfully used to induce exon skipping in a variety of genes and disorders including thalassaemic b-globin mutations, 7,8 Duchenne muscular dystrophy [9][10][11] and human immunodeficiency virus type 1/acquired immune deficiency syndrome. 12,13 More recently, we and others have shown that the U7 SmOPT tool can also be adapted to promote exon inclusion in the case of the human SMN2 gene, a potential therapeutic target for spinal muscular atrophy (SMA).…”