Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs

The T-->G mutation at nucleotide 705 in the second intron of the p-globin g ene creates an aberrant 5' splice site and activates a 3' cryptic splice si te upstream from the mutation. As a result, the IVS2-705 pre-mRNA is splice d via the aberrant splice sites leading to a deficiency of P-globin mRNA an d protein and to the genetic blood disorder thalassemia. We have shown prev iously that in cell culture models of thalassemia, aberrant splicing of P-t halassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activat ed by the mutation. To explore the possibility of using other snRNAs as vec tors for antisense sequences, U1 snRNA was modified in a similar manner. Re placement of the U1 9-nucleotide 5' splice site recognition sequence with n ucleotides complementary to the aberrant 5' splice site failed to correct s plicing of IVS2-705 pre-mRNA In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the hig hest levels of correction (up to 70%) in transiently and stably transfected target cells.