STABLE ALTERATION OF PRE-MESSENGER-RNA SPLICING PATTERNS BY MODIFIED U7 SMALL NUCLEAR RNAS

In several forms of beta-thalassemia, mutations in the second intron o f the beta-globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic b eta-globin pre-mRNAs are spliced almost exclusively via the aberrant s plice sites leading to a deficiency of correctly spliced beta-globin m RNA and, consequently, beta-globin, We have designed a series of vecto rs that express modified U7 snRNAs containing sequences antisense to e ither the aberrant 5' or 3' splice sites in the IVS2-705 thalassemic p re-mush. Transient expression of modified U7 snRNAs in a HeLa cell lin e stably expressing the IVS2-705 beta-globin gene restored up to 65% o f correct splicing in a sequence-specific and dose-dependent manner, C ell lines that stably coexpressed IVS2-705 pre-mRNA and appropriately modified U7 snRNA exhibited up to 55% of permanent restoration of corr ect splicing and expression of full-length beta-globin protein. This n ovel approach provides a potential alternative to gene replacement the rapies.