title: Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs.
abstract: In several forms of beta-thalassemia, mutations in the second intron of the beta-globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic beta-globin pre-mRNAs are spliced almost exclusively via the aberrant splice sites leading to a deficiency of correctly spliced beta-globin mRNA and, consequently, beta-globin. We have designed a series of vectors that express modified U7 snRNAs containing sequences antisense to either the aberrant 5' or 3' splice sites in the IVS2-705 thalassemic pre-mRNA. Transient expression of modified U7 snRNAs in a HeLa cell line stably expressing the IVS2-705 beta-globin gene restored up to 65% of correct splicing in a sequence-specific and dose-dependent manner. Cell lines that stably coexpressed IVS2-705 pre-mRNA and appropriately modified U7 snRNA exhibited up to 55% of permanent restoration of correct splicing and expression of full-length beta-globin protein. This novel approach provides a potential alternative to gene replacement therapies.
authors.affiliation: Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: L
authors.initials: L
authors.lastname: Gorman
authors.forename: D
authors.initials: D
authors.lastname: Suter
authors.forename: V
authors.initials: V
authors.lastname: Emerick
authors.forename: D
authors.initials: D
authors.lastname: Schümperli
authors.forename: R
authors.initials: R
authors.lastname: Kole
mesh_terms: D001483:Base Sequence; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D012326:RNA Splicing; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D016372:RNA, Antisense; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D005914:Globins