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Vol. 95, Issue 9, 4929-4934, April 28, 1998

Biochemistry
Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs

(gene therapy / antisense / genetic disease / thalassemia)

Linda Gorman^*, Daniel Suter, Victoria Emerick^*,, Daniel Schümperli, and Ryszard Kole^*,§

^* Lineberger Comprehensive Cancer Center and Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599; and  Abteilung für Entwicklungsbiologie, Zoologisches Institut der Universität Bern, Baltzerstrasse 4, CH-3012 Bern, Switzerland

Communicated by Mary Edmonds, University of Pittsburgh, Pittsburgh, PA, February 26, 1998 (received for review December 12, 1997)

In several forms of -thalassemia, mutations in the second intron of the -globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic -globin pre-mRNAs are spliced almost exclusively via the aberrant splice sites leading to a deficiency of correctly spliced -globin mRNA and, consequently, -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 -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 -globin protein. This novel approach provides a potential alternative to gene replacement therapies.

Copyright © 1998 by The National Academy of Sciences  0027-8424/98/954929-6$2.00/0
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