1. ¹Department of Genetics, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan
2. ²Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan
3. ³Department of Pediatrics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan

Correspondence: Toshiyuki Miyashita, Department of Genetics, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. E-mail: tmiyashita@nch.go.jp

Received 13 July 2007; Accepted 10 August 2007; Published online 13 September 2007.

Abstract

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy.