Ribozyme-mediated trans-splicing of a trinucleotide repeat.

Trinucleotide repeat expansions (TREs) are a recently described class of mutations characterized by a change in the size of the genomic fragment due to amplification of the repeated unit. A number of diseases have been attributed to TRE, including Huntington disease and myotonic dystrophy (DM), but attempts at genetic therapy have yet to prove successful. A potential therapeutic approach would be to repair the expanded repeat using the trans-splicing ability of group I intron ribozymes. We have used DM as a model to test this hypothesis. A group I intron ribozyme (DMPK-RZ1) was designed to modify the TRE at the 3' end of the human myotonic dystrophy protein kinase (DMPK) transcripts. DMPK-RZ1 was shown to ligate a small DMPK mRNA fragment, contained within the ribozyme, to a simple DMPK-target RNA in vitro. It also modified a larger target transcript, leading to replacement of twelve repeats with five repeats, both in vitro and in mammalian cells. Finally, this ribozyme successfully replaced the 3' end of endogenous DMPK mRNA in fibroblasts with a different 3' region. Ribozyme-mediated RNA repair may thus form a novel therapeutic strategy for diseases associated with repeat expansions.