Spliceosome-mediated RNA trans-splicing as a tool for gene therapy

We have developed RNA molecules capable of effecting spliceosome-mediated R NA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA ). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cult ured human cells, and H1299 human lung cancer tumors in athymic mice. Trans -splicing between a cancer-associated pre-mRNA encoding the p-subunit of hu man chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nont argeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PT M splice site. Competition between cis- and trans-splicing demonstrated tha t cis-splicing can be inhibited by transsplicing. RNA repair In a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, wh ich restored significant beta-galactosidase activity. These observations su ggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.