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

M Puttaraju, SF Jamison, SG Mansfield… - Nature …, 1999 - nature.com
M Puttaraju, SF Jamison, SG Mansfield, MA Garcia-Blanco, LG Mitchell
Nature biotechnology, 1999nature.com
We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-
splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-
splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299
human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-
mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre–trans-
splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted …
Abstract
We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre–trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis-and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant β-galactosidase activity. These observations suggest 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.
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