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

• Published in: Nature biotechnology Vol. 17; no. 3; pp. 246 - 252
• Main Authors: Garcia-Blanco, Mariano A, Mitchell, Lloyd G, Puttaraju, M, Jamison, Sharon F, Mansfield, S. Gary
• Format: Journal Article
• Language: English
• Published: New York, NY Nature 01.03.1999
• Subjects: Animals; beta-Galactosidase - metabolism; Biological and medical sciences; Chorionic Gonadotropin, beta Subunit, Human - genetics; DNA Primers; Exons; Fundamental and applied biological sciences. Psychology; Genetic Engineering; Genetic Therapy; Globins - genetics; HeLa Cells; Humans; Mice; Mice, Nude; Models, Biological; Molecular and cellular biology; Molecular genetics; Neoplasms, Experimental; Reverse Transcriptase Polymerase Chain Reaction; RNA Splicing - physiology; Sequence Analysis, DNA; Spliceosomes - genetics; Time Factors; Transcription. Transcription factor. Splicing. Rna processing; Transfection; Tumor Cells, Cultured; Spliceosome; Messenger RNA; Splicing; Trans effect; Mechanism of action; Gene therapy; Precursor; Medical application
• ISSN: 1087-0156; 1546-1696
• DOI: 10.1038/6986

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 beta-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 beta-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.