Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.
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.
Puttaraju
M
M
Intronn LLC, Durham, NC 27701, USA.
Jamison
S F
SF
Mansfield
S G
SG
Garcia-Blanco
M A
MA
Mitchell
L G
LG
D000818:Animals; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics; D017931:DNA Primers; D005091:Exons; D005818:Genetic Engineering; D015316:Genetic Therapy; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D008954:Models, Biological; D009374:Neoplasms, Experimental; D012326:RNA Splicing / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017381:Spliceosomes / Q000235:genetics*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
D018997:Chorionic Gonadotropin, beta Subunit, Human; D017931:DNA Primers; D005914:Globins; D001616:beta-Galactosidase