2
Duke University Medical Center, Durham,
NC 27710.
Correspondence should be addressed to Mariano A. Garcia-Blanco garci001@mc.duke.edu or Lloyd G. Mitchell lgm@intrex.netspliceosometrans-splicinggene therapyRNA repair
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 -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 -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.
-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 
