title: In vivo trans-splicing of 5' and 3' segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer.
abstract: We have developed a new paradigm of in vivo gene transfer termed "segmental trans-splicing" (STS), in which individual "donor" and "acceptor" DNA sequences, delivered in vitro or in vivo, generate pre-mRNAs with 5' and 3' splice signals, respectively, and complementary hybridization domains through which the two pre-mRNAs interact, facilitating trans-splicing of the two mRNA fragments. To demonstrate STS, we used alpha-cobratoxin, a neurotoxin that binds irreversibly to postsynaptic nicotinic acetylcholine receptors. Cells or animals receiving both donor and acceptor plasmids, but neither plasmid alone, yielded RT-PCR products with the correct sequence of mature alpha-cobratoxin mRNA, suggesting that trans-splicing had occurred. Mice receiving intravenous administration of > or = 7.5 microg donor + acceptor plasmids, but not either plasmid alone, died within 6 h. These data demonstrate that segmental trans-splicing occurs in vivo. This approach should permit the intracellular assembly of molecules hitherto too large to be accommodated within current gene transfer vectors.
authors.affiliation: Belfer Gene Therapy Core Facility, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Pergolizzi
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Ropper
authors.forename: Rachel
authors.initials: R
authors.lastname: Dragos
authors.forename: Alicia C
authors.initials: AC
authors.lastname: Reid
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Nakayama
authors.forename: Yadi
authors.initials: Y
authors.lastname: Tan
authors.forename: John R
authors.initials: JR
authors.lastname: Ehteshami
authors.forename: Struhan H
authors.initials: SH
authors.lastname: Coleman
authors.forename: Randi B
authors.initials: RB
authors.lastname: Silver
authors.forename: Neil R
authors.initials: NR
authors.lastname: Hackett
authors.forename: André
authors.initials: A
authors.lastname: Menez
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Crystal
mesh_terms: D000818:Animals; D003039:Cobra Neurotoxin Proteins / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000378:metabolism; D010957:Plasmids; D012322:RNA Precursors / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D020040:Trans-Splicing / Q000502:physiology*
chemical_list: D003039:Cobra Neurotoxin Proteins; D012322:RNA Precursors; D011978:Receptors, Nicotinic; C025154:alpha-cobratoxin