In vivo trans-splicing of 5' and 3' segments of pre-mRNA directed by corresponding DNA sequences delivered by gene transfer.
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.
Pergolizzi
Robert G
RG
Belfer Gene Therapy Core Facility, Weill Medical College of Cornell University, New York, New York 10021, USA.
Ropper
Alexander E
AE
Dragos
Rachel
R
Reid
Alicia C
AC
Nakayama
Katsutoshi
K
Tan
Yadi
Y
Ehteshami
John R
JR
Coleman
Struhan H
SH
Silver
Randi B
RB
Hackett
Neil R
NR
Menez
André
A
Crystal
Ronald G
RG
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*
D003039:Cobra Neurotoxin Proteins; D012322:RNA Precursors; D011978:Receptors, Nicotinic; C025154:alpha-cobratoxin