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A novel chimeric ribozyme vector produces potent inhibition of ICAM‐1 expression on ischemic vascular endothelium
Author(s) -
Sonnenday Christopher J.,
Warren Daniel S.,
Cooke Sara K.,
Dietz Harry C.,
Montgomery Robert A.
Publication year - 2004
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.697
Subject(s) - ribozyme , transfection , microbiology and biotechnology , biology , in vivo , viral vector , endothelium , expression vector , rna , gene , biochemistry , recombinant dna , endocrinology
Background Inhibition of intercellular adhesion molecule‐1 (ICAM‐1) expression can ameliorate the inflammation induced by ischemia‐reperfusion injury (IRI) in animal models. However, current strategies to reduce ICAM‐1 expression have been limited by the lack of stability, poor specificity, and the transient nature of synthesized regulatory molecules (antisense/ribozyme). Methods A chimeric expression vector was generated by fusing a ribozyme targeting sequence against ICAM‐1 to stabilizing stem‐loop structures and nuclear localization signals that are components of endogenous U1 small nuclear RNA. Oligonucleotide scanning was used to predict accessible sites for targeting within the rat ICAM‐1 transcript. Efficacy of the chimeric ribozyme vector was tested by transfection of rat aortic endothelial (RAE) cells ( in vitro ) and intraportal delivery in a rat hepatic IRI model ( in vivo ). Results Transfection of RAE cells with the chimeric ribozyme vector produced potent and specific inhibition of ICAM‐1 mRNA and protein levels by >65%. This reduction in ICAM‐1 expression was accompanied by a proportional decrease in neutrophil adhesion to RAE cells. In vivo intraportal delivery of the chimeric targeting vector to rats sustaining hepatic IRI produced a marked reduction in ICAM‐1 expression on liver endothelium after reperfusion. Conclusions A chimeric ribozyme vector effectively inhibited ICAM‐1 expression in vascular endothelial cells and in rat liver following IRI, demonstrating a novel gene targeting technique that may be ideally suited to clinical applications aimed at ameliorating IRI. Copyright © 2004 John Wiley & Sons, Ltd.