ALTERED MESSENGER-RNA SPLICING AND INHIBITION OF HUMAN E-SELECTIN EXPRESSION BY AN ANTISENSE OLIGONUCLEOTIDE IN HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS

We have characterized the mechanism of action of an antisense oligodeo xynucleotide (ASO) targeting human endothelial leukocyte adhesion mole cule, E-selectin. LSIS 4730, a 20-base ASO designed to be complementar y to a region in the 3'-untranslated region (3'-UTR) of human E-select in, is a potent and specific inhibitor of both mRNA and protein expres sion in human umbilical vein endothelial cells, Following treatment wi th ISIS 4730, a lower molecular weight mRNA (3300 bases) species was d etected by Northern blot analysis with a corresponding decrease in the mature E-selectin transcript (3875 bases). The ASO-induced low molecu lar weight mRNA is stable and remains in the nucleus. We demonstrate t hat ISIS 4730 targets E-selectin pre-mRNA in the nucleus and promotes cleavage of the pre-mRNA at the hybridization site, resulting in preve ntion of splicing of the last intron, The change in molecular weight o f the E-selectin transcript is the result of loss of the 3'-UTR due to ASO-mediated RNA cleavage and retention of the last intron, Cleavage of the E-selectin pre-mRNA appears to be due to endogenous RNase H or a related enzyme activity.