RIBOZYME-MEDIATED INHIBITION OF EXPRESSION OF LEUKOCYTE-TYPE 12-LIPOXYGENASE IN PORCINE AORTIC VASCULAR SMOOTH-MUSCLE CELLS

Activation of a Leukocyte-type 12-lipoxygenase (12-LO) has been propos ed to be an important mechanism for angiotensin II- and glucose-induce d vascular smooth muscle cell growth. Currently, no specific pharmacol ogical inhibitors for the leukocyte-type 12-LO are available to test t his hypothesis. We have therefore designed a chimeric DNA-RNA hammerhe ad ribozyme to produce cleavage at the first GUC sequence at nucleotid e 7 of porcine leukocyte 12-LO mRNA. The ribozyme was tested in vitro with a 206-base 12-LO mRNA as substrate. We observed that the ribozyme specifically and dose-dependently cleaved porcine leukocyte 12-LO mRN A at the predicted site under physiological temperature. Furthermore, we also efficiently delivered the ribozyme into porcine aortic vascula r smooth muscle cells by transfection with cationic liposomes. The rib ozyme caused a dose-dependent decrease in levels of porcine leukocyte- type 12-LO mRNA in these cells and was more potent than an antisense o ligonucleotide directed against porcine leukocyte 12-LO. The 12-LO rib ozyme also attenuated 12-LO protein levels in the cells. The action of the ribozyme was primarily a result of its catalytic activity, since a modified ribozyme that lacks catalytic activity showed reduced effec ts. This represents the first ribozyme directed against a mammalian LO pathway. These results demonstrate the potential utility of new riboz yme technology to generate novel agents for gene modulation experiment s to modify the development or progression of vascular disease in huma ns.