MODULATION OF HUMAN PRORENIN GENE-EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES IN TRANSFECTED CHO CELLS

Four phosphorothioate oligonucleotides whose sequences are complementary to the 5' untranslated region, the initiation codon or the coding region of human prorenin mRNA, were studied for their capacity to inhibit gene expression in stably transfected Chinese hamster ovary (CHO) cells constitutively producing human prorenin. In contrast to oligomers complementary to the initiation codon and the coding region, antisense oligomers directed towards the 5' untranslated region have no inhibitory effects. The intracellular delivery of a biotinylated phosphorothioate oligonucleotide (biotin-CATCCATGCTTCCCTC) was monitored in immunofluorescence studies. In the absence of a cationic liposome preparation, Lipofectin(TM), the oligomer failed to penetrate the cells. In the presence of Lipofectin(TM), the S-35-labelled oligomer entered the cells and was distributed in proportions of 54% to the nuclei and 35% to the cytosol. The effects of regular oligonucleotides and of 3'-end and/or 5'-end-modified phosphodiester oligonucleotides on prorenin production were tested. Terminal modification by biotinylation at the 5'-end and/or 3'-dodecyl esterification stabilized oligonucleotides towards exonucleases, but did not translate into a significant inhibition of prorenin production and did not improve the intracellular delivery and or stability of the oligomers. We have shown that it is possible to inhibit prorenin production intracellularly using specific antisense oligonucleotides. Stability and delivery are crucial factors in the design of potent and specific compounds directed at prorenin mRNA.