Antisense DNA mediated inhibition of renin activity: mechanism of action.

The potential of specific antisense oligonucleotides as regulators of gene expression has been evaluated in a number of pathophysiological conditions including hypertension. The selection of the target sequence has been, thus far, based on theoretical criteria such as melting temperature (Tm) of the synthetic oligonucleotides. Using renin as a model we have compared the efficacy of antisense oligonucleotides directed against the 5′-end of renin (ASR) and prorenin (ASProR). CHO-K1 cells transfected with eukaryotic expression vector pBK-CMV containing full length cDNA for renin were used to study the effects of ASR and ASproR on expressed renin activity. Uptake and distribution of ASR and ASproR was monitored by immunocytochemistry and fluorescence microscopy (confocal). Renin activity was measured by RIA. Both ASR and ASProR were effectively taken up by CHO-K1 cells distributing between nucleus and cytoplasm. Cells stably transfected with renin cDNA were treated with increasing concentration of ASR and ASProR. Dose dependent inhibition of renin activity was achieved with both the antisense oligonucleotides. ASProR was more inhibitory (IC50=2.7 μM) than ASR (IC50=6.0 μM). Random primer which served as control had an identical number of bases as ASR and ASProR and a similar Tm and uptake by cells but was without effect on renin activity. While similar intracellular distribution suggests a common mechanism of action (both transcriptional and translational inhibition), the variation in the potency of inhibition is suggestive of the unique physicochemical properties of these oligonucleotides and/or their differing site of action. ASR and ASProR were each 13 bases in length and of comparable Tm (37°C and 44°C respectively). However, ASProR is upstream to ASR and thereby closer to both the transcriptional and translational initiation sites. We conclude that with identical length and comparable Tm, the proximity of the antisense oligonucleotide to transcription and translation start sites determines their ultimate potencies as inhibitors.