Uptake and efflux of intact antisense phosphorothioate deoxyoligonucleotide directed against angiotensin receptors in bovine adrenal cells

Antisense oligonucleotide (AS-ODN) inhibition of angiotensin receptors (AT(1)-R) offers a potentially novel therapeutic approach for hypertension, left ventricular hypertrophy and other aspects of cardiovascular disease. To clarify questions concerning cellular uptake and retention of these oligos, we quantified the trafficking and stability of phosphorothioated modified AS-ODN to AT(1) receptor mRNA in adrenal cells, using visual and chromatographic analysis. The AS-ODN to AT(1) receptor mRNA was effective in significantly inhibiting AT(1) receptor binding in a dose dependent manner. FITC-labeled ODNs were used to determine the cellular uptake in bovine adrena cortex cells; using confocal microscopy, rapid cellular uptake of 15-mer ODNs was observed. Uptake is initially rapid (30 min to 4 h) followed by a slower uptake process 24h and after. The cellular accumulation of ODN involves a dynamic balance between influx and efflux processes. Efflux of FITC-ODN had a t(1/2) = 4.6 days. Uptake was time and dose dependent. No obvious degradation of intracellular ODNs occurred as shown by intact peaks for 15-mer ODN on thin layer chromatography. The results suggest that the AS-ODN to AT(1) receptor mRNA was resistant to cellular nucleases. The FITC-ODN accumulated mainly in the nucleus and remained there intact for up to 3 days. No significant change in target mRNA was observed by quantitative RT-PCR. Therefore the antisense inhibition mechanism of this ODN does not appear to stimulate RNase H or block transcription. Since the ODN accesses the nucleus, the results imply that the ODN inhibits specific mRNA transport into the cytoplasm. The data show that AS-ODN, for inhibition of AT(1) receptors, is rapidly taken up and stable in cells and produces specific inhibition of AT(1) receptors. (C) 1997 Elsevier Science Ltd.