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 hyperte nsion, left ventricular hypertrophy and other aspects of cardiovascula r disease. To clarify questions concerning cellular uptake and retenti on of these oligos, we quantified the trafficking and stability of pho sphorothioated modified AS-ODN to AT(1) receptor mRNA in adrenal cells , using visual and chromatographic analysis. The AS-ODN to AT(1) recep tor mRNA was effective in significantly inhibiting AT(1) receptor bind ing in a dose dependent manner. FITC-labeled ODNs were used to determi ne the cellular uptake in bovine adrena cortex cells; using confocal m icroscopy, rapid cellular uptake of 15-mer ODNs was observed. Uptake i s initially rapid (30 min to 4 h) followed by a slower uptake process 24h and after. The cellular accumulation of ODN involves a dynamic bal ance 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 degrada tion of intracellular ODNs occurred as shown by intact peaks for 15-me r ODN on thin layer chromatography. The results suggest that the AS-OD N to AT(1) receptor mRNA was resistant to cellular nucleases. The FITC -ODN accumulated mainly in the nucleus and remained there intact for u p to 3 days. No significant change in target mRNA was observed by quan titative 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 E lsevier Science Ltd.