IN-VITRO INHIBITION OF HEPATITIS-C VIRUS GENE-EXPRESSION BY CHEMICALLY-MODIFIED ANTISENSE OLIGODEOXYNUCLEOTIDES

We have explored different domains within the hepatitis C virus (HCV) 5' noncoding region as potential targets for inhibition of HCV transla tion by antisense oligodeoxynucleotides (ODNs). Inhibition assays were performed with two different cell-free systems, rabbit reticulocyte l ysate and wheat germ extract, and three types of chemical structures f or the ODNs were evaluated: natural phosphodiesters (beta-PO), alpha-a nomer phosphodiesters (alpha-PO), and phosphorothioates (PS), A total of six original ODNs, displaying sequence-specific inhibition ranging from 62 to 96%, that mapped in the pyrimidine-rich tract (nucleotides [nt] 104 to 127) and in the initiator AUG codon (nt 338 to 357) were i dentified, Two ODNs, which were targeted at the initiatory AUG (nt 341 to 367 and 351 to 377) and which had been previously described as act ive against genotype Ib and 2a sequences, were shown to exhibit inhibi tion of expression (>95%) of a type la sequence, Control experiments w ith the irrelevant chloramphenicol acetyltransferase sequence as a mar ker and randomized ODNs demonstrated that levels of inhibition associa ted with the use of PS compounds (of as much as 94%) were mainly due t o nonspecific effects, Both alpha- and beta-PO ODNs were found equally active, and no difference could be seen in the activity of beta-PO wh en it was tested in either rabbit reticulocyte lysate or wheat germ ex tract, suggesting that RNase H-independent mechanisms may be involved in the inhibitions observed, However, specific RNA cleavage products g enerated from beta-PO inhibition experiments could be identified, indi cating that, with these compounds, control of translation also involve s RNase II-dependent mechanisms, This study further delimits the exist ence of favorable target sequences for the action of ODNs within the H CV 5' noncoding region and indicates the possibility of using nuclease -resistant alpha-PO compounds in cellular studies.