ANTISENSE OLIGONUCLEOTIDE INHIBITION OF HEPATITIS-C VIRUS GENE-EXPRESSION IN TRANSFORMED HEPATOCYTES

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within t he 5' NCR. We have investigated inhibition of HCV gene expression usin g antisense oligonucleotides complementary to the 5 NCR, translation i nitiation codon, and core protein coding sequences. Oligonucleotides w ere evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (El). More than 50 oligonucleotides wer e evaluated for inhibition of HCV RNA and protein expression. Two olig onucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targete d to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV ge ne expression. ISIS 6095 and 6547 caused concentration-dependent reduc tions in HCV RNA and protein levels, with 50% inhibitory concentration s of 0.1 to 0.2 mu M. Reduction of RNA levels, and subsequently protei n levels, by these phosphorothioate oligonucleotides was consistent wi th RNase H cleavage of RNA at the site of oligonucleotide hybridizatio n. Chemically modified HCV antisense phosphodiester oligonucleotides m ere designed and evaluated for inhibition of core protein expression t o identify oligonucleotides and HCV target sequences that db not requi re RNase H activity to inhibit expression. A uniformly modified 2'-met hoxyethoxy phosphodiester antisense oligonucleotide complementary to t he initiator AUG reduced HCV core protein levels as effectively as pho sphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA l evels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modi fied antisense oligonucleotides can be used as tools to identify impor tant regulatory sequences and/or structures important for efficient tr anslation of HCV.