Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Abstract

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 the 5 NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5 NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were 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 (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5 NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 M. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. 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 modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.

Antisense Inhibition of Virus Infections

“…Hepatitis C virus gene expression was reported to be inhibited in stably transfected hepatocytes. The most active oligonucleotides were again targeted to sequences in the 5' untranslated region, reinforcing the importance of this region for regulating HCV expression (Hanecak et al, 1996). Phosphorothioate oligonucleotides were reported to inhibit RNA and protein production in a sequence-specific, length-dependent fashion.…”

Advances in hepatitis C: What is coming in the next 5 years?

“…Antisense oligonucleotides directed to these key regions of the HCV genome have been effective in preventing HCV replication. [39][40][41] Antisense oligonucleotides consist of DNA or RNA sequences specifically designed to bind an RNA target in order to form RNA-RNA (antisense RNA) or RNA-DNA (antisense DNA) hybrids. The formation of such hybrids can inhibit translation of HCV-RNA and/or RNA replication.…”

“…Phosphorothioate, methylphosphonate, and phosphotriester analogs of antisense oligonucleotides have been effective in increasing their resistance to nuclease and also in improving cellular uptake, stability and hybridization. [39][40][41] Ribozymes have been developed to inhibit HCV replication by cleaving the target HCV genomic RNA. 42,43 Ribozymes are naturally occurring short RNA molecules with endoribonuclease activity that are capable of catalyzing sequence-specific cleavage of RNA.…”

Emerging therapies of hepatitis B and C

“…94 The antisense strategy has been successfully applied in vitro to different viruses [95][96][97][98][99][100][101] including HBV infection [101][102][103][104] and HCV infection. [105][106][107][108][109][110] In vivo application of this approach has been shown to block DHBV 111 and woodchuck hepatitis virus replication. 112 While no tox-…”