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siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations

Masayoshi Konishi1, Catherine H Wu, Masahiko Kaito

  • 1Division of Gastroenterology & Hepatology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.

Journal of Viral Hepatitis
|October 21, 2006
PubMed

Insights

Long-term RNA interference (RNAi) using short interfering RNAs (siRNAs) against hepatitis C virus (HCV) did not eliminate viral RNA. However, HCV developed mutations conferring resistance to siRNA treatment.

Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • RNA interference (RNAi) is effective against hepatitis C virus (HCV) gene expression in short-term cultures.
  • The potential for HCV to develop resistance to long-term RNAi therapy is unknown.

Purpose of the Study:

  • To investigate if long-term RNA interference (RNAi) treatment against hepatitis C virus (HCV) leads to the emergence of resistant viral mutants.
  • To assess the complete elimination of HCV RNA and protein expression under sustained siRNA treatment.

Main Methods:

  • Huh7 cells harboring HCV replicons were treated with HCV-specific short interfering RNAs (siRNAs).
  • HCV RNA levels were quantified using real-time RT-PCR, and NS5A protein levels were measured by Western blot.
  • Sequencing of the siRNA target region was performed on viral clones before, during, and after siRNA treatment.

Main Results:

  • Four weeks of HCV-siRNA treatment inhibited HCV RNA by 50%, but levels rebounded to 85% after treatment cessation.
  • Mutations in the siRNA target region were observed in HCV replicons from treated cells, but not in controls.
  • HCV replicons with mutations in the target region exhibited resistance to the inhibitory effects of HCV-siRNA.

Conclusions:

  • Sustained RNA interference (RNAi) with siRNAs cannot completely eliminate hepatitis C virus (HCV) RNA and NS5A protein.
  • HCV develops resistance to siRNA therapy through mutations in the targeted sequence.
  • Emergence of drug-resistant HCV mutants is a significant concern for RNAi-based therapeutic strategies.

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