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New techniques increase efficiency of RNA interference to block HCV replication
  NEW YORK (Reuters Health) - German researchers describe new techniques for producing small interfering RNAs (siRNA) that degrade many common hepatitis C (HCV) genotypes. By targeting highly conserved portions of the HCV genome, they could prevent in vitro HCV replication and protect naïve cells from infection.
HCV genomes differ by up to 34% in their nucleotide sequences. The high degree of sequence diversity between HCV genotypes and the rapid evolution of escape mutations have limited the antiviral efficacy of RNA interference, Dr. Michael Frese, at the University of Heidelberg, and associates explain in their article, published in the Journal of Virology for April.
To address these shortcomings, the research team used endoribonuclease to develop esiRNAs directed against multiple highly conserved sites of the HCV genome or the 5' nontranslated region (5' NTR), an indispensable region for RNA translation and replication.
When the human hepatoma cell line Huh-7 was infected with different HCV genotypes, transfection with both types of esiRNAs decreased HCV RNA copies and HCV protein expression. They were as effective in cells containing subgenomic HCV replicons as in those encoding the entire viral protein core.
"It seems extremely unlikely that a viral genome can accumulate enough point mutations to escape the antiviral activity of siRNAs," Dr. Frese's group suggests.
They also chemically synthesized DNA oligonucleotides to generate 12 short hairpin RNA (shRNA) coding sequences, 11 of which targeted highly conserved sequence motifs within the 5' NTR or the early core coding region.
After transfecting Huh-7 cells using a retroviral vector, they found that shRNAs targeting domain IV efficiently induced RNA interference in three HCV genotypes. Only one of seven shRNAs targeting domains II or II was as effective, even though more than 90% of cells were transduced in each experiment.
In naïve Huh-7 cells, shRNA transduction also protected cells from HCV infections.
These techniques "may be used in future approaches to inhibit HCV replication in vivo," Dr. Frese and his associates conclude.
J Virol 2004;78:3436-3446.

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