The aim of this study was to characterize an adenovirus vector containing the gene for a small interfering RNA (siRNA) targeting the 59-untranslated region (UTR) of the hepatitis C virus (HCV) transcript. Viral production of AdSi73 was upscaled in human embryonic kidney (HEK) 293 cells, and virus particles were purified by a double cesium chloride centrifugation gradient and dialysis. Viral titer was determined by optical density measurement, and the presence of the siRNA gene was confirmed by polymerase chain reaction using sequence-specific primers. Cell cytotoxicity of AdSi73 was assayed in human hepatoma 7 (Huh-7) cells using the Trypan Blue Exclusion method to determine nontoxic titers of virus. AdSi73 was then infected in varying concentration at nontoxic levels into 5/15 replicon cell line stably expressing HCV replicon and grown in selection media supplemented with geneticin (G418) to select for cells expressing the HCV replicon. Twenty-four hours after infection, cells were fixed in acetone, and cells were immunostained for the viral protein NS3. The results of immunstaining showed a dose-dependent inhibition of NS3 expression. The findings of these studies demonstrated that the AdSi73 virus could be purified in a significant quantity and retain the siRNA gene. The data further showed that nontoxic levels of adenovirus could be infected into cells to elicit an inhibition of HCV protein expression. The findings of the current studies implicate that siRNA targeting the 59-UTR could provide a novel mechanism for treatment of HCV infection.
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