Targeted inhibition of hepatitis C virus–directed gene expression in human hepatoma cell lines  Catherine H. Wu, George Y. Wu  Gastroenterology  Volume 114, Issue 6, Pages 1304-1312 (June 1998) DOI: 10.1016/S0016-5085(98)70437-8 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 A schematic M-fold plot of nucleotides 1–341 of the HCV NTR showing the location of the target sequences for antisense oligonucleotides and their relationship to the translational start site. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 The effects of coadministration of oligonucleotides, HCV-marker construct CMV HCVluc, and a control CMV luc construct that lacks HCV-regulatory sequences. Huh7, asialoglycoprotein receptor–bearing cells were exposed to antisense or control oligonucleotides in increasing concentrations as described in Materials and Methods. After incubation, HCV-directed gene expression was determined by luciferase assays performed in triplicate, and results are expressed as means ± SD in picograms of luciferase per milligram of cell protein. (A) Concentration dependence of complexed anti-III oligonucleotide; (B) concentration dependence of complexed anti-IV oligonucleotide; (C) comparison of lipofected and complexed anti-III and sense-III oligonucleotides; and (D) comparison of lipofected and complexed anti-IV and sense-IV oligonucleotides. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of targeted antisense oligonucleotides on HCV-directed gene expression in SK Hep1 cells that lack asialoglycoprotein-receptor activity. Oligonucleotides were administered to SK Hep1 cells as described for Huh7 cells. After incubation, HCV-directed gene expression was determined by luciferase assays performed in triplicate, and results are expressed as means ± SD in picograms of luciferase per milligram of cell protein. (A) CMV HCVluc reporter construct; (B) CMVluc control reporter construct. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Administration of anti-III and sense-III oligonucleotides and their effects on constitutive expression of HCV gene markers. Huh7 cells were transfected with CMV HCVluc and an pSVneomycin plasmid and selected in medium containing G418. Cells from a stably transfected clone were treated for 24 hours with varying concentrations of anti-III or sense-III oligonucleotide–ASOR–PL complexes, anti-III complexes plus a 200-fold molar excess of ASOR, oligonucleotides alone, or equal concentrations of oligonucleotides in a lipofectamine mixture. After removal of treatment medium, cells were incubated with fresh medium without antisense oligonucleotide for 48 hours before being assayed for luciferase activity. (A) Lipofected anti-III oligonucleotide; (B) anti-III oligonucleotide alone; (C) complexed anti-III oligonucleotide; (D) a comparison of anti-III oligonucleotide administered by a complex, by lipofection, and simply added to the medium alone. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 Administration of anti-IV and sense-IV oligonucleotides and their effects on constitutive expression of HCV gene markers. Antisense oligonucleotides and controls were exposed to Huh7 cells and analyzed for luciferase expression as described in Figure 3. (A) Lipofected anti-IV oligonucleotide; (B) anti-IV oligonucleotide alone; (C) complexed anti-IV; (D) a comparison of anti-IV oligonucleotide administered by a complex, by lipofection, and simply added to the medium alone. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of complexed antisense oligonucleotides on viability and cell growth. Huh7 cells were exposed to oligonucleotides in the form of ASOR–PL complexes at the same concentrations and under the same conditions as for measurement of HCV NTR–directed luciferase expression. At regular intervals, the number of viable cells was determined by microscopically counting cells capable of trypan blue exclusion. All assays were performed in triplicate, and results are expressed as means ± SD. Effects of (A) 10 μmol/L and (B) 60 μmol/L oligonucleotides. Gastroenterology 1998 114, 1304-1312DOI: (10.1016/S0016-5085(98)70437-8) Copyright © 1998 American Gastroenterological Association Terms and Conditions