Department of Internal Medicine I, University of Bonn, Bonn, Germany.
J Hepatol. 2011 Jul;55(1):19-28. doi: 10.1016/j.jhep.2010.11.010. Epub 2010 Nov 30.
BACKGROUND & AIMS: In this study, adenoviral vectors encoding an antisense RNA complementary to the 5' non-coding region (5'NCR) of the HCV-genome were generated to inhibit HCV-RNA gene expression in cell culture and in vivo.
First and second-generation (with E4-deletion) adenoviruses encoding the HCV5'NCR in antisense direction (Ad-NCRas and Ad-E4del-NCRas) were generated. Inhibition of HCV gene expression was analyzed in hepatoma cells stably transfected with the HCV5'NCR cDNA fused to the firefly luciferase gene (NCRluc), as well as in the HCV subgenomic replicon (genotypes 1b and 2a) and the fully infectious HCV JFH-1 cell culture systems. For in vivo experiments, an adenovirus encoding the NCRluc-gene was injected intravenously to achieve a NCR-dependent luciferase-expression in the liver of C3H/HeNcrl-mice.
Forty eight hours after transduction with GFP-encoding adenoviruses, >85% of HepG2-, CCL13-and Huh7-cells expressed GFP. Surprisingly, GFP-expression of E4-deleted adenoviruses was considerably reduced at the same MOI. Using antisense first-generation adenoviruses (Ad-NCRas), a significant inhibition of the 5'NCR-dependent HCV-gene expression (54±19% in HepG2-cells and 66.2±15% in Huh7-cells) was achieved 48h after transduction. In Huh7-cells containing the HCV subgenomic replicons and in infectious HCV JFH-1 cell cultures, adenovirus-mediated transcription of antisense 5'NCR significantly blocked HCV-replication (40% and 76%, respectively). Corresponding to low transgene expression, the maximal inhibition reached with Ad-delE4-NCRas was 30%. In vivo, antisense adenoviral vectors also showed a significant inhibition (40%) of NCR-dependent luciferase expression compared to control adenoviruses (p<0.05).
The data indicate that HCV gene expression can be inhibited by antisense RNA encoding adenoviruses in the tested settings.
本研究构建了靶向 HCV 基因组 5'非编码区（5'NCR）的反义 RNA 的腺病毒载体，以在细胞培养和体内抑制 HCV-RNA 基因表达。
构建了第一代（含 E4 缺失）和第二代（含 E4 缺失）靶向 HCV5'NCR 的反义腺病毒（Ad-NCRas 和 Ad-E4del-NCRas）。通过转染含有 HCV5'NCR cDNA 和萤火虫荧光素酶基因（NCRluc）的肝癌细胞系、HCV 亚基因组复制子（基因型 1b 和 2a）和完全感染性 HCV JFH-1 细胞培养系统，分析 HCV 基因表达的抑制情况。为进行体内实验，将编码 NCRluc 基因的腺病毒静脉注射到 C3H/HeNcrl-mice 肝脏中，实现 NCR 依赖性的荧光素酶表达。
转染 GFP 编码腺病毒 48 小时后，HepG2、CCL13 和 Huh7 细胞中 GFP 表达率>85%。令人惊讶的是，同一 MOI 下 E4 缺失腺病毒的 GFP 表达显著降低。使用第一代反义腺病毒（Ad-NCRas），转染 48 小时后可显著抑制 5'NCR 依赖的 HCV 基因表达（HepG2 细胞中为 54±19%，Huh7 细胞中为 66.2±15%）。在含有 HCV 亚基因组复制子的 Huh7 细胞和感染性 HCV JFH-1 细胞培养物中，腺病毒介导的反义 5'NCR 转录显著抑制 HCV 复制（分别为 40%和 76%）。由于转染基因表达较低，Ad-delE4-NCRas 达到的最大抑制率为 30%。在体内，与对照腺病毒相比，反义腺病毒载体也可显著抑制 NCR 依赖的荧光素酶表达（40%，p<0.05）。
数据表明，在测试环境中，反义 RNA 编码的腺病毒可抑制 HCV 基因表达。
