Establishment Of A Mouse Model Long-term Expressing HCV IRES For Anti-HCV Drugs Evaluation

Hepatitis C represents a major health concern. It becomes persistent among 50% - 80% infected persons, and such patients may develop chronic hepatitis and cirrhosis, some may even progress to the stage of hepatocellular carcinoma. Today no therapy was proved to be enough valuable to Hepatitis C treatment. So the development of a new way for hepatitis C prevention and treatment is very desirable.Now the main strategy of treating hepatitis C is targeting to HCV genome and key proteins HCV expressed. The internal ribosome entry site (IRES) located in 5'-noncoding region of HCV genome, is a major potential drug target site in anti-HCV research. The HCV IRES is a double function molecule. HCV IRES can directly regulate the assembly of translation initiation complexes and control the cellular translation by the mechanism of cap-independent translation. On the other hand, HCV IRES can inhibite the PKR activity to decrease the effect of IFN-α. HCV IRES is also a highly structured and conserved RNA, which can resist to the viral "escape mutant" as a drug target site. So the research of drugs targeting to HCV IRES is very important.Recently great progress has gained on anti-HCV drugs such as aptamers and dsRNA (siRNA) et al, which have displayed good anti-virus effects in vitro or in cell lines. But for lacking of appropriate small animal models that can infect HCV, these drugs' effect, safety and toxicity in vivo can't be evaluated properly, which greatly delay these drugs' clinical use. So developing a mouse model for evaluating inhibitors targeting to HCV IRES is highly desirable.Here we report a mouse model, which can monitor the function of IRES activity in vivo. Instead of using traditional transgenic technique, the model is construted by hydrodynamic transfection coupling with phage integrase system.It can be used for evaluating the effect of shRNA and aptamer (targeting to HCV IRES) in vivo. The main content of our research will be discussed below:1. We constructed an eukaryotic expression vector pCI-attB-AAA-HCV IRES-Fluc-neo that can mediate persistent and high-level foreign gene expression in vivo. This vector had the liver-specific promoter AerApoEAAT, (AAA) and attB site that can be attached by phage integrase and the reportor gene Fluc expression can represent HCV IRES activity. To select the better promoter, pCI-attB-AAA-HCVIRES-Fluc-neo and pCI-attB-CMV-HCVIRES-Fluc-neo was transfected to mice respectively. Result indicated that AAA promoter made Fluc expression longer than CMV promoter (lasting around 20 d), in spite of its slightly lower priming efficiency than CMV promoter. Here we also found that the livers had no manifest damage after hydrodynamic transfection and different species mice had no significant difference on Fluc expression in this study.2.ΦC31 integrase(ΦC31-int ) has the ability, which can perform an efficient site-special chromosomal integration to get the long-term transgene expression of Fluc mediated by HCV IRES. In this study pCI-attB-AAA-HCV IRES-Fluc-neo was cotransfected with pCMVInt plasmid expressingΦC31-int into mice by hydrodynamics-based procedure and the bioluminescence imaging was used to real time quantitatively detect the dynamics of Fluc expression in mice livers. Study identified that Fluc got a long-term expression. After 30 d past cotransfection, the luciferase activity was keeping stable expression, even last 300 d. Mice from pCMV-int group, which had stable luciferase expression were operated a surgery of the two-thirds partial hepatectomy(PH) and were found that the Fluc expression recovered after 14 d past PH with lives regeneration. At the same time, we use nested-PCR to identify the integration of the Flue expression frame into mice chromosomal DNA with "pseudo-attP" sites. The result confirmed the hot site was mpsL1 located at mice chromosomal 2 generating two recombination junction sites (attR and attL), and there were 3-5 bp micro deletion near the core area TTG. 3. Based on the mouse model, the shRNAs and aptamer targeted to different HCV IRES sites were evaluated in vivo. By using pSilencer ^TM2.1-U6 neo and pAV7SL, we constructed shRNAs and aptamer expressing vectors: shIRES184, shIRES256 and A-256 targeting to HCVIRES'IIIb/IIId loops respectively. At HepG2, HepG2.9706 and mouse model, these drugs all displayed inhibitory effects on the Fluc expression mediated by HCV IRES, and if one or several nucleotides changed, inhibitory effect would almost lost. At the same time, study found that when combined shIRES184 with A-256, synergistic effect appeared, and when combined shIRES256 with A-256, antagonistic effect appeared. At mouse model the similar conclusion was gotten. The result also indicated that these shRNAs and aptamer needed some time (48 h in cell model and 3 d at mouse model) to transcription in vivo and piling up to get the inhibitory effection. When transfected repeatly, the similar dynamic inhibitory effection curve were gotten, which showed that this mouse model was steady.In summary, this study combined hydrodynamic transfection with phage integrase system to construct a long -term mouse model targeting to HCV IRES, and using this model to evaluate the anti-HCV drugs (shRNA and aptamer) and their combination use effect in vivo.This study would benefit for the further research on anti-HCV gene therapy.