Study Of Anti-HCV By SiRNAs & DNAzymes Targeting IRES And The Factors Affect The Function Of IRES

Hepatitis C Virus is the leading cause of chronic liver disease globally and is estimated to infect about 170 million people around the world. Chronic HCV infection frequently leads to liver fibrosis and cirrhosis, and is associated with the occurrence of hepatocellular carcinoma. Although IFN is the only approved anti-HCV therapeutic agent, it is effective in only 30% of HCV-infected individuals.As a Flaviviridae family member, HCV possesses a positive-sense, ssRNA genome of about 9600 nucleotides. The HCV genome consists of highly conserved 5'-and 3'-noncoding region, and a large open reading frame that encoding a polyprotein of approximated 3010 amino acids. The 5' NCR of the HCV genome is highly conserved region that contains an internal ribosome-entry site, which initiates translation by a cap-independent mechanism.RNA interference is a phenomenon in which small double-stranded RNA molecules induce sequence-specific degradation of homologous single-stranded RNA. It has been shown that siRNA with 3'- and 5'- terminal overhang of two bp can induce RNAi in mammal cell. DNAzymes are synthetic single-stranded DNA analogs of ribozymes. These Dz are short and have simple secondary structure, expected more stable than ribozymes. Our purpose is to take IRES as the target site, utilize RNAi and Dz activity for the treatment of HCV infected disease. We also study the influence factors on the function of IRES, such as IFN, alcohol, PTB, La antigen, and HCV non-structure protein et al.Since HCV could not be cultured in cells and animals, evaluating the effect of antiviral drugs is very difficult. It is important to construct the model for evaluating anti-HCV agents.In this study, we investigate the effect of siRNAs and DNAzymes on HCV IRES directed translation, using a stable transformed cell lines, HepG2.9706, which constitutively express viral RNA transcripts containing sequence encoding reporter protein (firefly luciferase). Luciferase activity in cell lysates was measured for quantitative determining antiviral effects within the cells. And hydrodynamics-based procedure for expressing high level of target protein in mice liver by systemic administration of plasmid DNA was used for evaluating the inhibitory effect of siRNAs and DNAzymes in mice.In the study, we synthesized siRNA by transcription in vitro with T7 RNA polymerase and expressed shRNA by vector. The results showed that both T7siRNAs and vector-express shRNA targeting to different domains of HCV IRES displayed inhibitory effects on the luciferase expression controlled by HCV IRES in a dose-dependent manner. siRNAs had specific inhibition to the target, if one nucleotide of T7siRNA changed, it showed almost no inhibitory effect. And the siRNA targeting HCV IRES showed no inhibition to EMCV IRES.DNAzyme is a catalytic molecules consisting of synthetic single-stranded DNA that specifically cleaves substrate RNA in a manner analogous to ribozymes. It has the potential advantages of being less sensitive to chemical and enzymatic degradation, easier to prepare and deliver to cell. It has three domains: a catalytic domain consisting of 15 nt flanks and two substrate recognition of the binding target RNA through Watson-Crick base pairing. Five DNAzymes 10-23 were designed to cleave the HCV IRES respectively and showed high activity in cleaving targets. The effect of arm length on DNAzymes was studied using Dz with arm lengths of 8nt, 1 Int and 14nt. And the result show the DNAzymes with shorter arm length had higher cleaving activity.At present, interferon(IFN- a ) is the only approved anti-HCV therapeutic agent. The inhibition of recombinant IFN-a to HCV IRES is evaluated in vitro and in vivo respectively. The result show IFN-a can inhibit the luciferase expression controlled by HCV IRES in a dose-dependent manner both in cell and in the liver of the mice. This implied that the inhibitory effect on IRES may be one kind of mechanism of the inhibition of IFN to HCV.Alcohol consumption accelerates liver damage and diminishes the anti-HCV effect of IFN-a...