| Hepatitis C is threatening human beings. There is about one percent of population all over the world suffering from the disease. The infectious rate in China is about 3%. What makes it difficult to cure lies in that it easily become chronic hepatitis, among which 20~4% may develop cirrhosis, end-stage liver disease and/ or hepatocellular carcinoma. People are seeking for strategies to inhibit HCV infection continuously even before the cDNA library of HCV RNA was set up by Choo in 1989. By now, IFN-α is believed to be the most effective therapeutic option to hepatitis C, but the efficacy is far beyond satisfaction because only 20-30% patients can acquire permanent response to IFN therapy. So it is necessary to find a more effective strategy. Lack of HCV replication cell lines and animal models have hampered development and screening of anti-HCV molecules seriously. Gene therapy threw light on the inhibition of HCV infection, and the favorite point is ribozyme. Ribozyme is a kind of small RNA molecule with catalytic function that can bind to specific target RNA via formation of base pairs and then cleave it at the specific site. Hammerhead ribozymes are the smallest and the best characterized among all the kinds of ribozymes. Hammerhead ribozymes consist of the catalytic active core (22nt) and flanking sequences. They recognize the "NUX" sequence, "N" can be A, C, U or G, "X" can be A, C, or U. The appropriate condition of reaction is at physiological temperature with the existence of Mg++(10mM). Some investigations have demonstrated that hammerhead ribozymes can cleave target RNA specifically and effectively. However, some deficiencies were found during their application. For example, they can't contact and react with substrates freely and effectively in vivo due to their different localizations in cells. The activities of ribozymes will change in vivo; they may loss their function in vivo while they can express efficient products and have effective catalytic activities in vitro, and unmodified ribozymes may be unstable both in cells and in sera. Furthermore, some strategies to improve the expression and stability of ribozymes may interfere with there catalytic activity. All of these have limited the anti-virus function of ribozymes. Efficient vectors must be construct to ensure that ribozymes will act as effectively in cells as in tubes. High levels of expression, stability, active conformation and correct cellular localization are the most important features for a ribozyme vector. U1 small nuclear RNA (U1 snRNA) which locates in nucleoplasm of eucaryotic cells only is just such a ideal ribozyme vector. This kind of small molecular RNA mainly participate in the splicing and mature process of hetrogeneous nuclear RNA (hn RNA, including pre-mRNA) due to its enzyme activity. Its sequence is highly conservative (95%). Human U1 snRNA (hU1 snRNA) consist of 163 nts and have m3G cap and four stem-loop structures. In cells each molar of U1 snRNA is combined with 7-8 kinds of proteins to form U1 small nuclear ribonucleoprotein (U1 snRNP), and exist and perform its function by this way. Up to now, study workers have successfully exploited the utilization of U1 snRNA as a vector of ribozymes and have cleaved HIV-1 RNA etc. with it successfully both in vitro and in vivo. Alessandro Michienzi has obtained conclusions in such a experiment as followes: ①U1 chimeric ribozyme (U1-Rz) has displayed high level of cleavage activity to HIV RNA no matter in vitro or in vivo; ② The U1-Rz are able to assemble into U1 snRNP-like particles, even though the efficiency is lower than that of wide type U1 snRNA. The facts indicate that neither U1 nor ribozyme in the chimera is interfered with each other in their active functions. The former studies have devised specific U1-Rz directed against pre-mRNA. We assumed that whether HCV can be served as an ideal target for U1-Rz. As a RNA virus, HCV genome can act as a template to replicate filial RNA and as an mRNA to direct translation of viral proteins. |
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