Inhibition Of HCV 5'NCR And Core Expression By ShRNA And Application Of New Gene Carrier HPhA

Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Effective therapies against viral infection are not available at present. There are some problems involved in the available techniques of HCV gene therapies, such as antioligonuclitide and ribozyme and deoxyribozyme. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections.RNA interference (RNAi) is a process during which double stranded RNA (dsRNA) induces the sequence-specific degradation of homologous single-stranded RNA. As a result, the expression of the target gene is suppressed. RNAi can therefore be used to develop specific dsRNA based gene therapeutics by knocking down specific genes such as the gene related to viral infection, carcinoma, and the others diseases.In cultured mammalian cells, the sequence-specific gene silencing can be accomplished by introducing small interfering RNA (siRNA) that is cleaved from dsRNA. Only RNA molecules<30 bases in length can be used to exclusively induce RNAi in mammalian cells because longer molecules also activate the nonspecific double-stranded RNA-dependent IFN response. With the advance of producing siRNA, several laboratories made progress in using RNAi to control viral infection and showed that siRNA can inhibit infection by polio, HIV, HCV and influenza virus. Recently it was reported that RNAi could also reduced HCV replication and expression in the post-transcriptional level.The likelihood of HCV developing escape mutations illustrates the importance of careful siRNA target sequence selection during the development of treatment strategies. Several conserved regions within the HCV5'noncoding region (5'-NCR) have been found to be viable targets for RNA interference and have potential in therapeutic RNA interference strategies. For example, the 5′UTR acts as an internal ribosome entry sequence, and its activity is determined by RNA structural characteristics. As such, the sequence does not tolerate nucleotide changes and is highly conserved between different HCV genotypes. Therefore, siRNA target sequences based on the 5′UTR region offer promise for siRNA-based treatments.We construct HCV-specific shRNA expression plasmid targeted 5'NCR and C region (Psilencirle A, B and D), luciferase-specific siRNA expression plasmid (Psilencirle E) and scrambled siRNA expression plasmid(Psilencirle C) by SilenCircleTM RNAi Transcription Kit. Then we transfect above plasmids and plasmids pCMV/T7-NCRC?-luc to HL-7702 cells, pCMV/T7-NCRC?-luc containing HCV 5'NCR, partial core region and luciferase sequences. The luciferase gene is utilized as a reporter gene, its expression can reflect the synthesis of HCV 5'NCR and core protein. The results show that Psilencirle A,B and D can inhibit the expression of HCV 5'NCR and core protein in HL-7702 cells, the inhibiting rate are 38%, 49%, 55% respectedly.An obvious challenge in the use of siRNA as a therapeutic agent is the development of suitable delivery methods. There have been recent advances in delivery of siRNA using peptide and polymeric vehicles and in vivo application through injection of a large volume of liquid siRNA solutions into the tail veins of mice. The latter treatment could induce RNA interference in the mouse liver and prevent Fas-mediated apoptosis. In our study we introduced the new transgene carrier HPhA (gene engineering production of jilin university) to transfect siRNA expression plasmid into HL-7702 cells.Gel electrophoresis mobility shift assays demonstrate that the purified HphA has high affinity to DNA. The HphA can increase plasmid electrophoresis mobilities with proper HphA/DNA mass ratios.We determined the toxicty of HphA to cells by MTT methods, and compared with LipofectamineTM 2000. The results shows that HphA almost don't affect the growth of cells in relative low concentration(20ug/ml), but LipofectamineTM 2000 cause relative cell viability obviously come down. Cells will die only when the concentration of HphA is higher than 100ug/ml . The toxicty of 1000ug/ml HphA to HL-7702 cells is similar with that of 40ug/ml LipofectamineTM 2000. Considering of the maximum amount of HphA per well is only 9ug when transfection, we can conclude that HphA is nontoxic to cells.HCV-specific siRNA expression plasmid mediated by HphA can inhibit the expression of HCV 5'NCR and core protein, its efficiency is similar with LipofectamineTM2000. The most important is that when medium containe10% fetal calf serum (FCS), the efficiency of HphA mediated transfection is similar with that without fetal calf serum, and LipofectamineTM2000 can't transfec plasmids into HL-7702 cells to express HCV 5'NCR and core protein ,when medium containe10% fetal calf serum (FCS). So we concluded that HphA is suitable to transfection in vivo, and it is a safe and effective transgene carrier and will play an import role in RNAi-based genetherapy.