Treatment Of HCV Core-positive Hepatocytes With The Transfer Of Recombinant Caspase-3 Using The 2'-5' Oligoadenylate Synthetase Gene Promoter

Introduction:Hepatitis C virus(HCV) infection is a major cause of chronic hepatitis,liver cirrhosis and hepatocellular carcinoma worldwide. The current standard therapy for chronic hepatitis C consists of a combination of pegylated IFN alpha(pegIFNα) and ribavirin.It achieves a sustained viral clearance in only 50-60%of patients.Moreover,this treatment is associated with substantial side effects,precluding its use by many individuals.Thus,current therapies are inadequate for the majority of patients,and there is an urgent need for the novel hepatitis C therapeutic strategies.It is generally thought that HCV infects only a small fraction of hepatocytes,approximately 1-20%as judged by the detection of HCV proteins or HCV RNA in liver biopsy samples.Therefore,induction of hepatic apoptosis is a potential approach for treatment of chronic hepatitis C.Activation of effector caspases is a central and ultimate step in many apoptosis pathways.Caspase-3 is the key executioner caspase,it exists as an inactive zymogen that is activated by upstream signals.Several groups have considered using the human caspase-3 gene as a novel form of anticancer gene therapy.However,overexpression of the wild-type caspase-3 in mammalian cells does not induce apoptosis,which is due to their inability to undergo autocatalytic processing without upstream caspase for activation.Recently,constitutively active recombinant caspase-3(re-caspase-3) has been was generated by making its small subunit preceding its large subunit.Unlike its wild-type counterpart that is the large subunit preceding the small subunit,the re-caspase-3 is capable of autocatalytic processing and inducing apoptosis independent of the upstream initiator caspase molecules.In addition,it could resist the effect of some apoptosis restraining genes.As caspase-3 is the most downstream executioner of apoptosis,the re-caspase-3 could be used at very low concentrations to induce apoptosis in target cells.However,if caspases-3 is transferred to normal tissues,it would be predicted to also undergo apoptosis,resulting in undesirable damage.To restrict induction of apoptosis to HCV infected cells and increase the safety of this approach,we needed to establish a HCV-specific caspase expression system.The HCV-core protein is currently considered to be a multifunctional protein that plays an important role in persistent infection and hepatocellular carcinogenesis.Naganuma et al found that HCV-core protein specifically activated the interferon(IFN)-inducible 2'-5'oligoadenylate synthetase(OAS) gene promoter in human hepatocyte cells,while the E1,E2,and NS5A proteins did not activate the OAS gene promoter.Moreover,the activation by the core protein is a general phenomenon,regardless of HCV genotype and strain.Therefore, utilization of the OAS gene promoter that is predominantly active in HCV-core positive hepatocytes would be an ideal system to restrict the cytotoxic caspase expression.Objective:Our current investigation attempts to construct an expression vector consisting of the re-caspase-3 under the OAS gene promoter(pGL3-OAS -re-caspase-3) and then investigate its effect on HCV-core positive liver cells.Methods:Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418.Expression of HCV-core was detected by RT-PCR and western blot.The OAS promoter sequences was amplified from the genomic DNA and inserted into pGL3-Basic vector.The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed.Recombinant caspase-3 gene was constructed by making its small subunit preceding its large subunit.An expression vector consisting of the re-caspase-3 under the OAS gene promoter(pGL3-OAS -re-caspase-3) was constructed and transfectd into cells to investigate its effect on HCV-core positive liver cells.Results:L02/core cell line stably expressing HCV-core protein was established.The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.The recombinant caspase-3 gene had enzymatic activity.The pGL3-OAS -re-caspase-3 construct induced apoptosis in HCV-core positive liver cells,but not in normal liver cells.Conclusion:1.We successfully cloned the 2'-5'oligoadenylate synthetase(OAS) gene promoter and demonstrated that it can be activated by HCV-core protein.2.We successfully constructed the expression vector consisting of recombinant caspase-3 gene under the OAS promoter, pGL3-OAS -re-caspase-3.3.For the first time,we demonstrated that pGL3-OAS -re-caspase-3 induced apoptosis in HCV-core positive liver cells significantly and specifically4.The present results strongly suggest that the transfer pGL3-OAS -re-caspase-3 is a novel targeting approach for the treatment of HCV infection.