A Study On Targeting Immuno-gene Therapy For Mice Lewis Lung Cancer

Background and Objective:Lung cancer is the first cancer killer nowadays.In spite of the continuous progress in fields of traditional treatments including surgery,chemotherapy,radiotherapy for lung cancer during the past decades,effects of traditional treatments for lung cancer is still far away from satisfaction.Immuno-gene therapy has become focus and expectable field of cancer research.One of the important strategies of immuno-gene therapy for cancer transduction of exotic antigen gene into tumor cells,so that exotic antigen could be expressed in cancer cells and immune reaction of host could be aroused.Possibility and feasibility of this immuno-gene therapy approach has been proved by different researches.Function and mechanisms of human immune reaction are very complicated,and excessive and extensive immune reaction will lead to serious immune related diseases.Ideally,therapeutic effect of cancer gene therapy should be highly tumor targeting.The most common and effective approach of targeting tumor gene therapy is to regulate expression of therapeutic gene at transcriptional level with tumor specific promoters,in which telomerase catalytic subunit(TERT)promoter is the most commonly used because of its strict tumor specificity.The present research is to explore possibility and approach of targeting immuno-gene therapy for lung cancer,using antigen gene of Much’s bacillus Ag85A driven by mice telomerase catalytic subunit(TERT)promoter.Methods:1.Mice TERT promoter(mTERTp)was cloned by polymerase chain reaction(PCR),using genomic DNA of mice as template.Then,mTERTp was inserted into plasmid pGL3-basic,so that Luciferase reporter plasmid driven by mTERTp(pGL3-mTERT)was constructed.Luciferase Assay was performed to investigate mTERTp transcriptional activity in various murine and human cancer cells and normal cells,including Murine Lewis lung cancer cells,Murine fibroblast NIH3T3,Human A549 lung adenocarcinoma cells,Human esophageal cancer EC-109 cells and Human embryonic lung fibroblasts MRC-5 cells.2.Much’s bacillus antigen gene Ag85A and Ag85B cDNA connected with P2A sequence(Ag85A-MYC-P2A-Ag85B-HA)were chemically synthesized,in which Myc and HA were tag sequences connected to Ag85A and Ag85B genes.Ag85A-MYC-P2A-Ag85B-HA sequence was cloned into the linearized lentiviral vector pLVX-EGFP-3FLAG-Puro so that Ag85A-Ag85B expression lentiviral plasmid driven by CMV promoter(pLVX-Ag85A-Ag85B-CMV)was constructed.Then CMV promoter in pLVX-Ag85A-Ag85B-CMV was removed and replaced by mTERTp,so that Ag85A-Ag85B expression lentiviral plasmid driven by mTERTp(pLVX-Ag85A-Ag85B-mTERTp)was constructed.3.Ag85A-Ag85B lentivirus driven by CMV promoter and mTERTp were packaged respectively.Then,Lewis lung cancer cells and NIH3T3 cells were infected with above Ag85A-Ag85B lentivirus,and expression of Ag85A and Ag85B in transfected cells was detected with western blot.4.Forty nine C57BL/6 mice were inoculated with Bacille Calmette-Guerin(BCG)vaccine subcutaneously.3 weeks later,mice were inoculated with Lewis lung cancer cells by subcutaneous injection in right axilla(2×106cells each mouse),and then divided into different groups 7 days later.Ag85A-Ag85B lentivirus was intravenously injected through tail veins,and therapeutic effect was investigated.Results:1.A piece of 331 bp mice TERT promoter(mTERTp)was successfully cloned,and its base sequence was proved complete same as that registered in GenBank by DNA sequencing.Luciferase assay demonstrated that mTERTp had transcriptional activities in murine Lewis lung cancer cells,human A549 lung adenocarcinoma cells and human esophageal cancer EC-109 cells,while no transcriptional activity murine fibroblast NIH3T3 and human embryonic lung fibroblasts MRC-5 cells.2.Ag85A-Ag85B express lentiviral plasmids driven by CMV promoter(pLVX-Ag85A-Ag85B-CMV)and mTERTp(pLVX-Ag85A-Ag85B-mTERTp)were successfully constructed,confirmed by enzyme digestion and DNA sequencing.Ag85A and Ag85B protein were detected in pLVX-Ag85A-Ag85B-CMV tranfected all kinds of cells in our research and pLVX-Ag85A-Ag85B-mTERTp tranfected cancer cells(including Lewis cells,A549 cells and EC-109 cells),while no Ag85A or Ag85B protein was detected in pLVX-Ag85A-Ag85B-mTERTp transfected normal cells(NIH3T3 cells,and MRC-5 cells).3.Neither pLVX-Ag85A-Ag85B-CMV nor pLVX-Ag85A-Ag85B-mTERTp lentivirus injection through tail vein displayed anti-tumor effect in BCG vaccinated Lewis burdened C57BL/6 mice.Conclusions:1.Mice TERT promoter had tumor specific transcriptional activity;2.Ag85A-Ag85B gene could selectively express in tumor cells,driven by mice TERT promoter;3.Ag85A-Ag85B express lentivirus intravenous injection did not display anti-tumor effect in BCG vaccinated Lewis burdened C57BL/6 mice.Further research is needed to reveal its reason.