Visualization Of Gene Therapy With A Liver Cancer-targeted Adeno-associated Virus 3 Vector

Background:Cancer has gradually become a major disease worldwide.There are approximately 700,000 deaths due to liver cancer each year worldwide,and about 55%occur in China,which is second only to lung cancer.However,it is easy to relapse or fail to achieve the desired effect after surgery,radiotherapy and chemotherapy.Traditional anticancer drugs generally have short effects and need to be administered multiple times.These problems have caused many problems in tumor treatment.However,the emergence of gene therapy has broken this limitation,provided a new method for liver cancer treatment,and brought hope for the survival of liver cancer patients.However,the lack of tumor targeting and low expression of the target gene in vivo are the main problems faced by clinical transformation of traditional tumor gene therapy,so finding safe and reliable high tumor targeting vectors is the focus of current tumor gene therapy research.At the same time,how to understand the expression distribution of therapeutic genes in tumor cells in vivo,how to perform early diagnosis and dynamic monitoring of the effects of targeted therapy,and how to combine drugs with targeting are still urgent problems to be solved.Objective:To evaluate the feasibility of a self-complementing recombinant adeno-associated virus 3?scrAAV3?vector targeting liver cancer and non-invasively monitor gene therapy of liver cancer.Methods:An scrAAV3-HSV1-TK-Kallistatin?ATK?gene drug was constructed,which contained the herpes virus thymidine kinase?HSV1-TK?reporter gene and human endogenous angiogenesis inhibitor?Kallistatin?gene for non-invasive imaging of gene expression.The virus was transfected into HepG2 cells.The experiment was divided into three groups,which were divided into experimental group?ATK group?,negative control group?scrAAV3-EGFP group?and control group?Control group?.Real-time quantitative PCR?RT-qPCR?and Western Blot analysis were used to identify the mRNA and protein expression levels of HSV1-TK and Kallistatin in the three groups.Cell proliferation experiments were performed to observe the proliferation ability of the three groups of HepG2 cells.Subcutaneous xenografted tumors of hepatoma in nude mice were generated for positron emission tomography/computed tomography?PET/CT?imaging.The ATK group was injected with the ATK gene through the tail vein,and an imaging agent was injected 2 weeks later.PET/CT imaging was performed at 1 hour after injection of the imaging agent.The control group was injected with phosphate-buffered saline at the same volume as the ATK gene drug.HE staining is used for pathological observation of tumor sections.HSV1-TK and Kallistatin expression was identified by immunofluorescence,RT-qPCR,and Western Blot.Results:In cell experiments,RT-qPCR and Western Blot analysis showed that compared with the two control groups,the mRNA and protein expression levels of HSV1-TK and Kallistatin were increased in the experimental group?P<0.05?,and between different control groups No difference in expression.The results of MTS cell proliferation experiments showed that the cell proliferation ability of the experimental group was significantly lower than that of the two control groups?P<0.05?,and there was no difference between the different control groups.In animal experiments,radioactivity on PET/CT images was significantly higher in the ATK group compared with the control group.¹⁸F-FHBG uptake values of left forelegs in ATK and control groups were 0.591±0.151%and 0.017±0.011%ID/g?n=5?,respectively?P<0.05?.After injection of the ATK gene drug,mRNA and protein expression of HSV1-TK and Kallistatin in subcutaneous xenograft tumors was detected successfully.In vitro analysis demonstrated significant differences in the expression of HSV1-TK and Kallistatin between ATK and control groups?P<0.05?.Conclusions:Cell experiments prove that ATK gene drugs can effectively transfect HepG2 cells and inhibit their proliferation.The results of animal experiments show that the scrAAV3 vector has a strong liver cancer-targeting ability,and the ATK gene drug can be used for targeted and non-invasive monitoring of liver cancer gene therapy.