The Application Of Recombinant AAV With Tumor Specific Promoters In Oncology Therapy

Background and ObjectiveTumor is a global safety and health problem.Gene therapy has become a popular method of tumor treatment.Human telomerase reverse transcriptase(h TERT)gene is highly expressed in tumor tissues,but is not expressed or rarely expressed in normal tissues.To target tumor cells more specifically and reduce nonspecific toxicity to normal cells,we optimized the human telomerase reverse transcriptase(h TERT)promoter to drive the effector gene only express in tumor tissues but not in normal tissues.In addition,adeno-associated virus have low immunogenicity,high safety,and mediating target genes long expression features.Our research will use the optimized tumor specific promoter combined with adeno-associated virus vector to get recombinant adeno-associated virus.This research aims to explore a safer and more effective method for tumor gene therapy.MethodsIn this study,we have constructed recombinant adeno-associated virus vector(p AAV-CB)containing CMV enhancer/chimeric promoter of chicken β-actin(CB),and recombinant adeno-associated virus vector(p AAV-ATM)containing specific promoter(ATM)prepared by the fusion of antioxidant response elements(ARE)and human telomerase reverse transcriptase(h TERT)promoter.In addition,Adeno-associated viruses are packaged through three-plasmids system.In order to demonstrate the tumor specificity of ATM promoter,normal cells and tumor cells were infected with r AAV in vitro,and the infection ability and efficiency of r AAV were detected by inverted fluorescence microscopy and flow cytometry.In vivo,the subcutaneous tumor model of C57BL/6N mice was injected with r AAV through tail vein and intratumoral injection,respectively.Under certain time conditions,the distribution and expression of r AAV in mice were monitored by small animal imaging machine.On the basis of verifying the tumor specificity of ATM promoter,recombinant adeno-associated virus vector(r AAV-ATM-B8R-flag),which was expressed by ATM promoter driven vaccinia virus(VV)antigen peptide B8 R,was constructed.Tumor treatment was performed by intratumoral injection of r AAV-ATM-B8R-Flag into the subcutaneous tumor model of C57BL/6N mice under the condition of VV pre-immunization.Results1.Recombinant adeno-associated virus vectors(r AAV-ATM-EGFP/ Luciferase,r AAV-ATM-Luciferase-flag,and r AAV-ATM-B8R-flag)were successfully constructed by molecular cloning techniques.2.The recombinant plasmid,packaging plasmid and helper plasmid were used to package adeno-associated viruses,and the titer of r AAV was determined by Q-PCR.3.In vitro recombinant adeno-associated virus infected normal liver cells and tumor cells with a certain number of infection(MOI=10^5).Tumor specificity of ATM promoter was verified by inverted fluorescence microscopy and flow cytometry.In vivo,the C57BL/6N mouse subcutaneous tumor model was injected with r AAV through tail vein and intratumoral injection of r AAV.Small animal imaging instrument showed that ATM promoter was not expressed in normal tissues,but only expressed in tumor tissues.4.Tumor gene therapy was performed using the tumor-specific promoter ATM.Under the condition of VV pre-immunization,the corresponding serotype of r AAV was injected into the subcutaneous tumor model of C57BL/6N mice to monitor the change of subcutaneous tumor volume.The experimental results showed that under this conditions,the tumor growth inhibition ability after virus injection was r AAV-ATM-B8R-flag > r AAV-ATM-Luciferase-flag> PBS.Conclusions1.Different serotypes of adeno-associated viruses have different ability to infect different tumor cells;2.ATM promoter is tumor-specific and could drive efficient gene expression;3.rAAV,which carries a tumor-specific promoter and the most immunogenic antigen B8 R of vaccinia virus(VV),can activate memory immune cells against VV,thus killing tumor cells and inhibiting tumor growth.