Precise Transduction Of Suicide Gene And Inhibition Of Bladder Tumor Growth By RAAV9-hUP?-TK-EGFP

Background: Bladder cancer(BC)is a common cancer of the human urinary system.The World Health Organization counted 573,000 new cases of bladder cancer and 212,000 deaths in 2020,and the increasing morbidity and mortality year by year places a huge burden on families and society.Bladder cancer is divided into non-invasive bladder cancer(low,medium and high risk)and invasive bladder cancer.The treatment methods of different grades of bladder cancer are also different,including surgery,chemotherapy,BCG intravesical instillation and radiotherapy.However,the high recurrence rate of bladder cancer and liver,lung,bone metastasis are the main reasons of reduced survival rate of patients.Therefore,there is an urgent need for a brand-new efficient and targeted therapeutic drug to improve the therapeutic effect and reduce toxic and side effects.Gene therapy,which refers to all approaches to treating diseases through gene-level interventions,is a promising therapeutic strategy that has received increasing attention.A large number of genes and non-coding RNAs are used in the study of bladder tumors,and these gene drugs may be a potentially effective treatment for bladder tumors in the future.Objective: According to the advantages of suicide gene HSV-TK,bladder tissue-specific promoter UP? and adeno-associated virus vector,a recombinant adeno-associated virus carrying bladder tissue-specific promoter gene and suicide gene was constructed to change the targeting of the virus,improve the conduction efficiency of suicide gene,and improve the anti-tumor effect of rAAV9-hUP?-TK-EGFP/GCV therapeutic strategy.To evaluate the therapeutic gene delivery ability and therapeutic effect of recombinant viral vectors in vitro and in vivo,and to provide a solid theoretical basis for us to develop targeted and efficient gene therapy drugs against bladder tumors.Methods: 1.Recombinant adeno-associated virus rAAV9-hUP?-TK-EGFP was designed and constructed.The recombinant viral vector was detected by PCR and gene sequencing to determine whether the UP? promoter gene sequence and HSV-TK gene sequence were successfully inserted into the recombinant viral vector;2.The targeting and safety of the recombinant adeno-associated virus vector were detected in vitro.After the recombinant viral vector was infected into different tissue cell lines,the degree of green fluorescence expression was observed under a fluorescence microscope,and the cell growth was observed and compared under a microscope to further confirm the safety of the recombinant virus;3.The targeting and safety of the recombinant adeno-associated virus vector penetrating GAGS layer to infect bladder epithelial cells were assessed.The recombinant virus was instilled through a catheter.One week later,the mice were sacrificed.The bladder,liver,kidney and brain were harvested.The green fluorescence expression in different tissues was compared under a fluorescence microscope after frozen section.The safety of intravesical instillation of the recombinant virus was assessed by HE staining;4.The expression of suicide gene protein was detected by Western blot after viral vector infection;5.Real Time Cel I Ana Iysis technique was used to dynamically examine the safety of suicide gene therapy strategy,and RTCA technique was used to real-time and dynamically evaluate the therapeutic effect of suicide gene therapy strategy;6.The therapeutic effect of recombinant adeno-associated virus rAAV9-hUP?-TK-EGFP/GCV treatment strategy was evaluated.After treatment,the subcutaneously implanted tumor tissues of the mice were harvested and weighed,explore the therapeutic mechanism by HE,immunofluorescence,and TUNEL staining.Results: 1.The recombinant adeno-associated virus rAAV9-hUP?-TK-EGFP was constructed,and the UP? promoter and HSV-TK gene sequences were confirmed by PCR and gene sequencing alignment;2.First,the recombinant viral vector was co-cultured with MB49,UMUC3,LNCaP,293 T,HuH7,and SGC7901 cells for 24 hours,and the green fluorescence expressed by bladder tumor cells was significantly stronger than that of non-bladder tumor cells under the fluorescence microscope,and then,the recombinant viral vector and negative control viral vector were infected with bladder tumor cells respectively,the green fluorescence expression of bladder tumor cells infected with the recombinant virus was significantly stronger than that of the negative control virus;3.After bladder tumor cells and non-bladder tumor cells were co-cultured with recombinant adeno-associated virus or PBS for 24 hours,the cell growth status of the two groups was compared under a microscope,it was found that the cell growth status of the two groups was good,the recombinant virus vector had no toxic and side effects on the cells;4.The recombinant viral vector can penetrate the GAGs layer on the medial side of the bladder and infect bladder epithelial cells in vivo;5,Compared with wild-type adeno-associated virus,the recombinant virus changes the hobby of the virus and enhances its ability to infect bladder epithelial cells.It was also confirmed that the human UP? promoter could also enhance the infection ability of viral vectors to murine bladder tumors;6.Intravesical instillation of recombinant rAAV9-hUP?-TK-EGFP viral vector or PBS via urinary catheter revealed no abnormalities,no bleeding or inflammation was found,indicating that the recombinant virus had low immunogenicity and high safety to the body;7.The targeting of recombinant virus was verified at the protein level,and it was found that the suicide gene was highly expressed in bladder tumor cells,the expression in non-bladder tumor cells was slight;8.CCK8 and real time cell analysis techniques detected the efficacy and safety of suicide gene therapy strategies,found that recombinant viral vectors,negative control viruses and drugs had no toxic and side effect on cell growth,but combination of recombinant viruses and drugs,the bladder cancer cells began to die and the growth curve shifted downward about 15 hours later,the proliferation of non-bladder tumor cells was not affected;9.The HSV-TK/GCV treatment strategy was confirmed to play a therapeutic role through the mechanism of apoptosis by HE staining,fluorescence labeling of BAX and caspase-3 apoptotic factors,and TUNEL staining.Conclusion: 1.UP? promoter can change the hobby of viral vector and enhance the infection ability of viral vector;2.rAAV9-hUP?-TK-EGFP viral vector can efficiently penetrate and conduct genes when the GAGs barrier layer is intact;3.Human UP? promoter has good histocompatibility with murine bladder epithelial cells;4.The green fluorescent expression gene carried by rAAV9-hUP?-TK-EGFP virus is highly expressed in the nucleus,the ability of virus to penetrate the cell membrane and nuclear membrane is strong,the therapeutic gene is expressed in the nucleus to facilitate therapeutic effect;5.rAAV9-hUP?-TK-EGFP viral vector has very good safety;6.rAAV9-hUP?-TK-EGFP viral vector can target delivery HSV-TK gene into bladder tumor cells,no expression of HSV-TK gene was detected in non-bladder tumor cells;7.HSV-TK/GCV treatment strategy confirmed to have a strong anti-tumor effect in vitro;8.HSV-TK/GCV treatment strategy had a significant therapeutic effect on mouse bladder tumor models;9.Apoptosis is one of the mechanisms of HSV-TK/GCV suicide gene therapy.