Construction Of Safer And High-efficient Recombinant Adenovirus For Tumor Gene Therapy

Recombinant adenovirus type5has been widely used in research of tumor genetherapy and achieved good tumor inhibitory effect in some such as breast cancer,bladder cancer, colon cancer and so on. However, almost recombinant adenovirusinjected into human body in the way to target tissues through the blood circulationenters liver instead. This bring great pressure to the liver. In this paper, based on theformer research data in our team, we designed a novel type of recombinant adenovirustype5vector to inhibit the growth of different tumor safely and effectively in vivo.Novel recombinant adenovirus type5vectors are modified based on the threeways of virus attaching to host cells to reduce the concentration of virus entering liver.Bringing RGD (Arg-Gly-Asp) peptide into HI-loop enhance the ability of a greatmany kinds of adenovirus for infecting most tumor cells(no CARs or less CARs).Mutation in knob region of adenovirus, which changes binding site of fiber and CAR,reduces the binding process they mediate. In this way the pressure of liver can bereduced. The adenovirus type5HVR5,7replaced with type26HVR5,7, make adecrease in the process of virus attaching to liver cells by changing HVR and FXbinding efficiency.Besides,by hTERT promoter regulating suicide gene HSV-TK, the ability ofanti-tumor of adenovirus type5is made sure to be realized. In addition, systemictoxicity is reduced and the safety in vivo is enhanced.In this paper, based on the construction of Ad5-RGD, we have gained theplasmids of Ad5-mu-RGD and Ad5-HVR26-mu-RGD. The generation of the virusAd5-mu-RGD-luc and Ad5-mu-RGD-hTERT-TK is going well. By now, we havecompleted the experiments of evaluating activity of infection and anti-tumor in vitroand analyzed the infection and distribution in vivo by utilizing imaging in vivo as wellas real-time fluorescent quantitative PCR. The results shows that in different CARlevel tumor cells，Ad5-mu-RGD-luc and Ad-luc which has no modified in skeletonhave different infection ability. This proves that in the process of Ad attach to target cells, the influence that RGD contacts to integrin on different cell surface make a bigdifference. Ad5-luc compared with Ad5-RGD-luc has a lower transduction efficiency.This demonstrates the RGD motif put into adenovirus knob HI-loop can widelyimprove adenovirus attaching to different tumor cells. In the process of the normalliver cell infection, the ability of Ad5-mu-RGD-luc is significantly lower than that ofAd5-RGD-luc, indicating that the point mutation in AB-loop Ser408, Pro409havingthe expected effects in reducing virus enrichment in liver, relieving the pressure thevirus bring to liver in the treatment of cancers. In vitro evaluation of antitumoractivity, the recombinant adenovirus carrying suicide gene have different levels ofantitumor tumor. The in vivo antitumor assay and hepatic tissue injury evaluation iscurrently going. Ad5-HVR26-mu-RGD has been completed its construction and thevirus packaging is being done.