The Construction Of Recombinant Oncolytic Adenovirus RAd-E1A-CDglyTK And The Study Of Its Anti-tumor Effect In Vitro And Vivo

Adenovirus as gene tranfer vectors with a great number of advantages,including its wide host range,high titers and high transduction efficiency has been used extensively in the context of cancer gene therapy.However,due to its non-target transduction and transient exogenous gene expression,recombinant replication defective adenovirus-based gene therapy in clinical trials has thus far yielded dissatisfactory results.In order to find a better adenovirus-based cancer gene therapeutic modality,we inserted the connecting fragment E1A-IRES,deriving from adenovirus 5 type E1A gene and human EMCV internal ribosome entry site fragment, into recombinant replication defective adenovirus,rAd-CDglyTK,and constructed a novel recombinant oncolytis adenovirus,rAd-E1A-CDglyTK,which contains adenovirus E1A gene,fusion suicide gene CDglyTK fragment and GFP reporter gene and can specificly replicate in the tumor cells.Firstly,we had the successfully constructed recombinant plasmid, prAd-E1A-CDglyTK,digested by Pmeâ… enzyme,then transfected into 293 cells with liposome.The successful package was verified by observing the expression of green fluorescence protein under the fluorescent microscope.After packed and proliferated in 293 cells,rAd-E1A-CDglyTK was purified through the method of cesium chloride density gradient ultracentrifugation.The existence of E1A gene and CDglyTK fragment was identified by PCR assay.The result of viral particle concentration tested by ultraviolet spectrophotometry was 9.47×10¹¹VP/ml and its infectious titre was 3.16×10¹⁰IU/ml tested through 50%tissue culture infectious dose assay.The virual ratio-activity,3.34%,was gained by the ratio of infectious titre and viral particle concentration,having achieve the SFDA rated ratio-activity requirement of the recombinant adenovirus clinical grade products.To investigate the antitumor effect of the novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,we chose two kind of tumor cell lines,human ovarian cancer SKOV3 cell line and human lung cancer A549 cell line.Firstly,we verified the specific replicating ability of rAd-E1A-CDglyTK in the two kind of tumor cells,observed its infectious efficiency to SKOV3 or A549 cells and determine the minimal multiplicity of infection that can lead to the infectious efficiency up to above 90%.With the minimal MOI virus dose to infect tumor cells,SKOV3 or A549 cells,and to observe the drug sensitivity to enzyme prodrug 5-FC or GCV by MTT assay,and then to determine the 50%inhibiting concentration of 5-FC or GCV.With the minimal MOI virus dose and the 50%inhibiting concentration of of 5-FC and/or GCV to observe the cell growth inhibiting ratio,and to compare the antitumor effect of the recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,with the recombinant replication defecient adenovirus,rAd-CDglyTK,as well as the combination effect with prodrug,5-FC and/or GCV.As a result,the data indicated that rAd-E1A-CDglyTK itself had a significant cytocidal effect on SKOV3 cells,and combined with the 50%inhibiting concentration of 5-FC or GCV,the growth inhibition ratio could be respectively up to(51.59±2.31)%and(49.61±2.90)%. rAd-E1A-CDglyTK combined with the 50%inhibiting concentration of 5-FC and GCV,the growth inhibition ratio could reach(71.68±2.63)%,which comparied with rAd-CDglyTK/5-FC+GCV(63.64±2.91)%,P＜0.01.The resemble data attained from the experiment of A549 cells.Therefore,we could easily have a conclusion that the recombinant oncolytic adenovirus combined with enzyme prodrugs,5-FC and GCV,can result in a better ant^tumor effect in vitro in comparison with the replication defecient adenovirus.To investigate the antitumor effect in vivo of the novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,we prepared Lewis lung cancer-bearing C57BL mouse model.The tumor-bearing mice were divided into seven groups with random and subsequently accepted different administration,including recombinant adenovirus,rAd-CDglyTK or rAd-E1A-CDglyTK,and enzyme prodrug 5-FC and/or GCV.From the tendency of average tumor weight changes and the comparision of tumor inhibition rate as well as the tumor pathological changes of different groups,to observe the antitumor effect of the recombinant oncolytic adenovirus combined with enzyme prodrugs,5-FC and GCV.The experimental data presented that rAd-E1A-CDglyTK could have a considerable antitumor effect on Lewis lung cancer-bearing C57BL mouse,rAd-E1A-CDglyTK combined with enzyme prodrugs, 5-FC and GCV,could achieve a siginificant antitumor effect,better than the replication defecient adenovirus,rAd-CDglyTK.In short,our study successfully constructed a novel recombinant oncolytic adenovirus,rAd-E1A-CDglyTK,which contains adenovirus E1A gene,fusion suicide gene CDglyTK fragment and GFP reporter gene and can specificly replicate in the tumor cells.And we verified that the insertion of E1A gene rendered rAd-E1A-CDglyTK a considerable antitumor effect in vitro and in vivo and combined with enzyme prodrugs,5-FC and GCV,the antitumor effect could be significantly increased,which was better than the antitumor effect of replication defecient adenovirus,rAd-CDglyTK.These results set up a reliable experimental basis for the research of adenovirus vectors for malignant tumor gene therapy.