The Application Of HSV-1 Oncolytic Virus In Cancer Precision Therapeutics And Mechanism Study Of Tumor Tolerance

Oncolytic virus therapy（OVT）has become a research hotspot in cancer therapy because of its safety,high efficiency,and fewer side effects.Herpes simplex virus type 1（HSV-1）,as the most widely studied oncolytic virus,has dozens of candidate drugs at the preclinical or clinical research stage.However,years of clinical trials have shown that the therapeutic effect of single oncolytic virus therapy is limited,and the oncolytic mechanism is not clear.Many proteins encoded by the host genes,namely the host dependency factor（HDF）,are essential for virus infection and proliferation.The defects in HDF can lead to the failure of viral infection at the cellular and animal levels.However,although the replication cycle of HSV-1 and the immune mechanism of HSV-1 have been intensively studied,the knowledge regarding the interaction between the virus and host,especially the role of cytoplasmic proteins involved in virus infection,replication and proliferation,is still limited.Whether HDF mutations in tumor patients have effects on the treatment of HSV-1 oncolytic virus has not been systematically studied.The purpose of this study is to explore the role of HDF mutations of HSV-1 virus in the precise treatment of oncolytic viruses and the related mechanisms of tumor tolerance.Methods:（1）Using the CRISPR/Cas9 technology,we constructed HSV-1-ΔICP34.5oncolytic virus with ICP34.5 gene deletion,a mouse colon cancer cell line MC38^Nectin1-/-with the deletion of the Nectin1 gene that acts as a HSV-1 virus receptor and inserted the immune activator protein NEO-2/15 into the ICP34.5 gene locus of HSV-1 oncolytic virus.（2）The mouse B16 melanoma model and MC38 colon tumor model were constructed and treated with HSV-1-ΔICP34.5 oncolytic virus to evaluate the therapeutic effect.（3）The mouse MC38 colon tumor model was constructed and treated with HSV-1-ΔICP34.5 and HSV-1-NEO-2/15 oncolytic virus to evaluate the therapeutic effect.（4）The human CRISPR whole-genome knockout library combined with high-throughput sequencing technology was used to screen the HDF of HSV-1 virus.（5）The candidate genes of HSV-1 virus HDF was determined by statistics,gene enrichment analysis,KEGG pathway analysis,PANTHER GO analysis and gene overlap analysis.And the CRISPR/Cas9 technology is used to prepare knockout cells for the possible HDF genes such as Nectin1,ENTPD1,HCFC1R1,EXT1,EXT2,EXTL3,Tm9sf3,and B3GALT6.（6）After virus infection,trypan blue cell counting method,q PCR and Western blotting were performed to detect the effect of HDF deletion on virus infection.（7）Through the membrane binding experiment of HSV-1 virus,the binding ability of virus and the host cell after HDF deletion of HSV-1 virus was evaluated.（8）Confocal microscopy imaging was used to observe the entry of HSV-1 virus into cells.（9）Use the TCGA database to analyze the mutation frequency of HSV-1 virus HDF in clinical tumor patients.Results:（1）HSV-1-ΔICP34.5 oncolytic virus treatment had a significant effect on the MC38 colon cancer in mice,while no obvious effect on B16 melanoma.Then we used HSV-1-ΔICP34.5 oncolytic virus to treat colon cancer of mice with deletion of the HSV-1virus receptor Nectin1 gene.The results showed that compared with the control group,the tumor volume of mice with Nectin1 gene deletion was not significantly reduced,which means that the oncolytic virus was ineffective for the treatment of MC38-ΔNectin1 colon tumor in mice.（2）In this study,we successfully prepared HSV-1-NEO-2/15 new oncolytic virus.Through the treatment of MC38 colon tumor in mice,our results show that NEO-2/15 can be used simultaneously with HSV-1 oncolytic virus,and the integration of NEO-2/15 can significantly enhance the therapeutic effect of oncolytic virus.（3）Human CRISPR whole genome knockout library combined with high-throughput sequencing technology,using statistical,gene enrichment analysis,KEGG pathway analysis,PANTHER GO analysis and gene overlap analysis and other analysis methods,we finally got 31 candidate HDF genes of HSV-1.Through gene knockout verification,we have confirmed that knocking out Nectin1,ENTPD1,HCFC1R1,EXT1,EXT2,EXTL3,Tm9sf3 and B3GALT6 at the cellular level can significantly enhance the ability of cells to resist HSV-1 virus infection.In addition,the results of KEGG analysis indicate that genes such as EXT1,EXT2,EXTL3 and B3GALT6 play the important roles in heparan sulfate synthesis pathway.The gene knockout results show that mutations in these genes can not only significantly reduce the infection level of the HSV-1 virus to the host,but also reduce the ability of the virus to bind to the host cell.The deletion of the ENTPD1 gene only affects the entry of HSV-1 virus into the host cell,and does not affect the binding of the virus to the cell membrane.（4）According to the cancer patient gene information provided by the TCGA database,most genes have mutations in at least one tumor,and the mutation frequency of some genes is more than 5%,which means that the HSV-1 oncolytic virus may not have a good therapeutic effect on these tumors.The good therapeutic effect implies the necessity of precise HDF detection before oncolytic virus treatment.Conclusion:In this study,based on the failure of oncolytic virus infection caused by HDF,we first propose the concept of oncolytic virus resistance,that is,when the mutation of oncolytic virus HDFs occurs in the patient,the therapeutic effect of the oncolytic virus will be hindered;or the effect of tumor treatment is significant at the beginning using an oncolytic virus,but as the heterogeneous differentiation during the proliferation of tumor cells,the expression level of HSV-1 HDFs in some tumor cells decreases or gene mutations occur,resulting in reduction or failure of HSV-1 virus proliferation in the host cell,which in turn affects the therapeutic effect of oncolytic viruses.Based on this concept,searching for new tumor markers is an important direction in the field of oncolytic virus-targeted therapy.Therefore,we have constructed a new HSV-1 oncolytic virus HSV-1-NEO-2/15,which can significantly enhance the therapeutic effect of tumors.In addition,the CRISPR whole-genome knockout library was used to screen multiple HDFs of HSV-1,and the functions of some genes were discussed in depth.In recent years,there are more and more clinical studies on oncolytic virus therapy.However,the precise medical detection of oncolytic virus has not been reported.This study confirmed that the HDFs mutation of HSV-1 virus can determine whether patients can benefit from oncolytic virus treatments,so as to reduce the delay for treating the disease and the waste of resources caused by improper treatment.In the future,in-depth studies on these proteins will provide a research basis for the development of HSV-1 oncolytic virus resistance test kits and the development of personalized treatment plans for the cancer patients.At the same time,based on these targets,new anti-HSV-1 virus drugs can be developed to provide a new solution to the potential infection risk caused by virus residues after oncolytic virus treatment,and to minimize the side effects.