Transcriptional Regulation And Ovarian Cancer Oncolytic Virus Engineering Of US12 Gene Of Herpes Simplex Viruses

Cancer immunotherapy was rated as the first of the top ten scientific breakthroughs in 2013,and oncolytic virus is an important branch of cancer immunotherapy,which has the advantages of good efficacy,less side effects and preventing recurrence.China approved the first oncolytic virus in the world in 2003.T-vec,the first oncolytic herpes simplex virus(oHSV)Drug approved by the Food and Drug Administration(FDA),was modified to significantly improve its specificity and targeting,and the results of phase ?and ? trials in patients with advanced melanoma were very encouraging.US 12 is an immediate early gene encoding a polymorphic protein—ICP47(infected cell polypeptide 47)that inhibits viral antigen presentation during HSV infection and promotes HSV immune escape.ICP47 can directly bind to transporter associated with antigen processing(TAP),restrict viral antigen transport,and lead to the occurrence of empty MHC-? molecules.HSV prevents cytotoxic T lymphocytes(CTLs)from killing HSV infected cells by blocking viral antigen from entering the ER,thereby leading to HSV immune escape and establishing the mechanism of infection in host cells.At present,the study on the regulation of US 12 gene transcription has not been reported.This study intends to analyze the transcriptional regulatory sequence of US 12 gene,and further construct the oHSV with the deletion of US 12,so as to provide theoretical and experimental basis for the verification of its killing effect on ovarian cancer cells and the development of new oncolytic anticancer drugs.Objective:To identified the transcriptional regulatory sequence(TRS)and transcriptional regulatory factor(TRF)of US 12 gene and then to construct an oHSV with US 12 gene deletion,so as to lay a foundation for the construction for the oncolytic therapy of ovarian cancer.Methods:1.Sequence alignment was used to determine the transcriptional regulatory sequence region of the US 12 gene of the new strain HSV-1-LXMW;2.Bioinformatics analysis of transcriptional regulatory sequences of US 12 gene;3.Engineering oHSV with US 12 gene deletion;4.Screening and identification of recombinant HSV.Results:Firstly,the transcriptional regulatory region sequence of US12 gene in HSV-1 strain(HSV-1-LXMW)isolated earlier by our research group was determined.Through phylogenetic evolution analysis,it was found that the transcriptional regulatory region sequence of HSV-1-LXMW was closely related to HSV-1-CR38 and HSV-1-17 from the UK.The transcriptional regulatory sequences and transcriptional regulatory factors(c-Rel,Elk-1,Pax-4,Oct-1,CF2-?,E74A,StuAp)of US12 of the 12 HSV strains were identified by the weight matrix software Matching.Studies have shown that c-Rel and Oct-1 have the biological functions in immune escape and virus replication during HSV infection,respectively.Based on the literature analysis,we further speculated the new mechanism of HSV-1 encephalitis,the c-Rel activated ICP47-mediated immune escape.Further verification by multi-sequence comparative analysis showed that the US12 transcriptional regulatory sequence of 9 HSV-1 strains contained 14 conserved regions,the US 12 transcriptional regulatory sequence of 3 HSV-2 strains contained 7 conserved regions,and the transcription factor HNF-4 was conserved in both HSV-1 and HSV-2.These findings add to a new understanding of the biology of HSV and transcriptional regulation in oncolytic virus therapy.At the same time,this research with the usage of the Chinese HSV-1-LXMW strain,analysis and identification of US12 transcriptional regulation,and partial building a ?US12-oHSV,laid a solid foundation for future application of virotherapy for ovarian cancer.Conclusions:1.Through transcriptional regulation region sequence analysis of US 12 gene,it was found that HSV-1-LXMW was closely related to HSV-1-CR38 and HSV-1-17 from the UK.2.The transcriptional regulatory sequences and transcriptional regulatory factors of 12 strains of HSV US 12 were identified and their functional conservatism was analyzed.Transcription factors c-Rel and Oct-1 have the biological functions of immune escape and virus replication during HSV infection,respectively.Based on the literature analysis,I further hypothesized the new mechanism of HSV-1 encephalitis,c-Rel activated ICP47-mediated immune escape.3.Through the CRISPR/Cas9 technical transformation of physi CAL separation of the Chinese HSV-1-LXMW strain,I partially engineered the new ?US12-oHSV for ovarian cancer therapy.