Oncolytic Engineering Of Herpes Simplex Virus Envelope Glycoprotein Targeting EGFR

Background: In recent years,with the continuous in-depth research on cancer,oncolytic virus therapy has become one of the treatment methods for patients with advanced cancer.In 2015,the United States approved the oncolytic herpes simplex virus talimogene laherparepvec for the clinical treatment of melanoma.The modification of the surface glycoprotein of Herpes simplex virus can specifically increase its ability to target abnormally expressed antigens(such as epidermal growth factor)on the tumor surface and reduce the virus' s toxicity to normal cells.At present,Oncorine is the only oncolytic virus drug that has been listed on the Chinese market since 2003.Oncolytic viruses have the advantages of high tumor killing,good targeting,small side effects,multiple ways to kill tumors and low cost.It is expected to become The most promising anti-cancer drugs.In order to develop high-efficiency,low-cost oncolytic herpes simplex virus drugs with independent intellectual property rights,comprehensive research is required.Purpose: Isolation and identification of Chinese HSV virus strains,complete genome sequencing and titer determination.The difference between the sequence of glycoprotein D on the surface of the new strain and the other 11 strains was further analyzed.The gene editing design of the surface glycoprotein D gene allows HSV-1 to specifically target EGFR-positive tumor cells,laying the foundation for the construction of a new type of o HSV to treat EGFR-positive tumors.Methods:(1)Use vero cells to culture potential HSV-1 and collect the virus suspension after most of the cells show cytotoxicity.Part of the virus suspension is extracted for virus DNA and sent for second-generation virus genome sequencing after preliminary characterization.Perform sequence comparison to determine the difference between the new strain HSV-1-g D and the other 11 strains.(2)Construct Crispr Cas9-related g RNA plasmids through molecular cloning technology,obtain and design EGFRtargeting single-chain antibody sequences to construct homologous recombination plasmid.(3)After co-transfection of Crispr Cas9-related vector plasmids,use end dilution method to select monoclonal cells,and verify the transfection efficiency by fluorescence observation and q PCR.Results:(1)A new Chinese human HSV-1 strain was isolated and identified,and it was found to be highly related to the earlier strain HSV-1-LXMW,and secondly to the HSV-1 strain 17.(2)Through the comparison and analysis of the US6 gene sequence of HSV-1 g D,it is found that it is closely related to HSV-1-LXMW,HSV-1-KOS and HSV-1-Patton.(3)Three HSV-1 g D g RNA plasmids were constructed to target the vicinity of the functional region of the g D sequence.(4)The infinite end dilution method was used to successfully select monoclonal cells,which laid the foundation for the construction of HSV-1 oncolytic virus.