Glycosylation Modification Of Herpes Simplex Virus Type 1 For Oncolytic Immunotherapy Targeting Hepatoma

Oncolytic immunotherapy is a promising strategy in the field of cancer treatment,owing to its ability to directly kill tumor cells and trigger a systemic immune response.Herpes simplex virus type I(oHSV)is the most commonly used oncolytic virus due to its capacity to selectively replicate in tumor cells,induce immunogenic cell death,promote humoral and cellular immune responses,and inhibit tumor growth.Despite its potential benefits,the current application of oHSV oncolytic immunotherapy is limited to local injection,mainly for surface tumors,due to the high density and pressure of solid tumors.Therefore,systemic administration through intravenous injection is a desirable approach for treating small tumors or metastases.However,intravenously administered oncolytic viruses face challenges such as poor tumor targeting,strong antiviral responses,and an immunosuppressive tumor microenvironment.To improve the effectiveness of targeted oncolytic immunotherapy for hepatocellular carcinoma,we developed a chemical engineering modification strategy by covalently attaching a galactose-polyethylene glycol(Glycosylated-PEG)polymer chain to herpes simplex oncolytic virus type 1(oHSV).This produced a new generation of Glycosylated-PEG-oHSV with improved tumor targeting,reduced antiviral response,and enhanced tumor penetration.The specific research contents of the study are outlined below:We determined the optimal covalent coupling ratio of the Glycosylated-PEG polymer to oHSV using flow cytometry and quantitative analysis of GFP expression in tumour cells infected with oHSV.Effect of Glycosylated-PEG polymer modification of oHSV on viral replication capacity by q PCR.We investigated the targeting effect of Glycosylated-PEG-oHSV on Hepa1-6 hepatocellular carcinoma cells with high expression of the desialoglycoprotein receptor using flow cytometry,while mouse embryonic fibroblast NIH/3T3 cell lines with low expression of the desialoglycoprotein receptor were used as controls.Additionally,we further investigated the targeting effect of Glycosylated-PEG-oHSV on Hep G2 hepatocellular carcinoma cells.To verify the targeted killing effect of Glycosylated-PEG-oHSV on Hepa1-6 tumor cells,we performed LDH assay kit、CCK-8 cell viability assays and Annexin V/PI flow cytometry apoptosis/necrosis staining.We also detected neutralizing antibodies using an ELISA kit.We established a subcutaneous tumor model of Hepa1-6hepatocellular carcinoma cells in mice and studied the distribution of Glycosylated-PEG-oHSV or oHSV labelled with fluorescent dye ICG-NHS by caudal vein injection using small animal fluorescence imaging system and confocal microscope system.We used q PCR to evaluate HSV-1 genomic DNA levels in tumors and major organs in vitro to further confirm the excellent tumor targeting ability of Glycosylated-PEG-oHSV in vivo.Moreover,we established a subcutaneous Hepa1-6 hepatocellular carcinoma therapeutic model to evaluate the efficacy of Glycosylated-PEG-oHSV in the treatment of hepatocellular carcinoma.We analyzed the maturation of antigen-presenting cells in the lymph nodes using flow cytometry and evaluated the number of regulatory T lymphocytes,the number of tumor-infiltrating CD8~+T cells,and the number of NK cells in the tumors.We evaluated the oncolytic effect of Glycosylated-PEG-oHSV on liver cancer and the mechanism of immune response.Finally,we verified the antitumor immune efficacy of Glycosylated-PEG-oHSV using H&E staining,immunohistochemistry,and immunofluorescence staining.The results of the experiments demonstrated that intravenously injected oHSV modified with Glycosylated-PEG significantly enhanced the targeting of liver cancer tissue and improved the penetration effect of oHSV into the tumor,while reducing oHSV infection and side effects on the brain tissue and other organs of mice.Moreover,Glycosylated-PEG-oHSV effectively reduced regulatory T cells and increased the number of infiltrating IFN-γ~+CD8~+T cells and NK cells,leading to inhibition of liver cancer tumor growth.Assessment of changes in body weight and pathological characteristics of major organs in mice during tumor treatment revealed that Glycosylated-PEG-oHSV was biologically safe.The targeted therapy strategy for liver cancer using galactose-polyethylene glycol(Glycosylated-PEG)polymer covalently coupled to oHSV in this study presents a promising approach for future clinical oncolytic immunotherapy of liver cancer.