The Construction Of A New Oncolytic Herpes Simplex Virus Expressing Murine Interleukin-15 With Gene Editing Technology

OVs(Oncolytic viruses)is an important immunotherapy strategy for cancer.Herpes simplex virus type II(HSV Ⅱ),modified by gene editing technique,can selectively replicate and proliferate in tumor cells and kill tumor cells directly.The anti-tumor immune mechanism of OVs: a)with the proliferation of virus in tumor,tumor microenvironment partially releasing the specific tumor antigen;b)tumor cells’ lysis to activate the specific anti-tumor immune response in tumor microenvironment.C)OVs expressed cytokines that enhance anti-tumor immune response in tumor microenvironment.Interleukin-15(IL-15)is a cytokine from 14 to 15 kDa.IL-15 enhances the antitumor activity of natural killer cells(NK)and macrophages through antibody dependent cell mediated cytotoxicity(ADCC)effects.Il-15 combination with antibodies to specific antigen of tumor cells,can up-regulate the expression of Fc receptor on NK cells and macrophages.After the antibody binds to the tumor antigen,the Fc fragment of the antibody binds to the Fc receptor to promote the killing of tumor cells by NK cells and macrophages.CRISPR/Cas9(clustered regular interspaced short palindromic repeat/CRISPR-associated protein 9)system has been proved to be an effective and accurate method of gene editing.CRISPR system is a complex of Cas9 protein and single guided RNA(sgRNA).sgRNA targets DNA,through base complementary pairing and Cas9 has nuclease activity.When the complex of sgRNA and Cas9 binds to the DNA target site,the DNA target is cut off.The target gene was knocked out through non-homologous recombination repair,and insert a target gene into the target locus through homologous recombination repair.In this study,we first demonstrated that the CRISPR / Cas9 system was able to edit the genes on oHSV2-hGM-CSF,and then used the system to construct the oHSV2-mIL-15CFP(a herpes simplex virus type II expressing mouse IL-15).The antitumor activity was evaluated in vitro and in vivo.In vitro,oHSV2-mIL-15 CFP upregulated the expression of PD-L1 in human and mouse tumor cells after viral infection,which laid the foundation for the combined application of oHSV2 of expressing IL-15 and PD-L1 antibody.In addition,non-homologous recombination repair can lead to false-positive virus in the process of virus gene editing.Finally,intratumoral injection of oHSV2-mIL-15 CFP inhibits tumor growth and prolonged the survival in CT26 mice model.These studies suggest that OVs expressing IL-15 may be potential therapeutic strategies for tumor immunotherapy.