Construction And Research On The Oncolytic Activity Of A Novel Herpes Simplex Virus Based On HTERT Promoter

Background:Oncolytic virotherapy, which uses oncolytic virus such as adenovirus, herpes simplex virus and Newcastle disease virus etc. to kill tumor cells directly, has been proved as a novel and useful strategy for cancer treatment in recent20years. Furthermore, tumor-specific replication-selective oncolytic virotherapy, which uses the virus that can induce cell death in tumor cells but not in normal cells, is a promising and safe way to treat cancer. For now, toxicity is a major obstacle in application of oncolytic virus. In order to solve this problem, based on oHSV1-17+, we have constructed a new oncolytic herpes simplex virus, oHSVl_hTERTp_ICP4, which is anticipated to selectively replicate in most cancer cells but not in normal somatic cells by using a tumor specific promoter to control ICP4that is critical for the transcriptional activation and replication of the virus. Here we evaluated the antitumor effect and tumor specificity of oHSV1_hTERTp_ICP4on series of tumor cells, in vitro and in vivo.Method:3recombinant oncolytic virus, oHSV1_hTERTp_ICP4, oHSV1_hTERTp_ICP4_CMVp_eGFP and oHSV1_hTERTp_ICP4_CMVp_Luc2, were constructed using plasmid construction and homologous recombination. In vitro cytopathic efficacy of oHSV1_hTERTp_ICP4was examined in several cell lines; and In vitro replication of oHSV1_hTERTp_ICP4in several cell lines was examined using titer determination; and cytopathic efficacy of oHSV1_hTERTp_ICP4for normal human blood cells was examined by using flow cytometry and confocol; and antitumor effects of oHSV1_hTERTp_ICP4were assessed in xenograft tumor models and in vivo imaging; and the effect of oHSV1_hTERTp_ICP4infection on the activity of hTERT promoter and mRNA expression of several transcription factors which may be invovled were identified by Quantitative real-time PCR; finally, the relationship between the cytopathic effect of oHSVl_hTERTp_ICP4and cell necrosis and apoptosis was examined.Results:（1） oHSV1_hTERTp_ICP4, oHSV1_hTERTp_ICP4_CMVp_eGFP and oHSVl_hTERTp_ICP4_CMVp_Luc2were constructed for the first time.（2） oHSVl_hTERTp_ICP4was cytopathic for most telomerase high activity cell lines with and achieved a similar oncolytic effect compared to oHSV1₁7+; but for some cell lines which show no or very low telomerase activity, oHSV1hTERTp ICP4showed no cytopathic effect, while oHSVl-17+was still cytopathic. On the other hand, and the consistency between cytopathic effect and replication of oHSV1_hTERTp_ICP4was clear and definite.（3） The results of flow cytometric analysis showed there were almost no green fluorescent cells found after infection of normal human blood cells with oHSV1_hTERTp_ICP4_CMVp_eGFP, while a small amount of green fluorescent cells were found after infection of oHSV1-17+-GFP. The results suggest that oHSV1_hTERTp_ICP4is much safer than oHSV1-17+. On the other hand, we simulated circulating tumor cells in cancer patients by incorporating Huh-7cells into normal human blood cells, and after infection of oHSV1_hTERTp_ICP4_CMVp_eGFP, the consistant expression of EpCAM and GFP showed the tumor specificity of oHSV1_hTERTp_ICP4again.（4） Antitumor effect of oHSV1_hTERTp_ICP4against BGC823xenograft tumor models was similar to that of oHSV1-17+, but the survival of the mice treated with oHSV1_hTERTp_ICP4were much longer compared to that treated with oHSV1-17+. On the other hand, there was a persistent replication of oHSVl_hTERTp_ICP4_CMVp_Luc2found in tumor, but the viruses in muscle and subcutaneous tissue were eliminated rapidly.（5） The upregulation of hTERT mRNA expression after infection of oHSV1_hTERTp_ICP4indicated the enhanced activity of hTERT promoter which may induce the replication of oHSV1_hTERTp_ICP4, but the former result showed that there were no significant virus replication and cytopathic effect. Furthermore, the mRNA expression of3cellular transcription factors（c-myc, Spl and E2F1） were identified. The results showed that E2F1mRNA expression is downregualted and indicated E2F1may be involved in regulation of hTERT promoter activity that was infected with oHSV1_hTERTp_ICP4.（6） In BGC823cells infected with different MOI of oHSV1_hTERTp_ICP4, the proportion of necrotic cells to proportion of apoptotic cells ratio increased in a MOI-dependent manner, and this result indicated that the cytopathic effect of oHSV1_hTERTp_ICP4is mainly caused by cell lysis and accompanying necrosis.Conclusions:For the first time, a novol oncolytic virus, oHSV1_hTERTp_ICP4, was constructed with the application of hTERT promoter. A better saftey oncolytic activity was proved in in vitro and in vivo experiments, and indicated that oHSV1_hTERTp_ICP4could be a powerful tool in cancer diagnosis and treatment.