Use of miRNA target sequences to control essential genes of HSV1-based oncolytic vectors

The principal aim of this research is to design a new oncolytic vector based on herpes simplex virus type 1 (HSV-1) that can selectively destroy the cancer cells, while reducing the risk of toxicity in surrounding tissue or in other tissues. One approach to design these vectors, in order to obtain a controlled viral replication and their spread only inside the tumor mass, is taking advantage of unique tissue-associated patterns of expression of microRNAs (miRNA). Recent studies have shown that some miRNAs are overexpressed and some are down-regulated in several cancer cells, compared with their normal tissues of origin, suggesting that these miRNAs may play a role as oncogenes or tumor suppressors in the tumorigenesis of various human cancers. In the last years, several studies revealed that the expression of miRNAs is deregulated in human hepatocellular carcinoma (HCC), the most common liver cancer of adults, in comparison with matched non-neoplastic tissue. Members of the miR-199 family, emerged in different studies, as frequently down regulated in Hepatocellular Carcinoma (HCC), ovarian, lung and pancreatic cancer. Based on these observations, we have developed a novel strategy for targeting these types of tumors by taking advantage of the differential miR-199 expression level between normal and cancer cells. In this study, we have constructed a miR199-controlled HSV1-based oncolytic designated HSVLuc27tmiR199gJHE vector to test the ability of miR-199 to regulate the infected cell protein 27 (ICP27) gene expression. ICP27 is an HSV1-immediate early protein (EI) that regulates the synthesis of early (E) and late (L) viral proteins. Inhibition of ICP27 expression blocks progression of the virus life cycle leading to an abortive infection with no yield of progeny virus. HSVLuc27tmiR199gJHE vector is a derivative of HSVLucgJHE, which contains the luciferase reporter gene under cytomegalovirus (HCMV) promoter and the GFP gene into the USS of HSV genome, between the TATA box and the coding sequence for the glycoprotein J (gJ). HSVLuc27tmiR199gJHE vector contains four copies of the miR-199 target site between the stop codon of ICP27 gene and the poly(A). Due to the complementarities of these target sites to miR-199, we have hypothesized that this miR could mediate the destruction of ICP27 mRNA in normal cells, inhibiting the viral replication. The ICP27 expression from HSVLuc27tmiR199gJHE vector was tested, in vitro, in a panel of human cancer cell lines (HepG2, Huh-7 and BxPC-3) that not expressing miR-199, HepG2miR-199 (HepG2 derived expressing constitutively miR-199) and normal cells (Vero). Mice models have been used to study toxicity, safety and efficacy of the target vector. In order to promote the antitumor effects we have generated another oncolytic vector expressing interleukin-12 (IL-12), cytokine widely known for its involvement in the antitumor response. The expression of the murine IL-12 has been regulated by human cytomegalovirus promoter (HCMV) to generate a positive control and by a hypoxia¬responsive element promoter (9XHRE) to allow its expression only in the tumor cells. The regulated expression of the IL-12 has promoted an increased therapeutic response to the tumor, allowing to considerably reducing the dose of virus administered into the animals.