Production of a replicating retroviral vector expressing Reovirus fast protein for cancer gene therapy

Abstract

Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.

Keywords: Cancer gene therapy; RCR vectors; Syncytium; p10(PuV) FAST.