451. Matrix Metalloproteinase-Targeted Oncolytic Sendai Virus Vector “Armed” with a Sucide Gene “Yeast Cytosine Deaminase”; Remarkable Combinational Effects

Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a non-segmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion (F) glycoprotein by a tissue-specific, trypsin-like protease. By deleting the viral matrix (M) protein, we had generated a genetically modified SeV that does not bud to mature virons, but is highly fusogenic and spreads extensively from cell to cell in a trypsin-dependent manner. Moreover, we had changed the tryptic cleavage site of the F glycoprotein of this vector to a site susceptible to MMPs. The resulting recombinant SeV vector was no longer activated by trypsin but spread efficiently in cultured cells supplemented with MMP2 or MMP9, and in human tumor cell lines expressing these MMPs. This vector spread extensively in tumor cells xenotransplanted to nude mice without disseminating to the surrounding normal cells, leading to the strong inhibition of the tumor growth in the mice. We call this vector as the "MMP-targeted oncolytic Sendai virus vector; SeV/F(MMP-sub)ΔM".In this study, we further generated an "armed" MMP-targeted oncolytic SeV vector with a suicide gene, yeast cytosine deaminase (yCD). The yCD activates the prodrug 5-FC to 5-FU and has been used for suicide gene therapy. This vector was expected to exert a potent anti-tumor effect with the combination of 5-FU conversion by yCD and syncytium formation by the genetically engineered F glycoprotein of SeV vector. To test this hypothesis, the killing effect against the human HT1080, U87, and SW620 tumor cells were analyzed in vitro after transduction with the vectors, the F gene-deleted SeV vector (SeV/ΔF), the yCD gene-carrying SeV/ΔF (yCD-SeV/ΔF), SeV/F(MMP-sub)ΔM, and the yCD gene-carrying SeV/F(MMP-sub)ΔM (yCD-SeV/F(MMP-sub)ΔM) by a tetrazolium salt (WST-1) based colorimetric assay. MMP-expressing tumor cells (HT1080, U87) were efficiently killed by transduction with SeV/F(MMP-sub)ΔM and yCD-SeV/F(MMP-sub)ΔM, but non-MMP-expressing tumor cells (SW620 et al.) were not. The addition of 5-FC further accelerated the killing effect only in the case of yCD-SeV/F(MMP-sub)ΔM. Furthermore, both the enhanced syncytium formation and the additive killing effect through the administration (i.p.) of the prodrug 5-FC were observed in the HT1080 tumor bearing nude mice in vivo by the administration of yCD-SeV/F(MMP-sub)ΔM.These result