Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases

Abstract

Malignant tumor cells often express matrix metalloproteinases (MMPs) at a high level to enable their dissemination and metastasis. Sendai virus (SeV), a nonsegmented negative strand RNA virus, spreads in the target tissues in vivo via cleavage activation of the viral fusion glycoprotein by a tissue-specific, trypsin-like enzyme. By deleting the viral matrix protein, we previously generated a recombinant SeV that does not bud to mature virions, but is highly fusogenic and spreads extensively from cell to cell in a trypsindependent manner. Here, we changed the tryptic cleavage site of the fusion glycoprotein of this virus to a site susceptible to MMPs. The resulting recombinant virus 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. Furthermore, the virus spread extensively in tumor cells xenotrasplanted to nude mice without disseminating to the surrounding normal cells, leading to the inhibition of the tumor growth in the mice. These results demonstrate the selective targeting and killing of human tumor cells by recombinant SeV technology and greatly advance the reemerging concept of oncolytic virotherapy, which currently appears to rely largely upon a natural preference of certain viruses for cancer cells.

Generation of optimized and urokinase-targeted oncolytic Sendai virus vectors applicable for various human malignancies

“…We examined three deleting mutants (Fct27, Fct14, Fct4), as shown in Figure 1a, and the fusion efficiency was quantified by counting the nuclei per formed syncytium. 8 As shown in Figure 1b, syncytia formation was observed only in cells with cotransfection of paired membrane glycoproteins (F and HN). The highest ratio of fusion activity, six times higher than that seen by wildtype F gene cotransfected (Po0.01), was seen in the use of a truncated clone (Fct14); therefore, we concluded that Fct14 should be used in our new design of an oncolytic SeV.…”

“…8 As expected, this new class of SeV vectors spread widely from cell to cell through fusion of the plasma membranes, forming syncytia among the MMPexpressing tumor cells in vitro, and led the tumor to extensive death in vivo, without releasing secondary vector particles. 8 Our subsequent studies examining the utility of the vector among various human cancer cell lines from different origins, however, showed that the efficacy of this prototype vector was relatively limited because of (1) the requirement for a relatively high expression level of MMPs for efficient cell death and (2) the limited numbers of MMP-expressing tumor cells, less than 20% (unpublished observation).…”

“…8 On the basis of this concept, in this study, we developed dramatically improved vectors by modifying the F gene sensitive to uPA and MMP associated with truncation of its cytoplasmic tail. These newly optimized vectors showed markedly enhanced cancer killing activity in a protease-dependent manner in vitro and in vivo, and, importantly, modification of amino-acid residues of the F gene for targeting uPA dramatically expanded the cellkilling spectrum of oncolytic SeV.…”

“…8,12,19,20 Their data show that those vectors can target the cytotoxicity of the fusogenic membrane glycoprotein in MMP-overexpressing tumor lines and xenografts, and maintain significant antitumor activity both in vitro and in vivo. MMPtargeted vectors, irrespective of the vectors' backbone, may be confronted with a similar difficulty in clinical settings.…”

“…The viral proteins were then analyzed by western blotting using an anti-F1 rabbit antiserum. Immunoblot analysis was carried out using a rabbit peptide antiserum against the F1 (dilution, 1:1000), 8 an anti-rabbit-horseradish peroxidase conjugate and the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).…”