Protease-activatable retroviral vectors offer the possibility of targeted gene transfer into cancer cells expressing a unique set of proteases as, for example, the matrix metalloproteases (MMPs). However, it is difficult to predict which substrate sequence will be optimally cleaved by a given tumour cell type. Therefore, we developed a novel approach that allows the selection of MMP-activatable retroviruses from libraries of viruses displaying combinatorially diversified protease substrates. Starting from a virus harbouring a standard MMP-2 substrate motif, after only two consecutive cycles of diversification and in vivo selection, MMP-activatable viruses were recovered. Biochemical characterization of the selected viruses revealed that their linker peptides showed a considerably increased sensitivity for MMP-2 cleavage, and interestingly also improved the particle incorporation rate of the Env protein. Owing to the optimized linker peptide, the selected viruses exhibited a greatly enhanced spreading efficiency through human fibrosarcoma cells, while having retained the dependency on MMP activation. Moreover, cell entry efficiency and virus titres were considerably improved as compared to the parental virus displaying the standard MMP-2 substrate. The results presented imply that retroviral protease substrate libraries allow the definition of MMP substrate specificities under in vivo conditions as well as the generation of optimally adapted tumour-specific viruses.

Viruses conditionally replicating in cancer cells form an attractive novel class of antitumoral agents. To engineer such viruses infectivity can be coupled with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukaemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumour-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on human fibrosarcoma or glioma cell lines resulted in the selection of about 10 virus clones, of which the three most frequent ones were shown to become activated by MMPs and to be replication competent on MMP-positive cells only. On these cells, the selected linker peptides improved the spreading by several orders of magnitude in vitro, as well as in tumour xenografts in vivo, approaching the kinetic of the unmodified wild-type virus. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type of interest. Gene Therapy (2005) 12, 918-926.

We report here a targeting method that exploits the expression pattern of cell surface proteases to induce gene delivery to specific tissues. We describe retroviral vectors harboring modified surface glycoproteins derived from an avian influenza virus hemagglutinin (HA) for which the cell entry properties, dependent on HA cleavage by producer cells, were conditionally blocked at a postbinding step by insertion of matrix metalloproteinase (MMP) substrates. We demonstrate that such vectors induce gene transfer, both in vitro and in mice harboring human tumor xenografts, only through contact with target cells expressing MMPs that cleave the substrate introduced into the recombinant HA. This selective gene transfer in MMP-rich cells was specifically inhibited by 1,10-phenanthroline, a broad-range MMP inhibitor. Importantly, such MMP-activatable vectors selectively transduced MMP-rich cells in heterogeneous populations containing MMP-rich and MMP-poor cells. These vectors will allow useful gene transfer applications into target cells exhibiting specific protease activities.