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A major concern of using viral gene therapy is the potential for uncontrolled vector propagation and infection that might result in serious deleterious effects. To enhance the safety, several viral vectors, including vectors based on Sindbis virus, were engineered to lose their capability to replicate and spread after transduction of target cells. Such designs, however, could dramatically reduce the therapeutic potency of the viral vectors, resulting in the need for multiple dosages to achieve treatment goals. Earlier, we showed that a replication-defective (RD) Sindbis vector achieved specific tumor targeting without any adverse effects in vivo. Here, we present a replication-competent Sindbis viral vector that has an hsvtk suicide gene incorporated into ns3, an indispensable non-structural gene for viral survival. The capability of viral propagation significantly increases tumor-specific infection and enhances growth suppression of tumor compared with the conventional RD vectors. Furthermore, in the presence of the prodrug ganciclovir, the hsvtk suicide gene serves as a safety mechanism to prevent uncontrolled vector propagation. In addition to suppressing vector propagation, toxic metabolites, generated by prodrug activation, could spread to neighboring uninfected tumor cells to further enhance tumor killing.

Our previous studies suggest that replication-defective Sindbis vectors might be promising agents for specific tumor targeting and detection. However, the effects of innate and/or adaptive anti-viral immunity, in particular, the IFN-I/STAT1 signaling pathway, may impact their therapeutic potential. Using a bioluminescent imaging system, we demonstrate that although most normal cells are not permissively transduced by replication-defective Sindbis vector, transduction of liver non-sinusoidal endothelial occurs the first time IFN-I/STAT1 signaling deficient mice are inoculated with the vector. Transduction of some cells is not surprising since STAT1 knockout animals show significant delay in IFN responses such as the production of IFN-alpha/beta and transcriptional activation of several anti-viral genes (IRF7, RIG-I, PKR, TLR3, USP18, ISG15). However, beyond the initial vector transduction, which resolves rapidly, secondary inoculums of Sindbis vectors do not transduce any liver cells, suggesting that an alternative antiviral pathway may protect against further transduction. Other known signaling pathways were examined using mice lacking functional TLR3, tumor necrosis factoralphaR or nuclear factor-kappa B (p50). Surprisingly, none of those pathways seem to play a significant role in anti-Sindbis responses. Thus it appears that in vivo, in contrast to the ready transduction of tumor cells, transduction of normal cells by replication-defective Sindbis vector is limited, possibly by a novel mechanism.