A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing

Herpes simplex virus thymidine kinase (HSVTK) with the guanosine analog ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system for gene therapy of cancer. Despite the broad application of the HSVTK/GCV approach, phosphorylation of GCV to its active state is inefficient such that high, myelosuppressive doses of GCV are needed to observe an antitumor effect. One strategy used to overcome the poor substrate specificity of HSVTK towards GCV ( K-m = 45 mu M) has been to create novel forms of TK with altered substrate preferences. Such mutant TKs have shown benefit and are currently in clinical use. We describe here a second strategy to increase the amount of intracellular triphosphorylated GCV by involving the second enzyme in the GCV activation pathway, guanylate kinase (GMK). As a means to overcome the bottleneck of prodrug activation from the monophosphate to the diphosphate, we sought to combine both the critical HSVTK and GMK activities together. In this report we describe the construction of a fusion or chimeric protein of HSVTK and guanylate kinase, show data that demonstrate it confers a similar to 175-fold decrease in IC50 compared to HSVTK alone in response to ganciclovir treatment in stably transfected C6 glioma cells and finally, we present biochemical evidence of a kinetic basis for this improved cell killing.