Fusion Enzymes Containing HSV-1 Thymidine Kinase Mutants and Guanylate Kinase Enhance Prodrug Sensitivity In Vitro and In Vivo

Herpes Simplex Virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We previously described two strategies to overcome this limitation: 1) generation of HSVTK mutants with improved GCV activation potential and 2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants (mutant 30 and SR39) were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, demonstrates significant reduction in IC(50) values of approximately 12,500-fold, 100-fold, and 125-fold compared to HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses reveal that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1mg/kg) that has no effect on wild type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2,500-fold or 25-fold compared to HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC(50) values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 appears to elicit stronger bystander killing effects where 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1mg/kg GCV. However, at a lower dose of GCV (0.1mg/kg), MGMK/SR39 appears to have slightly greater tumor growth inhibition properties compared to SR39 (P≤0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1mg/kg GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.