Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo

Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthes is and inhibition of cell proliferation. HSV-TK vectors have been successfu lly employed in cardiovascular and cancer gene therapy. Activation of GCV a nd ACV, after an initial phosphorylation step by the viral thymidine kinase , is carried out by guanylate kinase. We reasoned that coexpression of guan ylate kinase (CK) with HSV-TK would augment phosphorylation of CCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expres sing TK with CK (TKciteGK) was developed and tested on vascular smooth musc le cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significa ntly increased (P = 0.03). The TKciteGK construct was evaluated with three promoters: CMV, EF1 alpha, and SM22 alpha. TKciteGK expression driven by a CMV promoter induced cell killing more effectively than SM22 alpha or EF1 a lpha promoters in primary vsmcs. Based upon these in vitro findings, TKcite GK vectors with a CMV promoter were tested in two animal models of cardiova scular disease: balloon angioplasty and stent deployment in pig arteries. F ollowing vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to contro l vectors with GCV, In the angioplasty model, there was an 80% reduction in intima-to-media area ratio (P = 0.0002). These findings were paralleled in a stent model with 66% reduction in intimal lesions (P = 0.006). Coexpress ion of GK with TK increases cell killing and permits administration of CCV at lower doses. These modifications in TKciteGK vectors and GCV showed enha nced efficacy at lower prodrug doses, leading to improved safety for cardio vascular gene therapy.