Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo
Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo
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DOI：
10.1006/mthe.2001.0315
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发表时间：
2001-05-01
影响因子：
12.4
通讯作者：
Nabel, EG
中科院分区：
文献类型：
--
作者：
Aky端rek, LM;Nallamshetty, S;Nabel, EG
单纯疱疹病毒胸苷激酶（HSV-TK）使前药更昔洛韦（GCV）和阿昔洛韦（ACV）磷酸化，导致DNA合成中断和细胞增殖抑制。HSV-TK载体已成功应用于心血管和癌症基因治疗。GCV和ACV的活化，在通过病毒胸苷激酶的初始磷酸化步骤之后，通过鸟苷酸激酶进行。我们推断，鸟苷酸激酶（CK）与HSV-TK的共表达将增加CCV或ACV的磷酸化，导致细胞杀伤增加。为了验证这一假设，开发了表达TK和CK的载体（TKciteGK），并在体外和体内对血管平滑肌细胞（vsmcs）进行了测试。与HSV-TK载体相比，用TKciteGK转导并暴露于GCV的血管细胞的杀伤显著增加（P = 0.03）。用三种启动子：CMV、EF 1 α和SM 22 α评估TKciteGK构建体。在原代vsmc中，CMV启动子驱动的TKciteGK表达比SM 22 α或EF 1 α启动子更有效地诱导细胞杀伤。基于这些体外发现，在两种心血管疾病动物模型中测试了具有CMV启动子的TKciteGK载体：猪动脉中的球囊血管成形术和支架部署。血管损伤后，与GCV的对照载体相比，GCV的CMV-TKciteGK表达显著降低了vsmc增殖和内膜损伤形成。在血管成形术模型中，内膜与中膜面积比降低了80%（P = 0.0002）。这些结果在支架模型中得到证实，内膜病变减少66%（P = 0.006）。GK与TK的共表达增加了细胞杀伤，并允许以较低剂量给予CCV。TKciteGK载体和GCV中的这些修饰在较低的前药剂量下显示出增强的功效，从而提高了心血管基因治疗的安全性。
Herpes simplex virus-thymidine kinase (HSV-TK) phosphorylates the prodrugs ganciclovir (GCV) and acyclovir (ACV), leading to disruption of DNA synthesis and inhibition of cell proliferation. HSV-TK vectors have been successfully employed in cardiovascular and cancer gene therapy. Activation of GCV and ACV, after an initial phosphorylation step by the viral thymidine kinase, is carried out by guanylate kinase. We reasoned that coexpression of guanylate kinase (CK) with HSV-TK would augment phosphorylation of CCV or ACV, leading to increased cell killing. To test this hypothesis, a vector expressing TK with CK (TKciteGK) was developed and tested on vascular smooth muscle cells (vsmcs) in vitro and in vivo. Compared to HSV-TK vectors, killing of vascular cells transduced with TKciteGK and exposed to GCV was significantly 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 alpha promoters in primary vsmcs. Based upon these in vitro findings, TKciteGK vectors with a CMV promoter were tested in two animal models of cardiovascular disease: balloon angioplasty and stent deployment in pig arteries. Following vascular injury, expression of CMV-TKciteGK with GCV significantly reduced vsmc proliferation and intimal lesion formation compared to control 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). Coexpression of GK with TK increases cell killing and permits administration of CCV at lower doses. These modifications in TKciteGK vectors and GCV showed enhanced efficacy at lower prodrug doses, leading to improved safety for cardiovascular gene therapy.