Sutton M A, Berkman S A, Chen S H, Block A, Dang T D, Kattan M W, Wheeler T M, Rowley D R, Woo S L, Lerner S P
Scott Department of Urology, Baylor College of Medicine, Houston, TX 77030, USA.
Urology. 1997 Feb;49(2):173-80. doi: 10.1016/S0090-4295(96)00560-2.
To determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase gene (HSV-tk) and the prodrug ganciclovir (GCV) in a murine model of human transitional cell carcinoma.
We used a replication-defective adenoviral construct containing the beta-galactosidase gene (ADV/Rous sarcoma virus [RSV]-beta-gal) as a control or ADV/RSV-tk as the therapeutic vector under the transcriptional control of the RSV long-terminal repeat promoter. Transduction efficiency was assessed in vitro by infection of MBT-2 cells with ADV/RSV-beta-gal at various multiplicities of infection (MOI) utilizing 5-bromo-4-chlor-3-indolyl-beta-D-galactoside (X-gal) staining. Sensitivity of MBT-2 cells to the therapeutic vector was determined after infection with ADV/RSV-tk with or without GCV. Subcutaneous tumors were established in syngeneic C3H/He female mice with 5 x 10(5) MBT-2 cells. Optimal dosing of ADV/RSV-tk was determined by direct percutaneous tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate, similar, controlled experiments.
In vitro studies indicated greater than 95% transduction 96 hours after inoculation at an MOI of 3000 and a greater than 95% cell death rate with RSV-tk + GCV at an MOI of 61 or greater. In vivo experiments demonstrated an optimal viral dose of 3 x 10(8) plaque-forming units (pfu) and a greater than fourfold reduction in tumor growth for the animals treated with ADV/RSV-tk compared with control animals (P = 0.0013). Toxicity was limited to histologic evidence of hepatitis with ADV/RSV-tk doses greater than 3 x 10(8) pfu + GCV. Long-term survival of treatment animals was significantly increased over that of control animals (59%, P = 0.0001).
ADV/RSV-tk with GCV treatment results in efficient gene transfer in vitro and provides effective therapy in experimental murine bladder cancer by significantly inhibiting tumor growth and improving host survival.
在人移行细胞癌小鼠模型中确定使用腺病毒介导的单纯疱疹病毒胸苷激酶基因（HSV-tk）和前体药物更昔洛韦（GCV）进行自杀基因治疗的可行性、安全性和有效性。
我们使用一种含有β-半乳糖苷酶基因的复制缺陷型腺病毒构建体（ADV/劳斯肉瘤病毒[RSV]-β-gal）作为对照，或使用ADV/RSV-tk作为治疗载体，其受RSV长末端重复启动子的转录控制。通过利用5-溴-4-氯-3-吲哚基-β-D-半乳糖苷（X-gal）染色，以不同的感染复数（MOI）用ADV/RSV-β-gal感染MBT-2细胞，在体外评估转导效率。在用ADV/RSV-tk感染并添加或不添加GCV后，确定MBT-2细胞对治疗载体的敏感性。将5×10⁵个MBT-2细胞接种到同基因C3H/He雌性小鼠中建立皮下肿瘤。通过直接经皮肿瘤注射递增的病毒剂量并用GCV治疗，确定ADV/RSV-tk的最佳剂量。在单独的、类似的对照实验中确定治疗效果、长期生存率和毒性。
体外研究表明，在MOI为3000接种96小时后转导率大于95%，在MOI为61或更高时，RSV-tk + GCV处理后的细胞死亡率大于95%。体内实验表明，最佳病毒剂量为3×10⁸ 空斑形成单位（pfu），与对照动物相比，用ADV/RSV-tk治疗的动物肿瘤生长减少了四倍以上（P = 0.0013）。当ADV/RSV-tk剂量大于3×10⁸ pfu + GCV时，毒性仅限于肝炎的组织学证据。治疗动物的长期生存率比对照动物显著提高（59%，P = 0.0001）。
ADV/RSV-tk联合GCV治疗在体外可实现高效基因转移，并通过显著抑制肿瘤生长和提高宿主生存率，为实验性小鼠膀胱癌提供有效的治疗。
