Galipeau J, Li H, Paquin A, Sicilia F, Karpati G, Nalbantoglu J
Department of Medicine (Division of Hematology-Oncology), Lady Davis Institute for Medical Research, Montreal, Quebec, Canada.
Cancer Res. 1999 May 15;59(10):2384-94.
Direct in vivo tumor-targeting with "suicide" viral vectors is limited by either inefficient gene transfer (i.e., retroviral vectors) or indiscriminate transfer of a conditionally toxic gene to surrounding nonmalignant tissue (i.e., adenoviral vectors). Retrovectors pseudotyped with the vesicular stomatitis virus G protein (VSVG) may serve as a remedy to this conundrum. These retroviral particles differ from standard murine retroviruses by their very broad tropism and the capacity to be concentrated by ultracentrifugation without loss of activity. We propose that a VSVG-typed retrovector can be used for efficient and tumor-specific herpes simplex virus thymidine kinase (TK) gene delivery in vivo. To test this hypothesis, we developed a bicistronic retroviral vector that expresses TK and green fluorescence protein (pTKiGFP). The 293GPG packaging cell line was used to generate vTKiGFP retroparticles. In cytotoxicity assays, vTKiGFP-transduced human glioma cell lines were sensitized to the cytotoxic effects of gangciclovir (GCV) 10,000-fold. Subsequently, virus was concentrated by ultracentrifugation to a titer of 2.3 x 10(10) cfu/ml. We tested the antitumor activity of vTKiGFP retroparticles in a rat C6 glioma model of brain cancer. Concentrated retrovector stock (9 microl volume) was injected stereotactically in preestablished intracerebral tumor. Subsequently, rats were treated with GCV for 10 days. Control rats (no GCV) had a mean survival of 38 days (range, 20-52 days). Sections performed on postmortem brain tissue revealed large tumors with evidence of high efficiency retrovector transfer and expression (as assessed by GFP fluorescence). Fluorescence was restricted to malignant tissue. In the experimental group (GCV treated), 8 of 12 remain alive and well >120 days after glioma implantation. In conclusion, vTKiGFP is very efficient at transducing human glioma cell lines in vitro and leads to significant GCV sensitization. Recombinant retroviral particles can be concentrated to titers that allow in vivo intratumoral delivery of large viral doses. The therapeutic efficiency of this reagent has been demonstrated in a preclinical model of brain cancer.
使用“自杀”病毒载体进行直接的体内肿瘤靶向治疗受到低效基因转移（如逆转录病毒载体）或有条件毒性基因向周围非恶性组织的非特异性转移（如腺病毒载体）的限制。用水泡性口炎病毒G蛋白（VSVG）假型化的逆转录载体可能是解决这一难题的方法。这些逆转录病毒颗粒与标准鼠逆转录病毒不同，它们具有非常广泛的嗜性，并且能够通过超速离心浓缩而不丧失活性。我们提出，一种VSVG型逆转录载体可用于在体内高效且肿瘤特异性地递送单纯疱疹病毒胸苷激酶（TK）基因。为了验证这一假设，我们构建了一种表达TK和绿色荧光蛋白的双顺反子逆转录病毒载体（pTKiGFP）。利用293GPG包装细胞系产生vTKiGFP逆转录颗粒。在细胞毒性试验中，vTKiGFP转导的人胶质瘤细胞系对更昔洛韦（GCV）的细胞毒性作用敏感10000倍。随后，通过超速离心将病毒浓缩至滴度为2.3×10¹⁰ cfu/ml。我们在大鼠C6脑胶质瘤模型中测试了vTKiGFP逆转录颗粒的抗肿瘤活性。将浓缩的逆转录载体原液（9微升体积）立体定向注射到预先建立的脑内肿瘤中。随后，用GCV治疗大鼠10天。对照大鼠（未用GCV）的平均生存期为38天（范围为20 - 52天）。对死后脑组织进行的切片显示有大的肿瘤，有高效逆转录载体转移和表达的证据（通过GFP荧光评估）。荧光仅限于恶性组织。在实验组（用GCV治疗）中，12只大鼠中有8只在胶质瘤植入后120天以上仍存活且状况良好。总之，vTKiGFP在体外转导人胶质瘤细胞系方面非常有效，并导致显著的GCV敏感性。重组逆转录病毒颗粒可以浓缩至允许在体内肿瘤内递送大病毒剂量的滴度。该试剂的治疗效果已在脑癌临床前模型中得到证实。
