Cancer gene therapy by the in vivo transfer of cytokine-genes in to the artery that leads to tumors with fusogenic liposomes.

我们报道，肿瘤坏死因子(TNF) α在体内通过基因转移在动脉中产生导致肿瘤时，诱导了强烈的抗肿瘤免疫反应。我们使用右足部携带肉瘤- 18o肿瘤的小鼠模型，将包裹人tnf - α基因的融合性脂质体注入右股动脉。在这种条件下，人类tnf - α仅在通向肿瘤的动脉和肿瘤中检测到。将tnf - α基因注入右股动脉后，肿瘤生长明显消退，11只小鼠中有4只完全治愈。当tnf - α基因进入左股动脉或肿瘤时，或当给予荧光素酶基因时，均未观察到倒退。注射抗tnf - α、抗cd4或抗CD8单克隆抗体可抑制肿瘤消退，tnf - α处理小鼠肿瘤中CD8+ T细胞积累。这些结果表明，tnf - α在导致肿瘤的动脉中更局部地表达，通过增强抗肿瘤免疫反应有效地抑制肿瘤生长。此外，基因转移到中枢神经系统（CNS）的方法的发展对于基因治疗的基础研究和临床研究都很重要。阳离子脂质体（CL）因其安全性好、易于使用而成为极具吸引力的载体。然而，到目前为止，只有低成功率的报道。我们利用CL和基于T7 RNA聚合酶和T7 RNA聚合酶-以及T7启动子序列的荧光素酶基因的细胞质基因表达系统，成功地在体内获得了高转染效率。该系统的效率比使用含有劳斯肉瘤病毒启动子pRSVL的CL和荧光素酶基因的标准系统高2个数量级。此外，使用LLCMK2细胞进行的体外实验表明，转染后细胞质中基因表达迅速（在6小时内）。相比之下，pRSVL诱导荧光素酶表达需要24-48小时。少

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-18O tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD 8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed l … More ocally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. Additionally, Development of methodologies for gene transfer into the central nervous system (CNS) is important for fundamental research as well as clinical studies for gene therapy. Cationic liposomes (CL) are attractive vectors because of their safety and ease of use. However, to date only low rates of success have been reported. We succeeded in obtaining high transfection efficiencies into the newborn mouse brain in vivo by CL and a cytoplasmic gene expression system based on T7 RNA polymerase and T7 RNA polymerase- and the luciferase-gene with the T7 promoter sequence. This system showed an efficiency rate 2 orders of magnitude higher than the standard system, which used CL and luciferase genes with a Rous sarcoma virus promoter, pRSVL.In addition, in vitro experiments using LLCMK2 cells showed that cytoplasmic gene expression occurred rapidly (within 6 h) after transfection. In contrast, pRSVL required 24-48 h for induction of luciferase expression. Less