Improved Suicide Gene Therapy for Hepatic Cancers

描述(由申请人提供)：本提案的目的是审查 新一代肝内局部治疗的潜力 使用改进的胞嘧啶递送方法和形式的癌症 脱氨酶/5-氟胞嘧啶(CD/Fc)在酶/前药基因治疗中的应用现行基因 治疗方法至少有两个关键限制。第一个问题 产生足够的细胞毒性和放射增敏的能力，因为 没有足够的能力杀死邻近培养的肿瘤细胞 传递(“旁观者效应”)。从FC生成的FU已被演示给 有显著的旁观者效应。不幸的是，FC是一种低效的 细菌CD的底物与胞嘧啶相比，是天然的底物。至 开始克服这一限制，我们将开发酵母(Y)CD的使用 与以前使用的Fc相比，哪种FC是更有效的衬底 细菌(B)镉(目标1)，因此将在低于#年BCD的FC水平下工作 肠道细菌(用于降低全身毒性)。的第二个限制 当前版本的CD/FC系统是选择性的。初步数据使用 临床相关的肝内癌动物模型(BD9大鼠) 肝内K12腺癌)提示即使是(区域性的)肝脏 动脉注射BCD腺病毒产生显著的FU水平 正常肝脏，体内高浓度的PU，以及潜在的正常组织 毒性。为了克服这一限制，我们将构建一个腺病毒载体 使用酵母CD的肿瘤选择性启动子(目标2)。初步数据显示 CEA作为启动子的使用显著增强了 表情。此外，我们将加强区域病毒的传播，使用 血管手法(目标3)，包括渗透压(甘露醇)，机械 (微球)和激素(肾上腺素)方法，并用FOCUS进行预处理 电离辐射。初步数据显示，用甘露醇或 电离辐射可增加肿瘤CD的表达。这些改进 将被开发出来，然后在治疗试验中进行测试，比较大多数 积极的标准治疗，即肝动脉FUDR，效果最好 “增强型”CD/FC基因治疗(AIM 4)的MRI监测抗肿瘤效应 活着。裸鼠将用于治疗试验和所有更长期的研究 免疫原性可能会影响结果。虽然这个项目的目标是 为了了解如何设计更好的区域化疗放射治疗，我们的Long 术语目标是生成相关的信息和原则 适用于有效的、全身的、肿瘤选择性的基因治疗。

DESCRIPTION (provided by applicant): The goal of this proposal is to examine the potential for a new generation of regional treatments for intrahepatic cancers using improved methods of delivery and forms of cytosine deaminase/5-flucytosine (CD/FC) in enzyme/prodrug gene therapy. Current gene therapy approaches have at least two critical limitations. The first concerns the ability to generate sufficient cytotoxicity and radiosensitization because of an inadequate ability to kill adjacent cultured tumor cells that are not transduced ("bystander effect"). FU generated from FC has been demonstrated to have a significant bystander effect. Unfortunately, FC is an inefficient substrate for bacterial CD compared to cytosine, the natural substrate. To begin to overcome this limitation we will develop the use of yeast (y) CD for which FC is a far more efficient substrate compared to the previously utilized bacterial (b) CD (Aim 1) and, thus will work at FC levels lower than the bCD in gut bacteria (for lowered systemic toxicity). A second limitation of the current versions of CD/FC system is selectivity. Preliminary data using a clinically relevant animal model for intrahepatic cancer (BD9 rats bearing intrahepatic K12 adenocarcinoma tumors) suggest that even (regional) hepatic arterial infusions of bCD adenovirus produce significant FU levels in the normal liver, high systemic concentrations of PU, and potential normal tissue toxicity. To overcome this limitation, we will construct an adenoviral vector using a tumor selective promoter for yeast CD (Aim 2). Preliminary data suggest that the use of CEA as a promoter significantly enhances the specificity of expression. In addition, we will enhance regional delivery of virus using vascular manipulations (Aim 3), including osmotic (mannitol), mechanical (microspheres) and hormonal (epinephrine) methods, and pretreatment with focal ionizing radiation. Preliminary data show that pretreatment with mannitol or with ionizing radiation can increase tumor CD expression. These improvements will be developed and then tested in therapy trials comparing the most aggressive standard therapy, i.e., hepatic arterial FUdR, with the best "enhanced" CD/FC gene therapy (Aim 4) using MRI to monitor antitumor effects in vivo. Nude rats will be used in therapy trials and in all longer term studies where immunogenicity could affect results. Although the goal of this project is to understand how to design better regional chemoradiation treatments, our long term objective is to generate information and principles relevant and applicable to effective, systemic, tumor-selective gene therapy.