Improved Suicide Gene Therapy for Hepatic Cancers

描述（由申请人提供）：本提案的目标是审查 新一代肝内肿瘤局部治疗的潜力 使用改进的递送方法和胞嘧啶形式的癌症 脱氨酶/5-氟胞嘧啶（CD/FC）在酶/前药基因治疗中的应用。当前基因 治疗方法具有至少两个严重的局限性。第一个问题涉及 产生足够细胞毒性和放射增敏的能力， 没有足够的能力杀死邻近的培养肿瘤细胞， 转导（“旁观者效应”）。已证明FC产生的FU 具有显著的旁观者效应。不幸的是，FC是一个效率低下的 与天然底物胞嘧啶相比，细菌CD的底物。到 开始克服这一限制，我们将开发酵母（y）CD的用途， 与以前使用的相比，FC是更有效的底物 细菌（B）CD（目标1），因此将在低于bCD的FC水平下起作用， 肠道细菌（降低全身毒性）。第二个限制是 CD/FC系统的当前版本是选择性的。初步数据使用 临床相关的肝内癌动物模型（BD 9大鼠， 肝内K12腺癌肿瘤）表明，即使（区域）肝 bCD腺病毒的动脉输注可在患者中产生显著的FU水平。 正常肝脏、高PU全身浓度和潜在正常组织 毒性为了克服这一局限性，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（Aim 2）。初步数据表明 使用CEA作为启动子显著增强了 表情此外，我们亦会利用 血管操作（目标3），包括渗透（甘露醇），机械 （微球）和激素（肾上腺素）方法，以及用局灶性 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表达。这些改进 将被开发出来，然后在治疗试验中进行测试， 积极的标准治疗，即，肝动脉FUdR，最佳 “增强型”CD/FC基因治疗（目的4），使用MRI监测抗肿瘤作用， vivo.裸大鼠将用于治疗试验和所有长期研究 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局部放化疗治疗，我们长期以来 长期目标是提供有关信息和原则， 适用于有效的、全身性的、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.