GENE DELIVERY TO THE PRIMATE CNS--VIRAL VECTORS

描述(改编自申请人的摘要)：在灵长类中枢神经系统中， 拟议的研究旨在测试和比较基因治疗的可能载体。 来源于三组病毒：重组单纯疱疹病毒 单纯疱疹病毒(HSV)、腺病毒(Ad)和腺相关病毒(AAV)。尽管这些 在啮齿动物的中枢神经系统中已经研究了媒介，很少有研究涉及 安全、最佳输送、载体在特定细胞中表达的问题 种群、转基因表达的持续性和假定的功能 在灵长类中枢神经系统中系统地研究治疗基因。HSV、广告和 AAV载体已被证明能够包装和运送基因 啮齿动物中枢神经系统的序列及其利弊和选择性 每一种的内在特征都已被确定。隐藏着细胞的病毒库 标记基因，核定位的lacZ(LacZn1)或人神经胶质细胞系 衍生神经营养因子(GDNF)的生产和质量控制。 初步研究将涉及输送方法、转导效率、 从注射部位传播包括逆行运输，细胞 趋向性、载体持久性、转基因表达的稳定性和水平， 神经病理学和宿主细胞反应。系统性挑战与相关 还将研究病毒，以解决安全和免疫反应问题。 将选择一个载体将GDNF或失活突变体GDNF运送到 评估功能。GDNF具有良好的形态、生化和 对多巴胺神经元的功能影响，这与许多 具有更高的大脑功能和运动控制能力。我们将评估认知和 载体传递的GDNF在前额叶两个脑区的运动效应 MPTP诱导的大鼠大脑皮质和尾状核多巴胺耗竭 猴子。疗效的衡量标准将包括行为参数、分子 用酶联免疫吸附试验检测蛋白质、RT-PCR检测转基因基因的表达 载体DNA的聚合酶链式反应，多巴胺及其代谢物的生化分析， 神经解剖学和形态计量学分析、神经病理学和 放射自显影。 这些研究旨在提供有关病毒载体的基本信息 灵长类中枢神经系统通过综合的、比较的实验设计。这个 多巴胺缺乏对认知和运动的影响与GDNF的选择 在灵长类动物大脑中测试基因治疗功能结果的基因是 根据灵长类动物中这些效应的特征，以及 GDNF在啮齿动物和灵长类动物中的作用，包括GDNF基因 啮齿动物多巴胺耗竭模型的治疗。然而，主要的目标是 这些研究旨在为开发有效、安全的基因做出贡献。 未来广泛应用于人类中枢神经系统的给药系统 条件。

DESCRIPTION (Adapted from applicant's abstract): In the primate CNS, the proposed studies aim to test and compare possible vectors for gene therapy derived from three groups of viruses: recombinant herpes simplex virus (HSV), adenovirus (Ad) and adenoassociated virus (AAV). Although these vectors have been studied in the rodent CNS, few studies have addressed the issue of safety, optimal delivery, vector expression in specific cell populations, persistence of transgene expression, and function of putative therapeutic genes in a systematic fashion in the primate CNS. HSV, Ad and AAV vectors have been shown to be capable of packaging and delivering gene sequences to the rodent CNS, and advantages, disadvantages and selectivity inherent to each have been identified. Viral stocks harboring the cellular marker gene, nuclear localized lacZ (lacZnl) or human glial cell line derived neurotrophic factor (GDNF), will be produced and quality controlled. Initial studies will address methods of delivery, transduction efficiency, spread from the site of injection including retrograde transport, cellular tropism, vector persistence, stability and level of transgene expression, neuropathology and host cellular responses. Systemic challenge with related viruses also will be studied to address safety and immune responses. One vector will be selected to deliver GDNF or an inactive mutant GDNF to assess function. GDNF has well documented morphological, biochemical and functional effects on dopamine neurons, which are associated with many higher brain functions and with motor control. We will assess cognitive and motor effects of vector-delivered GDNF in two brain areas, the prefrontal cortex and the caudate nucleus, after MPTP-induced dopamine depletions in monkeys. Measures of efficacy will include behavioral parameters, molecular assays of transgene expression using ELISA for protein, RT-PCR for mRNA and PCR for vector DNA, biochemical assays of DA and its metabolites, neuroanatomical and morphometric analyses, neuropathology, and autoradiography. These studies aim to provide fundamental information on viral vectors in the primate CNS through an integrated, comparative experimental design. The choice of cognitive and motor effects of dopamine depletion and the GDNF gene to test functional outcomes of gene therapy in the primate brain are based on the well characterized nature of these effects in primates and the reported effects of GDNF in rodents and primates, including GDNF gene therapy in rodent dopamine depletion models. However, the major aim of these studies is to contribute to the development of effective and safe gene delivery systems for future application to the human CNS for a wide range of conditions.