GENE DELIVERY TO THE NERVOUS SYSTEM

基因输送至神经系统

• 批准号: 6112271
• 负责人: XANDRA OWENS BREAKEFIELD
• 金额: --
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6112271
• 关键词: Alphaherpesvirinae; Retroviridae; Schwann cells; astrocytes; cell growth regulation; congenital nervous system disorder; disease /disorder model; gene expression; gene targeting; genetic manipulation; genetic promoter element; glia; laboratory mouse; molecular pathology; neoplasm /cancer genetics; nervous system neoplasms; neurofibroma; neurogenetics; neurotrophic factors; newborn animals; tissue /cell culture; transfection /expression vector; virus replication

The goal of this project is to increase the ability of virus vectors to deliver genes to the postnatal nervous system and to use these vectors to alter cell fate in the nervous system. Two strategies will be undertaken to expand the range and longevity of gene delivery to neural cells in the mouse nervous system. In the first scheme, retrovirus vectors will be generated which can confer stable gene expression on astrocytic cells grafted into the brain. The phosphoglycerate kinase promoter will be used to regulate expression of brain-derived neurotrophic factor (BDNF) for degenerating dopaminergic neurons in newborn weaver mice. These vectors will also be used to genetically modify astrocytic lines capable of migrating in the brain. In addition, one of the astrocytic lines will be converted to a packaging cell line which can be used to deliver retrovirus vectors to endogenous neural cells in vivo. Retrovirus vectors will also be generated to aid in functional analysis of the NF2 gene product in Schwann cells and tumor cells. Further we will try to develop an animal model for neurofibroma formation by retrovirus-mediated delivery of antisense RNA to repress expression of neurofibromin or by homologous recombination to knock-out the normal NF1 allele in Schwann cells from heterozygous NF1 knock-out mice. The behavior of genetically altered Schwann cells will be evaluated in a peripheral nerve injury-repair model. In the second gene delivery scheme we will try to improve herpes vectors for delivery of genes to neurons. A "piggy back" system of herpes simplex virus vectors will be developed using a combination of amplicon and recombinant virus vectors to combine complementary features of both vector types and to make them mutually dependent on each other. This system will be designed to allow limited replication of the vectors in glia-derived cells using glia-specific promoters to regulate expression of critical genes. Amplicon vectors will be used to deliver NGF and Oct2 to neurons at the time of infection in order to encourage entrance of the virus into latency, at the same time making preparation of vector stocks feasible by using a tet operon system to control expression of these genes in culture. The recombinant virus vector will be deleted for genes contributing to neurovirulence including ICP4 (or ICP27), ribonucleotide reductase, gamma 34.5 and UL41. The herpes vector system will be evaluated for gene delivery, stability of transgene expression and pathogenicity to neural cells in the brain. These new gene delivery systems will provide a basis for therapeutic intervention in animal models of Parkinson's disease and neurofibromatosis.

该项目的目的是提高病毒向量的能力 将基因传递到产后神经系统，并使用这些向量 改变神经系统中的细胞命运。 将采取两种策略 扩大基因递送到神经细胞的范围和寿命 小鼠神经系统。 在第一个方案中，逆转录病毒向量将是 生成的，可以在星形细胞上赋予稳定的基因表达 嫁接到大脑中。 将使用磷酸甘油酸激酶启动子 调节脑衍生的神经营养因子（BDNF）的表达 新生儿编织小鼠中的多巴胺能神经元退化。 这些向量 还将用于基因修改能够的星形细胞系 在大脑中迁移。 此外，星形细胞系之一将是 转换为包装细胞系，可用于输送逆转录病毒 体内内源性神经细胞的向量。 逆转录病毒向量也将 生成以帮助对NF2基因产物的功能分析 雪旺细胞和肿瘤细胞。 此外，我们将尝试发展动物 通过逆转录病毒介导的递送神经纤维瘤形成的模型 反义RNA抑制神经纤维蛋白的表达或同源 重组以淘汰雪旺细胞中正常的NF1等位基因 杂合NF1敲除小鼠。 基因改变的行为 Schwann细胞将在外围神经损伤修复模型中进行评估。 在第二个基因输送方案中，我们将尝试改善疱疹向量 用于向神经元传递基因。 单纯疱疹的“背部背包”系统 病毒向量将使用扩增子的组合和 重组病毒向量结合了两个载体的互补特征 类型并使它们相互依赖。 这个系统将 设计以允许在胶质衍生中的载体的有限复制 使用胶质特异性启动子调节关键表达的细胞 基因。 扩增子向量将用于将NGF和OCT2传递给神经元 在感染时为了鼓励病毒进入 延迟，同时准备可行的矢量库存 使用TET操纵子系统控制这些基因在培养中的表达。 重组病毒载体将被删除，以促成 神经动力毒素包括ICP4（或ICP27），核糖核苷酸还原酶，伽马 34.5和UL41。 将评估疱疹矢量系统的基因 传递，转基因表达的稳定性和致病性对神经 大脑中的细胞。 这些新的基因输送系统将提供基础 用于治疗干预帕金森氏病动物模型和 神经纤维瘤病。