NEUROTROPHIC FACTOR GENE THERAPY FOR PARKINSONS DISEASE

描述：长期目标是开发临床应用的方法。 基因治疗在人类神经退行性疾病中的应用。 这 该提案的重点是离体和体内基因治疗，以提供有效的 神经营养因子（GDNF）对帕金森病（PD）多巴胺（DA）神经元的作用 大脑模型 单纯疱疹病毒扩增子（HSV）、腺病毒（Ad）和逆转录病毒 (RV)将为GDNF制备载体，移码突变GDNF作为对照， 和细胞标记基因，核定位LacZnl。 矢量将是 含有3种细胞启动子，鸡B-肌动蛋白 组成型，神经元启动子，NSE，或星形胶质细胞启动子，GFAP， 或病毒启动子。 启动子特异性，强度和持续时间 在HSV、Ad和RV载体的情况下的表达将在以下方面进行比较： 神经元和神经胶质的原代培养物，以及大鼠脑中的体内培养物。 向量 将用于在良好表征的大鼠中的体内和离体基因治疗 研究GDNF基因治疗对PD模型中受损DA神经元的影响。 成人脑和胎儿DA神经元移植到成人脑中。 的电池 DA依赖性纹状体功能的行为测试将用于比较 不同方法的有效性。 GDNF基因治疗的作用 还将与将重组GDNF注射到脑中的那些进行比较。 此外，含有神经保护性原癌基因的HSV载体， 在酪氨酸羟化酶启动子的控制下， 用在体内，以确定是否表达bcl-2在DA神经元的成人 大脑将它们从神经毒素MPP+引起的损伤中拯救出来。 方法 分子生物学，免疫细胞化学，神经形态计量学，原代神经元 培养、Elisa测定、生物测定、啮齿动物显微手术和行为测试 将被应用。 这些研究与临床直接相关。 在患有PD的人中的干预以及利用 胎儿移植物 更一般地说，他们承诺增加我们对 基因疗法治疗神经变性疾病的潜力和方法 用于从病毒载体获得CNS中的长期基因转基因表达。

DESCRIPTION: The long term goal is to develop methods for clinical application of gene therapy to human neurodegenerative diseases. This proposal focuses on ex vivo and in vivo gene therapy to provide the potent neurotrophic factor, GDNF, to dopamine (DA) neurons in the Parkinsonian (PD) brain model. Herpes simplex amplicon (HSV), adenoviral (Ad) and retroviral (RV) vectors will be made for GDNF , a frameshift mutant GDNF as a control, and the cellular marker gene, nuclear localized LacZnl. Vectors will be made containing each of 3 cellular promoters, the chicken B-actin constitutive, the neuronal promoter, NSE, or the astrocyte promoter, GFAP, or viral promoters. Promoter specificity, strength and duration of expression in the context of HSV, Ad, and RV vectors will be compared in primary cultures of neurons and glia, and in vivo in rat brain. Vectors will be used for in vivo and ex vivo gene therapy in well characterized rat models of PD to study effects of GDNF gene therapy on damaged DA neurons in the adult brain and fetal DA neurons grafted into adult brain. A battery of behavioral tests of DA-dependent striatal functions will be used to compare efficacy of the different approaches. The effects of gene therapy with GDNF will also be compared to those of injecting recombinant GDNF into the brain. In addition, HSV vectors harboring the neuroprotective proto-oncogene, bcl-2, under control of the tyrosine hydroxylase promoter will be made and used in vivo to determine whether expression of bcl-2 in DA neurons an adult brain rescues them from damage induced by neurotoxin MPP+. Methods in molecular biology, immunocytochemistry, neuromorphometry, primary neuronal culture, Elisa assay, bioassay, rodent microsurgery and behavioral testing will be applied. These studies are directly relevant to clinical intervention in humans suffering from PD and to clinical trials utilizing fetal grafts. More generally, they promise to increase our knowledge on the potential of gene therapy to treat neurodegenerative diseases and methods for obtaining long term gene transgene expression in CNS from viral vectors.