NEUROTROPHIC FACTOR GENE THERAPY FOR PARKINSONS DISEASE

描述：长期目标是开发用于临床的方法 基因治疗在人类神经退行性疾病中的应用。这 建议侧重于体外和体内的基因治疗，以提供强大的 神经营养因子对帕金森病患者多巴胺能神经元的影响 大脑模型。单纯疱疹病毒(HSV)、腺病毒(Ad)和逆转录病毒 (RV)载体为GDNF，作为对照移码突变体GDNF， 和细胞标记基因，核定位的LacZn1。向量将是 由3个细胞启动子组成的鸡B-肌动蛋白 构成，神经元启动子，NSE，或星形胶质细胞启动子，GFAP， 或者病毒推动者。启动子的特异性、强度和持续时间 在HSV、Ad和RV载体的上下文中的表达将在 原代培养神经元和神经胶质细胞，并在大鼠脑内进行活体培养。向量 将用于具有良好特性的大鼠的体内外基因治疗 大鼠帕金森病模型研究GDNF基因治疗对帕金森病大鼠DA神经元损伤的影响 成人脑和胎儿DA神经元移植到成人脑中。一组 将使用DA依赖的纹状体功能的行为测试来比较 不同方法的有效性。神经营养因子在基因治疗中的作用 也将与将重组GDNF注射到大脑中的方法进行比较。 此外，携带神经保护性原癌基因的HSV载体， Bcl2，在酪氨酸羟化酶启动子的控制下， 体内用来确定bcl2在成体DA神经元中的表达 大脑将它们从神经毒素MPP+引起的损伤中拯救出来。方法在 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、酶联免疫吸附试验、生物测定、啮齿动物显微外科手术和行为测试 将会被应用。这些研究与临床直接相关。 帕金森病患者的干预及临床试验 胎儿移植物。更广泛地说，它们承诺增加我们对 基因治疗治疗神经退行性疾病的潜力和方法 目的：从病毒载体中获得基因在中枢神经系统的长期表达。

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.