REGULATABLE SYSTEM FOR AMELIORATION OF NEURAL DYSFUNCTION

这项提议的目的是产生一种可调节的靶基因 其可以在所需时间在所选择的位置特定地表达。 我们计划在动物身上使用这个系统来测试基因的能力 对拯救神经退行性疾病的兴趣。 为了生成一个可监管的系统，我们将利用我们最近的 发现孕激素受体的一个羧基(C)末端突变体 对黄体酮无效，但能结合RU486和其他 对抗者。重要的是，这种截短形式的受体可以激活 在拮抗剂存在的情况下的靶基因。为了消除可能 内源性孕酮反应基因的激活我们将取代 孕酮受体这种突变形式的DNA结合域与 酵母转录因子GAL4 DNA结合域，允许 识别特定的GAL4结合位点。通过这种方式，只有可监管的 含有特定GAL4 DNA结合位点的靶基因将被激活 孕激素拮抗剂。因此，黄体酮的这种修饰形式 受体可能是目的基因表达的合适调节者。 黄体酮拮抗剂的存在。 为了测试这个可调节系统的有效性，我们将使用大鼠模型 刺激帕金森氏症和阿尔茨海默氏症的某些方面。 帕金森病涉及脑内多巴胺能神经元的广泛丢失 黑质。大鼠可产生帕金森氏症表型 用6-羟基多巴胺治疗并用来分析疗效 可调控的基因系统。为此，修改后的类固醇 受体调节剂将与可调节的靶标有能力共表达 表达酪氨酸羟基酶(TH)，这是一种转化酪氨酸的酶 对L-多巴，从而刺激水平的内源性多巴胺输入。 TH在大鼠纹状体内的可调控表达 帕金森病基因治疗效果评价模型 疾病。阿尔茨海默病是一种复杂的神经退行性疾病 不同的起源。最常见的缺陷是逐渐失去了 胆碱能功能。因此，我们希望使用监管机构 在穹隆海马伞中表达神经营养因子，如神经生长因子的系统 损毁大鼠并评估神经营养因子在还原中的能力 病变引起的胆碱能神经元变性。这种方法将是 有助于挽救与阿尔茨海默氏症相关的中枢胆碱能缺陷 病人。同样，神经营养因子的表达可能会增强 多巴胺能神经元的存活和维持。这件事的成功 新的方法可能会对基因疗法的发展产生影响 其他遗传和后天遗传缺陷。

The objective of this proposal is to generate a regulatable target gene which can be expressed specifically in a selected site at a desired time. We plan to use this system in animals to test the ability of the gene of interest to rescue neurodegenerative disorders. To generate a regulatable system, we will take advantage of our recent finding that a carboxyl (C)-terminal mutant of the progesterone receptor fails to respond to progesterone but is capable of binding RU486 and other antagonists. Importantly, this truncated form of the receptor can activate a target gene in the presence of antagonists. To eliminate possible activation of endogenous progesterone-responsive genes we will replace the DNA binding domain of this mutated form of the progesterone receptor with the yeast transcription factor GAL4 DNA binding domain, allowing recognition of specific GAL4 binding sites. In this way, only regulatable target genes containing specific GAL4 DNA binding sites will be activated by progestin antagonists. Thus, this modified form of the progesterone receptor would be a suitable regulator of the expression of target genes in the presence of progesterone antagonists. To test the validity of this regulatable system, we will use Rat models which stimulate aspect of Parkinson's disease and Alzheimer's disease. Parkinson's disease involves the extensive loss of dopaminergic neurons in the substantia nigra. Parkinsonian phenotype can be generated in rats using 6-hydroxydopamine treatment and used to analyze the effectiveness of the regulatable gene system. For this purpose, the modified steroid receptor regulator will be co-expressed with a regulatable target capable of expressing tyrosine hydroxylase (TH), an enzyme which converts tyrosine to l-dopa and thereby stimulates the level of intrinsic dopaminergic input. Regulatable expression of TH in the striata of rats should serve as a good model for assessing the therapeutic gene treatments for Parkinson's disease. Alzheimer's disease is a complex neurodegenerative disorder of heterogeneous origins. The most common defect is the gradual loss of cholinergic function. Therefore, we would like to use the regulatory system to express neurotrophic factors, such as NGF, in the fimbria-fornix lesioned rats and to assess the ability of neurotrophins in the reduction of lesion-induced cholinergic neuronal degeneration. This approach will be useful for rescuing central cholinergic defects associated with Alzheimer's patients. Similarly, the expression of neurotrophin may enhance the survival and maintenance of dopaminergic neurons. The success of this novel approach could have an impact ont he development of gene therapies for other inheritable and acquired genetic defects.