IMPROVED HSV VECTORS--GENE TRANSFER INTO NEROUS SYSTEM

描述：基因转移方法的进步创造了机会 用于人类神经疾病的基因治疗的发展，如 帕金森氏病(PD)。一个目标是减缓多巴胺能神经元的损失 这种神经保护性基因疗法的成功与否取决于 安全有效表达A基因的基因转移载体的研制 在特定神经元群体中长期有效的治疗性基因。 以单纯疱疹病毒(HSV)为基础的“扩增”载体 一种大的(9kb)酪氨酸羟化酶(TH)启动子，提供了高度的 多巴胺中选择性和相对长时间(10周)的基因表达 (DA)大鼠黑质神经元。但是，HSV扩增载体股票 还含有复制缺陷的HSV辅助病毒，它在一些 在感染条件下，特别是在感染的高度多元化的情况下， 细胞损伤和死亡。这个应用程序的总体目标有三个 折叠：目标1将比较几种互补的新方法，以减少和 或许可以消除辅助病毒相关的毒性，并确定它们是否 提供更高的效率和稳定性的扩增转基因基因 体外表达(在培养的原代神经元和星形胶质细胞中测量 扩增子和辅助子的滴度、lac阳性细胞数和 细胞毒性)和AIM 2将在体内使用Long 酪氨酸羟基酶启动子-b LacZ报告扩增子(传递病毒 纹状体与BGAL在DA神经元的表达(mRNA和蛋白) (TH阳性)在感染后1周、6周和16周的纹状体和SN中。 几个月。)。 最后，目标3将确定降低细胞毒性的方法 导致了一种更有效的基因疗法。申请人将建造新的 携带三个候选神经保护基因的扩增载体被认为是 通过不同的途径发挥作用，GDNF，BDNF和BCL-2，使用细胞毒性最小的 系统产生具有感染性的颗粒，并在 荧光金/6-OHDA损毁大鼠进行性帕金森病模型 基因表达特征(核糖核酸酶保护以测量载体mRNA 水平、酶联免疫吸附试验和免疫染色以测量基因产物)、神经保护 (荧光金标记神经元的定量)和神经化学变化 (多巴胺和代谢物的水平-DOPAC、HVA作为体内和 神经元间DA代谢及其对5-羟色胺能神经元的影响 5-HIAA)，在注射病毒后1个月和4个月。

DESCRIPTION: Advances in gene transfer methods have created the opportunity for development of gene therapy for human neurological disease such as Parkinsons disease (PD). One goal is to slow dopaminergic neuron loss and the success of such neuroprotective gene therapy is contingent on the development of safe and effective gene transfer vectors that can express a therapeutic gene over long periods of time in specific neuronal populations. The plasmid based herpes simplex virus (HSV) "amplicon" vectors accommodate a large (9kb) typrosine hydroxylase (TH) promoter and provide highly selective and relatively long term (10 weeks) gene expression in dopamine (DA) neurons in the rat substantia nigra. But, HSV amplicon vector stocks also contain replication defective HSV helper virus which under some conditions and particularly at high multiplicities of infection produces cellular injury and death. The overall goals of this application are three fold: Aim 1 will compare several complementary new methods to reduce and perhaps eliminate helper virus related toxicity and determine whether they provide increased efficiency and stability of amplicon transgene gene expression in vitro (in cultured primary neurons and astrocytes measuring titers of amplicon and helper, number of lac positive cells and cytotoxicity) and Aim 2 will test their efficacy in vivo using the long tyrosine hydroxylase promotor - b lacZ reporter amplicon (delivering virus to the striatum and scoring bgal expression (mRNA and protein) in DA neurons (TH positive) in the striatum and SN at 1,6, and 16 weeks after infection at several MOIs.). Finally Aim 3 will determine whether the approach of decreasing cytotoxicity leads to a more effective gene therapy. The applicant will construct new amplicon vectors carrying three candidate neuroprotective genes thought to work by different pathways, GDNF, BDNF and bcl-2, use the least cytotoxic system to produce infectious particles, and evaluate them in FluoroGold/6-OHDA lesioned rats, a progressive injury model of PD, examining gene expression characteristics (RNAse protection to measure vector mRNA levels, ELISA and immunostaining to measure gene products), neuroprotection (quantitation of FluoroGold labeled neurons) and neurochemical changes (levels of DA and metabolites-DOPAC, HVA as a measure of both intra and inter-neuron DA turnover and effects on serotonergic neurons with 5-HT, 5-HIAA) at 1 and 4 months after injection of viral stock.