IMPROVED HSV VECTORS--GENE TRANSFER INTO NEROUS SYSTEM

描述：基因转移方法的进步创造了机会， 用于开发人类神经系统疾病的基因治疗， 帕金森病（PD）。 一个目标是减缓多巴胺能神经元的损失， 这种神经保护基因疗法的成功取决于 开发安全有效的基因转移载体， 治疗基因在特定的神经元群体中长时间。 基于质粒的单纯疱疹病毒（HSV）“扩增子”载体适应 一个大的（9 kb）泰普罗辛羟化酶（TH）启动子，并提供高度 选择性和相对长期（10周）的多巴胺基因表达 (DA)大鼠黑质神经元。 但是，HSV扩增子载体储备 还含有复制缺陷型HSV辅助病毒， 条件下，特别是在感染的高多样性产生 细胞损伤和死亡。 本应用程序的总体目标有三个 倍：目标1将比较几个互补的新方法，以减少和 也许可以消除辅助病毒相关的毒性，并确定它们是否 提供扩增子转基因的提高的效率和稳定性 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阳性细胞的数量， 细胞毒性）和Aim 2将使用长的 酪氨酸羟化酶启动子- B lacZ报告扩增子（递送病毒 DA能神经元中bgal表达（mRNA和蛋白） (TH感染后1、6和16周， 几个MOI）。 最终目标3将确定降低细胞毒性的方法是否 从而带来更有效的基因治疗 申请人将新建 携带三个候选神经保护基因的扩增子载体被认为 通过不同的途径，GDNF，BDNF和bcl-2，使用最小的细胞毒性， 系统，以产生感染性颗粒，并在 FluoroGold/6-OHDA损伤大鼠，PD的进行性损伤模型，检查 基因表达特征（RNA酶保护以测量载体mRNA 水平，ELISA和免疫染色以测量基因产物），神经保护 （FluoroGold标记神经元的定量）和神经化学变化 （DA和代谢物-DOPAC、HVA水平作为体内和体内代谢物的测量， 神经元间DA转换和5-HT对多巴胺能神经元的影响， 5-HIAA）。

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.