GENE THERAPY OF BASAL FOREBRAIN CHOLINERGIC LESIONS

描述：（改编自申请人摘要）胆碱能损伤 基底前脑（CBF）系统在动物中使用免疫毒素产生鲁棒的 行为缺陷和模仿阿尔茨海默病（AD）的某些方面。这 模型是有用的测试乙酰胆碱的作用，在认知 方法和新的治疗策略，如基因治疗。的能力 控制基因表达是该技术的重要要求， 临床应用。本提案中探讨的病媒系统可能是 外生调控。该提案将研究恢复机制， CBF免疫毒素损伤动物的神经递质功能1） 通过胆碱补充增强移植后ACh释放，和2） 移植能够抑制或诱导胆碱转基因的细胞 乙酰转移酶（ChAT）。阻遏系统（四环素）和 诱导系统（蜕皮激素），以快速， 可逆和高度特异性的方式。 在第一系列实验中，研究人员将确定是否 不同剂量的外源性胆碱可以增加 和乙酰胆碱从基因工程成纤维细胞中的释放， 免疫毒性损伤脑血流后大鼠的皮质和海马。 一旦确定释放量增加， 将决定是否释放。 对于第二和第三系列实验，他们将使用可调节的 在施用后诱导或抑制ChAT表达的细胞系 强力霉素或Muristerone A。在免疫毒素损伤中， 将证明移植后这些细胞释放的乙酰胆碱， 新皮层和海马体对于行为的发生是必要的，也是充分的。 复苏这些可调控基因的作用将在空间上进行测试。 和非空间任务后，确定剂量和持续时间的外源性 诱导或抑制基因表达所需的化合物。如果成功， 实验将证明ACh的释放可以被外源性控制， 在动物中，并导致显著的行为改善。 在嫁接或直接转基因之前， 插入实验可以在人类中进行，以便能够 安全地终止基因产物的递送。这些方法不仅 适用于AD，但也适用于其他神经退行性疾病， 其他感兴趣的转基因。

DESCRIPTION: (adapted from applicant's abstract) Lesions of the cholinergic basal forebrain (CBF) system in animals using an immunotoxin produce robust behavioral deficits and mimic some aspects of Alzheimer's disease (AD). This model is useful for testing both the role of acetylcholine in cognitive processes and new therapeutic strategies such as gene therapy. The ability to control gene expression is an important requirement of this technology for clinical application. The vector systems being explored in this proposal may be exogenously regulated. This proposal will investigate mechanisms of restoring neurotransmitter function in animals with CBF immunotoxin lesions by 1) enhancing ACh release postgrafting via choline supplementation, and 2) transplanting cells capable of repressing or inducing the transgene for choline acetyltransferase (ChAT). The repressible system (tetracycline), and the inducible system (eccdysteroid), modulates gene expression of ChAT in a rapid, reversible and highly specific fashion. In the first series of experiments, the investigators will determine whether different doses of exogenously administered choline can augment the production and release of acetylcholine from genetically engineered fibroblasts grafted to the cortex and hippocampus of rats following immunotoxic lesions of the CBF. Once an increase in release has been determined, behavioral effects of this augmented release will be determined. For the second and third series of experiments, they will use the regulatable cell lines that either induce or repress ChAT expression after administration of doxycycline or Muristerone A, respectively. In their immunotoxin lesion they will demonstrate that acetylcholine released from these cells after grafting to the neocortex and hippocampus is both necessary and sufficient for behavioral recovery. The effects of these regulatable genes will be tested in a spatial and non-spatial task after determining the dose and duration of the exogenous compound needed to induce or repress gene expression. If successful, the experiments will demonstrate that release of ACh can be exogenously controlled in animals and results in significant behavioral improvement. The control of transmitter release is necessary before grafting or direct gene insertion experiments can be entertained in humans in order to be able to safely terminate delivery of the gene product. These approaches will not only be applicable for AD, but other neurodegenerative disorders and for delivering other transgenes of interest.