RNAi Gene Silencing of alpha-Synuclein for Parkinson's Disease

描述(申请人提供)：帕金森病(PD)是一种衰弱的神经退行性疾病，在美国有100多万人受到影响。路易体痴呆是仅次于阿尔茨海默病的第二大痴呆症。目前还没有治疗帕金森病或DLB的方法可以阻止或逆转神经退行性变的过程。该项目中的RNA干扰(RNAi)基因治疗方法具有阻止或逆转这些疾病的潜力。最近的研究表明，α-突触核蛋白(SNCA)在帕金森病和DLB以及其他被归类为a-突触核病症的神经退行性疾病的发生发展中发挥了作用。SNCA基因的三个点突变以及三个SNCA基因重复序列已被认为与家族性PD和DLB有关，这表明突变型和野生型SNCA都是这些疾病的治疗靶点。SNCA是神经元包涵体中的主要成分，称为路易小体，是帕金森病或DLB患者的神经病理特征。在帕金森病中，路易小体主要分布在黑质，那里有变性的多巴胺(DA)神经元。在DLB，路易小体存在于许多皮质和皮质下结构中。在这两种疾病中都发现了路易突起，即含有不溶性SNCA蛋白的神经元突起。在PD动物模型中，暴露于杀虫剂、鱼藤酮、神经毒素、MPTP和抑制蛋白质降解的药物等可导致DA神经元死亡，增加SNCA的表达并导致DA神经元中SNCA含有路易体样包涵体。此外，在一系列实验物种中，实验增加人SNCA的表达会导致DA神经元的病理和功能恶化。我们提出用基因治疗的方法抑制DA神经元SNCA表达可以防止DA神经退变的假设。腺相关病毒(AAV)是临床试验的首选载体，将用于人类SNCA在DA神经元异位表达导致DA细胞死亡的大鼠身上。在这个模型中，以及在依赖SNCA的细胞培养模型中，沉默SNCA表达的效果将通过形态计量学、组织学、神经化学和行为测试进行评估。此外，还将产生一个在可诱导启动子控制下具有沉默结构的载体，并将其作为临床基因治疗的可能安全策略进行测试。该项目的目标是生成支持临床试验开发的原则验证数据，使用一种新的基于RNAi的基因疗法来治疗PD和DLB患者。？

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a debilitating neurodegenerative disease that affects over i million individuals in the US. Dementia with Lewy Bodies (DLB) is second in occurrence only to Alzheimer's disease. There are presently no treatments for PD or DLB that can halt or reverse the neurodegenerative processes. The RNA interference (RNAi) gene therapy approach in this project has the potential of halting or reversing these diseases. Recent studies suggest a role for a-synuclein (SNCA) in the development of PD and DLB, and other neurodegenerative diseases classified as a-synucleinopathies. Three point mutations in the SNCA gene, as well as triplet SNCA gene repeats, have been linked to familial forms of PD and DLB, revealing both mutant and wild type SNCA as therapeutic targets for these diseases. SNCA is a main components in neuronal inclusions called Lewy bodies, the neuropathological hallmarks of individuals afflicted with PD or DLB. In PD, Lewy bodies are found predominantly in the substantia nigra where the degenerating dopaminergic (DA) neurons reside. In DLB, Lewy bodies are found in many cortical and subcortical structures. Lewy neurites, neuronal processes containing insoluble SNCA protein, are found in both diseases. Insults that lead to death of DA neurons in animal models of PD, including exposure to the pesticide, rotenone, the neurotoxin, MPTP, and drugs that inhibit protein degradation increase SNCA expression and result in SNCA containing Lewy body-like inclusions in DA neurons. Moreover, experimentally increasing expression of human SNCA in a range of laboratory species leads to DA neuronal pathology and functional deterioration. We propose to explore the hypothesis that inhibiting expression of SNCA in DA neurons using a gene therapy approach will prevent DA neurodegeneration. Adeno-associated virus (AAV), the vector of choice for clinical trials will be used in rats in which human SNCA is ectopically expressed in DA neurons leading to DA cell death. The effects of silencing SNCA expression in this model, as well as in an SNCA-dependent cell culture model, will be assessed using morphometry, histology, neurochemistry and behavioral testing. Also, a vector with the silencing construct under control of an inducible promoter will be generated and tested as a possible safety strategy for a clinical gene therapy. The goal of this project is to generate proof-of-principle data supporting the development of clinical trials using a novel RNAi-based gene therapy for individuals afflicted with PD and DLB. ?