Viral-based Therapeutic Approaches for Reversal of ALS Pathology

项目概要/摘要 肌萎缩侧索硬化症（amyotrophiclateralsclerosis，ALS）是一种以运动神经元死亡为特征的致死性疾病。一个关键的病理 一个特征是核转录和剪接调节因子Tar-DNA结合蛋白的胞质错误定位 分子量为43 kDa（TDP-43）。TDP-43沿着核输入/输出聚集在细胞质应激颗粒（SG）中 其毒性被认为是由于细胞质获得和细胞核功能丧失机制。 将其重新定位到细胞核有可能解决这两种形式的毒性。抑制SG的形成是一种 有希望的策略，并使用反义核酸下调SG相关蛋白Ataxin-2（Atxn 2） 在ALS的小鼠模型中，寡核苷酸（ASO）的抗肌萎缩强度和存活率。然而，ASO要求 频繁的CNS再给药，并且优选的方法是在一次治疗后实现敲低。 第二种方法是增强核输入，这是一种在二肽重复序列（DPR）毒性方面取得成功的策略 ALS体外模型。将这种策略扩展到非DPR形式的ALS有可能使广泛的 对疾病的影响此外，这两种方法之间的潜在协同作用具有巨大的治疗潜力。 如果成功，这些策略可以用于治疗绝大多数ALS。 在初步工作中，使用新型病毒载体递送的RNAi在关键基因中实现了Atxn 2的稳健敲低。 神经系统受ALS影响的区域。本提案的目的1是确定持续的Atxn 2敲除是否 在两种不同的小鼠模型中， 人症在其他初步工作中，过表达核输入因子的细胞系显示TDP-43减少。目的 2是测试增加核输入是否校正TDP-43定位并改善条件下的细胞存活 压力我的中心假设是，靶向TDP-43的细胞质聚集和细胞核丢失， 病毒介导的方法将导致持续的神经保护。这项工作完全符合NINDS的使命， 进一步了解我们的大脑和神经系统，并利用这些知识，以减轻负担， 疾病，专门针对神经学最具破坏性的痛苦之一。 阿马多博士是一位充满激情、训练有素的临床科学家，他的独特之处在于， 关于ALS她的导师贝弗利·戴维森博士，一位著名的神经退行性疾病专家， 基于载体的治疗方法的界限，以及她的咨询委员会， 神经学家和神经科学家将提供指导和指导，以确保她的成功， 热情的机构支持。宾夕法尼亚大学拥有无数的资源和设施， 是开启神经科学事业的绝佳之地阿马多博士将利用这5年的指导机会， 建立在她的基因治疗培训，并将其与她的临床专业知识，成为一个独立的，R 01资助的 为ALS患者开发新疗法的医生科学家。

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by death of motor neurons. A key pathologic feature is the cytoplasmic mislocalization of a nuclear transcription and splice regulator, Tar-DNA binding protein of 43kDa (TDP-43). TDP-43 is aggregated in cytoplasmic stress granules (SGs) along with nuclear import/export factors, and its toxicity is thought to be due to both cytoplasmic gain- and nuclear loss-of-function mechanisms. Relocating it to the nucleus has the potential to address both forms of toxicity. Inhibiting formation of SGs is one promising strategy, and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs strength and survival in a mouse model of ALS. However, ASOs require frequent CNS readministration, and a preferable approach would be to achieve knockdown after one treatment. A second approach is enhancing nuclear import, a strategy with success in dipeptide repeat (DPR) toxicity models of ALS in vitro. Extending this strategy to non-DPR forms of ALS has the potential to make a broad impact on the disease. In addition, potential synergy between the two approaches has great therapeutic potential. If successful, these strategies could be used to treat the vast majority of ALS. In preliminary work, RNAi delivered using a novel viral vector achieves robust knockdown of Atxn2 in the key areas of the nervous system affected in ALS. Aim 1 of this proposal is to determine if sustained Atxn2 knockdown in these regions reverses TDP-43 mislocalization and improves neuron survival in two distinct mouse models of ALS. In other preliminary work, cell lines overexpressing a nuclear import factor show reductions in TDP-43. Aim 2 is to test if augmenting nuclear import corrects TDP-43 localization and improves cell survival under conditions of stress. My central hypothesis is that targeting both cytoplasmic aggregation and nuclear loss of TDP-43 using viral-mediated approaches will result in sustained neuroprotection. This work fits squarely in NINDS’ mission to further our knowledge about the brain and nervous system and to use this knowledge to reduce the burden of disease, specifically targeting one of neurology’s most devastating afflictions. Dr. Amado is a passionate, highly-trained clinician-scientist uniquely poised to make a fundamental impact on ALS. Her mentor Dr. Beverly Davidson, a renowned neurodegenerative disease expert continually pushing the boundaries of vector-based therapeutics, and her advisory committee of deeply experienced and dedicated neurologists and neuroscientists, will provide the guidance and mentorship to ensure her success, backed by enthusiastic institutional support. The University of Pennsylvania, with its innumerable resources and facilities, is an outstanding place to launch a neuroscience career. Dr. Amado will use this 5-year mentored opportunity to build on her gene therapy training and merge it with her clinical expertise to become an independent, R01-funded physician-scientist developing novel therapies for patients with ALS.