Viral-based Therapeutic Approaches for Reversal of ALS Pathology

项目摘要/摘要 肌萎缩侧索硬化症(ALS)是一种以运动神经元死亡为特征的致命性疾病。一种关键的病理 特征是核转录和剪接调节蛋白Tar-DNA结合蛋白的细胞质错误定位 43 kDa(TDP-43)。TDP-43聚集在胞浆应激颗粒(SGS)中，伴随着核的输入/输出 其毒性被认为是由于细胞质获得和核功能丧失两种机制。 将其重新定位到原子核有可能解决这两种形式的毒性。抑制SGS的形成是其中之一 有希望的策略，并使用反义下调SG相关蛋白Aaxin-2(Atxn2) 寡核苷酸(ASOS)可延长ALS小鼠模型的强度和存活时间。然而，ASOS需要 频繁的中枢神经系统给药，更好的方法是在一次治疗后实现击倒。 第二种方法是加强核进口，这是一种在二肽重复(DPR)毒性方面取得成功的策略 ALS体外模型的建立。将这一策略推广到非DPR形式的ALS有可能使更广泛的 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肌萎缩侧索硬化。 在初步工作中，使用新型病毒载体传递的RNAi实现了对Key中Atxn2的强大击倒 肌萎缩侧索硬化症神经系统受影响的区域。该提案的目标1是确定持续的Atxn2击倒 在这些区域逆转TDP-43错误定位并改善两种不同的小鼠模型的神经元存活率 肌萎缩侧索硬化。在其他的初步工作中，过度表达核进口因子的细胞系显示TDP-43的表达减少。目标 二是测试增加核进口是否纠正了TDP-43的本地化，并在条件下提高了细胞存活率 压力的影响。我的中心假设是针对TDP-43的胞浆聚集和核丢失 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，并利用这些知识来减轻 疾病，特别是针对神经学最具破坏性的疾病之一。 阿马多医生是一位充满激情、训练有素的临床医生兼科学家，他准备做出根本性的影响。 在肌萎缩侧索硬化症上。她的导师贝弗利·戴维森博士是一位著名的神经退行性疾病专家，她不断地推动 以载体为基础的治疗的边界，以及她的咨询委员会，经验丰富和专注 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，并得到 热情的制度支持。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去处。Amado博士将利用这个为期5年的指导机会 以她的基因治疗培训为基础，并将其与她的临床专业知识相结合，成为一家独立的、由R01资助的 为肌萎缩侧索硬化症患者开发新疗法的内科科学家。

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.