A Novel Gene Therapy Approach to Prevent Alpha-synuclein Misfolding in Multiple System Atrophy

一种防止多系统萎缩中α-突触核蛋白错误折叠的新基因治疗方法

• 批准号: 10673418
• 负责人: Amanda L. Woerman
• 金额: $ 23万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673418
• 关键词: Adopted; Binding; Biological Models; Brain; Cell Line; Cells; Central Nervous System; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryoelectron Microscopy; Cultured Cells; Dependovirus; Disease; FYN gene; Genes; Goals; Greek; Human; In Vitro; Kinetics; Lipid Binding; Measures; Mediating; Membrane; Modeling; Molecular Conformation; Movement Disorders; Multiple System Atrophy; Mus; Mutation; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathogenicity; Patients; Point Mutation; Positioning Attribute; Prion Diseases; Prions; Process; Proteins; Research; Role; SNAP receptor; Sampling; Seminal; Sodium Chloride; Structure; Techniques; Terminal Disease; Testing; Therapeutic; Therapeutic Intervention; Transgenic Mice; Transgenic Organisms; Work; alpha synuclein; clinical candidate; experimental study; gene therapy; in vivo; innovation; knock-down; loss of function; novel; novel therapeutic intervention; prevent; prime editing; prion-like; protein aggregation; protein misfolding; therapeutic development; transmission blocking; transmission process; treatment strategy

Multiple system atrophy (MSA) is a prion-like movement disorder caused by misfolding and self- templating of the protein α-synuclein (α-syn), which spreads throughout the central nervous system to cause progressive degeneration. Similar to many other prion and prion-like neurodegenerative diseases, there are currently no therapeutics available that alter the course of disease for MSA patients. To interfere with α-syn self-templating, several groups have proposed various strategies for knocking down α-syn expression to reduce the amount of protein available as substrate. Unfortunately, these strategies may interfere with normal α-syn function in the brain, leading to loss-of-function deficits for MSA patients. Alternatively, MSA cannot propagate in transgenic (Tg) cells or mice expressing α-syn with the E46K mutation, raising the possibility of using gene therapy to generate conversion-incompetent α-syn to disrupt self-templating. However, to date, this approach has not been tested as a therapeutic intervention for MSA. The objective of the proposed work is to establish proof-of-concept that introducing a single residue change in the α-syn primary sequence can disrupt templated misfolding. We hypothesize that generating conversion-incompetent α-syn using CRISPR prime editing will reduce or prevent MSA propagation. Our approach will capitalize on our recent discovery that non- pathogenic α-syn mutations at residue K80 inhibit MSA propagation in vitro. In Aim 1, we will use CRISPR prime editing to insert our novel K80 mutations into Tg cells and mice expressing wild-type human α-syn prior to challenging the models with MSA patient samples. We have shown that MSA induces α-syn aggregation in unedited cells and mice expressing wild-type protein. We anticipate that successful gene editing will block transmission to these model systems. Cryo-electron microscopy has been used to resolve the structures of α- syn fibrils in MSA patient samples. This work has shown that misfolded α-syn adopts a Greek key conformation that is stabilized by a salt bridge between residues E46 and K80. In Aim 2, we will determine if our non-pathogenic K80 mutations exert their protective effectives by preventing salt bridge formation. We will also quantify the effect of these mutations on lipid binding and protein fibrillization. These orthogonal studies will determine if the K80 mutations are a viable clinical candidate for an MSA gene therapy. This work is innovative because it represents a paradigm-shift in how we approach gene therapies. Rather than focusing on correcting a disease-causing point mutation, we will establish proof-of-concept that gene therapy can be used to interfere with the self-templating disease mechanism underlying prion and prion-like neurodegenerative disorders. This work is significant because it has the potential to serve as a novel treatment strategy for patients with both sporadic and familial prion-like diseases. Through investigating the ability of conversion- incompetent α-syn to prevent MSA propagation, this work has the potential to transform the way we approach therapeutic development for neurodegenerative disease patients.

多系统萎缩（MSA）是一种因折叠和自我错误折叠和自我的运动障碍 蛋白质α-突触核蛋白（α-Syn）的模板，该核蛋白（α-Syn）在整个中枢神经系统中扩散以引起 进行性变性。类似于许多其他风ch和prion样神经退行性疾病，有 目前尚无治疗方法可以改变MSA患者的疾病病程。干扰α-syn 自我庆祝，几个小组提出了各种策略，以将α-Syn表达击倒 减少可作为底物的蛋白质量。不幸的是，这些策略可能会干扰正常 大脑中的α-Syn功能，导致功能丧失定义了MSA患者。另外，MSA不能 在具有E46K突变表达α-Syn的转基因（TG）细胞或小鼠中繁殖的可能性 使用基因疗法产生conversion依的α-syn来破坏自我训练。但是，到目前为止，这个 方法尚未作为MSA的治疗干预进行测试。拟议工作的目的是 建立概念概念，表明在α-Syn主要序列中引入单个居住地变化可能会破坏 模板错误折叠。我们假设使用CRISPR Prime产生不合时宜的α-Syn 编辑将减少或防止MSA传播。我们的方法将利用我们最近的发现，即 病原α-Syn突变在居住K80的体外抑制MSA传播。在AIM 1中，我们将使用CRISPR 将我们的新型K80突变插入TG细胞和表达野生型人α-syn的小鼠的主要编辑 用MSA患者样品挑战模型。我们已经表明，MSA诱导α-Syn聚集 未经编辑的细胞和表达野生型蛋白的小鼠。我们预计成功的基因编辑将阻止 传输到这些模型系统。冷冻电子显微镜已用于解决α-的结构 MSA患者样品中的合成原纤维。这项工作表明，不折叠的α-syn采用希腊钥匙 残留E46和K80之间的盐桥稳定的构象。在AIM 2中，我们将确定是否 我们的非致病K80突变通过防止盐桥形成行使其受保护的作用。我们将 还量化了这些突变对脂质结合和蛋白质纤维化的影响。这些正交研究 将确定K80突变是否是MSA基因治疗的可行临床候选者。这项工作是 创新性是因为它代表了我们如何处理基因疗法的范式变化。而不是专注于 纠正引起疾病的点突变，我们将确定可以使用基因治疗的概念证明 干扰幼儿园和类似神经退行性的自我训练疾病机制 疾病。这项工作很重要，因为它有可能作为一种新颖的治疗策略 患有零星和家族性王室样疾病的患者。通过调查转换的能力 - 无能防止MSA传播的α-Syn，这项工作有可能改变我们接近的方式 神经退行性疾病患者的治疗发育。