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

多系统萎缩（MSA）是一种朊病毒样运动障碍，由错误折叠和自我修复引起。 蛋白质α-突触核蛋白（α-syn）的模板，它遍布中枢神经系统， 进行性退化与许多其他朊病毒和朊病毒样神经退行性疾病相似， 目前没有可用于改变MSA患者的病程的治疗剂。干扰α-syn 自模板，几个小组提出了各种策略敲低α-syn表达， 减少可用作底物的蛋白质的量。不幸的是，这些策略可能会干扰正常的 大脑中的α-syn功能，导致MSA患者的功能缺失。或者，MSA不能 在表达具有E46 K突变的α-syn的转基因（Tg）细胞或小鼠中繁殖， 使用基因疗法来产生不能转化的α-syn来破坏自我模板。然而，到目前为止，这 该方法尚未被测试为MSA的治疗干预。拟议工作的目标是 建立概念验证，即在α-syn一级序列中引入单个残基变化可以破坏 模板错误折叠我们假设使用CRISPR引物产生转化不合格的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，非- 在残基K80处的致病性α-syn突变在体外抑制MSA增殖。在目标1中，我们将使用CRISPR 引物编辑将我们的新型K80突变插入Tg细胞和表达野生型人α-syn prior的小鼠中 用MSA患者样本挑战模型我们已经表明，MSA诱导α-syn聚集， 未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。低温电子显微镜已被用来解决结构的α- MSA患者样品中的顺式纤维。这项工作表明，错误折叠的α-syn采用希腊密钥 通过残基E46和K80之间的盐桥稳定的构象。在目标2中，我们将确定 我们的非致病性K80突变通过防止盐桥形成而发挥其保护作用。我们将 还量化了这些突变对脂质结合和蛋白质固定化的影响。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，因为它代表了我们如何对待基因疗法的范式转变。而不是专注于 纠正致病的点突变，我们将建立概念验证，基因治疗可以用于 干扰朊病毒和朊病毒样神经退行性疾病的自我模板机制 紊乱这项工作是重要的，因为它有可能作为一种新的治疗策略， 散发性和家族性朊病毒样疾病患者。通过对转化能力的考察- 不称职的α-syn防止MSA传播，这项工作有可能改变我们的方法 神经退行性疾病患者的治疗开发。

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.