Optimization of an in vivo base editing strategy to treat SOD1-linked ALS

项目总结 肌萎缩侧索硬化症(ALS)是一种进展迅速、瘫痪并最终致命的疾病。 由脊髓和大脑中运动神经元的选择性丧失引起。这个应用程序的首要目标是 Create Bio计划的优化轨道是提炼基因治疗的安全性和有效性 我们为由超氧化物歧化酶1的毒性、功能获得突变引起的各种形式的肌萎缩侧索硬化症开发的 (SOD1)，占该疾病所有家族性病例的20%。具体地说，我们开发了一种 利用CRISPR碱基编辑在体内灭活突变的SOD1蛋白的生产的方法，一个基因- 能够在DNA中引入精确的碱基替换的编辑方式，但不需要 突变DNA断裂，从而克服了传统基因实施面临的一大安全障碍-- 编辑核酸酶。特别是，当通过腺相关病毒传递到脊髓时，我们的SOD1- 靶向碱基编辑平台延长了存活时间，显著减缓了疾病的进展 SOD1连锁的进攻性ALS小鼠模型。 重要的是，与目前沉默SOD1的策略不同，SOD1以SOD1 mRNA为靶标，并可能具有 需要终生修复或有可能使内源RNA处理饱和的瞬时效应 途径，这可能会导致不良影响，我们的方法利用高度精确的DNA编辑 永久关闭突变SOD1生产的途径，只涉及一种治疗方法。因此， 由于其优势和体内疗效，我们现在的目标是改进这一策略，以实现以下最终目标 为肌萎缩侧索硬化症开发基因疗法。具体地说，通过优化其目标针对性、编辑能力、 药代动力学及其安全性，我们将通过引入自失活功能来确保这一点 为了方便其从单元格中清除，我们将开发一个高度优化的针对SOD1的CRISPR碱基编辑 该平台可用于永久有效地治疗SOD1相关的肌萎缩侧索硬化症。因此，通过利用：(1)a 高度创新的DNA编辑技术，能够克服基因沉默的限制 和(2)多学科研究团队，在肌萎缩侧索硬化症、AAV传递、基因编辑和 免疫学，这个项目不仅将导致ALS的优化治疗候选，毁灭性的， 衰弱和目前无法治愈的疾病几乎没有有效的治疗选择，但也为 使用基础编辑来安全地治疗神经疾病。

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic and ultimately fatal disease characterized by the selective loss of motor neurons in the spinal cord and brain. The overarching objective of this application to the Optimization Track of the CREATE Bio Program is to refine the safety and efficacy of a gene therapy that we developed for forms of ALS caused by toxic, gain-of-function mutations in superoxide dismutase 1 (SOD1), which account for up to 20% of all familial cases of the disease. Specifically, we have developed an approach to inactivate the production of the mutant SOD1 protein in vivo using CRISPR base editing, a gene- editing modality capable of introducing precise base substitutions in DNA, but without the requirement for a mutagenic DNA break, thereby overcoming a major safety hurdle facing the implementation of traditional gene- editing nucleases. In particular, when delivered to the spinal cord via adeno-associated virus, our SOD1- targeting base-editing platform prolonged survival and markedly slowed the progression of disease in a highly aggressive mouse model of SOD1-linked ALS. Importantly, as opposed to current strategies for silencing SOD1, which target SOD1 mRNA and can have a transient effect that requires a lifetime of redosing or can risk saturating endogenous RNA processing pathways, which could then lead to adverse effects, our approach harnesses a highly precise DNA editing pathway to permanently turn-off the production of mutant SOD1 and involves only a single treatment. Thus, because of its strengths and in vivo efficacy, we now aim to refine this strategy for the ultimate goal of developing a gene therapy for ALS. Specifically, by optimizing its targeting specificity, its editing capabilities, its pharmacokinetics and its safety, which we will ensure via the introduction of a self-inactivating functionality that facilitates its clearance from cells, we will develop a highly optimized SOD1-targeting CRISPR base editing platform that can be used to permanently and effectively treat SOD1-linked ALS. Thus, by capitalizing on: (1) a highly innovative DNA editing technology that has the capabilities to overcome the limitations of gene-silencing and (2) a multidisciplinary research team with complementary expertise in ALS, AAV delivery, gene-editing and immunology, this project will result in not only an optimized therapeutic candidate for ALS, a devastating, debilitating and currently incurable disorder with few effective treatment options, but also lay the foundation for using base editing to safely treat neurological disorders.