Development of a CRISPR-Cas13 Gene Therapy for SOD1-Linked ALS

项目摘要 肌萎缩侧索硬化症（ALS）是一种快速进展的麻痹性疾病，其特征是选择性的 脊髓和大脑中运动神经元的丧失。虽然大多数ALS病例是散发性的，但毒性功能获得性 超氧化物歧化酶1（SOD 1）的突变导致约20%的所有遗传形式的疾病。 鉴于其在ALS中的致病作用，反义寡核苷酸（ASO）和RNA干扰（RNAi）已经被广泛应用于治疗ALS。 用于沉默突变SOD 1蛋白的表达。然而，由于其短暂的生命周期， 将需要终生昂贵的侵入性给药，而RNAi易于诱导脱靶效应。 相反，虽然基因编辑技术，如CRISPR-Cas9，可以用于基因治疗， 由于突变的SOD 1，这些基因治疗策略的实施可能具有挑战性， DNA编辑器可以引入脱靶突变，并无意中产生新的突变SOD 1蛋白变体， 会危及他们的安全因此，仍然迫切需要能够安全有效地治疗癌症的疗法。 治疗ALS的SOD 1降低。 一种替代技术，几乎没有在细胞内诱导DNA损伤的风险，但仍然可以用于 有效降低SOD 1的是RNA靶向CRISPR-Cas 13效应子。CRISPR-Cas 13系统具有 DNA编辑CRISPR-Cas核酸酶的可编程性和多功能性特征，但对 因为它们不能切割DNA，所以诱导遗传毒性。此外，Cas 13蛋白表现出有利的 特异性相比，基因沉默技术和许多是紧凑的，足以适应在一个单一的腺， 相关病毒（AAV）载体，一种临床上有前途的基因递送载体，可以介导长期的细胞型逆转录病毒， 神经系统中的特定基因表达。因此，CRISPR-Cas 13有可能安全地和有效地治疗癌症。 在仅单次施用工程化病毒载体后持续沉默突变体SOD 1。然而，在这方面， Cas 13是否能在体内减少SOD 1并治疗该疾病仍是未知的。 该提案的总体目标是开发ALS的基因疗法。具体而言，我们建议 利用CRISPR-Cas 13 d技术降低体内突变的SOD 1，用于治疗SOD 1相关的ALS。支持 我们的初步研究表明，Cas 13蛋白比其他蛋白活性更高， 并且比临床前有希望的shRNA特异，它们可以通过AAV 9以高效率递送， 脊髓星形胶质细胞，它们可以有效地降低整个脊髓的突变SOD 1蛋白， 它们可以提供治疗益处。我们现在的目标是优化该平台的性能（具体目标1） 目的是测试其在ALS小鼠模型中的功效（具体目标2），并确定其作为基因的安全性 治疗剂（具体目标3）。因此，通过利用一种用于转录工程的创新技术， 可以克服传统基因沉默的局限性，我们将开发一种新的治疗ALS的方法， 目前无法治愈的疾病，几乎没有有效的治疗方案。

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic disorder characterized by the selective loss of motor neurons in the spinal cord and brain. While most cases of ALS are sporadic, toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) are responsible for ~20% of all inherited forms of the disease. Given its causative role in ALS, antisense oligonucleotides (ASOs) and RNA interference (RNAi) have been used to silence the expression of the mutant SOD1 protein. However, owing to their transient lifecycle, ASOs will require a lifetime of costly, invasive administrations, while RNAi is prone to inducing off-target effects. Conversely, while gene-editing technologies, such as CRISPR-Cas9, can be used be used to genetically inactivate mutant SOD1, the implementation of these strategies for gene therapy could prove challenging, as DNA editors can introduce off-target mutations and inadvertently create new, mutant SOD1 protein variants that can compromise their safety. Thus, there remains a crucial need for therapies that can safely and efficiently lower SOD1 for treatment of ALS. An alternative technology that holds little risk for inducing DNA damage within a cell but could still be used to efficiently lower SOD1 are RNA-targeting CRISPR-Cas13 effectors. CRISPR-Cas13 systems possess the programmability and versatility characteristic of DNA-editing CRISPR-Cas nucleases but pose limited risk for inducing genotoxicity since they are unable to cleave DNA. Moreover, Cas13 proteins display favorable specificity compared to gene silencing technologies and many are compact enough to fit within a single adeno- associated virus (AAV) vector, a clinically promising gene delivery vehicle that can mediate long-term, cell-type specific gene expression in the nervous system. Thus, CRISPR-Cas13 has the potential to safely and persistently silence mutant SOD1 following just a single administration of an engineered viral vector. However, it remains unknown whether Cas13 can be harnessed to reduce SOD1 in vivo and treat the disease. The overarching objective of this proposal is to develop a gene therapy for ALS. Specifically, we propose to harness CRISPR-Cas13d technology to lower mutant SOD1 in vivo for treatment of SOD1-linked ALS. In support of the feasibility of this objective, our preliminary studies have demonstrated that Cas13 proteins are more active and specific than a preclinically promising shRNA, that they can be delivered at high efficiencies by AAV9 to spinal cord astrocytes, that they can efficiently lower mutant SOD1 protein throughout the spinal cord, and that they can provide therapeutic benefit. We now aim to optimize the performance of this platform (Specific Aim 1) for the goal of testing its efficacy in mouse models of ALS (Specific Aim 2) and to determine its safety as a gene therapy agent (Specific Aim 3). Thus, by harnessing an innovative technology for transcriptional engineering that can overcome the limitations of traditional gene-silencing, we will develop a new therapy for ALS, a debilitating and currently incurable disorder with few effective treatment options.