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

项目总结 肌萎缩侧索硬化症(ALS)是一种进展迅速的瘫痪疾病，其特征是选择性 脊髓和大脑中运动神经元的丧失。虽然大多数肌萎缩侧索硬化症病例是散发性的、毒性的功能获得 在所有遗传性疾病中，约20%是由超氧化物歧化酶1(SOD1)基因突变引起的。 鉴于其在ALS中的致病作用，反义寡核苷酸(ASO)和RNA干扰(RNAi)已被 用于沉默突变的SOD1蛋白的表达。然而，由于其短暂的生命周期，ASOS 将需要终生昂贵的侵入性给药，而RNAi容易导致非靶向效应。 相反，虽然基因编辑技术，如CRISPR-Cas9，可以用来在基因上 失活突变体SOD1，这些基因治疗策略的实施可能被证明是具有挑战性的，因为 DNA编辑可以引入非靶标突变，并无意中创建新的突变型SOD1蛋白质变体 会危及他们的安全。因此，仍然迫切需要安全有效的治疗方法。 降低肌萎缩侧索硬化症的SOD1值。 一种替代技术，在细胞内诱导DNA损伤的风险很小，但仍可用于 有效降低SOD1的是RNA靶向CRISPR-Cas13效应器。CRISPR-CAS13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 诱导遗传毒性，因为它们不能切割DNA。此外，Cas13蛋白显示出良好的 与基因沉默技术相比，许多技术都足够紧凑，可以容纳在一个腺体中- 相关病毒(AAV)载体，一种临床上有希望的基因载体，可以介导长期的细胞型 神经系统中的特定基因表达。因此，CRISPR-CAS13有可能安全和 只给一次基因工程病毒载体后，持续沉默突变的SOD1。然而， 目前尚不清楚是否可以利用Cas13在体内减少SOD1并治疗该病。 这项提议的首要目标是开发一种治疗ALS的基因疗法。具体来说，我们建议 利用CRISPR-Cas13d技术在体内降低突变的SOD1用于治疗SOD1连锁的ALS。在支持中 对于这一目标的可行性，我们的初步研究表明，Cas13蛋白更具活性 而且比临床前有希望的shRNA更具特异性，它们可以由AAV9高效地输送到 脊髓星形胶质细胞，它们可以有效地降低整个脊髓中突变的SOD1蛋白，并且 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) 目的是在ALS小鼠模型上测试其有效性(特异性目标2)，并确定其作为基因的安全性 治疗剂(特效靶3)。因此，通过利用一种创新的转录工程技术， 为了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，一种使人虚弱的 而且目前无法治愈的疾病几乎没有有效的治疗选择。

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.