Development of a novel therapeutic strategy for treatment of SOD1-linked ALS by CRISPR/Cas9-mediated SOD1 promoter editing

肌萎缩侧索硬化症（amyotrophiclateralsclerosis，ALS）是一种由脑内运动神经元变性引起的致死性疾病 和脊髓，通常导致在发病后五年内死亡。虽然阿舒唑和依达拉奉 已经被批准用于治疗ALS，但它们的治疗功效似乎非常有限， 目前还没有有效的治疗方法。SOD 1突变与约20%的家族性和约1%的 散发性ALS患者。先前的研究表明突变的SOD 1导致运动神经元 通过“功能获得”机制来抑制退化。在过去的二十年里，各种治疗方法 通过小干扰RNA、短发夹RNA、microRNA、反义寡核苷酸和寡核苷酸来降低SOD 1水平的方法 已经在SOD 1-ALS小鼠模型中测试了寡核苷酸或小分子， 观察到的效果。其中一些方法目前正在临床试验中进行测试。然而， 这些候选药物降低脑脊液（CSF）中SOD 1的作用似乎有限（约10%）， 来自这些临床试验的初步数据未能显示出显著的治疗益处。基于这个理由， 需要在临床前研究中探索和测试替代和更有效的策略。以SOD 1为目标 更有效地，我们设计了一种转基因策略，使用 CRISPR-Cas9.我们发现，CRISPR-Cas9介导的SOD 1-exon 2编辑阻止了一种免疫缺陷病毒的发展。 两种SOD 1-ALS小鼠模型的临床ALS表型和病理学。所有SOD 1-G93 A转基因拷贝 被有效地编辑和灭活。虽然非常有效，但这种编码序列编辑可能会有一些 安全性问题，因为完全丧失SOD 1可能导致进行性痉挛性四肢瘫痪和轴性肌张力减退。 最佳的治疗策略将是有效地去除大部分SOD 1蛋白，以达到显著的治疗效果。 治疗效果，同时保持一小部分功能性SOD 1，以避免引起的不良反应 完全丧失了。为了实现这一目标，我们设计了一个策略来定位/编辑TATA框， 使用CRISPR-Cas9分析人SOD 1（hSOD 1）基因的启动子元件。我们在体外测试了这种策略， 细胞培养系统，我们发现TATA框编辑的等位基因丢失了超过70%的hSOD 1蛋白。在这 应用，我们提出了三个具体的目标，以测试这种TATA框编辑策略在两个SOD 1-ALS小鼠 模型在目标1中，我们将开发hSOD 1-TATA-Cas9单和hSOD 1-TATA-Cas9/SOD 1-G93 A双 转基因小鼠在目标2中，我们将表征TATA框编辑的SOD 1-ALS的表型和病理学。 小鼠在目标3中，我们将研究TATA框编辑的SOD 1-ALS小鼠和小鼠中的效率和安全性。 人体细胞启动子编辑代表了SOD 1-ALS的一种新的和更优化的治疗策略。如果 概念被证明，类似的策略可以适用于广泛的疾病，只要核心 相关基因的启动子元件，如TATA盒、CAT盒、GC盒等关键调控元件 可以鉴定启动子中的元件，并且可以获得合适的PAM。

Amyotrophic lateral sclerosis (ALS) is a fatal disorder caused by the degeneration of motor neurons in the brain and spinal cord, usually resulting in death within five years after disease onset. Although Riluzole and Edaravone have been approved for the treatment of ALS, their therapeutic efficacies appear to be very limited, and more effective therapies are not currently available. Mutations of SOD1 have been linked to ~20% of familial and ~1% of sporadic ALS patients. Previous studies have demonstrated that mutant SOD1 causes motor neuron degeneration through a “gain-of-function” mechanism. Over the past two decades, a variety of therapeutic approaches to decrease SOD1 levels by small interfering RNA, short hairpin RNA, microRNA, antisense oligonucleotides or small molecules have been tested in SOD1-ALS mouse models, with a variety of therapeutic effects observed. Some of these approaches are now being tested in clinical trials. However, the efficacy of these candidate drugs in reducing SOD1 in the cerebrospinal fluid (CSF) appeared to be limited (~10%), and preliminary data from these clinical trials failed to show significant therapeutic benefits. For this reason, alternative and more effective strategies need to be explored and tested in preclinical studies. To target SOD1 more effectively, we designed a transgenic strategy to edit the human SOD1 coding sequence (exon2) using CRISPR-Cas9. We found that CRISPR-Cas9-mediated editing of SOD1-exon2 prevented the development of a clinical ALS phenotype and pathology in two SOD1-ALS mouse models. All of the SOD1-G93A transgene copies were effectively edited and inactivated. Although very effective, this coding sequence editing may have some safety concerns, as a complete loss of SOD1 may lead to progressive spastic tetraplegia and axial hypotonia. An optimal therapeutic strategy would be to effectively remove the majority of SOD1 protein to reach significant therapeutic efficacy, while maintaining a small fraction of the functional SOD1 to avoid adverse effects caused by its complete loss. To reach this goal, we designed a strategy to target/edit the TATA box, the major core promoter element of the human SOD1 (hSOD1) gene using CRISPR-Cas9. We tested this strategy in vitro using a cell culture system, and we found that the TATA box-edited alleles lost over 70% of hSOD1 protein. In this application, we propose three specific aims to test this TATA box editing strategy in two SOD1-ALS mouse models. In aim 1, we will develop the hSOD1-TATA-Cas9 single and hSOD1-TATA-Cas9/SOD1-G93A double transgenic mice. In aim 2, we will characterize the phenotype and pathology of the TATA box-edited SOD1-ALS mice. In aim 3, we will study the efficiency and safety profiles in the TATA box-edited SOD1-ALS mice and in human cells. Promoter editing represents a novel and more optimal therapeutic strategy for SOD1-ALS. If the concept is proven, a similar strategy may be adapted to a broad spectrum of diseases, as long as the core promoter elements of the relevant genes, such as TATA boxes, CAT boxes, GC boxes and other key regulatory elements in the promoters could be identified and appropriate PAMs are available.