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

通过 CRISPR/Cas9 介导的 SOD1 启动子编辑开发治疗 SOD1 相关 ALS 的新治疗策略

• 批准号: 10172990
• 负责人: Han-Xiang Deng
• 金额: $ 37.48万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172990
• 关键词: 2 year old; ALS patients; Adverse effects; Alleles; Amyotrophic Lateral Sclerosis; Antisense Oligonucleotides; Brain; CRISPR/Cas technology; Cell Culture System; Cells; Cerebrospinal Fluid; Cessation of life; Chromosome 21; Clinical; Clinical Data; Clinical Research; Clinical Trials; Code; Cysteine; Data; Development; Disease; Elements; Event; Genes; Genetic Transcription; Goals; Guide RNA; Human; Human Cell Line; Human Genome; In Vitro; Lead; Link; Maps; Mediating; Messenger RNA; Methods; MicroRNAs; Minor; Modeling; Monitor; Morphology; Motor; Mus; Muscle Fibers; Muscle hypotonia; Mutation; Neuroglia; Neurons; Onset of illness; Pathologic; Pathology; Phenotype; Promoter Regions; Proteins; Regulatory Element; Reporting; Research; Riluzole; Safety; Skeletal Muscle; Small Interfering RNA; Spastic Tetraplegia; Spinal Cord; Structure; TATA Box; Testing; Therapeutic; Therapeutic Effect; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Treatment Efficacy; Work; amyotrophic lateral sclerosis therapy; base; design; disulfide bond; drug candidate; effective therapy; familial amyotrophic lateral sclerosis; gain of function; motor neuron degeneration; mouse model; mutant; novel; novel strategies; novel therapeutic intervention; oxidation; phenylmethylpyrazolone; preclinical study; prevent; promoter; protein aggregation; protein expression; small hairpin RNA; small molecule; sporadic amyotrophic lateral sclerosis; superoxide dismutase 1; therapeutic development; treatment strategy

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.

肌萎缩侧索硬化症(ALS)是一种由大脑运动神经元变性引起的致命疾病 和脊髓，通常在发病后五年内死亡。虽然利鲁唑和依达拉奉 已经被批准用于治疗肌萎缩侧索硬化症，但它们的治疗效果似乎非常有限，而且 目前还没有有效的治疗方法。SOD1突变与约20%的家族性疾病和~1%的家族性疾病有关 散发性肌萎缩侧索硬化症患者。先前的研究表明，突变的SOD1导致运动神经元 通过“功能增益”机制退化。在过去的二十年里，各种治疗方法 通过小干扰RNA、短发夹状RNA、微RNA、反义RNA降低SOD1水平的途径 寡核苷酸或小分子已经在SOD1-ALS小鼠模型中进行了测试，具有多种治疗方法 观察到效果。其中一些方法现在正在进行临床试验。然而，这种药物的功效 这些候选药物在降低脑脊液(CSF)中的SOD1方面似乎有限(~10%)，并且 这些临床试验的初步数据未能显示出显著的治疗效果。因为这个原因， 需要在临床前研究中探索和测试替代和更有效的策略。要以SOD1为目标 更有效地，我们设计了一种转基因策略来编辑人类SOD1编码序列(外显子2)，使用 CRISPR-CAS9。我们发现CRISPR-Cas9介导的SOD1-外显子2的编辑阻止了A 两种SOD1-ALS小鼠模型的临床ALS表型和病理。所有SOD1-G93A转基因拷贝 被有效地编辑和停用。虽然非常有效，但这种编码序列编辑可能会有一些 安全性方面的考虑，因为SOD1的完全丧失可能导致进行性痉挛四肢瘫痪和轴性低张力。 最佳的治疗策略将是有效地去除大部分SOD1蛋白以达到显著的 治疗效果，同时保持一小部分功能性SOD1，以避免引起的不良反应 因为它完全失去了。为了实现这一目标，我们设计了一种策略来定位/编辑主要核心Tata Box 用CRISPR-Cas9分析人SOD1(HSOD1)基因的启动子元件。我们在体外测试了这一策略 一个细胞培养系统，我们发现TATA盒编辑的等位基因丢失了70%以上的hSOD1蛋白。在这 应用程序，我们提出了三个具体目标来在两个SOD1-ALS小鼠上测试该TATA框编辑策略 模特们。在目标1中，我们将开发hSOD1-TATA-Cas9单项和hSOD1-TATA-Cas9/SOD1-G93A双项 转基因小鼠。在目标2中，我们将描述TATA盒编辑的SOD1-ALS的表型和病理特征 老鼠。在目标3中，我们将研究在TATA框编辑的SOD1-ALS小鼠和在TATA框编辑的SOD1-ALS小鼠中的有效性和安全性 人类细胞。启动子编辑为SOD1-ALS提供了一种新的、更理想的治疗策略。如果 概念是证明的，类似的策略可能适用于广泛的疾病，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 可以确定发起人中的元素，并且可以获得适当的PAM。