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

基本信息

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
项目类别：
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

项目摘要

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.

肌萎缩性侧索硬化症（ALS）是由大脑中运动神经元变性引起的致命疾病和脊髓，通常在疾病发作后五年内导致死亡。虽然Riluzole和Edaravone 已被批准用于治疗ALS，其治疗效率的外观非常有限，并且更多有效的疗法目前尚无可用。 SOD1的突变与约20％的家庭有关，约1％零星ALS患者。先前的研究表明，突变体SOD1引起运动神经元通过“功能获得”机制的变性。在过去的二十年中，各种治疗通过小干扰RNA，短发夹RNA，microRNA，反义降低SOD1水平的方法寡核苷酸或小分子已经在SOD1-ALS小鼠模型中进行了测试，并具有多种治疗性观察到的效果。现在，其中一些方法正在临床试验中进行测试。但是，有效性这些候选药物在减少脑脊液（CSF）中的SOD1方面似乎受到限制（〜10％），并且这些临床试验的初步数据未能显示出明显的治疗益处。为此原因，在临床前研究中需要探索和测试替代性和更有效的策略。靶标SOD1 更有效地，我们设计了一种转基因策略来使用使用人类SOD1编码序列（Exon2）编辑 CRISPR-CAS9。我们发现CRISPR-CAS9介导的SOD1-EXON2的编辑阻止了A的发展两个SOD1-AL小鼠模型中的临床ALS表型和病理。所有SOD1-G93A转换副本有效地编辑和灭活。尽管非常有效，但该编码序列编辑可能具有一些安全问题，因为SOD1的完全损失可能会导致进行性痉挛性四倍体和轴向肌张力低下。最佳疗法策略是有效去除大多数SOD1蛋白以达到重要治疗效率，同时保持一小部分功能SOD1以避免引起不良影响由于它的完全损失。为了实现这一目标，我们设计了一种策略来定位/编辑塔塔框，这是主要核心使用CRISPR-CAS9的人类SOD1（HSOD1）基因的启动子元素。我们使用一个细胞培养系统，我们发现塔塔盒编辑的等位基因损失了超过70％的HSOD1蛋白。在这个应用程序，我们提出了三个特定目的，以测试两种SOD1-ALS鼠标中的TATA盒编辑策略型号。在AIM 1中，我们将开发HSOD1-TATA-CAS9单和HSOD1-TATA-CAS9/SOD1-G93A双重转基因小鼠。在AIM 2中，我们将表征塔塔盒编辑的SOD1-ALS的表型和病理老鼠。在AIM 3中，我们将研究塔塔盒编辑的SOD1-ALS小鼠的效率和安全概况人类细胞。启动子编辑代表了SOD1-ALS的一种新颖且最佳的治疗策略。如果是概念已被证明，只要核心相关基因的启动子元素，例如tata盒，猫盒，GC盒和其他关键调节可以识别发起人中的元素并提供适当的PAM。