Therapeutic Genome Editing for Amyotrophic Lateral Sclerosis with a Compact, Hyper-accurate, and Versatile Cas9

使用紧凑、超准确且多功能的 Cas9 对肌萎缩侧索硬化症进行治疗性基因组编辑

基本信息

批准号：
9760509
负责人：
Alireza - Edraki
金额：
$ 2.25万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2019-12-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9760509
关键词：
Adult Alleles Amyotrophic Lateral Sclerosis Binding Biological Markers C9ORF72 CRISPR/Cas technology Cell Line Cells DNA DNA Binding DNA Sequence Dependovirus Development Dipeptides Disease Progression Event Excision Exhibits Exons Facial vein structure Future Gene Abnormality Genes Genetic Genome Goals Guide RNA Human Impairment In Vitro Individual Inherited Injections Introns Knock-out Lead Life Expectancy Loss of Heterozygosity Mammalian Cell Measures Mediating Molecular Abnormality Motor Neuron Disease Motor Neurons Mus Muscular Atrophy Mutation Neisseria meningitidis Nervous System control Neuraxis Neurodegenerative Disorders Neurons Nucleotides Pathogenicity Pathologic Patients Pharmaceutical Preparations Phenotype Point Mutation Proteins Quality of life RNA Signal Transduction Site Specificity Streptococcus pyogenes Survival Rate Testing Therapeutic Therapeutic Agents Tissues Transgenic Mice Viral Weight Western Blotting Work adeno-associated viral vector axonopathy base deep sequencing design experience flexibility gain of function gain of function mutation gene therapy genome editing improved in vivo motor control motor neuron function mouse model mutant nervous system disorder neuron loss nuclease particle prevent reduced muscle mass side effect superoxide dismutase 1 symptom management therapeutic gene therapeutic genome editing therapy development tool vector

项目摘要

Project Summary Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease in which the average survival rate from onset is 2-4 years, and only 10% of individuals live past 10 years. There is currently no cure for ALS, and available medications do not significantly prolong survival. Certain forms of ALS arise from gene abnormalities, such as a dominant, gain-of-function point mutation in superoxide dismutase 1 (SOD1G93A) and an intronic repeat expansion in C9ORF72 (chromosome 9 open reading frame 72). These mutations ultimately lead to neuronal death, decreased muscle mass, and loss of motor control. Gene editing approaches that eliminate ALS-causing mutations may prevent disease progression and improve the quality of life of some patients. Therapeutic gene editing has become a possible reality with the advent of CRISPR-Cas9 editing, which uses guide RNA (gRNA) and a bacterial Cas9 nuclease to recognize a specific DNA sequence, create a double- stranded break, and inactivate a target gene. However, currently-available Cas9 proteins are too large for efficient in vivo delivery and often induce unwanted gene editing at off-target sites. The goal of this project is to develop efficient and safe Cas9-based gene editing approaches to treat genetic cases of ALS. These approaches will use a recently-characterized Cas9 from a strain of Neisseria meningitidis (Nme2Cas9). Nme2Cas9 is compact, so it can be packaged with gRNA into a single vector (adeno-associated virus, AAV) for in vivo delivery. It is also hyper-accurate, with negligible off-target editing. Most importantly, Nme2Cas9 targeting relies on a unique DNA binding signal with a flexible sequence motif that provides a larger selection of potential target sites in the genome than what is available for other Cas9s. Using the SOD1G93A mouse model of ALS, Aim 1 will determine whether Nme2Cas9 can specifically target the mutant allele of the SOD1G93A gene while leaving the wild-type allele unscathed. To test this, Nme2Cas9 and a gRNA targeting SOD1G93A will be packaged into an all-in-one AAV9 vector, which targets the central nervous system (CNS), and injected via facial vein into SOD1G93A mice. This approach should knockout mutant SOD1 and reduce ALS phenotypes without inducing potential side effects associated with loss of normal SOD1 function. Aim 2 will use C9BAC transgenic mice expressing C9ORF72 expanded repeats to determine whether Nme2Cas9 can excise the intronic expansion in C9ORF72 without breaching the boundaries of flanking exons. An all-in-one AAV9 vector containing Nme2Cas9 and two gRNAs targeting each end of the repeat will be delivered by facial vein injection into C9BAC mice. Nme2Cas9-mediated excision of C9ORF72 repeats should ameliorate pathogenic biomarkers of ALS without altering normal C9ORF72 expression. Completion of the proposed aims will improve the potential of Cas9-based gene editing approaches for ALS treatment, and will guide the future development of therapies for genetically-defined neurological disorders.

项目概要肌萎缩侧索硬化症 (ALS) 是一种进行性运动神经元疾病，其平均存活率从发病开始为2-4年，只有10%的人能活过10年。目前 ALS 尚无治愈方法，并且现有的药物并不能显着延长生存期。某些形式的 ALS 是由基因异常引起的，例如超氧化物歧化酶 1 (SOD1G93A) 的显性功能获得性点突变和内含子在 C9ORF72（9 号染色体开放阅读框 72）中重复扩增。这些突变最终导致神经元死亡、肌肉质量减少和运动控制丧失。基因编辑方法可消除引起 ALS 的突变可能会阻止疾病进展并改善某些患者的生活质量。随着 CRISPR-Cas9 编辑技术的出现，治疗性基因编辑已成为可能的现实。引导 RNA (gRNA) 和细菌 Cas9 核酸酶识别特定 DNA 序列，创建双链链断裂，并使目标基因失活。然而，目前可用的 Cas9 蛋白对于有效的体内传递并经常在脱靶位点诱导不需要的基因编辑。该项目的目标是开发高效、安全的基于Cas9的基因编辑方法来治疗遗传性疾病 ALS 病例。这些方法将使用最近鉴定的来自脑膜炎奈瑟菌菌株的 Cas9 （Nme2Cas9）。 Nme2Cas9 结构紧凑，因此可以与 gRNA 一起包装成单个载体（腺相关载体）病毒，AAV）用于体内递送。它也是超准确的，脱靶编辑可以忽略不计。最重要的是， Nme2Cas9 靶向依赖于独特的 DNA 结合信号和灵活的序列基序，可提供与其他 Cas9 相比，基因组中潜在靶位点的选择范围更大。使用 ALS 的 SOD1G93A 小鼠模型，Aim 1 将确定 Nme2Cas9 是否可以特异性靶向 SOD1G93A 基因的突变等位基因，而野生型等位基因毫发无伤。为了测试这一点，Nme2Cas9 和靶向 SOD1G93A 的 gRNA 将被包装成一体式 AAV9 载体，该载体靶向中枢神经系统（CNS），并通过面静脉注射到 SOD1G93A 小鼠体内。这种方法应该敲除突变体 SOD1 并减少 ALS 表型，而不引起与正常 SOD1 丧失相关的潜在副作用功能。目标 2 将使用表达 C9ORF72 扩展重复序列的 C9BAC 转基因小鼠来确定是否 Nme2Cas9 可以切除 C9ORF72 中的内含子扩展，而不会破坏侧翼外显子的边界。包含 Nme2Cas9 和两个靶向重复序列两端的 gRNA 的一体化 AAV9 载体将是通过面静脉注射至 C9BAC 小鼠体内。 Nme2Cas9 介导的 C9ORF72 重复序列切除应该改善 ALS 的致病生物标志物而不改变正常的 C9ORF72 表达。完成拟议的目标将提高基于 Cas9 的基因编辑方法治疗 ALS 的潜力，并将指导遗传性神经系统疾病疗法的未来发展。