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.

项目摘要肌萎缩性侧索硬化症（ALS）是一种进行性运动神经元疾病，其中平均存活率从发病开始，只有2 - 4年，只有10％的人生活了10年。目前尚无ALS治疗，并且可用的药物不会显着延长生存率。某些形式的ALS来自基因异常，例如，超氧化物歧化酶1（SOD1G93A）中的主要功能点突变和内含子在C9orf72中重复扩展（9号染色体开放阅读框72）。这些突变最终导致神经元死亡，肌肉质量降低和运动控制丧失。消除的基因编辑方法引起ALS的突变可以防止疾病进展并改善某些患者的生活质量。随着CRISPR-CAS9编辑的出现，治疗基因编辑已成为现实引导RNA（GRNA）和细菌cas9核酸酶以识别特定的DNA序列，创建双重滞留的断裂，并使靶基因失活。但是，当前可用的cas9蛋白太大了有效的体内递送，通常会在脱离靶向位点诱导不需要的基因编辑。该项目的目的是开发高效且安全的基于CAS9的基因编辑方法来治疗遗传 ALS案件。这些方法将使用近代脑膜炎菌株的最近特征的cas9 （NME2CAS9）。 NME2CAS9是紧凑的，因此可以将GRNA包装到单个矢量中（与腺相关病毒，AAV）用于体内输送。它也是超准确的，具有可忽略的脱靶编辑。最重要的是， NME2CAS9的靶向依赖于具有柔性序列基序的独特DNA结合信号，该基序提供了基因组中潜在的目标位点的选择要比其他CAS9可用。使用ALS的SOD1G93A鼠标模型，AIM 1将确定NME2CAS9是否可以专门针对 SOD1G93A基因的突变等位基因毫发无损地离开野生型等位基因。为了测试这一点，NME2CAS9和A 靶向SOD1G93A的GRNA将被包装到一个靶向中枢神经的一为aav9载体中系统（CNS），并通过面部静脉注入SOD1G93A小鼠。这种方法应敲除突变体SOD1 并减少ALS表型，而不会引起与正常SOD1丢失相关的潜在副作用功能。 AIM 2将使用表达C9orf72的C9BAC转基因小鼠扩展重复序列，以确定是否是否 NME2CAS9可以在不突破侧面外显子边界的情况下对C9orf72中的内含子扩张进行切除。一个包含NME2CAS9和两个靶向重复端的GRNA的多合一AAV9向量将是通过面部静脉注射到C9BAC小鼠中。 NME2CAS9介导的C9orf72重复的切除应改善ALS的致病生物标志物，而不会改变正常的C9ORF72表达。完成拟议的目标将提高基于CAS9的基因编辑方法对ALS治疗的潜力，并将指导遗传定义神经系统疾病的疗法的未来发展。