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

项目摘要 肌萎缩侧索硬化症（ALS）是一种进行性运动神经元疾病， 从发病开始是2-4年，只有10%的人活了10年以上。目前还没有治愈ALS的方法， 现有的药物不能显著延长生存期。某些形式的ALS是由基因异常引起的， 如超氧化物歧化酶1（SOD 1G 93 A）中显性功能获得点突变和内含子 C9 ORF 72（9号染色体开放阅读框72）中的重复扩增。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。基因编辑方法可以消除 导致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编辑的出现，治疗性基因编辑已经成为可能的现实， 引导RNA（gRNA）和细菌Cas9核酸酶识别特定的DNA序列，产生双- 链断裂，和DNA靶基因。然而，目前可用的Cas9蛋白太大， 有效的体内递送，并且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有效和安全的基于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表征的来自脑膜炎奈瑟菌菌株的Cas9 （Nme2Cas9）。Nme 2Cas 9是紧凑的，因此它可以与gRNA一起包装到单个载体（腺相关病毒载体）中。 病毒，AAV）用于体内递送。它也是超准确的，可以忽略不计的脱靶编辑。最重要的是， Nme 2Cas 9靶向依赖于具有灵活序列基序的独特DNA结合信号，该序列基序提供了一种新的靶向机制。 基因组中潜在靶位点的选择比其他Cas9可用的更多。 使用ALS的S 0 D1 G93 A小鼠模型，Aim 1将确定Nme 2Cas 9是否可以特异性靶向ALS小鼠。 SOD 1G 93 A基因的突变等位基因，而使野生型等位基因不受影响。为了测试这一点，Nme 2Cas 9和一个 靶向SOD 1G 93 A的gRNA将被包装到靶向中枢神经系统的全合一AAV 9载体中。 系统（CNS），并通过面部静脉注射到SOD 1G 93 A小鼠中。这种方法可以敲除突变的SOD 1 并减少ALS表型而不诱导与正常SOD 1丧失相关的潜在副作用 功能目的2将使用表达C9 ORF 72扩增重复序列的C9 BAC转基因小鼠来确定是否 Nme 2Cas 9可以切除C9 ORF 72中的内含子扩增而不破坏侧翼外显子的边界。 包含Nme 2Cas 9和靶向重复序列的每个末端的两个gRNA的一体化AAV 9载体将被构建。 通过面部静脉注射递送到C9 BAC小鼠中。Nme 2Cas 9介导的C9 ORF 72重复序列的切除应 改善ALS的致病生物标志物而不改变正常C9 ORF 72表达。完成 提出的目标将提高基于Cas9的基因编辑方法治疗ALS的潜力，并将 指导未来开发遗传学定义的神经系统疾病的治疗方法。

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.