Silencing C9or72 with rAAV Mediated RNAi

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种进行性、不可治疗、一致致死的运动神经元疾病，有时与额颞叶痴呆（FTD）同时发生。ALS以散发（SALS）和家族（FALS）形式出现;约10%的病例以常染色体显性遗传特征传播。散发性ALS的原因尚不清楚。最近发现，约30-50%的FALS病例是由基因C9 ORF 72中非编码六核苷酸G4 C2扩增引起的。在10-20%的家族性FTD、10%的散发性FTD和约5%的SALS中也检测到这些扩增。这些统计数据将C9 ORF 72 G4 C2扩增定义为ALS最常见的原因。 在本研究中，我们建议使用rAAV型Rh 10引入microRNA沉默的C9 ORF 72的转录本的表达，窝藏冒犯G4 C2的扩展。在目标1中，我们将在体外筛选、鉴定和优化潜在的miRNA。在目的2中，我们将进一步表征我们的新型C9 ORF 72突变转基因小鼠的表型，并研究使用rAAVRh 10-C9 miR沉默突变C9转基因，从而减轻该小鼠的表型。在目的3中，我们将研究rAAVRhlO-C9 miR在非人灵长类动物模型中的递送和功效，作为将该疗法转化为临床应用的第一步。相关性：我们认为，这些研究将是高度相关的几个原因。(1)有效的ALS治疗存在迫切的未满足需求;该项目侧重于最常见形式的FALS，适用于SALS和FTD的某些病例。(2)本研究将开发使用鞘内rAAVRh 10作为CNS的基因治疗载体;鞘内途径是有利的，允许在CNS内广泛递送，其剂量比通过静脉内递送所需的剂量低一个数量级;并最大限度地减少外周暴露于病毒。(3)rAAV尚未用于人类神经退行性疾病。我们在这里提出开发的平台应该在广泛的神经系统疾病中具有广泛的适用性。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a progressive, untreatable, uniformly fatal motor neuron disorder that sometimes develops concurrently with frontotemporal dementia (FTD). ALS is encountered in both sporadic (SALS) and familial (FALS) forms; about 10% of cases are transmitted as autosomal dominant traits. The cause of sporadic ALS is not known. Recently it was discovered that about 30-50% of FALS cases are caused by expansions of a non-coding hexanucleotide G4C2 expansion in the gene C9ORF72. These expansions are also detected in 10-20% of familial FTD, 10% of sporadic FTD and in ~5% of SALS. These statistics define the C9ORF72 G4C2 expansion as the most common cause of ALS. In the present study, we propose to use rAAV type Rh10 to introduce a microRNA to silence expression of the transcripts of C9ORF72 that harbor the offending G4C2 expansion. In Aim 1, we will screen, identify and optimize potential miRNAs in vitro. In Aim 2, we will further characterize the phenotype of our novel C9ORF72mutant transgenic mouse and investigate use of rAAVRh10-C9miRs to silencing the mutant C9 transgene and thereby mitigate the phenotype in this mouse. In Aim 3 we will investigate delivery and efficacy of the rAAVRh10-C9miRs in a non- human primate model as first step to translating this therapy to clinical application. Relevance: We believe that these studies will be highly relevant for several reasons. (1) There is a compelling unmet need for effective ALS treatments; this project focuses on the most common form of FALS, with applicability as well to some cases of SALS and FTD. (2) This investigation will develop the use of intrathecal rAAVRh10 as a gene therapy vector for the CNS; the intrathecal route is advantageous, permitting widespread delivery within the CNS with doses that are an order-of-magnitude lower than are required via intravenous delivery; and minimizing peripheral exposure to virus. (3) rAAV has not been used in human neurodegenerative disorders. The platform we propose to develop here should have broad applicability across a breadth of neurological disorders.