Silencing C9or72 with rAAV Mediated RNAi

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种进行性的、无法治疗的、一致致命的运动神经元疾病，有时与额颞部痴呆(FTD)并发发展。ALS既有散发性(SALS)，也有家族性(FALS)；大约10%的病例是以常染色体显性遗传特征传播的。散发性肌萎缩侧索硬化症的原因尚不清楚。最近发现，大约30%-50%的FAL病例是由C9ORF72基因中非编码六核苷酸G4C2的扩展引起的。这些扩张也可见于10-20%的家族性FTD、10%的散发性FTD和~5%的SAL。这些统计数据将C9ORF72 G4C2扩展定义为ALS最常见的原因。在本研究中，我们建议使用rAAV型RH10来引入microRNA，以沉默含有有害的G4C2扩展的C9ORF72转录本的表达。在目标1中，我们将在体外筛选、鉴定和优化潜在的miRNAs。在目标2中，我们将进一步鉴定我们的新型C9ORF72突变转基因小鼠的表型，并研究使用rAAVRh10-C9miRs来沉默突变的C9转基因基因，从而减轻该小鼠的表型。在目标3中，我们将研究rAAVRh10-C9miRs在非人灵长类动物模型中的传递和疗效，作为将该疗法转化为临床应用的第一步。相关性：我们认为这些研究具有很高的相关性，原因有几个。(1)对有效的肌萎缩侧索硬化症的治疗存在迫切的未得到满足的需求；本项目侧重于最常见的FALS形式，也适用于SALS和FTD的一些病例。(2)这项研究将开发鞘内rAAVRh10作为中枢神经系统的基因治疗载体的应用；鞘内途径是有利的，允许在中枢神经系统内广泛传递，剂量低于静脉注射所需剂量的数量级；并最大限度地减少外周接触病毒。(3)rAAV尚未用于人类神经退行性疾病。我们在这里建议开发的平台应该在广泛的神经障碍领域具有广泛的适用性。

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.