Silencing C9or72 with rAAV Mediated RNAi

描述（由申请人提供）：肌萎缩性侧索硬化症（ALS）是一种进行性，不可治疗的，一致致命的运动神经元疾病，有时与额颞叶痴呆（FTD）并发。ALS有散发性（SALS）和家族性（FALS）两种形式；约10%的病例以常染色体显性性状传播。散发性肌萎缩性侧索硬化症的病因尚不清楚。最近发现，约30-50%的FALS病例是由基因C9ORF72中非编码六核苷酸G4C2扩增引起的。在10-20%的家族性FTD， 10%的散发性FTD和~5%的SALS中也检测到这些扩张。这些统计数据将C9ORF72 G4C2扩展定义为ALS的最常见原因。在本研究中，我们建议使用rAAV型Rh10引入一个microRNA来沉默含有违规G4C2扩增的C9ORF72转录本的表达。在Aim 1中，我们将筛选、鉴定和优化体外潜在的mirna。在Aim 2中，我们将进一步表征我们的新型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，从而减轻该小鼠的表型。在Aim 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：我们相信这些研究将是高度相关的几个原因。(1)对有效治疗ALS的迫切需求尚未得到满足；本项目侧重于最常见的FALS形式，也适用于SALS和FTD的某些情况。(2)本研究将开发鞘内rAAVRh10作为中枢神经系统的基因治疗载体；鞘内途径是有利的，允许在中枢神经系统内广泛给药，剂量比静脉给药所需的剂量低一个数量级；尽量减少周边接触病毒的机会。(3) rAAV尚未用于人类神经退行性疾病。我们建议在这里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.