Novel therapeutic strategies for motor neurone disease and frontotemporal dementia: moving towards gene therapy approaches

C9ORF72基因中六核苷酸重复序列的扩展是肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)最常见的已知原因，这是一种以神经元进行性死亡为特征的致命性成人脑部疾病，分别导致瘫痪和认知功能/人格特征改变。异常的C9ORF72重复转录产物是重复相关非AUG(RAN)翻译的底物，RAN翻译是一种非传统形式的翻译，产生在所有框架中都具有聚集特性的二肽重复蛋白(DPRs)，并且在缺乏规范起始密码子的情况下。越来越多的证据表明，在C9ORF72-ALS/FTD的细胞和动物模型中，dprs的积累是发病的主要驱动因素之一。因此，我们最近表明，降低dprs的表达水平可以在患者来源的运动神经元中提供神经保护，并抑制果蝇中与神经退行性相关的运动缺陷(Hautbergue.《自然通讯》2017年；8：16063)。值得注意的是，这些发现表明改变dprs的RAN翻译为体外和体内的神经保护提供了一种有效的治疗策略。为了支持这一点，研究人员已经申请了一项专利申请，将dprs拮抗剂用于通过基因治疗方法治疗C9ORF72-ALS/FTD(PCT/GB2017/051539)。C9ORF72重复转录本的RAN翻译涉及的机制仍然完全未知。因此，在没有目标的情况下，对RAN翻译进行治疗性操作是不可能的(S)。我们最近发现了两个与RAN翻译相关的因子，在这个提案中被命名为RTA1和RTA2。此外，这些因素中的任何一个的部分耗尽可以挽救DPR介导的神经细胞模型中的神经毒性和成年果蝇的神经退行性变相关的运动缺陷(未发表的数据)。目的：在这个PHD项目中，我们建议在临床前的C9ORF72-ALS/FTD小鼠模型中评估我们的基于腺相关病毒的基因治疗方法的新的神经保护策略的治疗效率。神经保护将在分子、神经元和运动功能水平上进行评估。目的：该研究项目基于多种前沿技术，包括分子和细胞生物学、生物信息学和动物研究，将为学生建立一个非常强大的课程。(1)产生编码RTA1-RNAi和RTA2-RNAi的腺相关病毒9(AAV9)盒。(2)利用微阵列技术验证AAV9病毒在C9ORF72-ALS/FTD神经细胞模型中的功能以及在全基因组水平上耗尽的特异性。(3)基因治疗计划将包括单剂脑池岩浆注射共表达绿色荧光蛋白(GFP)的AAV9病毒，以及在Ranum等团队建立的C9ORF72-ALS/FTD小鼠模型中进行对照-RNAi、RTA1-RNAi或RTA2-RNAi。注射的小鼠将被维持12个月，运动功能将通过转杆测试和足迹T台步分析进行监测。(4)组织学和分子生物学方法评估基因治疗方法在脊髓(颈/腰区)和脑(运动/非运动区)切片中的表达，以评估(I)DPR抗体检测dprs的表达；(Ii)Nissl和运动神经元标记物降钙素基因相关肽(CGRP)染色检测神经元数量；(Iii)qRT-PCR和Western blotting检测RTA1/2缺失情况。

Hexanucleotide repeat expansions in the C9ORF72 gene are the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a spectrum of fatal adult brain diseases characterised by the progressive death of neurons which respectively lead to paralysis and altered cognitive functions/ personality features.Abnormal C9ORF72 repeat transcripts are substrates of repeat-associated non-AUG (RAN) translation, an unconventional form of translation producing dipeptide repeat proteins (DPRs) with aggregating properties in all frames and in the absence of canonical start codons. A growing body of evidence pinpoints accumulation of DPRs as one of the primary driver of pathogenesis in cellular and animal models of C9ORF72-ALS/FTD. Accordingly, we recently showed that reducing the expression levels of DPRs confers neuroprotection in patient-derived motor neurons and suppresses neurodegeneration-associated motor deficits in Drosophila (Hautbergue et al. Nature Communications 2017;8:16063). Significantly, these findings showed that altering the RAN translation of DPRs provides a valid therapeutic strategy for neuroprotection in vitro and in vivo. Supporting this, the investigators have filed a patent application for the use of DPRs antagonists in the treatment of C9ORF72-ALS/FTD by gene therapy approaches (PCT/GB2017/051539).The mechanisms involved in the RAN translation of C9ORF72 repeat transcripts remain completely unknown. Therapeutically manipulating RAN translation has therefore not been possible in the absence of target(s). We recently uncovered two of the RAN-translation associated factors that are named RTA1 and RTA2 in this proposal. Moreover, partial depletion of either of these factors rescues DPR-mediated neurotoxicity in neuronal cell models and neurodegeneration-associated locomotor deficits in adult Drosophila (unpublished data).Aims: In this PhD program, we propose to evaluate the therapeutic efficiency of our novel neuroprotective strategies using a gene therapy approach based on Adeno-associated viruses in a pre-clinical mouse model of C9ORF72-ALS/FTD. Neuroprotection will be assessed at the molecular, neuronal and motor function levels. Objectives: The research project is based on diverse and cutting edge techniques including molecular and cellular biology, bioinformatics and animal research that will build a very strong curriculum for the student.(1) Produce Adeno-associated virus 9 (AAV9) encoding RTA1-RNAi and RTA2-RNAi cassettes. The RNAi cassettes are already engineered and tested.(2) Validate the functionality of AAV9 viruses in C9ORF72-ALS/FTD neuronal cell models and the specificity of depletion at genome-wide level using micro-arrays.(3) The gene therapy programme will involve single-dose cisterna magma injections of AAV9 virus co-expressing a Green Fluorescent protein (GFP) and either control-RNAi, RTA1-RNAi or RTA2-RNAi in a C9ORF72-ALS/FTD mouse model generated by the Ranum's group. Injected mice will be maintained for 12 months and motor function will be monitored using rotarod test and footprint catwalk analysis. One mouse from each treatment will be sacrificed a month post-viral delivery to evaluate gene transfer efficiency.(4) Histology and molecular assessment of the gene therapy approaches from spinal cord (cervical/lumbar regions) and brain (motor/non-motor regions) sections to evaluate (i) DPRs expression using DPR antibody, (ii) neuron counts using Nissl and motor neuron marker calcitonin gene-related peptide (CGRP) staining and (iii) RTA1/2 depletions using qRT-PCR and western blotting.