Development of SLIC, a methodology for synthetic lethal screening in the CNS

描述（由申请人提供）：挑战：鉴于大多数主要疾病缺乏有效的治疗方法，需要在神经退行性疾病研究领域提出新的假设。在简单的模式生物中，无偏倚的全基因组遗传筛选被用于以无偏倚的方式产生新的假设。合成致死基因筛选是一种基因筛选，在这种基因筛选中，一个没有毒性或毒性很小的突变通过额外的基因操作增强为现在有毒性的突变。因此，“合成致死”筛选被用来揭示与毒性突变基因相互作用的基因和途径。目前，由于成本过高和需要的动物数量，在小鼠身上进行这种基因筛选是不可行的。我们提出了一种合成致死性中枢神经系统（CNS）的策略，我们称之为SLIC。影响：使用我们的SLIC方法在小鼠中枢神经系统中进行无偏倚的全基因组筛选的能力将允许对神经退行性疾病的病因产生新的假设。潜在的影响将是探索潜在治疗靶点的无数新途径。一旦开发出来，我们的SLIC方法可以被其他研究人员应用于任何遗传诱导疾病的研究。方法：我们认为在大多数神经退行性疾病的小鼠模型中发现缺乏细胞死亡是一个独特的机会。我们建议在小鼠脑中进行合成致死筛选，以增强神经退行性疾病相关突变的毒性。SLIC合成致死筛选将结合使用i)慢竞争shRNA或诱导的条形码过表达文库；ii)将这些文库注入小鼠疾病模型和野生型幼崽的立体定位中枢神经系统；iii)孵育注射文库，使增强毒性的shrna或过表达基因导致细胞死亡；iv)对所有存活细胞中剩余的shrna和条形码过表达构建体进行测序和分析，以确定哪些构建体导致细胞死亡，从而“退出”病毒表征。我们将围绕以下具体目标开展工作：目标1。开发一种方法，允许在小鼠中枢神经系统（CNS）中进行合成致死筛选；目标2。使用Aim 1中开发的方法识别体内亨廷顿蛋白毒性增强因子

DESCRIPTION (provided by applicant): Challenge: New hypotheses in the neurodegenerative disease research field are needed, given the lack of effective therapeutics for most of the major diseases. In simple model organisms, unbiased, genome-wide genetic screens are used to generate new hypotheses in an unbiased way. Synthetic lethal genetic screens are genetic screens in which a mutation that has no or little toxicity is enhanced by an additional genetic manipulation into a mutation that now does. Thus 'synthetic lethal' screens are used to reveal genes and pathways that genetically interact with toxic mutations. Currently it is not feasible to conduct such genetic screens in mice, due to prohibitive cost and numbers of animals needed. We propose to develop a strategy for synthetic lethal in the central nervous system (CNS), which we term SLIC. Impact: The ability to perform unbiased, genome-wide screens in the mouse CNS with our SLIC methodology would allow for the generation of novel hypotheses regarding the etiology of neurodegenerative disease. The potential impact would be myriad new avenues to explore potential therapeutic targets. Once developed, our SLIC methodology could be applied by other researchers to the study of any genetically-induced disease. Approach: We view the lack of cell death found in most mouse models of neurodegenerative disease as a unique opportunity. We propose to conduct synthetic lethal screens in mouse brain, to enhance the toxicity of neurodegenerative disease-associated mutations. SLIC synthetic lethal screens will be conducted by combining the use of i) lentirival shRNA or inducible, barcoded overexpression libraries; ii) stereotaxic CNS injection of these libraries into mouse models of disease and wild-type littermates; iii) incubation of injected libraries, such that shRNAs or overexpressed genes that enhance toxicity will lead to cell death; iv) sequencing and analysis of the remaining shRNAs and barcoded overexpression constructs in all surviving cells, in order to determine which constructs have led to cell death, and thus 'drop out' of viral representation. Our work will be conducted along the following specific aims: Aim 1. To develop a methodology that allows for synthetic lethal screens in the mouse central nervous system (CNS); Aim 2. To identify, using the methodology developed in Aim 1, enhancers of Huntingtin toxicity in vivo