A new approach to modeling ALS based on TBK1 mutation in mice

项目总结 肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)是进行性的 神经退行性疾病。肌萎缩侧索硬化症会导致运动神经元退化和瘫痪。FTD原因 大脑皮层神经元变性导致人格改变和执行功能丧失。两者都有 疾病是不可阻挡的，以死亡告终。越来越多的因果基因突变正在被 其中许多病毒可导致这两种疾病。利用新发现的 了解疾病机制和开发治疗方法的ALS/FTD基因，动物 需要模特。然而，传统的基因过表达和转基因技术 淘汰赛速度慢、成本高，成为制约新模式的瓶颈 发展。此外，越来越多的人认识到一些患者有多种基因。 突变。这导致了一种假设，即一些ALS/FTD病例是由一种寡基因引起的 机制。因此，重要的是要研究不同的突变如何相互作用导致 动物中的疾病。目前，这是通过使不同的突变转基因小鼠品系杂交来完成的。 然而，这一过程很慢，因为双转基因或三转基因小鼠的产量较低，而且 将不同的转基因品系引入相同基因所必需的多世代杂交 背景资料。为了解决这些问题，我们提出了一种rAAV-鞘内基因传递方法 肌萎缩侧索硬化症转基因小鼠模型的构建这种方法能够传递基因 通过将rAAV单次注射到脑脊液中，在整个脊髓中进行转导。我们 计划提供针对新发现的ALS基因TBK1的rAAV以进行敲除。损失-- 在人类中，TBK1基因一个等位基因的功能突变是导致遗传性ALS的主要原因。 然而，这很难在小鼠身上建立模型，因为小鼠失去一个TBK1等位基因不会导致 一种明显的表型，但该基因的完全敲除会导致胚胎死亡。RNAi 基因敲除方法可以将基因表达沉默到正常水平的50%以下，我们的 以前的工作已经证明，特定基因的敲除可以实现基因亚型 活体表型。在这个方案中，我们将使用rAAV在脊髓中传递tbk1基因沉默。 电源线。我们将把rAAV注射到几个小鼠品系中，包括正常的野生型和突变型 出现ALS表型的转基因小鼠。我们将确定这种方法是否可以诱导 野生型小鼠的ALS表型和/或调节疾病表型和病理 突变的ALS转基因小鼠。如果成功，这项实验将建立一种新的小鼠模型 ALS和一种比传统的基因敲除法更快的构建方法 用于ALS小鼠模型和体内突变基因相互作用的研究。

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases. ALS causes motor neuron degeneration and paralysis. FTD causes cortical neuron degeneration leading to personality changes and loss of executive function. Both diseases are unstoppable ending in death. Increasing number of causal gene mutations are being identified and many of these can cause both diseases. To capitalize on the newly discovered ALS/FTD genes for understanding the disease mechanism and developing therapies, animal models are needed. However, the conventional transgenic technologies of gene overexpression and knockout are slow and expensive, thus becoming a bottleneck constraining new model development. Additionally, there is a growing recognition that some patients have multiple gene mutations. This has led to the hypothesis that some ALS/FTD cases are caused by an oligogenic mechanism. Thus, it will be important to investigate how different mutations interact to cause the disease in animals. Currently this is done by intercrossing different mutant transgenic mouse strains. However, this process is slow because of the low yield of double or triple transgenic mice and the multigenerational crosses that are necessary to bring different transgenic lines to the same genetic background. To solve these problems, we propose a rAAV-intrathecal gene delivery approach for construction of transgenic mouse models for ALS. This approach is capable of delivering gene transduction throughout the spinal cord by a single injection of rAAV into the cerebrospinal fluid. We plan to deliver rAAV targeting the newly discovered ALS gene TBK1 for knockdown. Loss-of- function mutations in one allele of TBK1 gene causes dominantly inherited ALS in humans. However, this is difficult to model in mice because loss of one TBK1 allele in mice does not cause an overt phenotype but a complete knockout of this gene causes embryonic lethality. RNAi knockdown approach can silence gene expression to below 50% of the normal level and our previous work has demonstrated that knockdown of specific genes can achieve gene hypomorphic phenotypes in vivo. In this proposal, we will use rAAV to deliver TBK1 gene silencing in the spinal cord. We will inject the rAAV into several mouse strains including the normal wild type and mutant transgenic mice that develop ALS phenotype. We will determine whether this approach can induce ALS phenotypes in the wild type mice and/or modulate the disease phenotypes and pathology in the mutant ALS transgenic mice. If successful, this experiment will establish a new mouse model for ALS and a new method that is faster than the conventional gene knockout approach for construction of mouse models for ALS and for studying mutant gene interactions in vivo.