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

项目摘要 肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）是进行性的 神经退行性疾病ALS会导致运动神经元变性和瘫痪。FTD原因 皮质神经元退化导致人格改变和执行功能丧失。两 疾病是不可阻挡的，最终导致死亡。越来越多的致病基因突变正在 这些疾病中的许多都可能导致这两种疾病。利用新发现的 ALS/FTD基因用于了解疾病机制和开发治疗方法，动物 需要模型。然而，传统的基因过表达和 淘汰赛是缓慢和昂贵，从而成为一个瓶颈制约新的模式 发展此外，越来越多的人认识到，一些患者具有多个基因， 突变。这导致了一些ALS/FTD病例是由寡基因突变引起的假设。 机制因此，重要的是研究不同的突变如何相互作用以引起突变。 动物疾病。目前，这是通过交叉不同的突变转基因小鼠品系来完成的。 然而，这一过程是缓慢的，因为双或三转基因小鼠产量低， 多代杂交是必要的，使不同的转基因品系相同的遗传 背景为了解决这些问题，我们提出了一种rAAV鞘内基因递送方法， ALS转基因小鼠模型的构建。这种方法能够将基因 通过将rAAV单次注射到脑脊液中，在整个脊髓中转导。我们 计划提供针对新发现的ALS基因TBK 1的rAAV用于敲除。损失- TBK 1基因的一个等位基因的功能突变导致人类显性遗传性ALS。 然而，这难以在小鼠中建模，因为小鼠中一个TBK 1等位基因的缺失不会导致 一个明显的表型，但该基因的完全敲除会导致胚胎死亡。RNAi 敲除方法可以沉默基因表达低于50%的正常水平，我们的研究表明， 以前的工作已经证明，敲除特定基因可以实现基因亚型 体内表型。在这个提议中，我们将使用rAAV在脊髓中传递TBK 1基因沉默， 线.我们将rAAV注射到几种小鼠品系中，包括正常野生型和突变型 转基因小鼠发展成ALS表型。我们将确定这种方法是否能诱导 通过在野生型小鼠中调节ALS表型和/或在野生型小鼠中调节疾病表型和病理， 突变型ALS转基因小鼠。如果成功，这项实验将建立一个新的小鼠模型， ALS和一种比传统基因敲除方法更快的新方法 用于ALS小鼠模型和研究体内突变基因相互作用。

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.