Transgenic Mouse Models of FUS/TLS-Mediated Amyotrophic Lateral Sclerosis

描述（由申请人提供）：本申请涉及广泛的挑战领域（15）：转化科学和特定的挑战主题，15-NS-104：早期治疗开发。肌萎缩侧索硬化症（ALS，Lou Gehrig's disease）是一种破坏性的神经退行性疾病，它会杀死大脑和脊髓中控制肌肉的神经细胞，导致3-5年内进行性虚弱和死亡。目前，没有任何治疗方法可以减缓疾病的进展，因此迫切需要开发新的ALS动物模型，以确定新的治疗策略。最近，一种新基因FUS/TLS的突变已被确定为导致约5%的家族性ALS病例。这些突变导致疾病的机制尚不清楚，但它们可能与其他已知ALS基因（如TDP-43，senataxin和dynactin）中的缺陷重叠。这些基因可能在RNA分子的加工、传递或调节中起作用，因此，这些功能的缺陷可能是ALS中运动神经元脆弱性的基础。突变型FUS/TLS与ALS的直接因果关系为开发新的体内ALS模型提供了机会，这将加速ALS新治疗靶点的鉴定。为了基于FUS/TLS突变快速生成新的和信息丰富的ALS模型，我们建议使用三种平行但互补的方法在转基因小鼠中表达正常或突变的FUS/TLS。在第一种方法中，我们将产生FUS/TLS（野生型和两种ALS突变体）的表达由天然基因组调控元件驱动的小鼠。我们设计的一个新方面将是纳入条件性敲除能力，这将使我们能够选择性地关闭突变基因表达，以确定突变蛋白质在哪些细胞类型中发挥最有效的作用。在第二种方法中，我们将产生FUS/TLS转基因表达仅在与在特定组织中表达Cre重组酶的小鼠杂交后激活的小鼠。这一策略将允许选择和繁殖创始动物，即使突变基因是剧毒的。此外，我们可以确定转基因表达是否通过细胞自主或非细胞自主机制引起运动神经元毒性。在第三种方法中，我们将使用四环素诱导策略在时间控制下在转基因小鼠中表达野生型和突变型FUS/TLS。在转基因表达的发育或出生后早期毒性的情况下，这种方法将使我们能够在不同年龄启动或沉默表达，从而使我们能够研究衰老在这种疾病中的作用以及突变基因沉默时这种疾病的可逆性。这些研究将指导未来治疗的发展。总之，这些互补的方法可能会产生一个或多个基于FUS/TLS突变的ALS动物模型，使它们可供我们和研究界研究疾病的机制和开发新的治疗策略。 公共卫生相关性：肌萎缩侧索硬化症（ALS，Lou Gehrig病）是一种破坏性的神经退行性疾病，其中大脑和脊髓中控制肌肉的神经细胞过早死亡。目前还没有真正有效的治疗方法来减缓这种疾病的无情进程。该项目将解决开发信息丰富的ALS小鼠模型的迫切需要，该模型将i）加速新治疗靶点的鉴定，ii）能够测试新的治疗策略以对抗ALS。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15-NS-104: Early-stage Therapy Development. Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is a devastating neurodegenerative condition that kills nerve cells in the brain and spinal cord that control the muscles, leading to progressive weakness and death within 3-5 years. Currently, no treatment can slow the progression of the disease, thus raising the urgency for developing new animal models of ALS that can be used to identify novel therapeutic strategies. Recently mutations in a new gene, FUS/TLS, have been identified to cause ~5% of familial ALS cases. The mechanism(s) by which these mutations cause the disease is not clear, but they may overlap with defects in other known ALS genes such as TDP-43, senataxin, and dynactin. These genes may function in the processing, delivery, or regulation of RNA molecules, and thus, defects in these functions may underlie motor neuron vulnerability in ALS. The direct causal link of mutant FUS/TLS to ALS provides an opportunity to develop novel in vivo ALS models that will accelerate the identification of new treatment target(s) for ALS. To generate new and informative ALS models rapidly based on FUS/TLS mutations, we propose to use three parallel but complementary approaches to express normal or mutant FUS/TLS in transgenic mice. In the first approach, we will produce mice in which expression of FUS/TLS (wild type and two ALS mutants) is driven by native genomic regulatory elements. A novel aspect of our design will be the incorporation of conditional knockout capabilities that will allow us to selectively turn off the mutant gene expression to determine in which cell types the mutant proteins exert their most potent effects. In the second approach, we will generate mice in which expression of FUS/TLS transgenes is activated only after crossing with mice that express Cre recombinase in specific tissues. This strategy will allow the selection and breeding of founder animals even if the mutant gene is highly toxic. Furthermore, we can determine whether transgene expression causes motor neuron toxicity by cell autonomous or non-cell autonomous mechanisms. In the third approach, we will express wild type and mutant FUS/TLS in transgenic mice under temporal control using a tetracycline-inducible strategy. In the case of developmental or early postnatal toxicity of transgene expression, this approach will allow us to initiate or silence expression at different ages, thus enabling us to investigate the role of aging in this disease and the reversibility of this disease when the mutant gene is silenced. These studies will guide the development of therapies in the future. Together, these complementary approaches are likely to produce one or more animal models of ALS based on FUS/TLS mutations, making them available for us and the research community in general to study the mechanism of the disease and to develop new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is a devastating neurodegenerative condition in which the nerve cells in the brain and spinal cord that control the muscles die prematurely. Currently no truly effective treatments exist to slow the relentless course of the disease. This project will address the urgent need to develop informative mouse models of ALS that will i) accelerate the identification of novel treatment target(s) and ii) enable the testing of new therapeutic strategies to combat ALS.