Establishing an FSHD-like mouse for therapeutic development

项目总结 面肩肱骨肌营养不良症(FSHD)是困扰男性和青少年的最常见的肌病 女性、儿童和成年人。在大多数临床FSHD病例中，肌肉无力不是 直到生命的第二个或第三个十年才明显，随后是一种进行性的病理，影响许多 日常生活的方方面面，从不会梳理自己的头发或遛狗到包括 不得不改变或放弃职业生涯，失去独立性，在约20%的FSHD患者中， 坐轮椅需要呼吸方面的帮助。目前还没有治疗方法 延缓、阻止或逆转疾病的进展。开发改进型产品的主要障碍 治疗缺乏基于DUX4表达的可靠的表型FSHD样动物模型 基因被广泛认为是FSHD病理生理学的关键调节因子。这个项目直接解决了这个问题 空虚。在FSHD中，DUX4-F1的表达增加，这可以作为DNA结合的转录 因子，改变肌肉的基因表达谱，并启动一系列事件，最终导致 FSHD病理生理学。因此，DUX4-FL的mRNA、DUX4-FL蛋白和下游靶点都是 极好的治疗发展目标。对于这个项目，我们已经成功地设计了一条生产线 含有人类DUX4-F1基因的转基因小鼠保持其天然基因结构。这些 通过Cre介导的重组，小鼠被证实正确表达剪接的DUX4-fl mRNA 当DUX4-fl在发育中的肌肉中表达时，会形成异常的肌肉表型。这 Data强烈支持最终一代的类似FSHD的模型鼠标；然而，精确的 产生有用的FSHD样表型的表达条件尚不清楚。在这里，我们 将使用一种在骨骼肌中表达他莫昔芬诱导的Cre的商业小鼠品系来 确定在一系列范围内产生可复制的FSHD样表型所需的条件 严肃性。一只成功的类似FSHD的小鼠最初看起来很健康，然后发展成一个渐进性的 基于包括握力在内的肌肉力量和功能的可量化指标的肌病表型 力量、轮杆和每个人在筋疲力尽之前所能跑的最大距离。 表型将通过FSHD样基因表达分析和组织病理学证实。 该项目的完成将为FSHD领域提供宝贵的工具，以便更好地了解 FSHD的致病进展及其机制。此外，这些型号将作为一种 针对DUX4-fl mRNA和蛋白AS的治疗策略的临床前测试资源 对FSHD的改良治疗。

PROJECT SUMMARY Facioscapulohumeral muscular dystrophy (FSHD) is the most prevalent myopathy afflicting males and females, children and adults. In the majority of clinical FSHD cases, muscle weakness is not noticeable until the second or third decade of life followed by a progressive pathology impacting many facets of everyday life, ranging from being unable to comb ones own hair or walk the dog to including having to change or abandon careers, loss of independence and, in ~20% of FSHD patients, becoming wheelchair bound an/or require aid in breathing. Currently there are no treatments to slow down, stop, or reverse disease progression. A major impediment to developing ameliorative treatments is the lack of a reliable phenotypic FSHD-like animal model based on expression of the DUX4 gene, widely considered the key mediator of FSHD pathophysiology. This project directly addresses this void. In FSHD, increased expression of DUX4-fl, which can function as a DNA-binding transcription factor, alters the gene expression profiles of muscles and initiates a cascade of events ultimately leading to FSHD pathophysiology. Thus, the DUX4-fl mRNA, DUX4- FL protein and downstream targets are all excellent targets for therapeutic development. For this project, we have successfully engineered a line of transgenic mice that contains the human DUX4- fl gene maintaining its native gene structure. These mice are validated to express correctly spliced DUX4-fl mRNA upon Cre-mediated recombination and develop an aberrant muscle phenotype when DUX4-fl is expressed in developing muscle. This data strongly supports the ultimate generation of an FSHD-like model mouse; however, the precise expression conditions that will result in a useful FSHD-like phenotype are still not known. Here, we will use a commercially available line of mice expressing tamoxifen-inducible Cre in skeletal muscles to determine the conditions required to generate reproducible FSHD-like phenotypes over a range of severities. A successful FSHD-like mouse will appear initially healthy, then develop a progressive myopathic phenotype based on quantifiable metrics of muscle strength and function including grip strength, rotarod, and the maximum distance each is capable of running before becoming exhausted. The phenotypes will be confirmed by FSHD-like gene expression analysis and histopathology. Completion of this project will provide the FSHD field with valuable tools for better understanding FSHD pathogenic progression and mechanisms. In addition, these models will serve as a resource for pre-clinical testing of therapeutic strategies targeting the DUX4-fl mRNA and protein as ameliorative treatments for FSHD.