Mechanisms of DUX4 mediated FSHD pathology

摘要 面肩肩周肌营养不良症(FSHD)是最常见的肌病之一，影响 所有年龄段的男性和女性，目前还没有治愈或治疗干预措施。这个 这个项目的长期目标是确定导致致病基因的表观遗传机制。 在FSHD中表达，并确定可作为治疗靶点的调节成分 发展。FSHD是一种复杂的遗传和表观遗传病，由染色质松弛引起 染色体4q35上的D4Z4大卫星重复序列导致基因异常表达 DUX4基因来自最远端重复单位。DUX4蛋白反过来激活一系列基因 在发育早期正常表达，在成人中错误表达时会导致病理 骨骼肌。这种建立的FSHD发病机制模型刺激了对DUX4- 基于治疗靶点，包括阻断DUX4表达的方法。虽然正常 D4Z4抑制的机制已经被很好地描述，对其影响因素知之甚少 以及促进DUX4异常激活的途径。在此之前，我们展示了 FSHD相关4q35基因座表观遗传失调的程度和稳定性是关键 DUX4表达、临床表现和FSHD病理严重程度的决定因素。因此， 靶向FSHD的表观遗传失调是一种潜在的强大的治疗途径。这样的一种 更好地了解促进DUX4的因素将极大地有助于采取这种方法 表达，每一个都代表了治疗抑制的潜在靶点。在基于候选人的 筛选，我们确定了几个表观遗传调控因子，它们具有新的功能，具有可用药结构域 DUX4的激活剂。在这里，我们将表征DUX4表达和DUX4表达的表观遗传促进剂 验证它们作为FSHD治疗靶点的潜力。我们将确定以下方面的全球影响 减少这些因子在骨骼肌健康和发育中的表达 患者细胞和新的动物模型。最后，我们将测试抑制这些的翻译潜力 FSHD患者成肌细胞来源的FSHD人异种移植小鼠模型中的DUX4调节因子 以及它们的内源性D4Z4阵列。DUX4调节子将被CRISPR抑制和 进行了特异性检测。该项目的成功完成将为我们提供对 FSHD的表观遗传失调机制及其翻译潜力的验证 体内治疗靶点。

SUMMARY Facioscapulohumeral Muscular Dystrophy (FSHD) is one of the most prevalent myopathies affecting males and females of all ages, and currently there is no cure or therapeutic intervention available. The long-term goal of this project is to determine the epigenetic mechanisms leading to pathogenic gene expression in FSHD, and identify regulatory components that are viable targets for therapeutic development. FSHD is a complex genetic and epigenetic disease caused by chromatin relaxation of the D4Z4 macrosatellite repeat array at chromosome 4q35, which leads to aberrant expression of the DUX4 gene from the distal-most repeat unit. The DUX4 protein, in turn, activates a host of genes normally expressed in early development, which cause pathology when mis-expressed in adult skeletal muscle. This established model of FSHD pathogenesis has stimulated the search for DUX4- based therapeutic targets, including approaches to block DUX4 expression. While normal mechanisms of D4Z4 repression have been well-characterized, very little is known about the factors and pathways responsible for facilitating aberrant activation of DUX4. Previously, we showed that the degree and stability of epigenetic dysregulation at the FSHD-associated 4q35 locus is the key determinant for DUX4 expression, clinical presentation, and severity of FSHD pathology. Thus, targeting epigenetic dysregulation in FSHD is a potentially powerful therapeutic avenue. Such an approach would be greatly aided by a better understanding of the factors facilitating DUX4 expression, each of which represents a potential target for therapeutic inhibition. In a candidate-based screen, we identified several epigenetic regulators with druggable domains that function as novel activators of DUX4. Here, we will characterize these epigenetic facilitators of DUX4 expression and validate their potential as therapeutic targets in FSHD. We will determine the global effects of reducing the expression of these factors on skeletal muscle health and development using primary patient cells and novel animal models. Finally, we will test the translational potential of inhibiting these DUX4 regulators in an FSHD human xenograft mouse model derived from FSHD patient myoblasts with their endogenous D4Z4 arrays. DUX4 regulators will be inhibited by CRISPR inhibition and assayed for specificity. Successful completion of this project will provide key insights into the mechanisms of epigenetic dysregulation in FSHD and validate the translational potential of novel therapeutic targets in vivo.