Reactivation of Epigenetically Silenced FMR1 as a Therapeutic Approach for Fragile X Syndrome

脆性X综合征(FXS)是最常见和最普遍的精神功能不全的遗传性形式 这种疾病是由 X连锁FMR1基因5‘非翻译区CGG重复扩增导致表观遗传学 沉默FMR1。 不是生产的。FMRP是一种RNA结合蛋白，通常会抑制大脑中的mRNA翻译；在其 缺乏时，蛋白质合成过多，从而导致疾病病理。表观遗传学的再激活 沉默的FMR1是一种有前途的治疗FXS的新方法，旨在纠正FXS的根本原因 疾病，而不是FMRP缺陷的次要下游后果。在预赛中 我的实验室进行了一项以候选人为基础的小规模筛选，以确定八个压抑性 促进FXS细胞中FMR1沉默的表观遗传调节因子(称为FMR1沉默因子，或FMR1- SFS)。短发夹状RNA(ShRNAs)或小分子抑制FMR1-SFS的表观遗传再激活 未分化诱导多能干细胞、神经前体细胞和有丝分裂后细胞中FMR1的沉默 来源于FXS患者的神经元。这些初步结果提供了重要的概念验证 重新激活表观遗传学沉默的FMR1基因作为FXS治疗方法的可行性。在这 应用程序我们建议使用基于候选对象的大规模无偏功能损失屏幕进行实验 通过重新激活FMR1发挥作用的药物。要确定FMR1重新激活的水平是否通过 ShRNA和小分子FMR1-SF抑制剂可能具有治疗作用，我们将进行几项 互补的方法。首先，我们将确定使油井正常化所需的最低FMRP水平- 表征FXS神经元的转录、翻译和形态异常。在这些 实验中，我们将在不同水平异位表达FMRP并分析其对FXS神经元的影响 表型。 人类FXS神经元。总而言之，拟议实验的结果将：(1)确定新的目标，其 神经元功能障碍，以及(3)检测shRNA和小分子FMR1-SF是否重新激活FMR1 抑制剂可以使人FXS神经元功能障碍的表型正常化。我们相信这一结果 本申请中提出的实验将对FXS治疗领域产生重大影响，并具有 有可能导致开发一类新的药物，可以改善这种毁灭性的疾病。

Fragile X Syndrome (FXS) is the most common inherited form of mental insufficiency and most prevalent monogenic cause of autism, occurring in ~1 in 4,000 males and ~1 in 5,000 females. The disease is caused by a CGG repeat expansion in the 5' untranslated region of the X-linked FMR1 gene that results in epigenetic silencing of FMR1. As a consequence, the product of FMR1, the fragile X mental retardation protein (FMRP), is not produced. FMRP is an RNA-binding protein that normally represses mRNA translation in the brain; in its absence, protein synthesis is excessive, which results in disease pathology. Reactivation of epigenetically silenced FMR1 is a promising new therapeutic approach for FXS that aims to correct the root cause of the disease rather than a secondary, downstream consequence of the FMRP deficiency. In preliminary experiments my laboratory has performed a small-scale candidate-based screen to identify eight repressive epigenetic regulators that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs by short hairpin RNAs (shRNAs) or small molecules reactivates epigenetically silenced FMR1 in undifferentiated induced pluripotent stem cells, neural progenitor cells, and post-mitotic neurons derived from FXS patients. These preliminary results provide important proof-of-concept regarding the feasibility of reactivating the epigenetically silenced FMR1 gene as a therapeutic approach for FXS. In this application we propose experiments using large-scale candidate-based and unbiased loss-of-function screens to identify additional FMR1-SFs, some of which may provide more desirable targets for the development of drugs that function by reactivating FMR1. To determine whether the level of FMR1 reactivation obtained with shRNA and small molecule FMR1-SF inhibitors is likely to be therapeutic, we will undertake several complementary approaches. First, we will determine the minimal level of FMRP required to normalize the well- characterized transcriptional, translational and morphological abnormalities of FXS neurons. In these experiments we will ectopically express FMRP at varying levels and analyze the effect on FXS neuronal phenotypes. Second, we will determine whether the level of FMR1 reactivation we obtain with shRNA and small molecule inhibitors of the FMR1-SFs we identify is sufficient to normalize dysfunctional phenotypes of human FXS neurons. In summary, the results of the proposed experiments will: (1) identify new targets whose inhibition leads to FMR1 reactivation, (2) determine the minimal levels of FMRP required to correct FXS neuronal dysfunctions, and (3) test whether FMR1 reactivation by shRNA and small molecule FMR1-SF inhibitors can normalize dysfunctional phenotypes of human FXS neurons. We believe the results of the experiments proposed in this application will have a major impact on the field of FXS therapeutics and have the potential to lead to development of a new class of drugs that can ameliorate this devastating disease.