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

脆性X综合征（FXS）是最常见的遗传形式的精神不全和最普遍的 孤独症的单基因原因，约4,000名男性中有1人，5,000名女性中有1人。这种疾病是由 X连锁FMR 1基因5'非翻译区的CGG重复扩增，导致表观遗传 FMR 1的沉默。因此，FMR 1的产物，脆性X智力低下蛋白（FMRP）， 不生产。FMRP是一种RNA结合蛋白，通常抑制大脑中的mRNA翻译; 缺乏，蛋白质合成过量，导致疾病病理。表观遗传学的重新激活 沉默的FMR 1是FXS的一种有前途的新治疗方法，旨在纠正FXS的根本原因。 疾病，而不是FMRP缺乏的继发性下游后果。初步 我的实验室进行了一个小规模的候选人为基础的筛选，以确定八个压抑 促进FXS细胞中FMR 1沉默的表观遗传调节因子（称为FMR 1沉默因子，或FMR 1- SF）。通过短发夹RNA（shRNAs）或小分子抑制FMR 1-SFs， 在未分化的诱导多能干细胞、神经祖细胞和有丝分裂后细胞中沉默FMR 1， 来自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FMR 1基因作为FXS治疗方法的可行性。在这 应用程序，我们提出了使用大规模的候选人为基础的实验和公正的功能丧失的屏幕 确定其他FMR 1-SF，其中一些可能为开发 通过重新激活FMR 1发挥作用的药物。为了确定FMR 1再激活水平是否与 shRNA和小分子FMR 1-SF抑制剂很可能是治疗性的，我们将进行几项研究。 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，我们将异位表达不同水平的FMRP，并分析其对FXS神经元的影响。 表型第二，我们将确定我们用shRNA获得的FMR 1再激活水平是否高于用siRNA获得的FMR 1再激活水平， 我们鉴定的FMR 1-SF的小分子抑制剂足以使FMR 1-SF的功能障碍表型正常化。 人类FXS神经元。总之，所提出的实验的结果将：（1）识别新的目标， 抑制导致FMR 1再激活，（2）确定纠正FXS所需的FMRP的最低水平 神经元功能障碍，以及（3）测试是否通过shRNA和小分子FMR 1-SF 抑制剂可以使人FXS神经元的功能障碍表型正常化。我们认为， 本申请中提出的实验将对FXS治疗领域产生重大影响， 有可能导致开发出一种新的药物，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.