Transcriptional Upregulation of the Epigenetically Repressed FXN Gene as a Therapeutic Approach for Friedreich Ataxia

Friedreich共济失调(FA)是一种以进行性神经损害为特征的常染色体隐性遗传病 系统和严重的心脏异常。这种疾病是由GAA·TTC三联体重复扩增引起的 FXN基因的第一个内含子(以下称为三重重复扩展(Tre)-FXN基因)，它抑制 FXN转录。FXN基因编码一种名为frataxin的蛋白质，它是一种普遍存在的核编码蛋白质 在铁代谢中起关键作用的线粒体蛋白。TrE-FXN的转录抑制导致 Frataxin水平降低，导致线粒体功能障碍，这是疾病的根本基础。 目前还没有有效的治疗FA的方法。抑制基因tre-FXN的转录上调 基因是治疗FA的一种潜在的治疗方法，它将纠正疾病的根本原因，而不是 Frataxin缺乏的继发性下游后果。在初步实验中，我们已经确定 10个表观遗传调节子和9个蛋白激酶，它们介导了tre-fxn基因的抑制，我们指的是 至AS FXN抑制因子(FXN-RFS)。短发夹状RNA或小分子对FXN-RFs的抑制作用 分子可恢复FA诱导的多能干细胞FXN mRNA和Frataxin的正常水平 FA神经元和心肌细胞，这是与疾病最相关的细胞类型。另外，我们发现， FXN-RF抑制上调TrE-FXN转录减轻FA特征性线粒体缺陷 神经元和心肌细胞。这些初步结果提供了重要的概念验证 上调tre-FXN转录作为FA治疗方法的可行性。我们假设在那里 还有其他尚未确定的FXN-RFs，它们可能为发展提供更可取的目标 通过上调tre-fxn转录发挥作用的药物。为此，我们将大规模放映一部 针对表观遗传调控因子的shRNA文库和一系列化学文库以鉴定新的FXN-RFS 和小分子FXN-RF抑制剂，并分析它们上调FA中tre-FXN转录的能力 神经元和心肌细胞。最有希望的小分子FXN-RF抑制剂将在 人源化FA小鼠模型上调tre-FXN和改善疾病症状。其中 FXN-RF抑制剂将在FA小鼠身上进行测试，这是我们在初步实验中确定的两种药物 在人体临床试验中确立了安全性。拟议的实验结果将提供：(1)集合 其抑制上调tre-FXN转录的有效和特征蛋白质靶点(FXN-RFs)， (2)一组经过验证和表征的小分子FXN-RF抑制剂，可为以下方面提供领先的候选药物 临床前开发，以及(3)确定最有希望的小分子FXN-RF 在人源化的FA小鼠模型中，抑制剂可以上调tre-FXN并改善疾病症状。 拟议的实验结果预计将对FA治疗学领域产生重大影响 并有可能导致开发一类新的药物来治疗这种毁灭性的疾病。

Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the FXN gene (hereafter called the triplet repeat expansion (TRE)-FXN gene), which represses FXN transcription. The FXN gene encodes a protein called frataxin, a ubiquitous, nuclear-encoded mitochondrial protein that plays a key role in iron metabolism. Transcriptional repression of TRE-FXN results in reduced levels of frataxin, leading to mitochondrial dysfunction, which is the underlying basis of the disease. Currently there are no effective treatments for FA. Transcriptional upregulation of the repressed TRE-FXN gene is a potential therapeutic approach for FA that would correct the root cause of the disease rather than a secondary, downstream consequence of the frataxin deficiency. In preliminary experiments, we have identified 10 epigenetic regulators and 9 protein kinases that mediate repression of the TRE-FXN gene, which we refer to as FXN Repressing Factors (FXN-RFs). Inhibition of FXN-RFs by short hairpin RNAs (shRNAs) or small molecules can restore normal levels of FXN mRNA and frataxin in FA induced pluripotent stem cells as well as FA neurons and cardiomyocytes, which are the cell types most relevant to the disease. In addition, we find that upregulating TRE-FXN transcription by FXN-RF inhibition mitigates characteristic mitochondrial defects of FA neurons and cardiomyocytes. These preliminary results provide important proof-of-concept regarding the feasibility of upregulating TRE-FXN transcription as a therapeutic approach for FA. We hypothesize that there are other, yet-to-be-identified FXN-RFs, which may provide more desirable targets for the development of drugs that function by upregulating TRE-FXN transcription. Toward this end, we will screen a large-scale shRNA library and a series of chemical libraries directed against epigenetic regulators to identify new FXN-RFs and small molecule FXN-RF inhibitors, and analyze their ability to upregulate TRE-FXN transcription in FA neurons and cardiomyocytes. The most promising small molecule FXN-RF inhibitors will be analyzed in a humanized mouse model of FA for upregulation of TRE-FXN and amelioration of disease symptoms. Among the FXN-RF inhibitors that will be tested in FA mice are two drugs we identified in preliminary experiments with established safety in human clinical trials. The results of the proposed experiments will provide: (1) a collection of validated and characterized protein targets (FXN-RFs) whose inhibition upregulates TRE-FXN transcription, (2) a set of validated and characterized small molecule FXN-RF inhibitors that may provide lead candidates for pre-clinical development, and (3) a determination of whether the most promising small molecule FXN-RF inhibitors can upregulate TRE-FXN and ameliorate disease symptoms in a humanized mouse model of FA. The results of the proposed experiments are expected to have a major impact on the field of FA therapeutics and have the potential to lead to development of a new class of drugs to treat this devastating disease.