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

表观遗传抑制 FXN 基因的转录上调作为弗里德赖希共济失调的治疗方法

• 批准号: 10650343
• 负责人: MICHELLE ALICE KELLIHER
• 金额: $ 36.64万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10650343
• 关键词: Asia; Cardiac Myocytes; Cells; Characteristics; Clinical; Clinical Trials; Collection; Defect; Development; Disease; EZH2 gene; Epigenetic Process; Event; Friedreich Ataxia; Genes; Genetic Transcription; Goals; Heart Abnormalities; Histopathology; Human; Introns; Knockout Mice; Laboratories; Mediating; Messenger RNA; Mitochondria; Mitochondrial Proteins; Motor; Mus; Nervous System; Neuromuscular Diseases; Neurons; Nuclear; Patients; Pharmaceutical Preparations; Play; Protein Kinase; Proteins; RNA interference screen; RNA library; Repression; Safety; Series; Spinal Ganglia; Symptoms; Testing; Therapeutic; Tissues; Transgenes; Trinucleotide Repeats; Undifferentiated; Up-Regulation; Validation; autosome; candidate validation; cell type; cytotoxicity; design; drug development; effective therapy; experimental study; fasudil; frataxin; gene repression; glucose tolerance; histone methyltransferase; humanized mouse; improved; induced pluripotent stem cell; inhibitor; insulin tolerance; iron metabolism; lead candidate; mitochondrial dysfunction; mouse model; novel drug class; preclinical development; reduce symptoms; rho; screening; small hairpin RNA; small molecule; small molecule inhibitor; small molecule libraries; therapeutic target; therapeutically effective

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.

弗里德赖希共济失调（FA）是一种常染色体隐性遗传疾病，其特征是神经系统的进行性损害。 系统和严重的心脏异常。这种疾病是由GAA·TTC三联重复扩增引起的， FXN基因的第一个内含子（下文称为三联体重复扩增（TRE）-FXN基因），其抑制 FXN转录。FXN基因编码一种名为frataxin的蛋白质， 线粒体蛋白，在铁代谢中起关键作用。TRE-FXN的转录抑制导致 共济失调蛋白水平降低，导致线粒体功能障碍，这是疾病的基础。 目前尚无有效的治疗方法。受抑制的TRE-FXN的转录上调 基因是一种潜在的治疗方法，可以纠正疾病的根本原因，而不是一种治疗方法。 是共济失调蛋白缺乏的继发性下游后果。在初步实验中，我们发现 10种表观遗传调节因子和9种蛋白激酶介导TRE-FXN基因的抑制，我们将其称为 FXN抑制因子（FXN-RFs）。通过短发夹RNA（shRNAs）或小RNA抑制FXN-RF 分子可以恢复FA诱导的多能干细胞中FXN mRNA和共济失调蛋白的正常水平， FA神经元和心肌细胞，这是与疾病最相关的细胞类型。此外，我们还发现 通过FXN-RF抑制上调TRE-FXN转录减轻FA的特征性线粒体缺陷 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 是其他尚未鉴定的FXN-RF，其可以为开发 通过上调TRE-FXN转录发挥作用的药物。为此，我们将进行大规模筛选 shRNA文库和一系列针对表观遗传调节因子的化学文库，以鉴定新的FXN-RFs 和小分子FXN-RF抑制剂，并分析它们在FA中上调TRE-FXN转录的能力。 神经元和心肌细胞。最有前途的小分子FXN-RF抑制剂将在一个 图10示出了用于上调TRE-FXN和改善疾病症状的FA的人源化小鼠模型。之间 将在FA小鼠中测试的FXN-RF抑制剂是我们在初步实验中鉴定的两种药物， 在人体临床试验中确立了安全性。建议的实验结果将提供：（1）收集 其抑制上调TRE-FXN转录的经验证和表征的蛋白质靶标（FXN-RF）， (2)一组经验证和表征的小分子FXN-RF抑制剂，可为以下药物提供主要候选物： 临床前开发，和（3）确定最有前途的小分子FXN-RF是否 抑制剂可以上调TRE-FXN并改善FA的人源化小鼠模型中的疾病症状。 这些实验的结果有望对FA治疗领域产生重大影响 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。