Mechanisms of Gene Silencing of Friedreich's Ataxia

描述（由研究者提供）：本申请旨在了解神经退行性疾病弗里德赖希共济失调 (FRDA) 的分子基础和新型疗法的开发。 FRDA 是三联体重复疾病之一，其中编码必需线粒体蛋白 frataxin 的 FXN 基因内 GAA.TTC 重复的扩增导致表观遗传转录沉默。 frataxin 的缺失会导致脊髓小脑共济失调并继发性心肌病，这是 FRDA 患者死亡的主要原因。目前尚无批准的 FRDA 治疗方法。由于GAA.TTC重复位于内含子中，并且不影响frataxin蛋白的序列，因此基因激活将具有治疗价值。我们确定 2-氨基苯甲酰胺类 HDAC 抑制剂的成员是 FXN 转录的有效激活剂。这些分子可穿过小鼠和犬科动物的血脑屏障，不表现出急性或慢性毒性，并在小鼠 FRDA 模型中增加大脑和心脏中的 FXN mRNA 和 frataxin 蛋白水平，以及在 Ib 期人体临床试验中经药物治疗的 FRDA 患者的循环淋巴细胞中的 FXN mRNA 和 frataxin 蛋白水平。虽然这些数据为这种治疗方法提供了概念证明，但我们目前的化合物存在药理学限制，妨碍了它们在慢性治疗中的使用。通过药物化学的努力，我们已经确定了解决这些局限性的新化合物，并且一种这样的分子正在被作为新的临床候选药物。在之前的申请期间，我们从 FRDA 患者体内产生了诱导多能干细胞 (iPSC)，并将这些细胞沿神经元谱系分化。我们使用这些细胞来模拟 FRDA，以研究 FXN 基因沉默和药物筛选。在本申请中，我们计划（1）优化 hiPSC 分化为感觉神经元（FRDA 中受影响的主要细胞类型）的方法，并通过全局基因表达研究和线粒体功能障碍标记物使用这些细胞来模拟该疾病。对于这些实验，我们将使用辅助依赖性腺病毒介导的同源重组来生成具有 GAA¿TTC 重复“校正”至正常长度的同基因细胞系。 (2) 由于心肌病是 FRDA 死亡的主要原因，我们还将在 FRDA iPSC 衍生的心肌细胞中模拟该疾病。 (3) 我们将使用这两种 FRDA 细胞模型来探究改进的 HDAC 抑制剂是否可以逆转 FRDA 基因表达特征和 FRDA 线粒体病理学。 (4) 最后，我们将使用神经元细胞和患者淋巴细胞来鉴定基因表达生物标志物，用于 FRDA 患者的 II 期疗效研究。我们的研究处于开发针对这种目前无法治疗的致命疾病的新型疗法的前沿。

DESCRIPTION (provided by investigator): This application is aimed at understanding the molecular basis for the neurodegenerative disease Friedreich's ataxia (FRDA) and development of novel therapeutics. FRDA is one of the triplet-repeat diseases, where expansion of GAA.TTC repeats within the FXN gene, encoding the essential mitochondrial protein frataxin, leads to epigenetic transcriptional silencing. Loss of frataxin results in a spinocerebellar ataxia with secondary cardiomyopathy, which is the major cause of death in FRDA patients. At present there is no approved therapy for FRDA. Since the GAA.TTC repeats are in an intron, and do not affect the sequence of frataxin protein, gene activation would be of therapeutic value. We identified members of the 2-aminobenzamide class of HDAC inhibitors as potent activators of FXN transcription. These molecules cross the blood brain barrier in mice and canines, exhibit no acute or chronic toxicity, and increase FXN mRNA and frataxin protein levels in the brain and heart in the mouse FRDA model, as well as in circulating lymphocytes in drug-treated FRDA patients in a Phase Ib human clinical trial. While these data provide a proof of concept for this therapeutic approach, our current compounds suffer from pharmacological limitations that preclude their use in chronic treatment. Through a medicinal chemistry effort, we have identified new compounds that have solved these limitations, and one such molecule is being taken forward as a new clinical candidate. During the previous application period, we generated induced pluripotent stem cells (iPSCs) from FRDA patients and differentiated these cells along the neuronal lineage. We have used these cells to model FRDA to study FXN gene silencing and for drug screening. In the present application, we plan to (1) optimize methods for the differentiation of hiPSCs to sensory neurons, the major cell type affected in FRDA, and to use these cells to model the disease through global gene expression studies and markers of mitochondrial dysfunction. For these experiments, we will use helper-dependent adenovirus-mediated homologous recombination to generate isogenic cell lines have the GAA¿TTC repeats "corrected" to normal lengths. (2) Since cardiomyopathy is the major cause of death in FRDA, we will also model the disease in FRDA iPSC-derived cardiomyocytes. (3) We will use these two FRDA cell models to ask if improved HDAC inhibitors can reverse FRDA gene expression signatures and FRDA mitochondrial pathology. (4) Lastly, we will use neuronal cells and patient lymphocytes to identify gene expression biomarkers to be used in Phase II efficacy studies in FRDA patients. Our studies are at the forefront of development of a novel therapeutic for this currently untreatable and lethal disease.