Mechanisms of Gene Silencing of Friedreich's Ataxia

Friedreich共济失调的基因沉默机制

• 批准号: 9128068
• 负责人: JOEL M. GOTTESFELD
• 金额: $ 42.11万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9128068
• 关键词: Acute; Affect; Afferent Neurons; Biochemistry; Biological Assay; Biological Markers; Blood - brain barrier anatomy; Brain; Canis familiaris; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cause of Death; Cell Line; Cell model; Cells; Chronic; Clinical; Clinical Trials; Data; Development; Disease; Disease model; Epigenetic Process; Exhibits; FDA approved; Friedreich Ataxia; Functional disorder; Gene Activation; Gene Expression; Gene Expression Profile; Gene Silencing; Genes; Genetic Transcription; Health; Heart; Histone Deacetylase Inhibitor; Human; Inherited; Introns; Length; Lymphocyte; Mediating; Messenger RNA; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mus; Neurodegenerative Disorders; Neurons; Pathology; Patients; Peripheral; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacodynamics; Phase; Preclinical Drug Evaluation; Proteins; Research Personnel; Secondary Myocardial Diseases; Spinal Ganglia; Spinocerebellar Ataxias; Testing; Therapeutic; Time; Toxic effect; Trinucleotide Repeats; adenoviral-mediated; base; cell type; design; effective therapy; efficacy trial; frataxin; homologous recombination; human subject; improved; induced pluripotent stem cell; member; mitochondrial dysfunction; mouse model; neuropathology; novel; novel therapeutics; patient population; research study; treatment duration

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.

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）的分子基础和新疗法的开发。FRDA是三重重复疾病之一，其中编码必需线粒体蛋白共济蛋白的FXN基因内GAA.TTC重复序列的扩增导致表观遗传转录沉默。共济失调蛋白的缺失导致脊髓小脑共济失调伴继发性心肌病，这是FRDA患者死亡的主要原因。目前尚无获批的FRDA治疗方法。由于GAA.TTC重复序列位于内含子中，并且不影响共济失调蛋白的序列，因此基因激活将具有治疗价值。我们鉴定了HDAC抑制剂的2-氨基苯甲酰胺类的成员作为FXN转录的有效激活剂。这些分子在小鼠和犬中穿过血脑屏障，没有表现出急性或慢性毒性，并且在小鼠FRDA模型中增加大脑和心脏中的FXN mRNA和共济失调蛋白水平，以及在Ib期人类临床试验中药物治疗的FRDA患者的循环淋巴细胞中增加FXN mRNA和共济失调蛋白水平。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受到药理学限制，无法用于慢性治疗。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，其中一种这样的分子正被作为新的临床候选物。在之前的应用期间，我们从FRDA患者中产生了诱导多能干细胞（iPSC），并使这些细胞沿着神经元谱系分化。我们已经使用这些细胞来模拟FRDA以研究FXN基因沉默和用于药物筛选。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，并使用这些细胞通过全局基因表达研究和线粒体功能障碍的标志物来模拟疾病。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产生具有“校正”为正常长度的GAA <$TTC重复的等基因细胞系。(2)由于心肌病是FRDA中死亡的主要原因，我们还将在FRDA iPSC衍生的心肌细胞中对该疾病进行建模。(3)我们将使用这两种FRDA细胞模型来询问改进的HDAC抑制剂是否可以逆转FRDA基因表达特征和FRDA线粒体病理。(4)最后，我们将使用神经元细胞和患者淋巴细胞来鉴定用于FRDA患者II期疗效研究的基因表达生物标志物。我们的研究处于开发这种目前无法治疗和致命疾病的新型治疗方法的最前沿。