NINDS Exploratory/Developmental Projects in Translational Research

描述（由申请人提供）：过氧化物酶体生物发生疾病（PBDs）是一组常染色体隐性神经退行性疾病，由PEX基因突变引起。我们的目标是评估一组新开发的药物的疗效，这些药物能够选择性地促进PEX基因中过早终止密码子（无义抑制化合物）的读取，作为pbd患者的治疗药物。其中一种无义抑制药物PTC-124正在临床试验中，分别用于治疗由CFTR和DMD无义突变引起的囊性纤维化和杜氏肌营养不良病例。另一系列基于母体化合物吲哚洛芬的类似物在细胞培养试验中显示出毫无意义的抑制活性，并且在啮齿动物中具有良好的毒性和生物利用度。实现这一目标的必要步骤是确定这些药物在过氧化物酶体组装（PEX）基因无义突变的PBD患者培养细胞中挽救过氧化物酶体结构和功能的能力。在本提案中，我们将对30名PBD患者的PEX基因进行突变分析，以扩展已知的具有致病无义突变的PBD患者亚群（Specific Aim 1）。这些患者体内都存在成纤维细胞，将用于过氧化物酶体功能的下游分析。与此同时，我们将对来自18名PBD患者的经药物处理的培养成纤维细胞进行一系列功能分析，这些患者先前已发现过氧化物基因无义突变（Specific Aim 2）。这包括生化、免疫定位、免疫印迹和基于报告基因的过氧化物酶体蛋白功能和组装的表征。如果这些无义抑制化合物能够有效地挽救培养成纤维细胞中的过氧化物酶体功能，我们将通过基于微阵列的药物处理细胞基因表达谱分析它们的脱靶效应。总的来说，这些研究将为无义突变抑制疗法对pbd的潜在疗效提供必要的初步评估，然后再开发用于临床环境的pbd治疗药物。

DESCRIPTION (provided by applicant): Peroxisomal biogenesis disorders (PBDs) are a group of autosomal recessive neurodegenerative disorders caused by mutations in PEX genes. Our goal is to evaluate the efficacy of a set of newly developed drugs capable of selectively promoting the read-through of premature stop codons (nonsense suppressor compounds) in PEX genes as therapeutic agents for a subset of individuals with PBDs. One such nonsense suppressor drug, PTC-124 is in clinical trials for the treatment of cystic fibrosis and Duchenne's muscular dystrophy cases caused by nonsense mutations in CFTR and DMD, respectively. Another series of analogs based on the parent compound indoprofen have shown nonsense suppressor activity in cell culture assays and have favorable toxicity and bioavailability profiles in rodents. A necessary step in achieving this goal is to determine the ability of these drugs to rescue peroxisome structure and function in cultured cells derived from PBD patients with nonsense mutations in peroxisome assembly (PEX) genes. In this proposal, we will conduct mutational analyses of PEX genes in thirty PBD patients to expand upon a known subset of PBD patients with disease-causing nonsense mutations (Specific Aim 1). Fibroblasts exist from each of these patients which will be used in downstream analyses of peroxisome function. In parallel, we will conduct a series of functional assays on drug-treated cultured fibroblasts derived from eighteen PBD patients with previously identified nonsense mutations in peroxin genes (Specific Aim 2). This includes biochemical, immunolocalization, immunoblot, and reporter gene-based characterization of peroxisome protein function and assembly. Should any of these nonsense suppressor compounds prove effective in rescuing peroxisome function in cultured fibroblasts, we will analyze their off-target effects through microarray-based gene expression profiling of drug-treated cells. Overall, these studies will provide a necessary initial evaluation of the potential efficacy of nonsense mutation suppressor therapies for PBDs prior to our long-term goal of developing therapeutic agents for PBDs that are used in clinical settings. PUBLIC HEALTH RELEVANCE: Project Narrative We are interested in developing therapies for patients with peroxisome biogenesis disorders (PBD), which are a group of inherited, often fatal, neurological diseases in which there is no current therapy. We will evaluate a series of drugs for their ability to rescue peroxisome functions in skin cells cultured from patients. These drugs have been shown to read through nonsense mutations in two other diseases, and preliminary chemical refinement and animal toxicity studies are in progress. Cellular rescue is a necessary preliminary step for the potential future application of these drugs to treat PBD caused by nonsense mutations.