NINDS Exploratory/Developmental Projects in Translational Research

描述(申请人提供)：过氧化体生物发生障碍(PBD)是一组常染色体隐性遗传性神经退行性疾病，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有效性，这些药物能够选择性地促进PEX基因中过早停止密码子(无意义抑制化合物)的阅读，作为治疗PBD患者子集的药物。其中一种无意义抑制药物PTC-124正在进行临床试验，分别治疗由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无意义的抑制活性，并在啮齿类动物中具有良好的毒性和生物利用度。实现这一目标的一个必要步骤是确定这些药物在PBD患者的培养细胞中拯救Peroxisome结构和功能的能力，这些细胞来自Peroxisome Assembly(PEX)基因的无义突变。在这项提案中，我们将对30名PBD患者进行PEX基因突变分析，以扩大具有致病无义突变的PBD患者的已知亚群(特定目标1)。这些患者体内都有成纤维细胞，这些成纤维细胞将用于过氧化物酶功能的下游分析。同时，我们将对来自18名先前发现Peroxin基因无义突变的PBD患者的药物处理后的培养成纤维细胞进行一系列功能分析(特定目标2)。这包括基于生化、免疫定位、免疫印迹和基于报告基因的过氧化体蛋白功能和组装的表征。如果任何这些无意义的抑制化合物被证明在拯救培养的成纤维细胞中的过氧化物体功能是有效的，我们将通过基于药物处理细胞的基因表达谱微阵列来分析它们的脱靶效应。总体而言，这些研究将在我们开发用于临床的PBD治疗药物的长期目标之前，对PBD的无意义突变抑制疗法的潜在疗效提供必要的初步评估。 公共卫生相关性：项目描述我们有兴趣为过氧化物体生物发生障碍(PBD)患者开发治疗方法，这是一组遗传性的、通常是致命的神经疾病，目前还没有治疗方法。我们将评估一系列药物拯救患者培养的皮肤细胞中过氧化物酶功能的能力。这些药物已经被证明可以读取另外两种疾病的无义突变，初步的化学提纯和动物毒性研究正在进行中。细胞拯救是这些药物未来可能应用于治疗由无义突变引起的PBD的必要的初步步骤。

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.