Mouse Model for Diseases of Protein Misfolding

描述（由申请人提供）：本项目旨在开发和表征人类蛋白质折叠疾病的两种原型转基因小鼠模型。这些模型需要将一类新的靶向药物，药物酮，用于人类（和动物）使用。错误折叠的突变蛋白由细胞质量控制系统（QCS）检测到，通常保留在内质网（ER）中进行再加工或降解；通常，这些突变会导致疾病。细胞培养的研究表明，这些突变体可以被靶向小分子（药酮）拯救，这些小分子进入细胞，作为重组突变体的模板，并允许它们通过质膜。许多突变体保留或恢复了它们的基本特性，如离子通道、酶或受体。由错误折叠引起的疾病（可能受益于这种方法）包括囊性纤维化、促性腺功能低下、尿囊性肾病、视网膜色素变性、高胆固醇血症、白内障、神经退行性疾病（亨廷顿氏症、阿尔茨海默氏症和帕金森症）、癌症和消化系统疾病。公平地说，几乎每个人都会在他或她的一生中受到蛋白质折叠疾病的影响，无论是直接的还是由于所爱的人的疾病。尽管如此，很少有模型系统，而且没有在小型实验动物中，允许将可用的体外数据翻译或从高通量筛选蛋白质拯救的“命中”测试到体内系统中。迄今为止，人类的治疗方法依赖于对临终病人的少量研究，使用的药物从未在动物模型中得到充分表征。这些模型需要解决药物安全性问题，优化治疗效果所需的给药模式，并作为新药临床前研究的测试模型。模式（途径、剂量和频率）尤其重要，因为这些药物的持续使用往往会抑制预期的活性，一旦发生挽救，就必须将其移除。此外，如果要将药物酮的使用转化为人类福祉，就需要为这些疾病建立一个方便的实验室模型，因为美国法律要求在正常人类志愿者之前进行动物试验。本研究将描述错误折叠的原型小鼠模型，依赖于生理上重要的GPCR（即促性腺激素释放激素受体）的异常良好的突变。关于促性腺激素释放激素受体（GnRHR）激活的机制，以及突变体E90K被认为导致疾病状态的生化机制，目前已有很多信息。这些信息有助于指导我们选择突变体E90K，并将有助于这个项目的成功。所使用的两种模型都是可用的，并且具有预测的基因型和必要的表型。

DESCRIPTION (provided by applicant): This project is designed to develop and characterize two prototypic genetically modified mouse models for human diseases of protein folding. These models are required to bring a novel class of target-specific drugs, pharmacoperones, to human (and animal) use. Misfolded mutant proteins are detected by the cellular quality control system (QCS) and are typically retained in the endoplasmic reticulum (ER) for either reprocessing or degradation; frequently, these mutants result in disease. Studies in cell cultures indicate that these mutants can be rescued by target-specific small molecules (pharmacoperones) which enter cells, serve as templates that refold the mutants, and permit their passage to the plasma membrane. Many mutants retain or regain their fundamental properties as ion channels, enzymes or receptors when re-routed correctly. Diseases caused by misfolding (which may benefit from this approach) include cystic fibrosis, hypogonadotropic hypogonadism, nephrogenic diabetes insipidus, retinitis pigmentosa, hypercholesterolemia, cataracts, neurodegenerative diseases (Huntington's, Alzheimer's and Parkinson's), cancers and digestive disorders. It is fair to say that virtually every person will be affected by protein folding diseases during his or her lifetime, either directly or due to the illness of a loved one. In spite of this, there are few model systems, and none in small laboratory animals, that allow the translation of available in vitro data or the testing of "hits" from high throughput screening on protein rescue into in vivo systems. To date, therapeutic approaches in humans have relied on a small number of studies in end-of-life patients, using drugs that have never been fully characterized in animal models. Such models are needed to address drug safety, the pattern of drug administration required to optimize therapeutic effectiveness, and serve as a test model for new drugs in preclinical studies. The pattern (route, dose and frequency) is particularly important as the persistence of these drugs frequently inhibits the desired activity, once rescue has occurred, so they must be removed. Moreover a convenient laboratory model for these diseases is needed if the use of pharmacoperones is to translate to human well-being, since US law requires animal testing prior to normal human volunteers. The present study will characterize prototypic mouse models of misfolding, relying on an unusually well-characterized mutant of a physiologically important GPCR (i.e. the gonadotropin releasing hormone receptor). There is much information available on the mechanism of activation of the gonadotropin releasing hormone receptor (GnRHR) and on the biochemical mechanism by which the mutant E90K is believed to cause the disease state. This information has been helpful in guiding our choices of mutant E90K and will contribute to the success of this project. The two models to be used are available and have the predicted genotype and necessary phenotype. PUBLIC HEALTH RELEVANCE: This project is designed to develop, characterize, compare and contrast two models for human diseases of protein folding. Diseases caused by misfolding include cystic fibrosis, hypogonadotropic hypogonadism, nephrogenic diabetes insipidus, retinitis pigmentosa, hypercholesterolemia, cataracts, neurodegenerative diseases (Huntington's, Alzheimer's and Parkinson's), particular cancers and a number of digestive disorders resulting from enzyme mutation. A convenient laboratory model for these diseases is needed if the use of pharmacoperones is to translate to human well-being, since US law requires animal testing prior to normal human volunteers.