Charcot-Marie-Tooth (CMT) Disease

检测开发和筛选技术集团(ADST)旨在通过研究和开发创新的检测(测试设计)和化学库筛选来推动治疗开发。Charcot-Marie-Tooth病是一组遗传性周围神经疾病，具有不同的遗传原因，导致运动/感觉异常，慢性疲劳/疼痛，特别是对肢体远端功能的不利影响。CMT通常被归类为主要的脱髓鞘(CMT1)或轴索(CMT2)，这种疾病是最常见的神经系统遗传性疾病之一。在Charcot-Marie-Tooth Association(CMTA)的财政支持下，并与CMT研究人员合作，我们正在设计并从功能上验证用于早期翻译研究的新型分析方法。 CMT类型1A型(与J.Svaren合作)。超过一半的遗传诊断的CMT病例是由影响关键髓鞘基因--外周髓鞘蛋白22(PMP22)的染色体重复引起的。由于PMP22表达水平的增加导致这种神经病变(被归类为CMT1A)，最简单的治疗策略是实现PMP22调节的相对微妙(2倍)的改变。原理验证研究表明，降低PMP22水平会对CMT1A啮齿动物模型产生有益影响。因此，我们开发了新的小分子筛选方法，以确定有效降低PMP22表达的化合物，并治疗CMT1A的根本原因。 我们设计了并在功能上验证了用于定量HTS的新的分析方法，该方法准确地反映了PMP22基因的生理调节。这项工作遵循迭代设计-构建-测试模式，使测试技术的进步与疾病生理学的关键方面相结合，以实现与超高通量测试平台兼容的最先进的生物测试。我们最初的实验设计使用PMP22调节元件驱动报告基因的表达作为PMP22基因表达的替代品。第二代分析现在利用突破性的基因组编辑方法在内源PMP22基因座插入报告者，这允许在天然染色质环境中对报告者进行生理调节。以这种方式创建的分析将能够识别转录和转录后效应(例如，miRNA介导的)。其中一种分析方法目前正在赛诺菲-Genzyme制药公司使用，同时努力为CMT1A疗法确定新的化学起点。 第三代检测设计结合了我们实验室开发的符合生物回路报告，以提高PMP22基因调控候选化合物选择的准确性。此外，我们还设计了一种检测PMP22蛋白翻译后命运的方法，方法是编辑PMP22基因位点以表达一个亲生物发光标签，从而使文库筛选成为我们基于转录的分析方法的简便和补充。这些分析已经被用于对40,000多种化合物进行筛选，以确定几种具有转录活性的化学类型。

Assay Development & Screening Technology group (ADST) is designed to advance therapeutic development through research and development of innovative assay (test designs) and chemical library screenings. Charcot-Marie-Tooth disease is a collection of inherited peripheral neuropathies with diverse genetic causes resulting in motor/sensory abnormalities, chronic fatigue/pain, and adverse impacts particularly on distal limb function. CMT is generally classified as primarily demyelinating (CMT1) or axonal (CMT2), and this disorder is one of the most common inherited diseases of the nervous system. Initiated with financial support from the Charcot-Marie-Tooth Association (CMTA) and in collaboration with CMT investigators we are designing and functionally validating novel assays for use in early stage translational research. CMT Type 1A (Collaboration with J. Svaren). More than half of the genetically diagnosed cases of CMT are caused by a chromosomal duplication affecting a critical myelin gene, Peripheral Myelin Protein 22 (PMP22). Since increased expression levels of PMP22 cause this neuropathy (classified as CMT1A), the simplest strategy for treatment is to achieve a relatively subtle (<2-fold) change in PMP22 regulation. Proof-of-principle studies have shown that reducing PMP22 levels leads to beneficial effects in rodent models of CMT1A. Therefore, we have developed novel assays for small molecule screening to identify compounds that effectively lower PMP22 expression and treat the root cause of CMT1A. We have designed and functionally validated novel assays for use in quantitative HTS that accurately reflect the physiological regulation of the PMP22 gene. This work follows an iterative design-build-test model enabling integration of advances in assay technology with key aspects of the disease physiology to achieve state-of-the-art bioassays compatible with ultra-high throughput testing platforms. Our initial assay designs using PMP22 regulatory elements driving expression of reporter genes as a surrogate of PMP22 gene expression. Second generation assays now utilize the groundbreaking approach of genome editing to insert reporters at the endogenous PMP22 locus, which allows physiological regulation of the reporter in the native chromatin environment. The assays created in this manner will be able to identify both transcriptional and post-transcriptional (e.g. miRNA-mediated) effects. One of these assays is now in use at the pharmaceutical company, Sanofi-Genzyme in a parallel effort to identify novel chemical starting points for a CMT1A therapeutic. A third-generation assay design incorporates a coincidence biocircuit reporter, developed in our laboratory to increase the accuracy of candidate compound selection for PMP22 gene modulation. In addition, we have designed an assay that examines the post-translation fate of PMP22 protein by editing the PMP22 gene locus to express a pro-bioluminescent tag allowing facile and complementary investigation of library screening to our transcriptionally based assays. The assays have been used to conduct a screen of over 40,000 compounds to identify several chemotypes with transcriptional activity.