Charcot-Marie-Tooth (CMT) Disease

分析开发和筛选技术组（ADST）旨在通过研究和开发创新的分析（测试设计）和化学库筛选来推进治疗开发。Charcot-Marie-Tooth病是一组遗传性周围神经病，具有不同的遗传原因，导致运动/感觉异常，慢性疲劳/疼痛，特别是对远端肢体功能的不利影响。 CMT通常分为原发性脱髓鞘（CMT 1）或轴突（CMT 2），这种疾病是神经系统最常见的遗传性疾病之一。 在Charcot-Marie-Tooth协会（CMTA）的财政支持下，并与CMT研究人员合作，我们正在设计和功能验证用于早期翻译研究的新型检测方法。 CMT Type 1A（与J. Svaren合作）。 超过一半的CMT基因诊断病例是由染色体重复引起的，影响了一个关键的髓鞘基因，外周髓鞘蛋白22（PMP 22）。 由于PMP 22的表达水平增加导致这种神经病（分类为CMT 1A），最简单的治疗策略是实现PMP 22调节的相对细微（<2倍）变化。 原理验证研究表明，降低PMP 22水平可在CMT 1A啮齿动物模型中产生有益作用。 因此，我们开发了用于小分子筛选的新型测定法，以鉴定有效降低PMP 22表达并治疗CMT 1A根本原因的化合物。 我们设计并功能验证了用于定量HTS的新检测方法，这些方法准确反映了PMP 22基因的生理调节。 这项工作遵循迭代设计-构建-测试模型，能够将检测技术的进步与疾病生理学的关键方面相结合，以实现与超高通量检测平台兼容的最先进的生物检测。 我们最初的试验设计使用PMP 22调控元件驱动报告基因的表达作为PMP 22基因表达的替代物。 第二代检测现在利用突破性的基因组编辑方法在内源性PMP 22基因座插入报告基因，这允许在天然染色质环境中对报告基因进行生理调节。 以这种方式创建的测定将能够鉴定转录和转录后（例如miRNA介导的）效应。 其中一种检测方法目前正在制药公司Sanofi-Genzyme使用，以确定CMT 1A治疗剂的新化学起点。 第三代检测设计结合了我们实验室开发的巧合生物电路报告基因，以提高PMP 22基因调控候选化合物选择的准确性。 最近，我们设计了一种检测方法，该方法通过编辑PMP 22基因位点以表达前生物发光标签来检查PMP 22蛋白的翻译后命运，从而允许对我们基于转录的检测方法进行文库筛选的简单和互补的研究。

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. Most recently 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.