Charcot-Marie-Tooth (CMT) Disease

点击翻译按钮获取中文摘要

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.