Development of Assays and the Identification of Targets and Molecules Modulating the Molecular Basis of Charcot Marie Tooth Disease Type 1A
Charcot Marie Tooth Disease (CMT) is a heterogeneous group of genetic disorders that disrupt the peripheral nervous system of 1:2,500 people of all backgrounds. CMT1A is the most frequent form, a demyelinating neuropathy caused by over-expression of the peripheral myelin protein 22 (PMP22) gene and its protein. The goals of the STAR initiative of the Charcot Marie Tooth Association (CMTA) are to identify medications that reduce PMP22 expression and treat patients with CMT1A. With CMTA support, we form the nexus of a translational program that integrates the expertise and efforts of leading laboratories studying CMT1A.
Together we have designed, developed, and optimized cell-based assays targeting CMT-based inherited peripheral neuropathy biology, and continue to refine our approaches with gene editing and stem cell technologies. These assays are being used in the pharmacological profiling of libraries of approved drugs and chemotypes derived from novel synthetic methodologies, and microorganisms. The assays further serve to aid in the identification of CMT1A modifying targets using gene silencing and chemical genomic strategies.
Assay Concepts Based on Knowledge of Biology
National Center for Advancing Translational Sciences
James Inglese, Ph.D.
University of Wisconsin
John Svaren, Ph.D.
Institute of Cell Biology-Department of Biology, ETH Zürich
Ueli Suter, Ph.D.
Max Planck Institute for Experimental Medicine
Klaus-Armin Nave, Ph.D.
University of Pennsylvania
Steven Scherer, M.D., Ph.D.
Wayne State University
Michael Shy, M.D.
Public Health Impact
CMT is the most commonly inherited genetic disorder of the peripheral nervous system, which affects more than 2.6 million people around the world without regard to age, sex, or ethnicity. CMT, which is currently incurable, slowly damages the nerve cells leading to the arms/hands and legs/feet and results in, among other things, muscle loss, sensory loss, and pain.
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- Sung-Wook Jang was selected from a competitive search as a CMTA-sponsored fellow in July 2009.
- In collaboration with the laboratory of Ueli Suter, a cell line based on the mouse Schwann cell, MSC80 cells, were engineered to express a two-color luciferase reporter driven from a 10Kb 5'UTR derived from the pmp22 gene. The cell line was studied for responsiveness to critical transcription factors known to be important for pmp22 expression. Based on insufficient signal modulation from Sox10 silencing and a strong differential response of the homologous luciferases traced to selective luciferase inhibition, the assay was not further considered.
- In collaboration with the laboratory of John Svaren, an assay was developed based on Svaren's identification of a unique region of the human pmp22 gene, which robustly regulates pmp22 expression. Utilizing a rat Schwann cell line, S16 cells, we designed a second generation orthogonal pair of cell lines based on the bioluminescent and fluorescent output of luciferase and β-lactamase, respectively. The assays robustly and accurately reflect the regulation of the pmp22 gene when key transcription factors are silenced.
- The S16 cell lines were screened against the NCGC pharmaceutical collection, resulting in the identification of four compounds, which reduced the levels of pmp22 expression. Results were confirmed on endogenous pmp22 using qPCR. Further studies are being conducted to explore the selectivity of the effect and examine these drugs in advanced models of the disease.
- Forward chemical genomics using annotated chemical probe libraries targeting specific druggable target classes is ongoing in collaboration with two pharmaceutical companies.