Abstract
Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and post-translational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.