Genome-Edited Coincidence and PMP22-HiBiT Fusion Reporter Cell Lines Enable an Artifact-Suppressive Quantitative High-Throughput Screening Strategy for <i>PMP22</i> Gene-Dosage Disorder Drug Discovery

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). <i>PMP22</i> overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of <i>PMP22</i> 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 <i>Pmp22</i> 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 <i>Pmp22</i> transcript levels. Moreover, some of these classes show pharmacological specificity in reducing <i>Pmp22</i> over another major myelin-associated gene, <i>Mpz</i> (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of <i>Pmp22</i> 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.