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.

中文翻译：

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基因组编辑的巧合和 PMP22-HiBiT 融合报告细胞系为 PMP22 基因剂量障碍药物发现提供伪影抑制定量高通量筛选策略

Charcot-Marie-Tooth 1A (CMT1A) 是最常见的遗传性周围神经病变形式，其特征是关键髓鞘基因周围髓鞘蛋白 22 (PMP22) 的遗传重复。PMP22过表达导致雪旺细胞分化异常，导致轴突丢失和肌肉萎缩。由于PMP22表达的调节是 CMT1A 治疗发现的主要目标，我们试图建立无偏见的方法，以识别这种疾病的治疗剂。使用基因组编辑，我们生成了准确监测Pmp22的巧合报告基因分析S16 大鼠雪旺细胞系中的转录水平，同时减少基于报告基因的假阳性。使用该测定法对 42 577 种化合物进行的定量高通量筛选 (qHTS) 揭示了降低内源性Pmp22转录水平的多种新型化学类别。此外，这些类别中的一些在减少Pmp22方面显示出药理学特异性，而不是另一个主要的髓鞘相关基因Mpz（髓鞘蛋白零）。最后，研究化合物介导的Pmp22减少是否转录物转化为降低的 PMP22 蛋白水平，我们编辑了 S16 基因组以生成使用 CRISPR/Cas9 表达 PMP22-HiBiT 融合蛋白的报告基因分析。总体而言，我们提出了一个筛选平台，该平台将编码基因组编辑的细胞系编码监测 PMP22 的转录和翻译后调控的报告基因与基于滴定的筛选（例如，qHTS）相结合，这可以有效地纳入 CMT1A 的药物发现活动。