title: Genome editing-enabled HTS assays expand drug target pathways for Charcot-Marie-tooth disease.
abstract: Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health , Rockville, Maryland 20850, United States.
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Patricia
authors.initials: P
authors.lastname: Dranchak
authors.forename: John J
authors.initials: JJ
authors.lastname: Moran
authors.forename: Sung-Wook
authors.initials: SW
authors.lastname: Jang
authors.forename: Rajini
authors.initials: R
authors.lastname: Srinivasan
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Santiago
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Guha
authors.forename: Natalia
authors.initials: N
authors.lastname: Martinez
authors.forename: Ryan
authors.initials: R
authors.lastname: MacArthur
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Cost
authors.forename: John
authors.initials: J
authors.lastname: Svaren
mesh_terms: D002607:Charcot-Marie-Tooth Disease / Q000235:genetics*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans