title: Genome-Engineering Tools to Establish Accurate Reporter Cell Lines That Enable Identification of Therapeutic Strategies to Treat Friedreich's Ataxia.
abstract: Friedreich's ataxia is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin. This deficiency results from expansion of a trinucleotide repeat in the first intron of the frataxin gene. Because this repeat expansion resides in an intron and hence does not alter the amino acid sequence of the frataxin protein, gene reactivation could be of therapeutic benefit. High-throughput screening for frataxin activators has so far met with limited success because current cellular models may not accurately assess endogenous frataxin gene regulation. Here we report the design and validation of genome-engineering tools that enable the generation of human cell lines that express the frataxin gene fused to a luciferase reporter gene from its endogenous locus. Performing a pilot high-throughput genomic screen in a newly established reporter cell line, we uncovered novel negative regulators of frataxin expression. Rational design of small-molecule inhibitors of the identified frataxin repressors and/or high-throughput screening of large siRNA or compound libraries with our system may yield treatments for Friedreich's ataxia.
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland University of Basel, Basel, Switzerland.
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Villaseñor
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
authors.forename: Loren
authors.initials: L
authors.lastname: Miraglia
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
authors.forename: Angelica
authors.initials: A
authors.lastname: Romero
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
authors.forename: Buu
authors.initials: B
authors.lastname: Tu
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland University of Basel, Basel, Switzerland Uppsala University, Department of Medical Biochemistry and Microbiology, BMC Uppsala, Sweden.
authors.forename: Tanel
authors.initials: T
authors.lastname: Punga
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland University of Basel, Basel, Switzerland.
authors.forename: Philip
authors.initials: P
authors.lastname: Knuckles
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland University of Basel, Basel, Switzerland.
authors.forename: Stephan
authors.initials: S
authors.lastname: Duss
authors.affiliation: Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.
authors.forename: Tony
authors.initials: T
authors.lastname: Orth
authors.affiliation: Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland University of Basel, Basel, Switzerland marc.buehler@fmi.ch aorth@gnf.org.
authors.forename: Marc
authors.initials: M
authors.lastname: Bühler
mesh_terms: D002461:Cell Line, Transformed; D055808:Drug Discovery; D005621:Friedreich Ataxia / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D015870:Gene Expression; D017930:Genes, Reporter; D005818:Genetic Engineering; D057166:High-Throughput Screening Assays; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D034741:RNA, Small Interfering
keywords: Friedreich’s ataxia (FRDA); PRKD1; SBF1; drug discovery; frataxin (FXN) gene; genome engineering; high-throughput screening (HTS); triplet repeat expansion disorder (TRED); zinc-finger nuclease