Phenotypic Screening for Friedreich Ataxia Using Random shRNA Selection.
Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are no proven effective treatments. FRDA is caused by decreased expression and/or function of the protein frataxin. Frataxin chaperones iron in the mitochondrial matrix and regulates the iron-sulfur cluster (ISC) assembly complex. ISCs are prosthetic groups critical for the function of the Krebs cycle and the mitochondrial electron transport chain. Decreased expression of frataxin is associated with decreased ISC assembly, mitochondrial iron accumulation, and increased oxidative stress, all of which contribute to mitochondrial dysfunction. In media with beta-hydroxybutyrate (BHB) as carbon source, primary FRDA fibroblasts grow poorly and/or lose viability over several days. We screened a random, short-hairpin-RNA (shRNA)-expressing library in primary FRDA fibroblasts and identified two shRNAs that reverse the growth/viability defect in BHB media. One of these two clones increases frataxin expression in primary FRDA fibroblasts, either as a vector-expressed shRNA or as a transfected short-interfering RNA (siRNA).
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The Penn Medicine/CHOP Center of Excellence for Friedreich's Ataxia Research, Philadelphia, PA, USA.
M Grazia
MG
Cotticelli
Department of Molecular Medicine and Medical Biotechnologies, University of Naples "Federico II," Naples, Italy.
Fabio
F
Acquaviva
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Shujuan
S
Xia
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The Penn Medicine/CHOP Center of Excellence for Friedreich's Ataxia Research, Philadelphia, PA, USA.
Avinash
A
Kaur
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Yongping
Y
Wang
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The Penn Medicine/CHOP Center of Excellence for Friedreich's Ataxia Research, Philadelphia, PA, USA wilsonr@mail.med.upenn.edu.
Robert B
RB
Wilson
D020155:3-Hydroxybutyric Acid / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003470:Culture Media; D005621:Friedreich Ataxia / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays; D006801:Humans; D033862:Iron-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D017422:Sequence Analysis, DNA; D000096923:Frataxin
D020155:3-Hydroxybutyric Acid; D003470:Culture Media; D033862:Iron-Binding Proteins; D034741:RNA, Small Interfering; D000096923:Frataxin
Friedreich ataxia; library; shRNA; siRNA; therapeutics