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).
['Cotticelli|M Grazia|MG|', 'Acquaviva|Fabio|F|', 'Xia|Shujuan|S|', 'Kaur|Avinash|A|', 'Wang|Yongping|Y|', 'Wilson|Robert B|RB|']
D020155:3-Hydroxybutyric Acid D001483:Base Sequence D002478:Cells, Cultured D003470:Culture Media D005621:Friedreich Ataxia D055785:Gene Knockdown Techniques D059014:High-Throughput Nucleotide Sequencing D057166:High-Throughput Screening Assays D006801:Humans D033862:Iron-Binding Proteins D008969:Molecular Sequence Data D010641:Phenotype D034622:RNA Interference D034741:RNA, Small Interfering D017422:Sequence Analysis, DNA
Friedreich ataxia shRNA library siRNA therapeutics