Upregulation of expression from the FRDA genomic locus for the therapy of Friedreich ataxia.

Background Friedreich ataxia is a slowly progressive neurodegenerative disease caused by reduced expression of frataxin as a result of a GAA repeat expansion in the first intron of the FRDA gene. We report here the development of a sensitive cellular assay for frataxin expression from the intact FRDA locus that should facilitate the identification of potentially therapeutic pharmacological agents to treat Friedreich ataxia. Methods PAC and BAC clones containing the entire human FRDA functional genomic sequence were identified and shown to express FRDA mRNA. The GET Recombination system was used to insert cassettes consisting of the gene encoding EGFP linked to a kanamycin/neomycin resistance determinant into a BAC clone containing the entire FRDA gene and surrounding regions. Results Two in-frame fusions between the FRDA gene and a gene coding for enhanced green fluorescent protein (EGFP) were constructed. One fusion is within exon 2 of the FRDA gene. The other is at the end of exon 5a, containing the entire frataxin protein fused to EGFP. Both constructs were shown to drive the expression of EGFP from the regulatory elements of the FRDA locus, with the frataxin-EGFP fusion proteins targeted to the mitochondria. Stable cell lines containing the EGFP fusion in exon 5a were produced. Enhancement of FRDA gene expression by hemin and butyric acid was demonstrated. Conclusions Expression studies with FRDA-EGFP fusion constructs will facilitate delineation of regulatory elements determining the tissue and developmental specificity of FRDA gene expression. These constructs should also facilitate screening for pharmacological compounds that can modulate the expression of the FRDA gene in a clinically relevant manner. Copyright © 2002 John Wiley & Sons, Ltd.