RESUMO
Friedreich ataxia (FRDA) is due to a triplet repeat expansion in FXN, resulting in deficiency of the mitochondrial protein frataxin. Resveratrol is a naturally occurring polyphenol, identified to increase frataxin expression in cellular and mouse models of FRDA and has anti-oxidant properties. This open-label, non-randomized trial evaluated the effect of two different doses of resveratrol on peripheral blood mononuclear cell (PBMC) frataxin levels over a 12-week period in individuals with FRDA. Secondary outcome measures included PMBC FXN mRNA, oxidative stress markers, and clinical measures of disease severity. Safety and tolerability were studied. Twenty-four participants completed the study; 12 received low-dose resveratrol (1 g daily) and 12 high-dose resveratrol (5 g daily). PBMC frataxin levels did not change in either dosage group [low-dose group change: 0.08 pg/µg protein (95% CI -0.05, 0.21, p = 0.21); high-dose group change: 0.03 pg/µg protein (95% CI -0.10, 0.15, p = 0.62)]. Improvement in neurologic function was evident in the high-dose group [change in Friedreich Ataxia Rating Scale -3.4 points, 95% CI (-6.6, -0.3), p = 0.036], but not the low-dose group. Significant improvements in audiologic and speech measures, and in the oxidative stress marker plasma F2-isoprostane were demonstrated in the high-dose group only. There were no improvements in cardiac measures or patient-reported outcome measures. No serious adverse events were recorded. Gastrointestinal side-effects were a common, dose-related adverse event. This open-label study shows no effect of resveratrol on frataxin levels in FRDA, but suggests that independent positive clinical and biologic effects of high-dose resveratrol may exist. Further assessment of efficacy is warranted in a randomized placebo-controlled trial.
Assuntos
Antioxidantes/uso terapêutico , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Estilbenos/uso terapêutico , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , F2-Isoprostanos/sangue , Feminino , Análise de Fourier , Humanos , Proteínas de Ligação ao Ferro/genética , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Resveratrol , Resultado do Tratamento , Adulto Jovem , FrataxinaRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. We previously generated BAC-based transgenic mice containing an FXN-EGFP genomic reporter construct in which the EGFP gene is fused in-frame immediately following the final codon of exon 5a of the human FXN gene. These transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Fxn gene, to generate mice homozygous for the Fxn knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Fxn knockout mutation was observed. Rescue mice displayed normal behavioral and histological parameters with normal viability, fertility and life span and without any signs of aberrant phenotype. Immunoblotting demonstrated the production of full-length frataxin-EGFP fusion protein that appears to act as a bifunctional hybrid protein. This study shows frataxin replacement may be a viable therapeutic option. Further, these mice should provide a useful resource for the study of human FXN gene expression, frataxin function, the evaluation of pharmacologic inducers of FXN expression in a whole-animal model and provide a useful source of cells for stem cell transplantation studies.
Assuntos
Técnicas de Inativação de Genes , Genes Reporter/genética , Genômica , Proteínas de Fluorescência Verde/genética , Proteínas de Ligação ao Ferro/genética , Mutação , Animais , Teste de Complementação Genética , Homozigoto , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , FrataxinaRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ataxia de Friedreich/tratamento farmacológico , Proteínas de Ligação ao Ferro/genética , Linhagem Celular , Ataxia de Friedreich/genética , Biblioteca Gênica , Genes Reporter , Genômica , Células HeLa , Humanos , Expansão das Repetições de Trinucleotídeos , Regulação para Cima , FrataxinaRESUMO
Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Modelos Animais de Doenças , Camundongos , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/mortalidade , Animais , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ordem dos Genes , Vetores Genéticos/genética , Genótipo , Humanos , Metaboloma , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Camundongos Knockout , Camundongos Transgênicos , TransgenesRESUMO
OBJECTIVE: Friedreich ataxia (FA) is the most common ataxia and results from an expanded GAA repeat in the first intron of FXN. This leads to epigenetic modifications and reduced frataxin. We investigated the relationships between genetic, epigenetic, and clinical parameters in a large case-control study of FA. METHODS: Clinical data and samples were obtained from individuals with FA during annual visits to our dedicated FA clinic. GAA expansions were evaluated by polymerase chain reaction (PCR) and restriction endonuclease digest. DNA methylation was measured using bisulfite-based EpiTYPER MassARRAY (Sequenom, San Diego, CA). FXN expression was determined using real-time reverse transcriptase PCR. Significant correlations between the different parameters were examined using the nonparametric Spearman rank correlation coefficient, as well as univariate and multivariate regression modeling. RESULTS: Characteristic DNA methylation was identified upstream and downstream of the expansion, and validated in an independent FA cohort. Univariate and multivariate analyses showed significant inverse correlations between upstream methylation and FXN expression, and variation in downstream methylation and age of onset. FXN expression also inversely correlated with the Friedreich Ataxia Rating Scale score, an indicator of disease severity. INTERPRETATION: These novel findings provide compelling evidence for the link between the GAA expansion, the DNA methylation profile, FXN expression, and clinical outcome in FA. Epigenetic profiling of FXN could be used to gain greater insight into disease onset and progression, but also as a biomarker to learn more about specific treatment responses and pharmacological mechanism(s). This work also highlights the potential for developing therapies aimed at increasing frataxin levels to treat this debilitating disease.
Assuntos
Metilação de DNA/genética , Ataxia de Friedreich/genética , Marcadores Genéticos/genética , Proteínas de Ligação ao Ferro/genética , Expansão das Repetições de Trinucleotídeos/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Progressão da Doença , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sequências Repetitivas de Ácido Nucleico , Adulto Jovem , FrataxinaRESUMO
BACKGROUND: Friedreich ataxia (FRDA) is the most common form of hereditary ataxia characterized by the presence of a GAA trinucleotide repeat expansion within the first intron of the FXN gene. The expansion inhibits FXN gene expression resulting in an insufficiency of frataxin protein. METHODOLOGY/PRINCIPAL FINDING: In this study, computational analyses were performed on the 21.3 kb region upstream of exon 1 of the human FXN gene and orthologs from other species in order to identify conserved non-coding DNA sequences with potential regulatory functions. The conserved non-coding regions identified were individually analyzed in two complementing assay systems, a conventional luciferase reporter system and a novel Bacterial Artificial Chromosome (BAC)-based genomic reporter. The BAC system allows the evaluation of gene expression to be made in the context of its entire genomic locus and preserves the normal location and spacing of many regulatory elements which may be positioned over large distances from the initiation codon of the gene. CONCLUSIONS/SIGNIFICANCE: The two approaches were used to identify a region of 17 bp located approximately 4.9 kb upstream of the first exon of the FXN gene that plays an important role in FXN gene expression. Modulation of FXN gene expression was found to be mediated by the action of the Oct-1 transcription factor at this site. A better understanding of cis-acting regulatory elements that control FXN gene expression has the potential to develop new strategies for the upregulation of the FXN gene as a therapy for FRDA.
Assuntos
Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro/genética , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Cromossomos Artificiais Bacterianos/genética , Biologia Computacional , Sequência Conservada/genética , DNA/genética , DNA Intergênico , Genes Reporter/genética , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Dados de Sequência Molecular , Fator 1 de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas/genética , Deleção de Sequência/genética , FrataxinaRESUMO
Friedreich ataxia is a neurodegenerative disorder caused by the expansion of a GAA trinucleotide repeat sequence within the first intron of the FXN gene. Interruptions in the GAA repeat may serve to alleviate the inhibitory effects of the GAA expansion on FXN gene expression and to decrease pathogenicity. We have developed a simple and rapid PCR- and restriction enzyme-based assay to assess the purity of GAA repeat sequences.
Assuntos
Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Reação em Cadeia da Polimerase/métodos , Expansão das Repetições de Trinucleotídeos , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Reação em Cadeia da Polimerase/economia , FrataxinaRESUMO
Friedreich ataxia (FRDA) is an autosomal recessive disorder characterised by neurodegeneration and cardiomyopathy. It is caused by a trinucleotide (GAA) repeat expansion in the first intron of the FXN gene that results in reduced synthesis of FXN mRNA and its protein product, frataxin. We report the generation of induced pluripotent stem (iPS) cell lines derived from skin fibroblasts from two FRDA patients. Each of the patient-derived iPS (FA-iPS) cell lines maintain the GAA repeat expansion and the reduced FXN mRNA expression that are characteristic of the patient. The FA-iPS cells are pluripotent and form teratomas when injected into nude mice. We demonstrate that following in vitro differentiation the FA-iPS cells give rise to the two cell types primarily affected in FRDA, peripheral neurons and cardiomyocytes. The FA-iPS cell lines have the potential to provide valuable models to study the cellular pathology of FRDA and to develop high-throughput drug screening assays. We have previously demonstrated that stable insertion of a functional human BAC containing the intact FXN gene into stem cells results in the expression of frataxin protein in differentiated neurons. As such, iPS cell lines derived from FRDA patients, following correction of the mutated gene, could provide a useful source of immunocompatible cells for transplantation therapy.
Assuntos
Linhagem Celular , Ataxia de Friedreich/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Reprogramação Celular , Ataxia de Friedreich/genética , Humanos , Proteínas de Ligação ao Ferro/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Teratoma/patologia , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
BACKGROUND: Methylmalonic aciduria is an autosomal recessive inborn error of the propionate metabolic pathway. One form of this disorder is caused by mutations in methylmalonyl-coenzyme A mutase (MCM), resulting in reduced levels of enzyme activity. The pharmacological up-regulation of residual mutase activity is one approach to advance treatment strategies for individuals affected by this disorder. We describe the construction, characterization and use of a cellular genomic reporter assay for MCM expression that will potentially identify therapeutic pharmacological agents for methylmalonic aciduria treatment. METHODS: Homologous recombination was used to insert an enhanced green fluorescent protein (EGFP) cassette inframe before the last codon of exon 13 of the MCM gene (MUT) in a BAC clone. The construct was used to generate stable HeLa cell lines. EGFP expression was measured by flow cytometry and the real-time reverse transcriptase-polymerase chain reaction was used to quantify changes in MUT gene mRNA levels. RESULTS: The genomic reporter assay used to screen a selection of compounds. Cisplatin, zidovudine and adefovir were found to increase the levels of MCM mRNA and EGFP expression, providing support for the possible efficacy of these pharmacological compounds in treating methylmalonic aciduria. CONCLUSIONS: This assay has the potential of being used in high-throughput screening of chemical libraries for the identification of novel compounds that specifically modulate the expression of MCM.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Erros Inatos do Metabolismo/terapia , Metilmalonil-CoA Mutase/deficiência , Metilmalonil-CoA Mutase/genética , Adenina/análogos & derivados , Adenina/farmacologia , Cisplatino/farmacologia , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Erros Inatos do Metabolismo/genética , Organofosfonatos/farmacologia , RNA Mensageiro/análise , Zidovudina/farmacologiaRESUMO
The development of homologous recombination methods for the precise modification of bacterial artificial chromosomes has allowed the introduction of disease causing mutations or fluorescent reporter genes into human loci for functional studies. We have introduced the EGFP gene into the human PRPH-1 locus to create the Peripherin-EGFP (hPRPH1-G) genomic reporter construct. The hPRPH1-G reporter was used to create transgenic mice with an intrinsically fluorescent peripheral nervous system (PNS). During development, hPRPH1-G expression was concomitant with the acquisition of neuronal cell fate and growing axons could be observed in whole embryo mounts. In the adult, sensory neurons were labeled in both the PNS and central nervous system, while motor neurons in the spinal cord had more limited expression. The fusion protein labeled long neuronal processes, highlighting the peripheral circuitry of hPRPH1-G transgenic mice to provide a useful resource for a range of neurobiological applications.
Assuntos
Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Sistema Nervoso Periférico/metabolismo , Animais , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hibridização In Situ , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteínas do Tecido Nervoso/metabolismo , Neurônios Aferentes/metabolismo , Periferinas , Proteínas Recombinantes de Fusão/metabolismo , Recombinação GenéticaRESUMO
Using the lipofection reagent LipofectAMINE 2000 we have examined the delivery of plasmid DNA (5-200 kb) to mouse embryonic stem (mES) cells by flow cytometry. To follow the physical uptake of lipoplexes we labeled DNA molecules with the fluorescent dye TOTO-1. In parallel, expression of an EGFP reporter cassette in constructs of different sizes was used as a measure of nuclear delivery. The cellular uptake of DNA lipoplexes is dependent on the uptake competence of mES cells, but it is largely independent of DNA size. In contrast, nuclear delivery was reduced with increasing plasmid size. In addition, linear DNA is transfected with lower efficiency than circular DNA. Inefficient cytoplasmic trafficking appears to be the main limitation in the nonviral delivery of large DNA constructs to the nucleus of mES cells. Overcoming this limitation should greatly facilitate functional studies with large genomic fragments in embryonic stem cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Transfecção/métodos , Animais , Sequência de Bases , Linhagem Celular , DNA Recombinante/química , DNA Recombinante/genética , DNA Recombinante/metabolismo , Citometria de Fluxo , Corantes Fluorescentes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Indicadores e Reagentes , Lipídeos , Lipossomos , Camundongos , Plasmídeos/química , Plasmídeos/genética , TiazóisRESUMO
Friedreich ataxia is an autosomal recessive neurodegenerative disorder caused by a GAA trinucleotide expansion in the first intron of the Friedreich ataxia gene (FRDA) that causes reduced synthesis of frataxin, a mitochondrial protein likely to be involved in biosynthesis of iron-sulfur clusters. This leads to increased oxidative stress, progressive loss of large sensory neurons, and hypertrophic cardiomyopathy. To elucidate the mechanisms regulating FRDA expression and to develop an in vivo assay for agents that might upregulate FRDA expression in a therapeutically relevant manner, we have generated transgenic mice with a BAC genomic reporter construct consisting of an in-frame fusion between FRDA and the gene coding for enhanced green fluorescent protein (EGFP). Production of full-length frataxin-EGFP fusion protein was demonstrated by immunoblotting. EGFP expression was observed as early as day E3.5 of development. Most tissues of adult transgenic mice were fluorescent. The level of FRDA-EGFP expression in peripheral blood, bone marrow, and cells obtained from enzymatically disaggregated tissues was quantitated by flow cytometry. There was a twofold increase in EGFP expression in mice homozygous for the transgene when compared to hemizygous mice. These transgenic mice are a valuable tool for the examination of spatial and temporal aspects of FRDA gene expression and for the preclinical evaluation of pharmacological inducers of FRDA expression in a whole-animal model. In addition, tissues from these mice should also be valuable for stem cell transplantation studies.
Assuntos
Modelos Animais de Doenças , Ataxia de Friedreich/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Ligação ao Ferro/genética , Animais , Cromossomos Artificiais Bacterianos , Estudos de Avaliação como Assunto , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Immunoblotting , Hibridização in Situ Fluorescente , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transgenes/genética , FrataxinaRESUMO
Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.
Assuntos
Fibroblastos/patologia , Ataxia de Friedreich/patologia , Ferro/análise , Linfócitos/patologia , Mitocôndrias/química , Adolescente , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibroblastos/química , Corantes Fluorescentes , Humanos , Peróxido de Hidrogênio/toxicidade , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Proteínas Reguladoras de Ferro/metabolismo , Ativação Linfocitária , Linfócitos/química , Masculino , Receptores da Transferrina/análiseRESUMO
Three independent transgenic mouse lines were generated with the human Friedreich ataxia gene, FRDA, in an 188-kb bacterial artificial chromosome (BAC) genomic sequence. Three copies of the transgene per diploid mouse genome were integrated in a single site in each mouse line. Transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Frda gene, to generate mice homozygous for the Frda knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Frda knockout mutation was observed in all three lines. Rescued mice displayed normal behavioral and biochemical parameters. RT-PCR analysis demonstrated that human FRDA mRNA is expressed in all the lines. The relative expression of the human FRDA and mouse Frda genes showed a similar pattern in different tissues in all three lines, indicating position-independent control of expression of the human FRDA transgene. However, large differences in the human:mouse mRNA ratio were observed between different tissues in all three lines. The human transgene is expressed at much higher levels in the brain, liver, and skeletal muscle than the endogenous gene, while expression of the human transgene in blood is only 25-30% of the mouse gene. These studies will facilitate the development of humanized mouse models of Friedreich ataxia through introduction of a GAA trinucleotide expansion or specific known point mutations in the normal human FRDA locus and the study of the regulation of gene expression from the FRDA locus.
Assuntos
Cromossomos Artificiais Bacterianos , Ataxia de Friedreich/genética , Ataxia de Friedreich/fisiopatologia , Camundongos Knockout/genética , Camundongos Transgênicos/genética , Mutação/genética , Animais , Feminino , Dosagem de Genes , Genes Letais , Teste de Complementação Genética , Homozigoto , Humanos , Hibridização in Situ Fluorescente , Locomoção , Masculino , Camundongos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transgenes/fisiologiaRESUMO
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.