Rescue of the Friedreich ataxia knockout mutation in transgenic mice containing an FXN-EGFP genomic reporter.

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.

Pharmacological screening using an FXN-EGFP cellular genomic reporter assay for the therapy of Friedreich ataxia.

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.

Long range regulation of human FXN gene expression.

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.

Gene induction for the treatment of methylmalonic aciduria.

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.

Transgenic mice expressing the Peripherin-EGFP genomic reporter display intrinsic peripheral nervous system fluorescence.

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.

Flow-cytometric analysis of mouse embryonic stem cell lipofection using small and large DNA constructs.

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.

Chromatin-binding regions of EBNA1 protein facilitate the enhanced transfection of Epstein-Barr virus-based vectors.

Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBVbased vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficiency by up to 10-fold. Moreover, a variant of EBNA1 comprising two copies of domain A fused to the DBD enhances DNA transfection to an even greater extent than wild-type EBNA1. We therefore propose that EBNA1-mediated transfection of EBV-based vectors is dependent on the presence of chromatin- binding regions and the DBD, but not the NLS.

Evaluation of an FRDA-EGFP genomic reporter assay in transgenic mice.

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.

Friedreich ataxia: from genes to therapies?

Most cases are caused by a single mutation, paving the way for therapeutic advances for this fatal disease.

Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment.

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity?

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.

Human BAC-mediated rescue of the Friedreich ataxia knockout mutation in transgenic mice.

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.

Friedreich ataxia-update on pathogenesis and possible therapies.

Friedreich ataxia is the most-common inherited ataxia. Since the causative genetic basis was described in 1996, much has been learnt about the pathogenesis from human, animal, and yeast studies. This has led to the development of rational therapeutic approaches. In this review, the current state of knowledge regarding the pathogenesis of Friedreich ataxia is presented and possible therapeutic strategies based on this knowledge are discussed.

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.

Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette.

A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus. We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus. The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it. Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q). Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones. The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.

Wnt-4 regulation by the Wilms' tumour suppressor gene, WT1.

The Wilms' tumour suppressor gene, WT1, encodes multiple nuclear protein isoforms, all containing four C-terminal zinc finger motifs. WT1 proteins can both activate and repress putative target genes in vitro, although the in vivo relevance of these putative target genes is often unverified. WT1 mutations can result in Wilms' tumour and the Denys-Drash Syndrome (DDS) of infantile nephropathy, XY pseudohermaphroditism and predisposition to Wilms' tumour. We have established stable transfectants of the mouse mesonephric cell line, M15, which express WT1 harbouring a common DDS point mutation (R394W). A comparison of the expression profiles of M15 and transfectant C2A was performed using Nylon-based arrays. Very few genes showed differential expression. However Wnt-4, a member of the Wnt gene family of secreted glycoproteins, was downregulated in C2A and other similar clones. Doxycycline induction of WT1-A or WT1-D expression in HEK293 stable transfectants also elicited an elevation in Wnt4 expression. Wnt4 is critical for the mesenchyme-to-epithelial transition during kidney development, making it an attractive putative WT1 target. We have mapped human Wnt-4 gene to chromosome 1p35-36, a region of frequent LOH in WT, have characterized the genomic structure of the human Wnt-4 gene and isolated 9 kb of immediate promoter. While several potential WT1 binding sites exist within this promoter, reporter analysis does not strongly support the direct regulation of Wnt4 by WT1. We propose that Wnt-4 regulation by WT1 occurs at a more distant promoter or enhancer site, or is indirect.