Hady Wardan

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

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.

Humanized β-Thalassemia Mouse Model Containing the Common IVSI-110 Splicing Mutation

Splicing mutations are common causes of β-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal β-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for β-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human β-globin (huβ-globin) locus. We examined heterozygous murine β-globin knock-out mice (muβth-3/+) carrying either the IVSI-110 or the normal huβ-globin locus. Our results show a 90% decrease in huβ-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal huβ-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable β-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.

Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation

A greater understanding of the regulatory mechanisms that govern γ‐globin expression in humans, especially the switching from γ‐ to β‐globin, which occurs after birth, would help to identify new therapeutic targets for patients with β‐hemoglobinopathy. To further elucidate the mechanisms involved in γ‐globin expression, a novel fluorescent‐based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183‐kb intact human β‐globin locus under the control of Gγ‐ or Aγ‐globin promoter and β‐globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage‐specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)‐mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ‐globin‐linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ‐globin gene sup pression.—Chan, K. S. K., Xu, J., Wardan, H., McColl, B., Orkin, S., Vadolas, J. Generation of a genomic reporter assay system for analysis of y‐ and β‐globin gene regulation. FASEB J. 26, 1736‐1744 (2012). www.fasebj.org

siRNA-mediated reduction of α-globin results in phenotypic improvements in β-thalassemic cells

Reduction of α-globin expression has long been known to improve anemia in β-thalassemia. This study shows that siRNA-mediated reduction of α-globin results in phenotypic improvements in β-thalassemic cells. β-thalassemia is an inherited hemoglobinopathy caused by defective synthesis of the β-globin chain of hemoglobin, leading to imbalanced globin chain synthesis. Excess α-globin precipitates in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anemia. Decreased α-globin synthesis leads to milder symptoms, exemplified in individuals who co-inherit α- and β-thalassemia. In this study, we investigated the feasibility of utilizing short-interfering RNA (siRNA) to mediate reductions in α-globin expression. A number of siRNA sequences targeting murine α-globin were tested in hemoglobinized murine erythroleukemic cells. One highly effective siRNA sequence (si-α 4) was identified and reduced α-globin by approximately 65% at both the RNA and the protein level. Electroporation of si-α 4 into murine thalassemic primary erythroid cultures restored α :β-globin ratios to balanced wild-type levels and resulted in detectable phenotypic correction. These results indicate that siRNA-mediated reduction of α-globin has potential therapeutic applications in the treatment of β-thalassemia.

Development of a novel bacterial artificial chromosome cloning system for functional studies.

Bacterial artificial chromosome (BAC) cloning systems currently in use generate high quality genomic libraries for gene mapping, identification, and sequencing. However, the most commonly used BAC cloning systems do not facilitate functional studies in eukaryotic cells. To overcome this limitation, we have developed pEBAC190G, a new BAC vector that combines the features of the first generation PAC/BAC vectors with eukaryotic elements that facilitate the transfection, episomal maintenance, and functional analysis of large genomic fragments in eukaryotic cells. A number of different cloning strategies may be used to retrofit genomic fragments from existing libraries into the new vector. The system was tested by the retrofitting of a 170kb NotI genomic fragment from the RPCI-11 BAC library into the NotI site of pEBAC190G. Clones from any eukaryotic genomic library harboured in this vector can be transferred from bacteria directly to eukaryotic cells for functional analysis.

An in vivo model for analysis of developmental erythropoiesis and globin gene regulation

Expression of fetal γ‐globin in adulthood ameliorates symptoms of β‐hemoglobinopathies by compensating for the mutant β‐globin. Reactivation of the silenced γ‐globin gene is therefore of substantial clinical interest. To study the regulation of γ‐globin expression, we created the GG mice, which carry an intact 183‐kb human β‐globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ‐globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta–gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human β‐globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase‐1 in GG primary erythroid cells increases the proportion of eGFP+ cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced > 3‐fold after treatment of erythroid precursors with epigenetic drugs known to induce γ‐globin expression, demonstrating the suitability of the Gγ‐globin eGFP reporter for evaluation of γ‐globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the β‐globin locus and for discovery of novel therapies for the P‐hemoglobinopathies.—McColl, B., Kao, B. R., Lourthai, P., Chan, K., Wardan, H., Roosjen, M., Delagneau, O., Gearing, L. J., Blewitt, M. E., Svasti, S., Fucharoen, S., Vadolas, J. An in vivo model for analysis of developmental erythropoiesis and globin gene regulation. FASEB J. 28, 2306–2317 (2014). www.fasebj.org

A 191-kb genomic fragment containing the human α-globin locus can rescue α-thalassemic mice

A 191-kb human bacterial artificial chromosome (BAC) containing the human α-globin genomic locus was used to generate transgenic mice that express, exclusively, human α-globin (huα-globin). Expression of huα-globin reaches a level of 36% of that of endogenous mouse α-globin (muα-globin) on a heterozygous mouse α-thalassemia background (muα-globin knockout, muα+/−). Hemizygous transgenic mice carrying the huα-globin locus on a heterozygous knockout background (huα+/0, muα++/−−) demonstrated complementation of most hematologic parameters. By crossing huα+/0, muα++/−− mice, we were able to generate mice entirely dependent on huα-globin synthesis. Breeding and fluorescent in situ hybridization studies demonstrate that only mice homozygous for the transgene were able to rescue embryonic lethal homozygous muα-globin knockout embryos (muα−−/−−). Adult rescued mice produce hemoglobin at levels similar to wild-type mice, with partial red cell complementation based on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW) measurements. Significant erythrocythemia above wild-type levels seems to be the main compensatory mechanism for the normalization of the hemoglobin levels in the rescued animals. Our studies demonstrate that the huα-globin locus in the 191-kb transgene contains all the necessary elements for the regulated expression of huα-globin in transgenic mice. This animal model should be valuable for studying the mechanisms regulating huα-globin production and for development of therapeutic strategies for β-thalassemia based on downregulation of α-globin expression.

Gene induction for the treatment of methylmalonic aciduria.

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.