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RoFAR awards over 1.3 million Swiss Francs to fund ground-breaking anaemia research in the USA, Canada and Italy
The award winners with institution and the description of their projects are:
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Prof. Clara Camaschella
University Vita-Salute San Raffaele, Italy
GLRX5 deficiency as a model of anaemia responsive to iron chelation |
New models of inherited anaemias characterised by red cells of small size and excess of total body iron have been recently recognised based on animal models (mice and fish) with similar defects. Absence of the protein enzyme glutaredoxin-5 (GLRX5) was first described in fish models, where it is incompatible with life, because of the development of severe anaemia before birth. Reduction of GLRX5 has been recognised so far in a single patient of middle age with severe anaemia and iron overload. The story of this patient is unusual, because of the paradoxical effect of blood transfusions that worsened anaemia, which was significantly ameliorated only by therapeutic iron removal. This project is proposed to facilitate the study of this new type of anaemia. It aims to develop in vitro models of different cells (hepatocytes, erythroblasts) in which the effect of artificially decreasing GLRX5 enzyme activity is analysed, as well as to understand the molecular mechanisms induced by drug-mediated iron removal.
Using recombinant DNA technology, the gene expression of this enzyme will be down-regulated in hepatic and erythroid cell lines, and the effect on cellular iron changes and the ability of the cell to respond to these changes will be evaluated, analysing proteins of iron metabolism in the different cell compartments.
The project might reveal new physiological mechanisms of cellular iron regulation and their derangements in GLRX5 deficiency, with potential therapeutic implications for patients affected by rare congenital and acquired anaemias secondary to defects of GLRX5 or related enzymes implicated in the same pathway.
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Dr Madeleine Carreau
Laval University, Canada
Fanconi anaemia proteins as regulators of genes involved in haematopoietic stem cell function
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Children affected by Fanconi anaemia (FA) suffer from a life-threatening haematological failure, congenital abnormalities and a predisposition to cancer such as leukaemia. Although many genes associated with this disease have been identified, their function remains unknown. We know that when mutated or de-regulated, these Fanconi genes affect the normal development of haematopoietic stem cells leading to their progressive exhaustion. These stem cells are responsible for the generation of all blood cell types, thus their exhaustion leads to anaemia and bone marrow failure.
Our main objective is to identify the function of Fanconi genes and their role in haematopoiesis, specifically those related to stem cell function such as their maintenance and renewal. We have obtained data linking Fanconi genes to a signalling pathway well- known for its role in embryonic stem cell development, the Notch1/HES1 pathway. We have found that HES1 is a molecular partner of FA genes. We propose to characterise the role of this novel FA-HES1 interaction in haematopoietic stem cell function.
We believe that understanding the molecular basis of the Fanconi anaemia disease will lead to the development of new therapeutic approaches aimed at increasing haema-topoietic stem cell numbers through regulation of their maintenance and renewal. We also believe that knowing molecular partners of FA genes and their role in regulating haematopoietic stem cells should help design new therapies for blood disorders such as aplastic anaemia. Since deregulation of FA genes cause leukaemia, knowing their functions will inevitably help the design of new drugs that specifically target leukaemia cells.
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Dr Wenbin Deng
University of California, Davis, USA
Protective effects of erythropoietin against hypoxic-ischaemic injury to developing oligodendrocytes |
Periventricular leukomalacia (PVL) is the predominant form of brain injury in the premature infant and the most common cause of cerebral palsy, yet no therapy currently exists for this serious human disorder. Injury to the oligodendrocyte precursor (termed “pre-OL”) is a major factor in PVL. We have recently shown that erythropoietin (EPO) attenuates pre-OL injury in vitro. This proposal aims to expand upon the preliminary results and further investigate the protective effect of EPO in pre-OL injury in vivo. We will characterise the oligodendrocyte-specific developmental regulation of the EPO receptor in the rodent white matter and in the human parietal white matter by using post-mortem human tissue. We will evaluate the effect of EPO on hypoxic-ischemic pre-OL injury in an animal model of PVL that we have recently established. We will determine the mechanisms of EPO protection against pre-OL injury. Completion of this project will help to define the translational potential of EPO as a therapeutic agent for pre-OL injury underling PVL, for which no therapy presently exists. The scientific knowledge to be acquired through this project is of likely benefit to the care of children with PVL leading to cerebral palsy.
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Dr Adam Goldfarb
University of Virginia School of Medicine, USA
Iron regulation of erythropoiesis: characterisation of a novel signalling pathway
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Human iron deficiency impairs the marrow response to the red cell growth factor erythropoietin (EPO). Conversely, intravenous iron infusion enhances the bone marrow response to EPO, even in anaemia patients with adequate pre-existing iron stores. Iron regulation of EPO-driven red cell production affects proliferation and differentiation of early bone marrow progenitors, prior to the onset of haemoglobin production. Thus, while iron is essential for all cells, red cell precursors manifest an exquisite sensitivity to iron deficiency, most likely as a rationing mechanism to protect other, more vital iron-dependent functions. Using a system with human bone marrow precursors cultured in defined levels of iron and EPO, we have confirmed the existence of a critical threshold of iron deprivation, at which erythroid progenitors display proliferative and maturation blockade while other blood cell types remain unaffected. Extensive pharmacologic and genetic screening for components of this iron response pathway have identified the aconitase enzymes as a critical signalling node. Mitochondrial and cytosolic aconitase (mAcon & cAcon) interconvert citrate and isocitrate as a key step in the Krebs cycle. The aconitase enzymes within red cell precursors are more sensitive to iron deprivation than aconitase enzymes in other cell types. Providing cells with the product of the aconitase enzymes, isocitrate, restores the growth and development of red cell precursors subjected to iron deprivation. In addition, treatment of mice with isocitrate enhances their red cell haemoglobin production. In an effort to identify how the aconitase enzymes are regulated within red cell precursors, we identified PKCa as a kinase whose activity is regulated by iron deficiency and which appears to control the activity of the aconitase enzymes. This project will delineate the function of PKCa in the response of red cell precursors to iron deficiency. This pathway has direct relevance for future clinical approaches to EPO-unresponsive chronic anaemias.
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Dr Véronique Lefebvre
Cleveland Clinic Foundation, USA
Erythropoiesis control by Sox6 and erythropoietin signalling
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This project is designed to advance understanding of major mechanisms involved in red cell formation. We will focus on the gene called Sox6, which encodes a protein that helps several cell types activate the genes that they need to fulfil specialised functions. We recently found that these cell types include red cell precursors (called erythroid cells). We showed that Sox6 helps these cells survive and proliferate, and is crucial in the final steps of their conversion into mature red cells. Mice that lack Sox6 have mild anaemia under normal conditions of life, but are unable to quickly produce mature red cells when subjected to severe blood loss, and consequently, many die within a few days. We also found that Sox6 works together with the hormone called erythropoietin (or in short, EPO), which has a key role in stimulating erythroid cell survival and proliferation and thereby in adjusting red cell production to body need, but Sox6 also works beyond EPO to ensure red cell timely maturation. The first aim of this project is to identify mechanisms whereby Sox6 acts in erythroid cells. We will specifically ask how Sox6 works together and beyond EPO to control expression of the gene for Bcl-xL, a protein essential for erythroid cell survival. The second aim is to identify the hormones and intracellular proteins that control expression of the Sox6 gene in erythroid cells. In both aims, we will identify the DNA sequences that are needed for expression of the Bcl-xL and Sox6 genes in vivo, and we will identify the proteins that bind to these sequences. By greatly increasing our understanding of the molecular mechanisms underlying red cell formation, we expect our study to provide solid foundations for uncovering the causes of various forms of anaemia diseases and for designing better therapies for these diseases.
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Prof. John G. Quigley
University of Illinois at Chicago, USA
FLVCR protein trafficking and the regulation of haem export
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FLVCR (Feline Leukemia Virus subgroup C Receptor) function, the export of haem, is required to permit normal erythropoiesis. Inhibition of this function leads to a block in erythroid differentiation, and severe anaemia. Cell membrane expression of FLVCR, similar to other membrane transporters is likely regulated through control of its trafficking to and from the cell surface via interactions with a PDZ domain-containing protein. A yeast two hybrid screen using the cytoplasmic tail of FLVCR identified a candidate protein, which contains a PDZ domain. The proposed experiments involve verification of interactions between FLVCR and the candidate protein, and character-isation of the interacting protein. In addition, the effects of deletions of domains of the interacting protein on FLVCR expression and trafficking in heterologous cells, including polarised cells, will be examined. An overriding aim however, is to determine the role of trafficking on FLVCR haem export function in epithelial and erythroid cell lines. This will improve our understanding of haem transport, relevant to diseases characterised by haemolysis and release of free (toxic) haem, including the haemolytic anaemias, sickle cell disease, the thalassaemias, and malaria, situations where FLVCR likely functions to protect cells from haem toxicity.
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Dr Li Zhong
University of Florida College of Medicine, USA
Recombinant parvovirus vectors for gene therapy of Fanconi anaemia
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Fanconi anaemia (FA) is an autosomal recessive disease that is characterised by congenital abnormalities, defective haematopoiesis, a high risk of developing acute myeloid leukaemia and certain solid tumours, and cellular sensitivity to cross-linking agents. Since none of the currently used therapeutic approaches is curative, the development of a safe and effective gene therapy approach involving the introduction of a functional FA gene into haematopoietic stem cells (HSCs) followed by autologous stem cell transplantation could achieve long-term benefits. Parvovirus vectors, including the non-pathogenic adeno-associated virus (AAV) and parvovirus B19 vectors have gained attention as a useful alternative to the more commonly used retrovirus- and adenovirus-based vectors in human gene therapy.
Our hypothesis is that by using novel serotype AAV and recombinant parvovirus B19 vectors and strategies that have been developed by us and others, highly efficient transduction of FA complementation C (FancC) gene in primary murine and human HSCs can be achieved. The optimal recombinant parvovirus vector-mediated FancC transduction will prove to be safe and effective in therapeutic correction of FA in animal models in vivo. The following specific aims will be pursued in this proposal:1)To develop novel serotype AAV and recombinant parvovirus B19 vectors containing FancC cDNA driven by haematopoietic cell-specific promoters to achieve high-efficiency expression of FancC gene in murine and human HSCs and alleviate complications of FA in vitro; 2)To evaluate the efficacy of novel serotype AAV and parvovirus B19 vectors for efficient transduction and long-term expression of FancC gene to allow functional reconstitution and therapeutic correction of FA-C in mice in vivo. The safety of these vectors will also be evaluated.
These studies will provide new insights into the development of safe and effective parvovirus-based vectors, and help to evaluate the in vivo efficacy and safety of these vectors prior to their potential use in gene therapy of human haematopoietic diseases, particularly in FA syndrome.
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