RoFAR awards 5 new anaemia-related research projects proposed by experienced investigators in Canada, Denmark, Switzerland and the USA
The award winners with their institutions and a description of the projects are:
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Dr Jen-Tsan Chi
Duke Medical Center, Durham, USA
Genomic analysis of erythrocyte microRNA to dissect the heterogeneity of sickle cell anaemia |
Anaemia disorders affect hundreds of millions of people and present a huge challenge. Although we have gained significant understanding about how these diseases occur and many treatment options are available, we still cannot explain many aspects of these diseases. For example, different patients with the same anaemia vary significantly in their anaemia severity and response to treatment. The understanding of similar issues in human cancers has benefited tremendously from the analysis of gene expression with gene chips. Although a similar genomic analysis of human red blood cells (RBCs) may provide useful information, such an analysis has been deemed impossible since these cells are thought to not contain any genetic material to be analysed by microarrays. Recently, our laboratory has found that the traditional view that no genetic material is present in the erythrocyte is incorrect. Even though mature erythrocytes do not have the large-sized RNA present in all human cells, they do contain an abundant and diverse collection of microRNA. MicroRNA is a class of small size RNA which has been recently recognised to play an important functional role in many important processes in human cells, including the formation and destruction of red blood cells. Since microRNA in the circulating RBC is likely to be a product accumulated during earlier stages of differentiation, the analysis of the RBC microRNA composition with microarrays may allow the analysis of human anaemia disorders with the modern genomic tools and advanced bioinformatics. In this proposal, we plan to use microarrays to analyse the erythrocytes from sickle cell anaemia with varying manifestation in their severity and treatment response. This will allow us to compare the gene expression before and after hydroxyurea treatment and conduct functional studies to define the roles of erythrocyte microRNA in determining the severity and manifestation of sickle cell anaemia.
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Prof. Tarik Möröy
Institut de recherches cliniques de Montréal (IRCM), Canada
Regulation of erythropoiesis by the zinc finger transcription factor and chromatin regulator Gfi1b
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The formation of mature red blood cells originates from so-called blood stem cells that reside in the bone marrow. These stem cells differentiate and transform into the typical red cells that are released into the bloodstream. This process is governed in part by a group of proteins known as transcription factors. Recently, the transcription factor Gfi1b has become the centre of attention, since a lack of this protein causes a severe defect in the formation of red blood cells in mice. Experiments with cells in culture have confirmed a similar role for Gfi1b in humans. Gfi1b acts on the DNA and regulates the expression of specific genes that are required for erythropoiesis. We have gathered preliminary data and have generated new tools which will allow us to address the role of Gfi1b in erythroid differentiation and function and to advance our knowledge on this critical protein significantly. We have identified a number of Gfi1b target gene candidates in primary erythroid cells from the mouse that we wish to validate in our project. Moreover, we have established a mouse model in which the Gfi1b gene can be switched off at will at any given time point, which will allow us to decipher more precisely the biological function of Gfi1b during the maturation of red blood cells. Another aspect of our project touches on the sensitivity of red blood cells to cellular damage caused by oxygen radicals. Such damage can lead to haemolysis and a potentially life-threatening anaemia. We have evidence that Gfi1b can interact with another transcription factor, Foxo3a, which has a proven role in the response of red blood cells to oxygen-induced damage. We intend to investigate whether and how these two proteins enable erythrocytes to deal with this particular challenge represented by oxidative damage.
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Prof. Per Soelberg Sørensen
Rigshospitalet, Copenhagen, Denmark
Double-blind crossover study to assess the effects of erythropoietin on brain pathology as shown by magnetic resonance imaging in patients with multiple sclerosis
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Multiple sclerosis (MS) is a disabling chronic disease of the brain and spinal cord, most frequently seen in young people. Whereas several treatments are available for the relapsing-remitting form of MS, there is no effective treatment for the progressive forms of this disease. Erythropoietin (EPO) has been found to be part of a highly potent endogenous neuroprotective system in the brain and might, hence, have beneficial effect on progressive MS. In a small open study, high doses of EPO showed a beneficial effect in four patients with progressive MS, and the treatment was safe.
In this project we will study the effect of EPO in patients with progressive MS in a controlled clinical trial. In 56 patients with progressive MS, EPO or placebo (inactive salt water) will be administered weekly intravenously for 24 weeks: weekly for 12 weeks and bi-weekly for 12 weeks. The 24-week treatment period will be followed by a 24-week observation period. The effect of EPO compared with placebo on the patients’ disability, including hand function, gait and cognitive function will be studied. Further, we will follow disease activity seen on magnetic resonance imaging of the brain and the progression of disease symptoms during EPO and placebo treatment. Finally we will study the effects of EPO on the deviations in the immune system seen in MS. Neither the patients, nor the physicians and nurses who examine the patients, will be aware of which treatment the patients receive.
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Dr Oliver Speer
University Children's Hospital Zurich, Switzerland
The role of microRNAs in childhood anaemia
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MicroRNAs are a family of approximately 22-nucleotide non-coding RNAs that regulate gene expression post-transcriptionally by targeting mRNAs in sequence-specific manner. MicroRNAs are involved in the regulation of gene expression during proliferation, apoptosis, development and differentiation. Little is known about the physiological role of human microRNAs, even less about their clinical relevance.
Foetal haemoglobin (HbF) synthesis is high at birth, followed by a decline and switch to adult haemoglobin (HbA). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in thalassaemia and sickle cell disease (SCD). Pharmacological agents, for example hydroxyurea (HU), can reactivate HbF expression. However, the effects in patients are heterogeneous and the mechanisms are not well understood.
We have first evidence for a post-transcriptional regulation of the HbF expression in SCD patients during HU therapy. Therefore, we started early experiments and observed different microRNA patterns between patients with high HbF levels and healthy individuals. Additionally, we have identified novel microRNA candidates within the ß globin cluster and have detected in red blood cells (RBCs) members of the protein machinery (miRISC) needed for the microRNA-induced translational repression. Thus, we hypothesise that microRNAs are involved in the post-transcriptional regulation of globin expression.
In the proposed project, we will isolate miRISC from paediatric patients with thalassaemia, SCD (+/- HU), congenital or acquired anaemias, and from healthy infants and children. These miRISC will contain microRNAs and target-mRNAs. Among these targets we expect the mRNAs for HbF and g- and a globin. We will quantify both microRNAs and mRNAs (including globin mRNAs) co-isolated together with the miRISC. Thus, we will obtain both microRNA and target-mRNA pattern from patients and healthy subjects. Newly identified microRNAs as well as microRNA-target interactions will be validated by inhibiting microRNAs or transfecting microRNAs into young RBCs followed by quantitation of globin transcripts and haemoglobin. With the proposed project we aim to understand functions of microRNAs during haemoglobin expression. These new insights might lead to better therapies or even drug development for the treatment of anaemia in children and infants.
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Prof. Don Wojchowski
Maine Medical Center Research Institute, Scarborough, USA
Defining of core EPO receptor response circuits and erythropoietic role of TRIBs in primary bone marrow proerythroblasts
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Important new actions of erythropoietin (EPO), and erythroid stimulating agents (ESAs), are being increasingly realised in the contexts of ischemic cytoprotection, angiogenesis and the anaemia of cancer and chemotherapy. To understand how EPO exerts these effects, to identify potentially new candidate targets for anti-anemia agents and to discern between beneficial vs. potentially adverse properties of emerging ESAs, an improved understanding of EPO receptor (EPOR) action mechanisms is needed. Towards revealing new EPO effects, we have applied a unique combination of ex vivo erythroid progenitor cell development systems, knocked-in EPO receptor alleles, and global transcriptome profiling approaches. These aggressive investigations have led to the discovery of several intriguing new EPO/EPOR response circuits and action modes. This includes sharp EPO/EPOR modulation of novel cell surface adhesion/migration factors, cell cycle regulators and survival factors. This proposed project builds on these new discoveries. Via Aim 1, proposed studies will extend our transcriptome based investigations to define core EPOR circuits in primary erythroid progenitor cells from mice, and humans. Aims 2 and 3 will then focus on two of our newly discovered EPO/EPOR response factors TRIBBLES-3 and TRIBBLES-2 (TRIB3 and TRIB2) as important regulators of erythroid progenitor cell expansion. Aim 2 specifically will employ unique knock-out models to assess TRIB3 and -2 regulation of EPO-dependent pro-erythroblast development. Via co-immunoprecipitation and mass spectrometry approaches, erythroid targets of TRIB3 also will be defined (Aim 3). TRIB3 is regulated further via an EPOR PY343/STAT5 axis, which we have shown to be critical for erythropoiesis during anaemia and/or embryogenesis. Overall, proposed investigations should advance a molecular understanding of EPO/EPOR-regulated erythropoiesis, and provide important new information, resources, reagents and models for present and future studies of key erythropoietic regulators.
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