By working to identify drugs that promote erythropoietin-responsive precursors, Assistant Professor Johan Flygare hopes to open up new opportunities for the treatment of patients suffering from anaemia

Targeting genes to treat anaemia

Can you summarise the existing treatments that are available to patients with anaemia?

Today there are three major strategies for treating patients with anaemia. Firstly, to treat the underlying cause, such as giving the patient iron if the anaemia is caused by iron defi ciency; secondly, to give the patient new red blood cells by transfusing red blood cells from a blood donor; and thirdly, to treat the patient

with a drug (erythropoietin) that stimulates production of red blood cells.

Can you tell us how your research is working to improve the existing treatments?

Our research focuses on improving anaemia treatment based on all three principles with the main focus on developing a new family of drugs. We are studying the underlying mechanisms that cause anaemia in patients with Diamond-Blackfan anaemia (DBA). Today, these patients have but few good treatment options. By discovering the details of the underlying cause we hope to be able to develop disease-specifi c treatments for these forms of anaemia. We are also developing cell culture methods that could allow large-scale production of red blood cells in a bioreactor, with the ultimate aim of being able to produce red blood cells, in a factory setting, that could be used for transfusion to patients. This would bypass the limitations relating to available blood donors and reduce the risk of infection with blood-borne pathogens. In addition, we are focusing our laboratory on the development

of better drugs for anaemia treatment. Currently, the only drugs used to increase red blood cell production in anaemic patients are recombinant erythropoietin (Epo) analogues. These drugs are based on research that in the 1980s identifi ed erythropoietin as the main regulator of red blood cell production. Erythropoietin is, however, mostly effective in anaemic patients suffering low endogenous Epo production, while other types are completely or relatively resistant to Epo treatment. The Epo-resistant anaemias include bone marrow failure syndromes such as DBA that do not respond even to high doses of Epo. Other more common forms of anaemia are only relatively Epo-resistant, such as those associated with cancer and infl ammation that often respond relatively poorly to Epo only if given in high doses. Due to the limitations of Epo therapy, blood transfusions are often used for treatment of Epo-insensitive and Epo-resistant anaemias. New erythropoiesis stimulating agents that promote Epo-responsive precursor cell production in these patients would therefore be of signifi cant socioeconomic andmedical value.

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JOHAN FLYGARE

Untangling the mystery behind anaemiaIdentifying genetic factors that lead to Diamond-Blackfan Anaemia offers researchers at Lund University a unique opportunity to understand the pathogenic mechanisms driving anaemia

Drug-induced stimulation of BFU-E progenitors

represents a conceptually new therapeutic window

for treating erythropoietin-resistant anaemia

ASSISTANT PROFESSOR JOHAN FLYGARE’S STUDENTS IN THE LAB

PRESENTLY, ONE IN four people around the globe suffer from some form of anaemia, making the condition one of the largest health problems in the world and putting a tremendous burden on the global economy. Anaemia is a diagnostic term used to describe a condition of having a reduced number of red blood cells and thus a reduced oxygen-carrying capacity of the blood. Since most cells in our bodies absolutely depend on red blood cells for respiration, anaemic patients experience weakness and shortness of breath and this can, if untreated, lead to death. Anaemia is in principle caused either by impaired production or increased destruction of red blood cells. There are, however, few good treatment options available to sufferers of anaemia, but some new research taking place at a laboratory in Sweden is hoping to develop a suite of disease-specifi c treatments to relieve many patients of their symptoms.

Research leader Assistant Professor Johan Flygare has recently arrived from the Whitehead Institute for Biomedical Research in Boston to coordinate this work in the Faculty of Medicine at Lund University in Sweden, which is Scandinavia’s largest institution for education and research. From Flygare’s perspective this is an intellectually stimulating environment where the research infrastructure is world-class: “I believe this is a great location to establish translational research programmes, and a place where you are given resources and the room to develop as an independent scientist”. He hopes that his research work into the underlying mechanisms causing anaemia will help support the growing body of scientifi c research now coming out of Sweden.

THE IMPORTANCE OF ERYTHROPOIETIN

Red blood cells are the most abundant cell type in our bodies and are absolutely essential for transporting oxygen and carbon dioxide through our whole body. Every second, under normal conditions, 2 million red blood cells are synthesised by the human body. Since both too

few and too many red blood cells in the blood cause disease, it is extremely important that new red blood cells are produced in exactly the right amounts at all times. Erythropoiesis is the name given to this tightly regulated process of making new red blood cells, which is critical in order for the human body to function properly. The main focus of Flygare’s research team is to study mechanisms regulating erythropoiesis to identify new drugs and target genes for the treatment of Diamond-Blackfan anaemia (DBA) and other forms of anaemias where Epo treatment is not effective.

Normal or ‘steady-state’ erythropoiesis alone is not able to correct the red blood cell defi ciency

seen during severe chronic anaemia. This steady-state erythropoiesis is regulated by Epo, which is produced in the kidneys when the blood fails to deliver enough oxygen. The increased Epo levels then stimulate specifi c precursor cells in the bone marrow to produce more red blood cells. However, under maximum Epo stimulation these Epo-responsive precursor cells can only produce between 30 and 60 red blood cells each. This capacity to increase red blood

cell production is enough to maintain steady-state red blood cell levels. According to Flygare, during severe chronic anaemia the number of Epo-responsive precursor cells in the bone marrow ultimately limits the therapeutic effect of Epo: “Such conditions, where the demand for new red blood cells exceeds the capacity of Epo, leads to activation of a second line of defence against anaemia; the mechanisms of stress erythropoiesis”.

IMPROVING KNOWLEDGEOF EPO!INDEPENDENT MECHANISMS

One strand of Flygare’s research aims at understanding how Epo-independent mechanisms regulate the self-renewal of early committed red cell progenitors during stress erythropoiesis. During conditions of stress-erythropoiesis, new Epo-responsive

How important is it to move away from recombinant Epo analogues and explore, characterise and identify other drugs for anaemia treatment?

Epo is an excellent drug for many types of anaemia, such as for kidney failure patients who have become anaemic because the kidneys stopped producing Epo. I do not argue that we should move away from Epo treatment in the patients that respond. The idea is instead to develop drugs that allow treatment for the millions of people with forms of anaemia that respond poorly to Epo treatment.

To what extent would you suggest that your fi eld of study represents a new paradigm for treating Epo-resistant anaemia?

All current drugs for anaemia treatment are Epo analogues that act on Epo-responsive red blood cell precursors in the bone marrow. Our group is instead trying to identify drugs that promote production of these Epo-responsive precursors. Such drugs would open a new therapeutic window for improved treatment of a large group of anaemias that are not treatable with recombinant Epo therapy.

In terms of your genetic screening work, how important is your collaboration with Professor David Root at the Broad Institute, MA, USA?

David has many years experience in developing and performing similar types of screens to ours. He has generated the libraries used in the screens and assists in analysing the results, and the collaboration is fundamental for the execution of the project.

GENES FOR ANAEMIA TREATMENT

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INTELLIGENCENEW DRUG TARGET GENES FOR ANAEMIA TREATMENT

OBJECTIVES

The work is based on the hypothesis that new drugs for anaemia treatment can be developed based on Epo-independent mechanisms regulating self-renewal of early committed red cell (BFU-E) progenitors during stress erythropoiesis. While Epo-dependent erythropoiesis is well studied, relatively little is known of the Epo-independent mechanisms that promote BFU-E progenitor self-renewal and increased production of Epo-responsive precursor cells during stress erythropoiesis.

KEY COLLABORATORS

Professor David Root, Director of the RNA Interference Platform; Project leader of the RNAi Consortium (TRC) at the Broad Institute, Cambridge MA, USA

FUNDING

Ragnar Söderberg Foundation • Diamond-Blackfan Anemia Foundation • Lund Medical Faculty

CONTACT

Johan Flygare MD PhDAssistant Professor

Lund University, Faculty of MedicineDivision of Molecular Medicine and Gene TherapyBMC A12221 84Lund, Sweden

T +46 46 22 20 442E johan.fl ygare@med.lu.se

http://www.med.lu.se/labmedlund/molecular_medicine_and_gene_therapy/

ASSISTANT PROFESSOR JOHAN FLYGARE was awarded an MD and a PhD in Molecular Medicine and Gene Therapy from Lund University, Sweden in 2007. He is now an Assistant Professor at Lund’s Department of Molecular Medicine and Gene Therapy. Prior to this appointment, he worked as a Postdoctoral Fellow at the Whitehead Institute for Biomedical Research, Cambridge MA, USA.

precursor cells are produced from stem cell-like erythroid progenitor cells known as Burst Forming Unit-Erythroid (BFU-E) cells. Flygare says that while Epo-dependent erythropoiesis is well-studied, relatively little is known of the Epo-independent mechanisms that promote BFU-E progenitor self-renewal and increased production of Epo-responsive precursor cells during stress erythropoiesis. He believes that a deeper understanding of the mechanisms regulating BFU-E self-renewal and the production of Epo-responsive precursor cells could result in the development of drugs that stimulate the physiological mechanisms of stress erythropoiesis: “Drug-induced stimulation of BFU-E progenitors represents a conceptually new therapeutic window for treating erythropoietin-resistant anaemia”. Unlike Epo, which only can be used to maximise erythrocyte output from steady-state erythropoiesis, these drugs could become useful in treating relatively Epo-resistant anaemias, including patients with bone marrow failure disorders such as DBA, trauma, sepsis, cancer and others that fail to respond to Epo.

THE VALUE OF GENETICAND CHEMICAL SCREENING

Flygare’s work is also aimed at identifying a genetic factor or drug that ‘rescues’ DBA. The research team has several reasons for studying this relatively rare form of anaemia. Patients with DBA suffer from a severe defi ciency of Epo-responsive precursor cells in the bone marrow. The few precursor cells that are present, however, respond normally to Epo and produce normal red

blood cells. DBA is therefore a good example of an Epo-resistant anaemia that they believe can be treated with a drug which increases production of Epo-responsive precursor cells. Flygare explains that his group are therefore using DBA cells in several of their genetic and chemical screens in the hope that a factor which rescues red blood cell production in DBA also can increase red cell production in other forms of anaemia. Another reason for the focus on DBA is that uncovering the poorly understood underlying mechanisms causing anaemia in these patients could potentially reveal important knowledge about regulation of red blood cell precursor cells. The research has hypothesised that detailed understanding of the pathogenic mechanism in DBA could lead to the development of new drugs, not only for DBA but possibly also for other more common forms of anaemia.

The laboratory is now carrying out genetic and chemical screens to identify new drug target genes that can be used to enhance production of Epo-responsive precursor cells, and one example is their work using bone marrow cells from a mouse with DBA. High-throughput screens are performed to identify factors able to rescue formation of red blood cells from these progenitor cells. Flygare says that similar screens are underway to identify factors able to rescue red blood cell production in other forms of Epo-resistant anaemias based on the mechanisms of stress erythropoiesis: “These very exciting experiments were initiated less than six months ago and we anticipate it will take one or two years to identify new drug target candidates”.