MICA: Identification of compounds capable of de-repressing zeta-globin in order to treat patients with severe alpha-thalassaemia

Haemoglobin (Hb) is the major component of the protein found in red blood cells, it gives blood its red colour and is responsible for carrying oxygen around the body. Haemoglobin is made up of four chains: 2 alpha chains and 2 beta chains. Mutations in the genetic code (the DNA) responsible for producing these chains can lead to decreased production of the alpha chains, a condition called alpha-thalassaemia (mutation of the beta-chains, causes beta-thalassemia). There are four alpha-globin genes in humans, each of which normally contributes to alpha-globin production. If one or two genes are affected by mutations, then this produces a mild anaemia without any symptoms. However, if three alpha-globin genes are affected, this can lead to a severe anaemia needing regular transfusions. If all four alpha-globin genes are affected, this means that no functional haemoglobin can be produced, and such patients die in the womb approximately 4-6 months after conception (a condition called Barts Hydrops fetalis).

Mild alpha-thalassaemia provides some protection against the infectious disease malaria. In certain parts of the world, alpha-thalassaemia is therefore very common: the carrier frequency is 4-8% in Southern China and Hong Kong and at a similar level in the Thai, Filipino and Vietnamese populations, however, it is most common in Northern Thailand where up to 14% of the population are carriers. Severe alpha-thalassemia is therefore a major global health problem with at least 26,000 at-risk pregnancies annually and because of migration this is now a global health problem. At the moment, treatments for alpha-thalassaemia are limited to blood transfusion and occasionally, bone marrow transplantation. Bone marrow transplantation can be very dangerous, with up to 1/5 patients dying because of the procedure itself. Regular blood transfusions also cause serious medical problems long-term. In addition, it is normally recommended that Bart's Hydrops fetalis babies are aborted because they are so unwell prior to being born. We therefore urgently need new treatments for severe forms of alpha-thalssaemia.

When babies develop, they initially produce a different globin chain, similar to alpha-globin for the first 8 weeks after conception; this is called zeta-globin. We know this is capable of substituting for alpha-globin in adults. Unfortunately however, it is normally switched off after eight weeks. If we could find a way of turning it back on in adult red blood cells, this would treat patients with severe forms of alpha-thalssaemia and could allow them to live a normal life.

In this project, we aim to identify chemical compounds which could be used as medicines to switch zeta-globin back on, thereby treating patients with severe alpha-thalassaemia. To help undertake this, we have made a mouse, where zeta-globin is "tagged" by a fluorescent protein. This means that we have a sensitive and specific way of identifying blood cells where zeta-globin is turned on, as when zeta-globin is on, the red blood cell glows. This allows us to add lots of different compounds to red blood cells from the mouse, and see quickly and easily which ones cause the red blood cells to glow. Once we have identified potential compounds, we will make sure they also turn zeta-globin back on in human cells and try and understand how they are turning zeta-globin back on by doing additional experiments. We will perform the initial screen of compounds by collaborating with a pharmaceutical company called AstraZeneca, as they have particular expertise in developing medicines. Longer-term, we would aim to test the compounds, initially in mouse models of alpha-thalassaemia, then in human cellular systems and if that is successful, in people.

Haemoglobin in adults is composed of two alpha and two beta-globin chains and production of these chains is finely balanced under normal circumstances. In alpha-thalassaemia, there is decreased or absent production of alpha-globin; this leads to the formation of non-functional tetramers of beta-globin chains causing ineffective erythropoiesis and anaemia.
In humans there are four alpha-globin genes, arranged as linked pairs on chromosome 16 (HBA2 and HBA1). Disruption of three genes leads to HbH disease: this can cause profound anaemia and extramedullary haematopoiesis. Deletion of four alpha-genes leads to the Hb Bart's Hydrops Fetalis syndrome (BHFS) which is fatal in utero due to failure to produce functional haemoglobin. Treatment options for HbH and BHFS are limited to blood transfusion and allogeneic bone marrow transplantation; it is normally recommended that fetuses with BHFS are terminated. Novel therapeutic strategies are consequently urgently required.
In addition to the alpha-globin genes, humans and mice have the embryonic alpha-like chain zeta-globin gene (HBZ) lying up-stream, which is only expressed during the early embryonic period. Zeta-globin is capable of substituting for alpha-globin, and its expression rescues severe alpha-thalassaemia in pre-clinical models. De-repression of zeta-globin is consequently an attractive potential therapy for severe alpha-thalassaemia.
The alpha-globin locus is highly conserved between mouse and human, therefore we have developed a mouse line in which a fluorescent reporter replaces the zeta-globin coding sequence. This allows high-throughput screening for compounds capable of de-repressing zeta-globin in primary adult murine red blood cells using flow cytometry. We plan to validate identified compounds in both primary human and mouse erythroid cells and investigate the mechanism by which these compounds operate.

The alpha-globin gene locus (which contains zeta-globin) has served as a model to establish the general principles by which mammalian genes are normally regulated and how this is perturbed in human genetic disease. Determining compounds capable of de-repressing zeta-globin and understanding the mechanisms by which these will therefore benefit researchers who are more generally interested in the principles of gene regulation, as well as researchers directly interested in regulation of the globin gene loci and haemoglobinopathies. It is possible that this research into gene regulation may benefit disciplines quite separate from haemoglobin disorders in the future including fields as diverse as cancer biology, autoimmune disease and inherited genetic disorders. All investigators applying here attend international conferences on both haemoglobin and more generally, gene regulation and results would be expected to disseminated and discussed with peers there.

Alpha-thalassemia is one of the most commonly inherited single gene disorders with several thousand sufferers world-wide. Therefore if we are able to identify a clinically applicable compound to reactivate the zeta-globin gene it would be of great benefit to this patient group. Our research will also be of interest to researchers into other thalassaemias generally because of similarities between the alpha- and beta-globin loci in terms of regulation. If we could identify different modalities of derepressing genes in the alpha and beta-globin loci generally there is a global patient population of tens of millions who stand to benefit. We will ensure that researchers in other disciplines and globally will be aware of research pertinent to their field to facilitate rapid translation of this research.

As is the rule in the host laboratory, we will publish research in high impact general journals where possible and will target specific relevant findings to appropriate conferences or speciality journals. We will publish open-access articles to ensure the widest possible readership. Locally, the work will be presented regularly both to the groups working directly on gene regulation and alpha-globin and more widely to the entire research community within the Weatherall Institute of Molecular Medicine.

The principle of collaboration between industry and academia is an important one. A successful project here will provide a model for future collaborations and drug development across a whole range of fields.