Stefano Rivella

Therapeutic haemoglobin synthesis in β-thalassaemic mice expressinglentivirus-encoded human β-globin

The stable introduction of a functional β-globin gene in haematopoietic stem cells could be a powerful approach to treat β-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal β-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human β-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human β-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine α-globin and two human βA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In β-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Identification of erythroferrone as an erythroid regulator of iron metabolism

Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbbth3/+ mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.

The cHS4 Insulator Increases the Probability of Retroviral Expression at Random Chromosomal Integration Sites

ABSTRACT Retroviruses are highly susceptible to transcriptional silencing and position effects imparted by chromosomal sequences at their integration site. These phenomena hamper the use of recombinant retroviruses as stable gene delivery vectors. As insulators are able to block promoter-enhancer interactions and reduce position effects in some transgenic animals, we examined the effect of an insulator on the expression and structure of randomly integrated recombinant retroviruses. We used the cHS4 element, an insulator from the chicken β-like globin gene cluster, which has been shown to reduce position effects in transgenic Drosophila. A large panel of mouse erythroleukemia cells that bear a single copy of integrated recombinant retroviruses was generated without using drug selection. We show that the cHS4 increases the probability that integrated proviruses will express and dramatically decreases the level of de novo methylation of the 5′ long terminal repeat. These findings support a primary role of methylation in the silencing of retroviruses and suggest that cHS4 could be useful in gene therapy applications to overcome silencing of retroviral vectors.

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinsons disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in O2 homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Non-transfusion-dependent thalassemias

Non-transfusion-dependent thalassemias include a variety of phenotypes that, unlike patients with beta (β)-thalassemia major, do not require regular transfusion therapy for survival. The most commonly investigated forms are β-thalassemia intermedia, hemoglobin E/β-thalassemia, and α-thalassemia intermedia (hemoglobin H disease). However, transfusion-independence in such patients is not without side effects. Ineffective erythropoiesis and peripheral hemolysis, the hallmarks of disease process, lead to a variety of subsequent pathophysiologies including iron overload and hypercoagulability that ultimately lead to a number of serious clinical morbidities. Thus, prompt and accurate diagnosis of non-transfusion-dependent thalassemia is essential to ensure early intervention. Although several management options are currently available, the need to develop more novel therapeutics is justified by recent advances in our understanding of the mechanisms of disease. Such efforts require wide international collaboration, especially since non-transfusion-dependent thalassemias are no longer bound to low- and middle-income countries but have spread to large multiethnic cities in Europe and the Americas due to continued migration.

Reducing TMPRSS6 ameliorates hemochromatosis and β-thalassemia in mice

β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe(-/-)) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB(th3/+) mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.

Decreased hepcidin mRNA expression in thalassemic mice

Beta thalassemia major is a congenital haemolytic anaemia resulting from the lack of synthesis of the b-globin chain, a major component of haemoglobin A. In the absence of lifelong transfusions the disease is lethal. When adequate transfusion support is provided the patients suffer from iron overload that can lead to endocrine deficiencies, cirrhosis and cardiac failure that frequently lead to their demise. The iron overload results from ‘transfusional’ iron as well as from an inadequate high intestinal absorption despite high levels of body iron. The understanding of the deranged regulation of intestinal iron absorption is of utmost importance and may lead to rational therapy aimed at decreasing iron overload. Hepcidin, a recently identified anti-microbial peptide expressed in the liver was shown to play a role in conditions associated with both iron overload and iron deficiency. Hepcidin inhibits iron absorption in the proximal small bowel and the release of iron from macrophages. In healthy individuals as well as in murine models hepcidin expression is increased when iron stores are elevated and decreased as a result of iron deficiency anaemia and hypoxia (Nicolas et al, 2001; Park et al, 2001; Pigeon et al, 2001; Ganz, 2003). Thalassemia major represents a unique situation where anaemia, which is expected to decrease hepcidin expression, coexists with iron overload, which ought to increase it. Therefore, it was of interest to study which of these factors has an upper hand in hepcidin expression regulation. We used the recently described severe anaemia model of C57Bl/6 Hbb mice (May et al, 2002) that exhibit anaemia, abnormal red cell morphology, splenomegaly and develop spontaneous hepatic iron deposition (demonstrated by Gomori iron stain of liver tissue), and C57Bl/6 control mice in an attempt to understand the inadequately enhanced absorption of iron in thalassemia. RNA extracted from the liver of adult mice was analysed by quantifiable real time reverse transcription polymerase chain reaction (RT-PCR) with specific primers for the following iron metabolism-related genes: hepcidin, iron-regulated gene 1 (IREG1), neutral gelatinase-associated lipocalin (NGAL), haemochromatosis (HFE), transferrin receptors 1 and 2 (TfR1 and TfR2). The expression levels were normalized to b-actin. Interestingly, despite iron overload the expression levels of both hepcidin and TfR1 were significantly lower (0Æ38-fold) in the thalassemic mice. HFE was moderately decreased (0Æ7-fold), while NGAL was significantly increased (2Æ45-fold). TfR2 and IREG1 did not change significantly (Fig 1). This would suggest that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia. Recently, decreased expression of hepcidin was found in hereditary haemochromatosis in association with elevated levels of nontransferrin bound iron. The elevated expression of NGAL, an alternative iron delivery vehicle, supports the role of nontranferrin bound iron in the abnormal iron regulation in thalassemia. The decreased HFE expression level is similar to the finding in hereditary haemochromatosis (Bridle et al, 2003). The exact mechanism remains to be elucidated. These preliminary findings call for confirmation in human thalassemia patients. If supported, a new therapeutic approach to iron overload states such as thalassemia can be devised based on hepcidin administration or other interventions aimed at overcoming the inadequate response to iron overload associated with elevated levels of non-transferrin bound iron. Fig 1. Expression of iron regulatory genes in the liver of a mouse model of b-thalassemia. cDNA was prepared from the total RNA extracted from the liver of three C57B1/6 Hbb mice and three normal C57B1/6 wild-type mice. Gene expression of six iron regulatory genes was measured using specific primers for quantifiable reverse transcription polymerase chain reaction. Bars represent the average fold change in mRNA expression of each gene in C57B1/6 Hbb mice (black bars), when compared with control mRNA levels in C57B1/6 wild-type mice (grey bars). Each bar represents the average of 2–4 independent experiments, normalized to corresponding b-actin expression. correspondence

Ineffective erythropoiesis and thalassemias

Purpose of reviewIn thalassemia, ineffective erythropoiesis is characterized by apoptosis of the maturing nucleated erythroid cells. New studies also suggest that limited erythroid cell differentiation plays a role in the development of ineffective erythropoiesis. This would further exacerbate anemia and increase iron absorption. Recent findingsDuring erythroid differentiation and maturation, it is critical that the components of hemoglobin are made in stoichiometric amounts. It is, therefore, conceivable that factors that modify this process intrinsically or extrinsically will also affect erythropoiesis. Several proteins have the potential to alter erythroid replication and differentiation in conditions of ineffective erythropoiesis. Elevated erythropoietin levels increase the number of erythroid precursors bearing a phosphorylated form of Jak2. This, in a pathological condition, may contribute to limited erythroid differentiation. Unbalanced synthesis of globins and heme modifies the activity of the heme-regulated inhibitor kinase, affecting proliferation and differentiation of the erythroid precursors. In addition, inefficient elimination of reactive oxygen species, which are increased under conditions of iron overload, may also hamper erythropoiesis. SummaryUse of Jak2 inhibitors may limit the overproduction of immature erythroid cells in thalassemia, with the potential of reversing extramedullary hematopoiesis and preventing splenectomy. In addition, preventing iron overload and formation of reactive oxygen species may also be beneficial in limiting tissue damage and ineffective erythropoiesis.