Laura Breda

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.

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.

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

Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera

β‐Thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β‐thalassaemia, we observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in β‐thalassaemia might suppress liver hepcidin expression. Sera from β‐thalassaemia and haemochromatosis (C282Y mutation) patients were compared with those of healthy individuals regarding their capacity to induce changes the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β‐thalassaemia major patients induced a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared with sera from healthy individuals. A significant correlation was found between the degree of downregulation of HAMP and HFE2 induced by β‐thalassaemia major sera (r = 0·852, P < 0·0009). Decreased HAMP expression was also found in HepG2 cells treated with sera from β‐thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients induced an increase in HAMP expression, which correlated with transferrin (Tf) saturation (r = 0·765, P < 0·0099). Our results suggest that, in β‐thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption in these patients.

Role of Iron in Inducing Oxidative Stress in Thalassemia: Can It Be Prevented by Inhibition of Absorption and by Antioxidants?

Abstract: The pathophysiology of thalassemia is, to a certain extent, associated with the generation of labile iron in the pathological red blood cell (RBC). The appearance of such forms of iron at the inner and outer cell surfaces exposes the cell to conditions whereby the labile metal promotes the formation of reactive oxygen species (ROS) leading to cumulative cell damage. Another source of iron accumulation results from increased absorption due to decreased expression of hepcidin. The presence of labile plasma iron (LPI) was carried out using fluorescent probes in the FACS. RNA expression of hepcidin was measured in two models of thalassemic mice. Hepcidin expression was also measured in human hapatoma HepG2 cells following incubation with thalassemic sera. LPI was identified and could be quantitatively measured and correlated with other parameters of iron overload. Hepcidin expression was downregulated in the livers of thalassemic mice, in major more than in intermedia. Thalassemic sera down regulated hepcidin expression in HepG2 liver cells. A possible way to decrease iron absorption could be by modulating hepcidin expression pharmacologically, by gene therapy or by its administration. Treatment with combination of antioxidants such as N‐acetylcysteine for proteins and vitamin E for lipids in addition to iron chelators could neutralize the deleterious effects of ROS and monitored by quantitation of LPI.

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients. We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S). Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

Gene Therapy for Hemoglobinopathies: Progress and Challenges

Hemoglobinopathies are genetic inherited conditions that originate from the lack or malfunction of the hemoglobin (Hb) protein. Sickle cell disease (SCD) and thalassemia are the most common forms of these conditions. The severe anemia combined with complications that arise in the most affected patients raises the necessity for a cure to restore hemoglobin function. The current routine therapies for these conditions, namely transfusion and iron chelation, have significantly improved the quality of life in patients over the years, but still fail to address the underlying cause of the diseases. A curative option, allogeneic bone marrow transplantation is available, but limited by the availability of suitable donors and graft-vs-host disease. Gene therapy offers an alternative approach to cure patients with hemoglobinopathies and aims at the direct recovery of the hemoglobin function via globin gene transfer. In the last 2 decades, gene transfer tools based on lentiviral vector development have been significantly improved and proven curative in several animal models for SCD and thalassemia. As a result, clinical trials are in progress and 1 patient has been successfully treated with this approach. However, there are still frontiers to explore that might improve this approach: the stoichiometry between the transgenic hemoglobin and endogenous hemoglobin with respect to the different globin genetic mutations; donor cell sourcing, such as the use of induced pluripotent stem cells (iPSCs); and the use of safer gene insertion methods to prevent oncogenesis. With this review we will provide insights about (1) the different lentiviral gene therapy approaches in mouse models and human cells; (2) current and planned clinical trials; (3) hurdles to overcome for clinical trials, such as myeloablation toxicity, insertional oncogenesis, and high vector expression; and (4) future perspectives for gene therapy, including safe harbors and iPSCs technology.

Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers

Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.

Aromatic polyamidines inhibiting the Tat-induced HIV-1 transcription recognize structured TAR-RNA

We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.

Recent trends in the gene therapy of β-thalassemia.

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.