Carla Casu

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.

The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis

In hereditary hemochromatosis, mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition, HFE potentially modulates cellular iron uptake by interacting with transferrin receptor, a crucial protein during erythropoiesis. However, the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this, we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second, we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly, we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake, whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary, we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis, and impairing transferrin-bound iron uptake by erythroid cells. Moreover, our results provide novel suggestions to improve the treatment of hemochromatosis.

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Background: FGF-23, a bone-derived hormone, regulates phosphate and vitamin D in the kidney. Results: Genetic and pharmacological manipulations of FGF-23 alter erythropoiesis and HSC frequency both in young adult age and embryonically. Conclusion: Fgf-23 regulates erythropoiesis through Epo and independent of vitamin D. Significance: These findings provide a new target for treating blood disorders associated with bone and renal defects. Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.

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.

Minihepcidin peptides as disease modifiers in mice affected by β-thalassemia and polycythemia vera

In β-thalassemia and polycythemia vera (PV), disordered erythropoiesis triggers severe pathophysiological manifestations. β-Thalassemia is characterized by ineffective erythropoiesis, reduced production of erythrocytes, anemia, and iron overload and PV by erythrocytosis and thrombosis. Minihepcidins are hepcidin agonists that have been previously shown to prevent iron overload in murine models of hemochromatosis and induce iron-restricted erythropoiesis at higher doses. Here, we show that in young Hbb(th3/+) mice, which serve as a model of untransfused β-thalassemia, minihepcidin ameliorates ineffective erythropoiesis, anemia, and iron overload. In older mice with untransfused β-thalassemia, minihepcidin improves erythropoiesis and does not alter the beneficial effect of the iron chelator deferiprone on iron overload. In PV mice that express the orthologous JAK2 mutation causing human PV, administration of minihepcidin significantly reduces splenomegaly and normalizes hematocrit levels. These studies indicate that drug-like minihepcidins have a potential as future therapeutics for untransfused β-thalassemia and PV.

Combination of Tmprss6-ASO and the iron chelator deferiprone improves erythropoiesis and reduces iron overload in a mouse model of beta-thalassemia intermedia

Beta-thalassemia is one of the most frequently inherited disorders caused by mutations in the beta globin gene or its promoter, leading to reduced or absent beta globin synthesis. Ineffective erythropoiesis (IE) and consequent extramedullary hematopoiesis, splenomegaly and systemic iron overload are major features of this disease. The disease course can be associated with severe anemia and need for lifelong transfusion therapy (thalassemia major, TM) or relatively less severe anemia (non-transfusion-dependent thalassemia, NTDT, or thalassemia inter-media, TI). Patients affected by beta-thalassemia intermedia do not require chronic blood transfusions for survival. However, transfusion-independence is still associated with a variety of serious clinical morbidities.1–3 In NTDT the master regulator of iron homeostasis, hepcidin (Hamp), is chronically repressed.4–7 Therefore, patients absorb abnormally high levels of iron, requiring iron chelation to prevent the clinical sequelae associated with iron overload. Iron homeostasis needs to be carefully regulated in order to avoid toxicity due to its excess. If untreated, iron overload leads to organ failure and death. For this reason, in beta-thalassemia and other iron-related disorders, the management of iron overload has become the main focus. Chelation therapy, however, does not target the mechanism responsible for abnormal iron absorption, which is low levels of Hamp expression and synthesis. It has been shown that in mice affected by NTDT (Hbbth3/+ or th3/+), second generation antisense oligonucleotides (Tmprss6-ASO) or lipid nanoparticle (LNP)-formulated siRNAs can reduce the expression of transmembrane serine protease Tmprss6, one of the major suppressors of hepcidin expression.8,9 Suppression of Tmprss6 led to an increase in hepcidin synthesis and hemoglobin levels. These observations were also associated with a net reduction in splenomegaly, iron overload, transferrin saturation (TfSat), formation of insoluble membrane-bound globins (hemichromes) and reactive oxygen species (ROS).9 Thus, we hypothesized that the simultaneous use of the iron chelator deferiprone (DFP) with Tmprss6-ASO (Tmprss6-ASO+DFP) could combine the positive effects of Tmprss6-ASO on erythropoiesis and iron absorption with the chelation benefit on organ iron content. In this study, 3- to 4-month-old Hbbth3/+ females were treated with 50 mg/kg of Tmprss6 antisense oligonucleotide (Tmprss6-ASO, twice a week for 6 weeks) or Tmprss6-ASO in combination with the oral iron chelator DFP dissolved in the drinking water at 1.25 mg/ml, using either a commercial diet (normally used in the facility where animals were housed) containing 200 ppm of iron, or a physiological diet containing 35 ppm of iron. The majority of the animals available were treated using the commercial diet and just a few animals per group received the physiological one. With both diets we obtained the same trend in behavior, but considering that the numbers were not comparable, we decided to show only the data obtained from the 200 ppm diet.

Decreased hepcidin expression in murine β-thalassemia is associated with suppression of Bmp/Smad signaling.

To the editor: β-thalassemia is a genetic disorder of hemoglobin production characterized by ineffective erythropoiesis and anemia.[1][1] Iron overload, a major source of morbidity, results from inappropriately low expression of the gene encoding hepcidin ( Hamp1 ).[1][1] Hamp1 controls plasma

A preclinical approach for gene therapy of β-thalassemia

Lentiviral‐mediated β‐globin gene transfer successfully treated β‐thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different β‐globin mutations found in patients. Most mutations can be classified as β0 or β+, based on the amount of β‐globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two‐phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human β‐globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooleys anemia patients.