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200 billion red blood cells (RBCs) are produced every day, requiring more than 2 × 10 15 iron atoms every second to maintain adequate erythropoiesis. These numbers translate into 20 mL of blood being produced each day, containing 6 g of hemoglobin and 20 mg of iron. These impressive numbers illustrate why the making and breaking of RBCs is at the heart of iron physiology, providing an ideal context to discuss recent progress in understanding the systemic and cellular mechanisms that underlie the regulation of iron homeostasis and its disorders. [ABSTRACT FROM AUTHOR]

Erythroblasts possess unique characteristics as they undergo differentiation from hematopoietic stem cells. During terminal erythropoiesis, these cells incorporate large amounts of iron in order to generate hemoglobin and ultimately undergo enucleation to become mature red blood cells, ultimately delivering oxygen in the circulation. Thus, erythropoiesis is a finely tuned, multifaceted process requiring numerous properly timed physiological events to maintain efficient production of 2 million red blood cells per second in steady state. Iron is required for normal functioning in all human cells, the erythropoietic compartment consuming the majority in light of the high iron requirements for hemoglobin synthesis. Recent evidence regarding the crosstalk between erythropoiesis and iron metabolism sheds light on the regulation of iron availability by erythroblasts and the consequences of insufficient as well as excess iron on erythroid lineage proliferation and differentiation. In addition, significant progress has been made in our understanding of dysregulated iron metabolism in various congenital and acquired malignant and non-malignant diseases. Finally, we report several actual as well as theoretical opportunities for translating the recently acquired robust mechanistic understanding of iron metabolism regulation to improve management of patients with disordered erythropoiesis, such as anemia of chronic inflammation, β-thalassemia, polycythemia vera, and myelodysplastic syndromes. [ABSTRACT FROM AUTHOR]

Iron is essential for the growth and proliferation of cells, as well as for many biological processes that are important for the maintenance and survival of the human body. However, excess iron is associated with the development of cancer and other pathological conditions, due in part to the pro-oxidative nature of iron and its damaging effects on DNA. Current studies suggest that iron depletion may be beneficial for patients that have diseases associated with iron overload or other iron metabolism disorders that may increase the risk for cancer. On the other hand, studies suggest that cancer cells are more vulnerable to the effects of iron depletion and oxidative stress in comparison to normal cells. Therefore, cancer patients might benefit from treatments that alter both iron metabolism and oxidative stress. This review highlights the pro-oxidant effects of iron, the relationship between iron and cancer development, the vulnerabilities of the iron-dependent cancer phenotype, and how these characteristics may be exploited to prevent or treat cancer. [ABSTRACT FROM AUTHOR]

Abstract: β-Thalassemia and Polycythemia vera are genetic disorders which affect the synthesis of red blood cells, also referred to as erythropoiesis. Although essentially different in clinical presentation – patients with β-thalassemia have an impairment in β-globin synthesis leading to defective erythrocytes and anemia, while patients with Polycythemia vera present with high hemoglobin levels because of excessive red blood cell synthesis – both pathologies may characterized by lasting high erythropoietic activity, i.e. chronic stress erythropoiesis. In both diseases, therapeutic strategies targeting chronic stress erythropoiesis may improve the address phenotype and prevent secondary pathology, such as iron overload. The current review will address the basic concepts of these strategies to reduce chronic stress erythropoiesis, which may have significant clinical implications in the near future. [Copyright &y& Elsevier]

The β-thalassemias and sickle cell disease are severe congenital anemias that are caused by the defective synthesis of the β chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a regulated β-globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling transgene expression, which ideally should be erythroid-specific, differentiation stage-restricted, elevated, position-independent, and sustained over time. Using lentiviral vectors, we recently demonstrated that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated expression of the β-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in β-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors provide a paradigm for improving vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy. [ABSTRACT FROM AUTHOR]

Shows that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. Speculation that the stable introduction of a functional beta-globin gene in hemotopoietic stem cells could be a powerful approach to treat beta-thalassemia and sickle-cell disease; Findings of experiments with beta-thalassemic mice; Conclusion that such therapy should be of benefit to patients with severe defects in hemoglobin production.

The author discusses the study by Z. Sui and colleagues on the roles for Tropomodulin (Tmod)-3 in enucleation, survival and erythroid proliferation. He mentions that the study showed that Tmod3 is needed in both macrophages and erythroblasts to form correctly erythroid islands. He states that the observations may also recommend a role of macrophages in strengthening the program in erythroid cells that integrates cell-cycle exit having enucleation.

Inherited monogenic disorders such as beta-hemoglobinopathies (BH) are fitting candidates for treatment via gene therapy by gene transfer or gene editing. The reported safety and efficacy of lentiviral vectors in preclinical studies have led to the development of several clinical trials for the addition of a functional beta-globin gene. Across trials, dozens of transfusion-dependent patients with sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) have been treated via gene therapy and have achieved reduced transfusion requirements. While overall results are encouraging, the outcomes appear to be strongly influenced by the level of lentiviral integration in transduced cells after engraftment, as well as the underlying genotype resulting in thalassemia. In addition, the method of procurement of hematopoietic stem cells can affect their quality and thus the outcome of gene therapy both in SCD and TDT. This suggests that new studies aimed at maximizing the number of corrected cells with long-term self-renewal potential are crucial to ensure successful treatment for every patient. Recent advancements in gene transfer and bone marrow transplantation have improved the success of this approach, and the results obtained by using these strategies demonstrated significant improvement of gene transfer outcome in patients. The advent of new gene-editing technologies has suggested additional therapeutic options. These are primarily focused on correcting the defective beta-globin gene or editing the expression of genes or genomic segments that regulate fetal hemoglobin synthesis. In this review, we aim to establish the potential benefits of gene therapy for BH, to summarize the status of the ongoing trials, and to discuss the possible improvement or direction for future treatments. [ABSTRACT FROM AUTHOR]

The author presents his views on the expression of the genes encoding erythroid cells secreting alpha and beta-globin chains. He informs that erythroid cells are equipped with protein quality control responses that eliminate the excess of alpha-chains produced in beta thalassemia to a certain extent. He suggests that erythroid cell death/apoptosis is associated with formation of alpha-chains.

An introduction to the journal is presented in which the editor discusses an article on the close relationship of erythropoieses and iron metabolism and another on clinical observations in sickle cell anemia and myelodysplastic syndromes.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual’s lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP–messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti–human CD117/LNP–based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT. [ABSTRACT FROM AUTHOR]

Hepcidin agonists are a new class of compounds that regulate blood iron levels, limit iron absorption, and could improve the treatment of hemochromatosis, b-thalassemia, polycythemia vera, and other disorders in which disrupted iron homeostasis causes or contributes to disease. Hepcidin agonists also have the potential to prevent severe complications of siderophilic infections in patients with iron overload or chronic liver disease. This review highlights the preclinical studies that support the development of hepcidin agonists for the treatment of these disorders. [ABSTRACT FROM AUTHOR]

An abstract of the article "Optimization of Globin Lentiviral Vector Design for the Treatment of ͒-Thalassemia," by Leszek Lisowski, Stefano Rivella and Michel Sadelain is presented.

• β-THAL is one of the most prevalent blood disorders in some regions of the world. • However, given its prevalence, in the EU and the US it is classified as a rare disease. • A wide array of therapeutic approaches has been developed over the past decades. • β-THAL is a learning case on the potential global impact of Orphan Legislations. • Gene therapy for β-THAL is promising, but scenarios for global access are missing. In many countries, β-thalassemia (β-THAL) is not uncommon; however, it qualifies as a rare disease in the US and in European Union (EU), where thalassemia drugs are eligible for Orphan Drug Designation (ODD). In this paper, we evaluate all 28 ODDs for β-THAL granted since 2001 in the US and the EU: of these, ten have since been discontinued, twelve are pending, and six have become licensed drugs available for clinical use. The prime mover for these advances has been the increasing depth of understanding of the pathophysiology of β-THAL; at the same time, and even though only one-fifth of β-THAL ODDs have become licensed drugs, the ODD legislation has clearly contributed substantially to the development of improved treatments for β-THAL. [ABSTRACT FROM AUTHOR]

Iron is one of the most abundant elements in the Earth and a fundamental component of enzymes and other proteins that participate in a wide range of biological processes. As the human body has no mechanisms to eliminate the excess of iron, its metabolism needs to be tightly controlled in order to avoid all the sequelae associated with high iron levels. Iron overload is the main cause of morbidity and mortality in beta thalassemia. The master regulator of iron homeostasis, hepcidin, is chronically repressed in this disorder, leading to increased intestinal iron absorption and consequent iron overload. Many groups have focused on obtaining a better understanding of the pathways involved in iron regulation. New molecules have recently been synthesized and used in animal models of dysregulated iron metabolism, demonstrating their ability to target and reduce iron load. Antisense oligonucleotides, as well as lipid nanoparticle-formulated small interfering RNAs and minihepcidins peptides, are novel agents that have already proved to be efficient in modulating iron metabolism in mouse models and are therefore promising candidates for the treatment of patients affected by iron disorders. [ABSTRACT FROM AUTHOR]

Significance: In this review, the dual nature of both iron and reactive oxygen species (ROS) will be explored in normal and cancer cell metabolism. Although iron and ROS play important roles in cellular homeostasis, they may also contribute to carcinogenesis. On the other hand, many studies have indicated that abrogation of iron metabolism, elevation of ROS, or modification of redox regulatory mechanisms in cancer cells, should be considered as therapeutic approaches for cancer. Recent Advances: Drugs that target different aspects of iron metabolism may be promising therapeutics for cancer. The ability of iron chelators to cause iron depletion and/or elevate ROS levels indicates that these types of compounds have more potential as antitumor medicines than originally expected. Other natural and synthetic compounds that target pathways involved in ROS homeostasis also have potential value alone or in combination with current chemotherapeutics. Critical Issues: Although ROS induction and iron depletion may be targets for cancer therapies, the optimal therapeutic strategies have yet to be identified. This review highlights some of the research that strives to identify such therapeutics. Future Directions: More studies are needed to better understand the role of iron and ROS in carcinogenesis not only as cancer promoters, but also as cytotoxic agents to cancer cells and cancer stem cells (CSCs). Moreover, the structure-activity effects of iron chelators and other compounds that increase ROS and/or disrupt iron metabolism need to be further evaluated to assess the effectiveness and selectivity of these compounds against both cancer and CSCs. Antioxid. Redox Signal. 20, 1917-1924. [ABSTRACT FROM AUTHOR]

β-thalassemia encompasses a group of monogenic diseases that have in common defective synthesis of β-globin. The defects involved are extremely heterogeneous and give rise to a large phenotypic spectrum, with patients that are almost asymptomatic to cases in which regular blood transfusions are required to sustain life. As a result of the inefficient synthesis of β-globin, the patients suffer from chronic anemia due to a process called ineffective erythropoiesis (IE). The sequelae of IE lead to extramedullary hematopoiesis (EMH) with massive splenomegaly and dramatic iron overload, which in turn is responsible for many of the secondary pathologies observed in thalassemic patients. The processes are intimately linked such that an ideal therapeutic approach should address all of the complications. Although β-thalassemia is one of the first monogenic diseases to be described and represents a global health problem, only recently has the scientific community started to focus on the real molecular mechanisms that underlie this disease, opening new and exciting therapeutic perspectives for thalassemic patients worldwide. [ABSTRACT FROM AUTHOR]

Polycythemia Vera (PV) is a chronic myeloproliferative neoplasm resulting from an acquired driver mutation in the JAK2 gene of hematopoietic stem and progenitor cells resulting in the overproduction of mature erythrocytes and abnormally high hematocrit, in turn leading to thromboembolic complications. Therapeutic phlebotomy is the most common treatment to reduce the hematocrit levels and consequently decrease thromboembolic risk. Here we demonstrate that, by using the iron restrictive properties of the antisense oligonucleotides against Tmprss6 mRNA, we can increase hepcidin to achieve effects equivalent to therapeutic phlebotomy. We provide evidence that this less invasive approach could represent an additional therapeutic tool for the treatment of PV patients. [ABSTRACT FROM AUTHOR]

Significance: Iron is an essential element required for sustaining a normal healthy life. However, an excess amount of iron in the bloodstream and tissue generates toxic hydroxyl radicals through Fenton reactions. Henceforth, a balance in iron concentration is extremely important to maintain cellular homeostasis in both normal hematopoiesis and erythropoiesis. Iron deficiency or iron overload can impact hematopoiesis and is associated with many hematological diseases. Recent Advances: The mechanisms of action of key iron regulators such as erythroferrone and the discovery of new drugs, such as ACE-536/luspatercept, are of potential interest to treat hematological disorders, such as β-thalassemia. New therapies targeting inflammation-induced ineffective erythropoiesis are also in progress. Furthermore, emerging evidences support differential interactions between iron and its cellular antioxidant responses of hematopoietic and neighboring stromal cells. Both iron and its systemic regulator, such as hepcidin, play a significant role in regulating erythropoiesis. Critical Issues: Significant pre-clinical studies are on the way and new drugs targeting iron metabolism have been recently approved or are undergoing clinical trials to treat pathological conditions with impaired erythropoiesis such as myelodysplastic syndromes or β-thalassemia. Future Directions: Future studies should explore how iron regulates hematopoiesis in both benign and malignant conditions. Antioxid. Redox Signal. 35, 415–432. [ABSTRACT FROM AUTHOR]

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe -/-mouse model as well as β-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/-mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in HbbtH3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in β-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania. [ABSTRACT FROM AUTHOR]

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis. Maria Feola et al., elucidate the compensatory role of pleckstrin-2 in ineffective erythropoiesis in β-thalassemic mice by reducing cofilin-mediated apoptosis and enhancing enucleation by activating RacGTPases. These findings could support future therapeutic interventions. [ABSTRACT FROM AUTHOR]

Iron–sulfur (Fe-S) clusters are required for mitochondrial function. Fe-S cluster synthesis occurs in the mitochondria and iron uptake is required for mitochondrial biogenesis. However, Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfR1), whereas lack of the Fe-S cluster stimulates TfR1 expression. Yet, it is unclear whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, whether this negatively modulates TfR1 expression. We manipulated peroxisome proliferator-activated receptor-gamma coactivator-1α expression to control mitochondrial biogenesis in a variety of cell types, including erythroid cells. We demonstrated that Fe-S cluster synthesis increases with mitochondria biogenesis but does not interfere with increasing TfR1 expression. In fact, TfR1 expression is stimulated through alternative means to meet iron requirement for mitochondria biogenesis. Furthermore, under enhanced mitochondria biogenesis, increased Fe-S cluster synthesis inhibits the function of iron-regulating protein (IRP)1 and hence stimulates the expression of 5′-aminolevulinate synthase 2 (ALAS2), a target of IRP1 and rate-limiting enzyme in erythroid heme biogenesis. Increased ALAS2 expression leads to enhanced heme production, hemoglobinization, and erythropoiesis. Therefore, our study also provides a mechanism to link mitochondrial biogenesis with erythropoiesis and has a potential therapeutic value in the treatment of blood disorders. [ABSTRACT FROM AUTHOR]

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia. [ABSTRACT FROM AUTHOR]

Severe anemia and iron deficiency are common complications in chronic kidney disease. The cause of renal anemia is multifactorial and includes decreased erythropoietin (Epo) production, iron deficiency, and inflammation, and it is currently treated with injections of synthetic Epo. However, the use of recombinant Epo has several adverse effects. We previously reported that high fibroblast growth factor 23 (FGF23) levels in mice are associated with decreased red blood cell production, whereas genetic inactivation of Fgf23 results in expansion of the erythroid lineage. The present study is the first to show that high FGF23 levels in a mouse model of renal failure contribute to renal anemia, and inhibiting FGF23 signaling stimulates erythropoiesis and abolishes anemia and iron deficiency. Moreover, we show that inhibition of FGF23 signaling significantly decreases erythroid cell apoptosis and influences the commitment of hematopoietic stem cells toward the erythroid linage. Furthermore, we show that blocking FGF23 signaling attenuates inflammation, resulting in increased serum iron and ferritin levels. Our data clearly demonstrate that elevated FGF23 is a causative factor in the development of renal anemia and iron deficiency, and importantly, blocking FGF23 signaling represents a novel approach to stimulate erythropoiesis and possibly improve survival for millions of chronic kidney disease patients worldwide.--Agoro, R., Montagna, A., Goetz, R., Aligbe, O., Singh, G., Coe, L. M., Mohammadi, M., Rivella, S., Sitara, D. Inhibition of fibroblast growth factor 23 (FGF23) signaling rescues renal anemia. [ABSTRACT FROM AUTHOR]

Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing wild-type or K8R Fpn, and mature human red blood cells.Weconclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders. (Blood. 2018;131(8):899-910) [ABSTRACT FROM AUTHOR]

The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly- L -lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders. [Display omitted] • Hematopoietic stem cell gene therapy for DNA repair disorders is challenging. • BaEV-Rless pseudotyped lentiviral vector could solve the issue by cytokine depletion. • Although the titer is too low, novel virus production protocol is needed. • We modified protocol focusing on syncytium and detachment of transfected 293 T cells. • Our novel protocol resulted in a 10-fold titer increase compared to basic protocol. [ABSTRACT FROM AUTHOR]

Biogenesis of mammalian red blood cells requires nuclear expulsion by orthochromatic erythoblasts late in terminal differentiation (enucleation), but the mechanism is largely unexplained. Here, we employed high-resolution confocal microscopy to analyze nuclear morphology and F-actin rearrangements during the initiation, progression, and completion of mouse and human erythroblast enucleation in vivo. Mouse erythroblast nuclei acquire a dumbbell-shaped morphology during enucleation, whereas human bone marrow erythroblast nuclei unexpectedly retain their spherical morphology. These morphological differences are linked to differential expression of Lamin isoforms, with primary mouse erythroblasts expressing only Lamin B and primary human erythroblasts only Lamin A/C. We did not consistently identify a continuous F-actin ring at the cell surface constriction in mouse erythroblasts, nor at the membrane protein-sorting boundary in human erythroblasts, which do not have a constriction, arguing against a contractile ring-based nuclear expulsion mechanism. However, both mouse and human erythroblasts contain an F-actin structure at the rear of the translocating nucleus, enriched in tropomodulin 1 (Tmod1) and nonmuscle myosin IIB. We investigated Tmod1 function in mouse and human erythroblasts both in vivo and in vitro and found that absence of Tmod1 leads to enucleation defects in mouse fetal liver erythroblasts, and in CD34+ hematopoietic stem and progenitor cells, with increased F-actin in the structure at the rear of the nucleus. This novel structure, the "enucleosome,"maymediatecommoncytoskeletalmechanismsunderlying erythroblast enucleation, notwithstanding the morphological heterogeneity of enucleation across species. [ABSTRACT FROM AUTHOR]

Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed Tf R1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice. [ABSTRACT FROM AUTHOR]

Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adeninefed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted. [ABSTRACT FROM AUTHOR]

Background: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on β-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning.Methods: Erythroid precursor cells were obtained from 72 patients, mostly β-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC.Results: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from β-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers.Conclusion: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Hbbth3/+ 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. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology. [ABSTRACT FROM AUTHOR]

The thalassemia syndromes (α- and β-thalassemia) are the most common and frequent disorders associated with ineffective erythropoiesis. Imbalance of α- or β-globin chain production results in impaired red blood cell synthesis, anemia, and more erythroid progenitors in the blood stream. While patients affected by these disorders show definitive altered parameters related to erythropoiesis, the relationship between the degree of anemia, altered erythropoiesis, and dysfunctional iron metabolism has not been investigated in both α-thalassemia carriers ( ATC) and β-thalassemia carriers ( BTC). Here, we demonstrate that ATC have a significantly reduced hepcidin and increased soluble transferrin receptor levels but relatively normal hematological findings. In contrast, BTC have several hematological parameters significantly different from controls, including increased soluble transferrin receptor and erythropoietin levels. These changes in both groups suggest an altered balance between erythropoiesis and iron metabolism. The index s Tf R/log ferritin and (hepcidin/ferritin)/s Tf R are, respectively, increased and reduced relative to controls, proportional to the severity of each thalassemia group. In conclusion, we showed in this study, for the first time in the literature, that thalassemia carriers have altered iron metabolism and erythropoiesis. [ABSTRACT FROM AUTHOR]

Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin αmu-globin2/βhu-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Patients surviving sepsis develop anemia, but the molecular mechanism is unknown. Here we observed that mice surviving polymicrobial gram-negative sepsis develop hypochromic, microcytic anemia with reticulocytosis. The bone marrow of sepsis survivors accumulates polychromatophilic and orthochromatic erythroblasts. Compensatory extramedullary erythropoiesis in the spleen is defective during terminal differentiation. Circulating tumor necrosis factor (TNF) and interleukin (IL)-6 are elevated for 5 d after the onset of sepsis, and serum high-mobility group box 1 (HMGB1) levels are increased from d 7 until at least d 28. Administration of recombinant HMGB1 to healthy mice mediates anemia with extramedullary erythropoiesis and significantly elevated reticulocyte counts. Moreover, administration of anti-HMGB1 monoclonal antibodies after sepsis significantly ameliorates the development of anemia (hematocrit 48.5 ± 9.0% versus 37.4 ± 6.1%, p < 0.01; hemoglobin 14.0 ± 1.7 versus 11.7 ± 1.2 g/dL, p < 0.01). Together, these results indicate that HMGB1 mediates anemia by interfering with erythropoiesis, suggesting a potential therapeutic strategy for anemia in sepsis [ABSTRACT FROM AUTHOR]

Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non–erythroid Krüppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krüppel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation. [ABSTRACT FROM AUTHOR]

Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis, with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In β-thalassemia, unequal production of α- and β-globin chains in erythroid precursors causes apoptosis and inhibition of late-stage erythroid differentiation, leading to anemia, ineffective erythropoiesis (IE), and dysregulated iron homeostasis. Here we used a murine model of β-thalassemia intermedia (Hbbth1/th1 mice) to investigate effects of a modified activin receptor type MB (ActRIIB) ligand trap (RAP-536) that inhibits Smad2/3 signaling. In Hbbminh1 mice, treatment with RAP-536 reduced overactivation of Smad2/3 in splenic erythroid precursors. In addition, treatment of Hbbth1/th1 mice with RAP-536 reduced α-globin aggregates in peripheral red cells, decreased the elevated reactive oxygen species present in erythroid precursors and peripheral red cells, and alleviated anemia by promoting differentiation of late-stage erythroid precursors and reducing hemolysis. Notably, RAP-536 treatment mitigated disease complications of IE, including iron overload, splenomegaly, and bone pathology, while reducing erythropoietin levels, improving erythrocyte morphology, and extending erythrocyte life span. These results implicate signaling by the transforming growth factor-β superfamily in late-stage erythropoiesis and reveal potential of a modified ActRIIB ligand trap as a novel therapeutic agent for thalassemia syndrome and other red cell disorders characterized by IE. [ABSTRACT FROM AUTHOR]

Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases. [ABSTRACT FROM AUTHOR]

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). Bonederived 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. [ABSTRACT FROM AUTHOR]