Thalassemic Mice Research Articles

Running exercise alleviates trabecular bone loss and osteopenia in hemizygous β-globin knockout thalassemic mice.

A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice and to determine whether endurance running (60 min/day, 5 days/wk for 12 wk in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1-2 mo old) showed growth retardation as indicated by smaller body weight and femoral length than their wild-type littermates. A decrease in BMD was more severe in female than in male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines IL-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice compared with those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, whereas running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia.

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia.

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Phenotypic comparison of four thalassemia model mice reconstructed from cryo-banked embryos

A major clinical feature of patients with thalassemia is growth retardation due to anemia, therefore, the hematological parameters, weanling weight and post-weanling weight of pups obtained from vitrified warmed embryo transfers were studied for the first time in this report. Two-cell embryos of four transgenic (TG) thalassemic mouse lines (BKO, 654, E2, and E4) were produced by breeding four lines of TG thalassemic males to wild-type (WT) females (C57BL/6J) and were cryopreserved by vitrification in straws using 35% ethylene glycol. After transfer of vitrified-warmed embryos, hematological parameters, spleen index, weanling and post-weanling weight were determined in TG and WT viable pups. In the BKO and 654 mice significantly abnormal hematological parameters and spleen index were observed compared to WT, E2 and E4 mice. The weanling and post-weanling weights of BKO and 654 pups were significantly less than that of the age-matched WT pups. However, no significant differences in weanling and post-weanling weight were found between WT and E2-TG or E4-TG pups. In conclusion, the four transgenic mice lines produced from cryopreserved embryo transfer retain the phenotype of the natural breeding mice indicating that these banked embryos are appropriate for thalassemia model productions.

First US Phase I Clinical Trial Of Globin Gene Transfer For The Treatment Of Beta-Thalassemia Major

▪To date, the only curative therapeutic approach for beta-thalassemia major has been allogeneic stem cell transplantation (SCT) for patients with HLA-matched siblings. For the majority of patients who do not have a matched sibling, allogeneic SCT is associated with major risks of morbidity and mortality. The stable transfer of a functional globin gene into the patient's own hematopoietic progenitor cells (HPCs) yields a perfectly matched graft that does not require immunosuppression to engraft. We previously demonstrated successful globin gene therapy in murine thalassemia models, using a lentiviral vector that encodes the human ß-globin promoter and arrayed regulatory elements uniquely combined to achieve high level and erythroid-specific globin expression. In vivo in thalassemic mice, the vector termed TNS9.3.55, increased hemoglobin levels by an average 4-6 g/dL per vector copy. We obtained in 2012 the first US Food and Drug Administration (FDA) approval to proceed to a clinical study in adult subjects with beta-thalassemia major (NCT01639690). We have to date enrolled 5 patients and recently treated the first three, administering the transduced HPCs after non-myeloablative conditioning. Engraftment data are available for the first two patients. Patient 3 was recently infused with CD34+ cells and is at this time too early to evaluate.Patient 1 is a 23 year old female with a ß039 – IVS1,110 mutation. Patient 2 is an 18 year old female with a ß039 – IVS1,6 mutation. Both patients underwent mobilization of peripheral blood stem cells (PBSCs) with filgrastim and mobilized 25 x 10^6 and 9.9 x 10^6 CD34 cells/Kg respectively. CD34+ PBSCs were transduced with the lentiviral vector TNS9.3.55 encoding the normal human beta-globin gene. The average vector copy number (VCN) in bulk CD34+ cells for these two patients was respectively 0.39 and 0.21 copies per cell. Both patients underwent non-myeloablative cytoreduction with busulfan administered at 2 mg/Kg/dose Q12H x 4 doses (total 8 mg/Kg), followed by reinfusion of 11.8 x 10^6 and 8.4 x 10^6 CD34+ cells/Kg, respectively. Both patients tolerated cytoreduction well and recovered their blood counts. While they continue to be transfusion dependent, both patients show a gradual rise in vector copy number in peripheral blood white blood cells and neutrophils, steadily increasing by 1-2% every month, reaching an average VCN of 5-7% 3-6 months after transplantation.In summary, patients with thalassemia major underwent safe and effective mobilization followed by excellent transduction of mobilized CD34+ cells. The transplant non-myeloablative conditioning was well tolerated, and followed by rapid engraftment and gradual rise in VCN. Continued clinical and molecular monitoring is on-going and will be presented. Disclosures:No relevant conflicts of interest to declare.

Steady State Differences In Metabolic Properties Of Bone Marrow Versus Spleen Erythroid Cells

Erythropoiesis in adult mice occurs principally in the bone marrow, while stress erythropoiesis is mostly localized in the spleen. Although both bone marrow and spleen produce fully functional mature red blood cells, we noticed that the rate of maturation of erythroid precursors (based on the ratio of mature/immature erythroid cells) is over 10 times higher in the spleen as compared to the bone marrow in C57BL6 mice (3.7 ± 0.6 in the spleen as compared to 0.4 ± 0.1 in the bone marrow, n ≥ 10 mice). Cell cycle analysis of erythroid precursors revealed that bone marrow erythroid cells cycle up to 3 times more than their spleen counterparts (88 ± 1% vs 25 ± 7% of cells in the S phase, n=3). As reactive oxygen species (ROS) influence cell cycle, we measured ROS levels by flow cytometry using the CM-H2DCFDA probe. To our surprise, we found ROS levels to decrease (rather than increase) progressively in the bone marrow erythroid cells as they mature and accumulate hemoglobin. Interestingly, the levels of ROS were twice as high in the spleen erythroid cells as compared to erythroid cells in the bone marrow. As mitochondria are a major site of ROS production we measured mitochondrial mass by flow cytometry using Mitotracker Green. Mitochondrial mass was found to be two fold lower in the spleen erythroid cells as compared to the bone marrow. In agreement with these findings, qRT-PCR expression analysis of different antioxidant enzymes such as gluthathione peroxidases Gpx1 and Gpx4 showed higher levels in the bone marrow as compared to the spleen erythroid precursors. In particular, Gpx1 expression increased ten fold during erythroid maturation in the bone marrow while in the spleen the expression of Gpx1 did not change significantly. Together these results suggest erythroid metabolic profile is distinct in the spleen as compared to the bone marrow at the steady state. In order to compare homeostatic versus stress erythropoiesis we analyzed bone marrow and spleen from Foxo3-/- and Th3/+ thalassemic mice, two models of ineffective erythropoiesis with different degrees of severity and splenomegaly. As anticipated Foxo3-/- and Th3/+ erythroid precursors displayed decreased rate of maturation as compared to wild type cells in both bone marrow (0.3 ± 0.02 and 0.12 ± 0.01 for Foxo3-/- and Th3/+ respectively, n ≥ 10) and spleen (2.1 ± 0.3 and 0.42 ± 0.1 for Foxo3-/- and Th3/+ respectively, n ≥ 10 mice per group) and cell cycle analysis showed an increased number of cells in the S phase in both Foxo3-/- and Th3/+ spleen erythroid cells as compared to wild type (63 ± 2% and 79 ± 2% respectively, n=3). Unexpectedly however, ROS levels in both Foxo3-/- and Th3/+ spleen erythroid cells were decreased as compared to their wild type counterparts. The observed inverse correlation between cell cycling and ROS levels was further supported by expression analysis of Gpx1 and Gpx4, the levels of which were increased in Foxo3-/- as compared to wild type spleen erythroid cells. Collectively our results highlight the different erythroid metabolic conditions in the bone marrow versus spleen under both homeostatic and disease states. Further investigations elucidating differences in metabolic conditions and properties of erythroid cells in bone marrow versus spleen should improve our understanding of generation of erythroid cells under stress and the production of erythroid cells in vitro. Disclosures:No relevant conflicts of interest to declare.

Plerixafor+G-CSF-Mobilized Blood From Thalassemic Mice Provides High Yields Of Long-Term Repopulating Cells With Enhanced Engrafting Capacity : Implications For Human Stem Cell Gene Therapy

A durable therapeutic effect of gene therapy in β-thalassemia largely depends on the infusion of high numbers of genetically-modified hematopoietic stem cells (HSCs) with an enhanced engrafting potential.These requirements are necessary to compensate for the lack of a selective advantage of the thalassemic HSCs over the endogenous unmodified stem cells, especially if a nonmyeloablative conditioning is used. The higher HSC yields acquired by the mobilized peripheral blood and their faster time to engraftment as compared to bone marrow,qualifies mobilized blood cells as the preferable HSC source for genetic modification.To determine the optimal mobilization strategy for thalassemia, thalassemic (Hbbth345.2+) mice were mobilized with G-CSF or Plerixafor or Plerixafor+G-CSF and the yields of hematopoietic stem (Lin-sca-1+ckit+ cells-LSK cells) and progenitor cells (CFU-GM) were assayed by flow cytometry (FCM) and methylcellulose cultures and calculated in absolute numbers. C-kit+ progenitors mobilized by all the above approaches, were compared in terms of migration capacity towards SDF-1a, cell cycle status and CXCR4 and CD26 expression. To investigate the engrafting and long-term repopulation capacity of cells mobilized by different modes, whole blood from mobilized thalassemic 45.2+mice was infused into lethally irradiated PepBoy (45.1+) mice at a 3:1 (donors:recipient) ratio or as equal number of LSK cells, along with a fixed number of competitor 45.1+ cells (0.5x106). Donor chimerism was tested monthly by the frequency of 45.2+ cells in the recipients' blood by FCM analysis. Six months after primary transplantation, 2x106bm cells from the competitively engrafted mice were injected into lethally-irradiated secondary recipients in a noncompetitive manner.Plerixafor+G-CSF yielded significantly higher numbers of LSKs and CFU-GM over G-CSF-alone or Plerixafor-alone (p≤0.004) while Plerixafor-alone didn't further impove mobilization over G-CSF-alone. After competitive transplantation, Plerixafor+G-CSF-mobilized cells achieved earlier hematological reconstitution and increased donor chimerism over G-CSF- or Plerixafor-alone-mobilized cells (p≤0.05). To test whether the higher levels ofengraftment simply reflect the presence of higher numbers of stem/progenitor cells in the Plerixafor+G-CSF-mobilized graft (p<0.05) rather than an inherent qualitative difference of these cells, equal numbers (1X104) of LSK cells from each mobilization group was transplanted in a competitive manner. Plerixafor+G-CSF-mobilized LSKs showed again significantly higher engraftment levels than the single-agent mobilized LSKs (p<0.05), thus implying a qualitative benefit of this type of graft. This benefit was also evident after secondary transplantation, as the Plerixafor+G-CSF-cell secondary recipients exhibited the same or even higher levels of engraftment compared to primary engraftment, in contrast to the reduced secondary donor chimerism of mice that underwent transplantation from Plerixafor- or G-CSF- bone marrow primary chimeras, respectively. Plerixafor-mobilized cells showed decreased competiveness over G-CSF- or Plerixafor+G-CSF cells both in primary and secondary transplantations, a finding which could be attributed to the mobilization by Plerixafor of more actively proliferating cells compared to G-CSF or the Plerixafor+G-CSF combination (S/G2/M:G0+G1 ratio, p≤0.03) as well as to the higher CD26 expression in c-kit+ Plerixafor-mobilized progenitors (p=0.04).In conclusion, Plerixafor+G-CSF-mobilized peripheral blood represents an optimal graft for gene therapy of β-thalassemia because of the higher HSC-yields, the faster hematological recovery and the superiority in long-term engraftment over single-agent-mobilized blood cells. The therapeutic implications however, are not restricted to thalassemia but are of obvious relevance to all other stem cell gene therapy applications. Disclosures:No relevant conflicts of interest to declare.

Cardiomyocyte ultrastructural damage in β-thalassaemic mice

β-thalassaemia is a hereditary anaemia resulting from the absence or reduction in β-globin chain production. Heart complications related to iron overload are the most serious cause of death in these patients. In this report cardiac pathology of β-thalassaemic mice was evaluated by light and electron microscopy. The study was carried out in thalassaemic mice carrying human β-thalassaemia mutation, IVSII-654 (654), transgenic mice carrying human β(E) -globin transgene insertion (E4), thalassaemic mice with human β(E) -globin transgene insertion (654/E4) and homozygous thalassaemic mice rescued by the human β(E) -globin transgene (R), which is generated by cross-breeding between the 654 and E4 mice. Histology showed iron deposition in cardiac myocytes of 654 and R mice, but the ultrastructural damage was observed only in the R mice when compared with the wild type, 654, E4 and 654/E4 mice. Histopathological changes in the cardiomyocytes of the R mice included mitochondrial swelling, loss of myofilaments and the presence of lipofuscin, related to the increased level of tissue iron content. The progressive ultrastructural pathology in R mice cardiomyocytes is consistent with the ultrastructural pathology previously studied in patients with thalassaemia. Thus, this R thalassaemic mouse model is suitable for in vivo pathophysiological study of thalassaemic heart.

In vivo activation of the human -globin gene: the therapeutic potential in -thalassemic mice

β-thalassemia and sickle cell disease are widespread fatal genetic diseases. None of the existing clinical treatments provides a solution for all patients. Two main strategies for treatment are currently being investigated: (i) gene transfer of a normal β-globin gene; (ii) reactivation of the endogenous γ-globin gene. To date, neither approach has led to a satisfactory, commonly accepted standard of care. The δ-globin gene produces the δ-globin of hemoglobin A2. Although expressed at a low level, hemoglobin A2 is fully functional and could be a valid substitute of hemoglobin A in β-thalassemia, as well as an anti-sickling agent in sickle cell disease. Previous in vitro results suggested the feasibility of transcriptional activation of the human δ-globin gene promoter by inserting a Kruppel-like factor 1 binding site. We evaluated the activation of the Kruppel-like factor 1 containing δ-globin gene in vivo in transgenic mice. To evaluate the therapeutic potential we crossed the transgenic mice carrying a single copy activated δ-globin gene with a mouse model of β-thalassemia intermedia. We show that the human δ-globin gene can be activated in vivo in a stage- and tissue-specific fashion simply by the insertion of a Kruppel-like factor 1 binding site into the promoter. In addition the activated δ-globin gene gives rise to a robust increase of the hemoglobin level in β-thalassemic mice, effectively improving the thalassemia phenotype. These results demonstrate, for the first time, the therapeutic potential of the δ-globin gene for treating severe hemoglobin disorders which could lead to novel approaches, not involving gene addition or reactivation, to the cure of β-hemoglobinopathies.

EPO signaling and hematopoietic expansion as causes of osteoporosis in a thalassemia mouse model

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

CNTO 530 Increases Expression of HbA and HbF in Murine Models of β-Thalassemia and Sickle Cell Anemia

CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies. Keywords: Erythropoiesis, Flow Cytometry, Fusion protein, Hematology, Ion Exchange Chromatography, Pharmacodynamics

1,25‐Dihydroxyvitamin D3‐induced intestinal calcium transport is impaired in β‐globin knockout thalassemic mice

Besides being a common haematological disorder caused by a reduction in β-globin production, β-thalassemia has been reported to impair body calcium homeostasis, leading to massive bone loss and increased fracture risk. Here, we demonstrated that heterozygous β-globin knockout thalassemic mice had a lower rate of duodenal calcium absorption compared with the wild-type littermates, whereas the epithelial electrical parameters, including transepithelial resistance, were not affected, suggesting no change in the epithelial integrity and permeability. Daily subcutaneous injection of 1 µg kg(-1) 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] for 3 days enhanced the duodenal calcium absorption in wild-type, but not in thalassemic mice. Although β-thalassemia increased the mRNA level of divalent metal transporter-1, an iron transporter in the duodenum, it had no effect on the transcripts of ferroportin-1 or the principal calcium transporters. In conclusion, β-thalassemia impaired the 1,25(OH)2 D3 -dependent intestinal calcium absorption at the post-transcriptional level, which, in turn, contributed to the dysregulation of body calcium metabolism and β-thalassemia-induced osteopenia.

Resveratrol Induces Erythroid Maturation by Activating FOXO3 and Improves in Vivo Erythropoiesis in Normal and Beta -Thalassemic Mice

AbstractAbstract 3191Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities. High concentrations of resveratrol (50 μM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 μM) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 μM). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/− mice (2 months of age) for 6 months. In resveratrol Hbb3th+/− treated mice increased Hb levels (8.3±0.6 vs 10.3±0.5 g/dL, n=12; P<0.05) and decreased reticulocyte count (33.9±0.8 vs 23.7± 8.2 %, n=12; P<0.05) were observed. Significant increased MCV (34.6±0.6 vs 41.6± 5.4 fL, n=12; P<0.05) and MCH (9.7± 0.6 vs 12.8 ± 2.1 pg, n=12; P<0.05) were also noted. Flow cytometric evidence of decreased ineffective erythropoiesis and reduced spleen/body weight ratio were also observed. These data indicate that resveratrol affects erythroid maturation both in vitro and in vivo, and that these effects have possible therapeutic relevance for the treatment of thalassemias. Disclosures:Cappellini:Novartis Pharmaceuticals: Honoraria, Research Funding.

Hematopoietic Progenitors and Erythropoietin Affect Osteoblast Function and Lead to Osteoporosis in a Thalassemia Mouse Model

Abstract 3261Osteoporosis affects up to 75% of thalassemia patients. The th3/+ mouse has hematological and bone abnormalities similar to humans with β thalassemia intermedia. In this mouse, we documented decreased bone formation as a cause of bone loss. We used the th3/+ mouse to determine mechanisms by which abnormal erythropoiesis affects osteoblast function and leads to bone loss in thalassemia. Methods:Mesenchymal stem cells (MSCs) from wild type (wt) and th3/+ mice were differentiated into osteoblasts in osteogenic media. MSC co-cultures with various hematopoietic progenitors from wt and th3/+ animals were differentiated to osteoblasts similarly. Osteogenesis was determined by Colony Forming Units for Osteoblasts (CFU-O) numbers and qPCR for osteogenesis-related genes (genes that encode for Runx2, bone sialoprotein, osterix, osteocalcin expressed through osteoblast matuaration), with four experiments performed in triplicates. Results:CFU-O numbers were decreased by 40% in MSC cultures from th3/+ mice compared to wt, while gene expression was 40 to 60% lower, indicating alterations in MSC fate that led to decreased osteogenesis. Treatment of MSC cultures with erythropoietin (EPO) antibody (EPO Ab) or soluble EPO receptor (s EPOR) reversed the decreased CFU-O numbers and expression of osteogenesis-related genes in th3/+ animals, indicating that EPO signaling guides MSC fate in thalassemia. Consistently, qPCR analysis of MSCs and osteoblasts showed increased EPO and EPOR expression in th3/+ versus wt controls.To determine the role of hematopoietic progenitors on MSC fate, we isolated various progenitors and co-cultured them with MSC from wt animals. Lin-Sca+ ckit+ (LSK) precursors from th3/+ mice yielded 50% fewer CFU-O and decreased expression of osteogenic genes by 70%. In contrast, thalassemic erythroid precursors (CD71+Ter119- cells) and mature erythroid cells (Ter119+) had little to no effect on osteogenesis respectively. LSK-MSC co-cultures from wt and th3/+ mice were then treated with EPO Ab to determine the role of EPO on LSK-MSC interactions. Indeed, a reversal of the suppressed osteogenesis (determined by CFU-O numbers and qPCR results) was seen in th3/+ with such treatment, indicating a role of EPO signaling in LSK-MSK interactions. In support of these findings, EPOR and EPO expression in thalassemia LSK was increased two fold as compared to wt. Since Bone Morphogenic Proteins (BMPs) potentiate both erythroid expansion and osteogenesis, we examined BMP-2,-4,-6 expression in LSK from wt and th3/+ mice by qPCR. Expression of all three cytokines was decreased by 60–70% in th3/+, and reversed when thalassemic LSK cells were treated with EPO-Ab, suggesting that EPO affects LSK-MSC interaction by regulating BMP expression in LSK.In summary, we observed decreased osteogenesis as result of altered MSC differentiation in the th3/+ thalassemia mouse. MSC fate is regulated by both hematopoietic progenitors and EPO. Specifically, LSK but not mature erythroid cells interact with MSCs in th3/+ to decrease osteogenesis. Our data support a dual role of EPO on osteogenesis in thalassemia. First, EPO produced by thalassemic MSCs alters osteogenesis via direct autocrine EPO signaling. Second, EPO also guides MSC-LSK interactions by regulating expression of BMPs -2,-4 and 6 in hematopoietic progenitors. Therefore, we conclude that EPO through an extra-hematopoietic action leads to bone loss in thalassemia. Disclosures:Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Modulation of Activin Signaling by RAP-011 (ActRIIA-IgG1) Improve Anemia, Increases Hemoglobin Levels and Corrects Ineffective Erythropoiesis in β-Thalassemia

AbstractAbstract 247 Background:β-thalassemia is associated with ineffective erythropoiesis, accelerated erythroid differentiation and apoptosis resulting in anemia and iron overload. The molecular mechanism involved is still incompletely understood. Members of the TGF-β superfamily participate in both proliferation and differentiation of erythroid progenitors. However, the role of these molecules in models of ineffective erythropoiesis has not been addressed so far. RAP-011 is a ligand trap consisting of the extracellular domain of ActRIIA linked to mouse IgG1 Fc domain. We aimed to study the role of ActRIIA signaling in the ineffective erythropoiesis of β-thalassemia and to evaluate the therapeutic impact of RAP-011. Methods:Hbbth1/th1 mice (a model of β-thalassemia intermedia) were subcutaneously treated with RAP-011 (10mg/kg body weight) twice a week for 30–60 days and biological and biochemical parameters were followed. Results:RAP-011 treatment significantly increased hemoglobin levels, red blood cell counts, MCV, MCH and hematocrit with a concomitant decrease in bilirubin levels and reticulocyte counts (since 10 days of treatment and sustained until day 60 of follow up). Flow cytometry analysis showed that RAP-011 significantly decreased late basophilic and polychromatic erythroblast cell numbers in both bone marrow and spleen indicating that RAP-011 corrects ineffective erythropoiesis. We next evaluated the expression of putative ActRIIA ligand(s) in β-thalassemia. Increased expression of Growth Differentiation Factor 11 (GDF11) was observed in cultured erythroblasts and in spleen sections of thalassemic mice. RAP-011 treatment decreased these elevated GDF11 levels in both bone marrow and spleen. We further investigated how BMP/Activin signaling was involved in ineffective erythropoiesis. Anti-GDF11 antibodies, follistatin (activin and GDF11 antagonist) and dorsomorphin (a small molecule inhibitor of SMAD1/5/8 phosphorylation) reduced differentiation, induced FAS-L expression and apoptosis in erythroblasts both in vivo and in vitro whereas noggin (a BMP-2/4 antagonist) had no effect on erythroblast differentiation. Altogether, these data suggest that Activin/BMP signaling controls erythroblast differentiation and targeting BMP type II /activin type II receptors can decrease ineffective erythropoiesis of β-thalassemia. Summary:Sotatercept (a humanized version of RAP-011) is currently in phase II clinical trials for treatment of anemia in patients with Myeloma Bone Disease and End Stage Renal Disease and data from our non-clinical findings support a newly initiated β-thalassemia clinical trial. Our results suggest that sotatercept would be a potential therapeutic tool to improve anemia, increase hemoglobin levels and correct ineffective erythropoiesis and its side effects in β-thalassemic patients. Disclosures:Daniel:Celgene Corporation: Employment. Chopra:Celgene Corp: Employment, Equity Ownership. Sung:Celgene: Employment.

Ferric iron uptake into cardiomyocytes of β-thalassemic mice is not through calcium channels

Iron-overload cardiomyopathy is a major cause of death in thalassemic patients. However, pathways of non-transferrin-bound iron (NTBI) uptake into cardiomyocytes under iron-overload conditions are still controversial. We previously demonstrated that Fe2+ uptake in thalassemic cardiomyocytes is mainly mediated by T-type calcium channels (TTCCs). However, direct evidence regarding Fe3+ uptake, the other form of NTBI, in thalassemic cardiomyocytes has never been investigated. Hearts from genetic-altered β-thalassemic mice and adult wild-type (WT) mice were used for cultured ventricular cardiomyocytes. Blockers for L-type calcium channel (LTCC), TTCC, transferrin receptor1 (TfR1), and divalent metal transporter1 (DMT1) were used, and quantification of cellular iron uptake was performed by the acetoxymethyl ester of calcein fluorescence assay. Cellular uptake of Fe3+ under iron-overload conditions in cultured ventricular myocytes of thalassemic mice was greater than that of WT cells (P < 0.01). The iron chelator, deferoxamine, could prevent Fe3+ uptake into cultured cardiomyocytes. However, blockers of TfR1, DMT1, LTCC, and TTCC could not prevent Fe3+ uptake into cardiomyocytes. Our findings indicated that, unlike Fe2+, Fe3+ uptake in cultured thalassemic cardiomyocytes is not mainly mediated by TfR1, DMT1, LTCC, and TTCC, suggesting that another alternative pathway could play a major role in Fe3+ uptake in thalassemic cardiomyocytes.

Mitochondrial calcium uniporter blocker prevents cardiac mitochondrial dysfunction induced by iron overload in thalassemic mice

Iron-overload induced cardiomyopathy is a major cause of morbidity and mortality in thalassemic patients. Previous studies suggest that cardiac mitochondrial dysfunction may be involved in the pathogenesis of cardiomyopathy in thalassemia. We tested the hypothesis that iron overload causes dysfunction of cardiac mitochondria isolated from thalassemic mice. Cardiac mitochondria were isolated from the heart tissue of genetically-altered, β-thalassemic mice (HT) and adult wild-type mice (WT). Ferrous iron (Fe(2+)) at various concentrations (0-5 μg/ml) was applied to induce iron toxicity. Pharmacological interventions, facilitated by mitochondrial permeability transition pore (mPTP) blocker, CsA, and mitochondrial Ca(2+) uniporter (MCU) blocker, Ru360, were used to study their respective effects on cardiac mitochondrial dysfunction. Cardiac mitochondrial ROS production, mitochondrial membrane potential changes, and mitochondrial swelling were determined. Iron overload caused increased ROS production, mitochondrial depolarization, and mitochondrial swelling in a dose-dependent manner in WT and HT cardiac mitochondria. CsA decreased only ROS production in WT and HT cardiac mitochondria, whereas Ru360 completely prevented the development of cardiac mitochondrial dysfunction by decreasing ROS, mitochondrial depolarization, and swelling in both WT and HT cardiac mitochondria. Ru360, an MCU blocker, provides protective effects by preventing ROS production and mitochondrial depolarization as well as attenuating mitochondrial swelling caused by Fe(2+) overload. These findings indicate that the MCU could be a major portal for Fe(2+) entry into cardiac mitochondria. Therefore, blocking MCU may be an effective therapy to prevent iron-overload induced cardiac mitochondrial dysfunction in patients with thalassemia.

T‐type calcium channel blockade improves survival and cardiovascular function in thalassemic mice

Iron-overload cardiomyopathy is a major cause of morbidity and mortality in patients with thalassemia. However, the precise mechanisms of iron entry and sequestration in the heart are still unclear. Our previous study showed that Fe(2+) uptake in thalassemic cardiomyocytes are mainly mediated by T-type calcium channels (TTCC). Nevertheless, the role of TTCC as well as other transporters such as divalent metal transporter1 (DMT1) and L-type calcium channels (LTCC) as possible portals for iron entry into the heart in in vivo thalassemic mice under an iron-overload condition has not been investigated. An iron-overload condition was induced in genetically altered β-thalassemic mice and adult wild-type mice by feeding them with an iron diet (0.2% ferrocene w/w) for 3 months. Then, blockers for LTCC (verapamil and nifedipine), TTCC (efonidipine), and DMT1 (ebselen) as well as iron chelator desferoxamine (DFO) were given for 1 month with continuous iron feeding. Treatment with LTCC, TTCC, DMT1 blockers, and DFO reduced cardiac iron deposit, cardiac malondialdehyde (MDA), plasma non-transferrin-bound iron, and improved heart rate variability and left ventricular (LV) function in thalassemic mice with iron overload. Only TTCC and DMT1 blockers and DFO reduced liver iron accumulation, liver MDA, plasma MDA, and decreased mortality rate in iron-overloaded thalassemic mice. DMT1, LTCC, and TTCC played important roles for iron entry in the thalassemic heart under an iron-overloaded condition. Unlike LTCC blocker, TTCC blocker provided all benefits including attenuating iron deposit in both the heart and liver, reduced oxidative stress, and decreased mortality in iron-overloaded mice.

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.

The Regulation of Hepcidin in β-Thalassemia

Abstract 901β-Thalassemia is a genetic disorder characterized by reduced or absent β-globin synthesis. Complications include anemia, ineffective erythropoiesis (IE), splenomegaly and iron overload. Homeostatic iron levels are maintained by the hepatic hormone hepcidin (HAMP), which degrades the iron exporter ferroportin. Iron-responsive transcriptional control of HAMP is normally mediated by the bone morphogenetic protein (BMP)/SMAD pathway. Despite the high organ iron load associated with β-thalassemia, low Hamp levels have been reported in patients with this disorder. HAMP levels are also low relative to organ iron load in mouse models of β-thalassemia. Although iron overload is a central component of the pathogenesis of this disease, the factors contributing to inadequate HAMP expression have not been fully described. We previously showed that moderate transgenic overexpression of HAMP increased hemoglobin levels, improved erythropoiesis, and decreased both splenomegaly and iron overload in a mouse model of β-thalassemia intermedia (th3/+). In order to define the pathways controlling HAMP transcription in β-thalassemia, initial studies examined HAMP expression in the context of BMP/SMAD pathway members and targets genes. qRT-PCR analyses confirm decreased HAMP expression in 2 month old th3/+ mice and indicate a significantly larger decrease in mice with β-thalassemia major (th3/th3) relative to wild-type (+/+) controls. A significant decrease in the common SMAD pathway member, SMAD4, is also evident in both strains. TMPRSS6, the transcript encoding the protease matriptase-2, is also decreased in th3/th3 mice. In contrast, at 5 months of age the transcriptional profile of these genes in th3/+ mice is indistinguishable from that of wild-type mice. By 12 months of age, there is a trend toward increased HAMP transcription corresponding to increases in the BMP/SMAD targets ID1 and ATOH8. Examination of the effect of a low-iron diet on the transcriptional levels of these genes over time in th3/+ mice indicates no significant differences from +/+ mice after the data were normalized to liver iron concentrations. In contrast, comparison of the transcriptional profile of these genes with +/+ mice fed a high-iron diet indicates that HAMP expression is blunted in thalassemic mice. Preliminary Western blot analyses indicate that phosphorylation of SMAD 1/5/8 in th3/+ and th3/th3 liver samples, although comparable to that in +/+ samples, is lower than the levels observed in mice fed a high-iron diet. These data indicate that iron sensing through the BMP/SMAD pathway is intact, but somewhat blunted in thalassemic mice. Thus an alternative mechanism, possibly mediated by the IE that is characteristic of this disease, is likely responsible for the suppression of HAMP. In order to gain support for this hypothesis, qRT-PCR analyses were performed on mice overexpressing transgenic erythropoietin (TgEpo) to examine the effect of erythropoiesis on HAMP expression. The data indicate suppression of HAMP transcription despite clear activation of the BMP/SMAD pathway, as evidenced by increases in BMP6 and ID1. Of note, SMAD7 transcription was not detected in these animals. Compared to +/+ mice, phlebotomized mice also show decreased transcription of HAMP and SMAD4 without corresponding decreases in the BMP/SMAD pathway targets ID1 and ATOH8. TMPRSS6 transcription was also decreased, but FURIN expression was increased. FURIN, a known hypoxia target gene, was also transcribed at a significantly higher rate in th3/th3 mice than in either th3/+ or +/+ mice. qRT-PCR data indicate that this is mediated by HIF-2α, as evidenced by decreased transcription of the HIF-1α target, pyruvate dehydrogenase kinase 1, and increased transcription of the HIF-2α target, erythropoietin (EPO), in th3/th3 liver samples. HIF2-α expression in th3/th3 liver lysates was confirmed by Western blot analysis. Collectively, these data indicate that HAMP expression in thalassemia is suppressed through a mechanism distinct from a BMP/SMAD feedback loop and provide support for the existence of an ‘erythroid factor’. Results also suggest the increased suppression of HAMP observed in th3/th3 mice is attributable to the activation of HIF2-α, which in turn would be expected to aggravate the production of an erythroid factor through the upregulation of EPO. Disclosures:No relevant conflicts of interest to declare.