Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo.

High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34(+) human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in beta-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in beta-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin. (Blood. 2001;97:3776-3782)

Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA.

OBJECTIVE: A new intramuscular DNA electrotransfer method for erythropoietin (EPO) expression was evaluated in the natural mouse model of human beta-thalassemia (Hbb-thal1) in terms of its ability to reverse the anemia and improve the thalassemic features of erythrocytes. MATERIALS AND METHODS: Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used. RESULTS: This procedure induced very high hematocrit levels in beta-thalassemic mice compared to nonelectrotransferred mice. The hematocrit increase was dose dependent, still increased 4 months after injection of plasmid DNA, and associated with a high transgenic EPO blood level in all mice (up to 2500 mU/mL of plasma). EPO gene electrotransfer not only led to a long-lasting and dose-dependent increase in the hematocrit but also to a 100% increase in the lifespan of erythrocytes of thalassemic mice. This was related to a nearly complete reestablishment of alpha/beta globin chain balance, as demonstrated by a marked decrease in unpaired alpha globin chain. Eight months after the first electrotransfer of pCMV-mEPO plasmid, reinjection of the same construct raised the hematocrit to a level close to that observed following the first electrotransfer. CONCLUSION: This is the first description of the use of plasmid DNA to achieve long-term improvement in a mouse model of a human genetic disorder.

Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors.

Erythropoiesis is positively regulated by stem cell factor, interleukin 3, and erythropoietin, which synergize to allow the production of hemoglobinized red blood cells from erythroid progenitors. In contrast, interferon gamma, tumor necrosis factor alpha, and transforming growth factor B(1), (TGF-beta(1)) are powerful inhibitors of erythropoiesis. Interferon gamma and alpha act principally by inducing apoptosis. The aim of this study was to elucidate the mechanisms by which TGF-beta(1) inhibits erythropoiesis. We used an in vitro serum-free system of human red blood cell production. From a virtually pure population of CD36(+) erythroid progenitors, stem cell factor, interleukin 3, and erythropoietin allowed massive proliferation (x300) and promoted terminal red blood cell differentiation. We show here that TGF-beta(1) (2 ng/mL) inhibited the growth of CD36(+) cells by 15-fold. TGF-beta(1) markedly accelerated and increased erythroid differentiation as assessed by hemoglobin and glycophorin expression. Furthermore, May-Grünwald-Giemsa staining and ultrastructural analysis revealed that TGF-beta(1) induced full differentiation toward normal enucleated red cells even in the absence of macrophages. This acceleration of erythroid differentiation did not modify the pattern of hemoglobin chains expression from adult or fetal erythroid progenitors. Analysis of apoptosis, cell cycle and Ki-67 expression showed that TGF-beta(1) inhibited cell proliferation by decreasing the cycle of immature erythroid cells and accelerating maturation toward orthochromatic normoblasts that are not in cycle. We showed that TGF-beta(1) is a paradoxical inhibitor of erythropoiesis that acts by blocking proliferation and accelerating differentiation of erythroid progenitors.

Formation of dense erythrocytes in SAD mice exposed to chronic hypoxia: evaluation of different therapeutic regimens and of a combination of oral clotrimazole and magnesium therapies.

We have examined the effect of hydroxyurea (HU), clotrimazole (CLT), magnesium oxide (Mg), and combined CLT+Mg therapies on the erythrocyte characteristics and their response to chronic hypoxia in a transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days with 1 of the following regimens (administered by gavage): control (n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5), Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU induced a significant increase in mean corpuscular volume and cell K content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and reduced density in SAD mice. After 21 days of treatment, the animals were exposed to hypoxia (48 hours at 8% O(2)) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell dehydration. These hypoxia-induced changes were blunted in either HU- or Mg-treated SAD mice and were completely abolished by either CLT or CLT+Mg treatment, suggesting a major role for the Gardos channel in hypoxia-induced dehydration in vivo.

Improvement of mouse beta-thalassemia upon erythropoietin delivery by encapsulated myoblasts.

The goal of the present study was to analyze if sustained delivery of elevated doses of recombinant erythropoietin (Epo), by genetically modified and immunoprotected allogenic cells, was able to correct the chronic anemia, characteristic of a spontaneous mouse model of beta-thalassemia (Hbb thal 1). Mouse C2C12 myoblast cells were transfected with a plasmid containing the mouse Epo cDNA and a mutated dihydrofolate reductase (DHFR) gene for gene amplification upon administration of increasing doses of methotrexate. In order to immunoprotect the transplanted cells, the stably modified cells were loaded into polyethersulfone microporus hollow fibers which were implanted subcutaneously into Hbb thal 1 mice. An increase in hematocrit starting 2 weeks after implantation was associated with elevated blood levels of Epo and an improved red blood cell phenotype. The latter indicated an improvement of cell morphology and membrane defects, in particular a reduced amount of free alpha hemoglobin chain, the hallmark of globin chain imbalance in beta-thalassemia. A reduction of reticulocyte count contrasting with the increase in hematocrit was also observed suggesting an improved erythrocyte survival. We conclude that the phenotype can be durably improved in some beta-thalassemic mice upon in vivo delivery of recombinant Epo by polymer encapsulated cells. Sustained elevated delivery of recombinant Epo holds promise for the treatment of beta-thalassemia-associated chronic anemia.

[Erythropoietin gene: regulation and therapeutic concern]. / Gène de l'érythropoïétine: régulation et intérêt thérapeutique.

The erythropoietin (Epo) is a model of proteins expressed upon hypoxia, regulated at the transcriptional and post transcriptional levels. The Hypoxia Induced Factor (HIF-I) activates the transcription of genes which exhibit the Hypoxia Regulatory Element (HRE). In addition the mRNA is stabilized upon hypoxia, increasing the hormone synthesis. The Epo gene is a convenient reporter gene to develop gene delivery systems and the in vivo regulation of the transgene. The beta thalassemia and acquired chronic anemias may benefit from the Epo gene transfer and expression, if it becomes safe, tunable and inexpensive.

Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.

We have examined whether the secretion of erythropoietin (Epo) from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to Epo. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine Epo cDNA is expressed under the control of the murine phosphoglycerate kinase promoter. "Neo-organs" containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit (> 80%) and elevated serum Epo concentration (ranging from 60 to 408 milliunits/ml) were observed in recipient animals over a 10-month observation period. Hematocrit values measured in recipient mice varied according to the number of implanted Epo-secreting fibroblasts (ranging from 2.5 to 20 x 10(6)). The implantation of neo-organs containing Epo-secreting fibroblasts appeared, therefore, as a convenient method to achieve permanent in vivo delivery of the hormone. We estimated that the biological efficacy of the approach may be relevant for the treatment of human hemoglobinopathies.

Retrovirus-mediated transfer of the erythropoietin gene in hematopoietic cells improves the erythrocyte phenotype in murine beta-thalassemia.

Repeated injections of large doses of erythropoietin (Epo) have been shown to be of benefit in the treatment of murine and human beta-thalassemia. To determine whether Epo gene therapy could replace this treatment for long-term periods, lethally irradiated beta-thalassemic (Hbbd3th haplotype) and normal DBA/2J (Hbbd haplotype) mice were grafted with syngeneic bone marrow cells infected with a retroviral vector carrying the Epo cDNA. In normal mice, dysregulated Epo production induced elevated serum Epo levels (176 +/- 68 mU/mL), high hematocrit levels (73% +/- 8%), and elevated beta-minor globin chain synthesis. In contrast, in thalassemic mice, moderate increases in the hematocrit levels (from 33% +/- 1% to 43% +/- 9%), associated with limited increases in the initially elevated Epo levels (from 83 +/- 22 to 190 +/- 230 mU/mL), were recorded 2 months after transplantation. In mice in which the hematocrit increased most, from 33% +/- 1% before transplantation to 49% +/- 10%, the retroviral Epo gene expression induced a striking improvement of the beta-thalassemic syndrome. These mice exhibited normal or near-normal beta/alpha-globin chain synthesis ratios, induced by the activation of the beta-minor chain. This led to the elimination of the high amounts of unpaired alpha chains in erythrocytes and finally reduced the reticulocyte count despite the permanent Epo stimulation. These results show that efficient Epo gene expression corrects the erythrocyte phenotype of the mouse beta-thalassemic syndrome. However, the incidence of lethal polycythemia or of transient improvements indicates that the present strategy is only the first step toward such indirect gene therapy.

Improvement of mouse beta thalassaemia by hydroxyurea.

The present report provides evidence that hydroxyurea (HU) improves the beta thalassaemic phenotype in mice receiving 200 mg/kg/d for 30 d. The haematocrit rose from 29 +/- 3% at day 0 to 37 +/- 4% at day 30 (P < 0.05), despite myelosuppression and decreased reticulocyte counts. The beta minor/alpha ratio of globin chain synthesis increased from 0.78 at day 0 to 0.97 at day 30 (P < 0.001). Membrane defects improved: the proportion of bound alpha chains decreased, the proportion of spectrin and ankyrin increased and red cell deformability also increased.

Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes.

While red cells from individuals with beta thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess alpha-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired alpha-hemoglobin chains, purified alpha-hemoglobin chains were entrapped within normal erythrocytes. These "model" beta-thalassemic cells generated significantly (P < 0.001) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of alpha-hemoglobin chain autoxidation and appearance of membrane-bound globin. In addition, alpha-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001) enhanced in the alpha-hemoglobin chain-loaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001) in the alpha-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped alpha-hemoglobin chains. In summary, entrapment of purified alpha-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess alpha-hemoglobin chains can be examined.

Alpha- and beta-haemoglobin chain induced changes in normal erythrocyte deformability: comparison to beta thalassaemia intermedia and Hb H disease.

The alpha- and beta-thalassaemias are characterized by decreased erythrocyte deformability. To determine what effects excess alpha- and beta-haemoglobin (globin) chains have on cellular and membrane deformability, purified haem-containing alpha- and beta-chains were entrapped within normal erythrocytes. Entrapment of purified alpha-chains in normal erythrocytes resulted in a significant decrease in cellular and membrane deformability similar to that observed in beta-thalassaemia intermedia. The decreased deformability was correlated with alpha-chain membrane deposition, an alteration in membrane proteins and a decrease in membrane reactive thiol groups. These changes in membrane and cellular deformability were time dependent and closely correlated with membrane alpha-chain deposition. The membrane changes and the loss of membrane deformability appeared to account for the loss of cellular deformability in the alpha-chain loaded cells. While both beta-chain loaded and Hb H erythrocytes demonstrated a significant loss of cellular deformability, this loss was less pronounced than in the alpha-chain loaded and beta-thalassaemic cells and may arise from either the increased intracellular viscosity of the beta-chain loaded cells or to the smaller amount of membrane bound globin. In summary, these studies demonstrate that alteration of cellular and membrane deformability occurs very rapidly and as a direct consequence of the autoxidation and membrane binding of the unpaired globin chains.

Erythropoietic protoporphyria in the house mouse. A recessive inherited ferrochelatase deficiency with anemia, photosensitivity, and liver disease.

A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features infrequent in the human, this mutation may represent a model for the human disease, especially in its severe form.

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.

Improvement of mouse beta-thalassemia by recombinant human erythropoietin.

Homozygous beta thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 +/- 0.6 g/dL to 10.5 +/- 0.4 g/dL (P = .002) and hematocrit increased from 29.2% +/- 0.9% to 34.1% +/- 1.9% (P = .0014). The beta minor/alpha globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 +/- 0.07) and day 4 (0.81 +/- 0.04) (P = .01) and reached a minimum ratio (0.67 +/- 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in beta thalassemic mice treated by rhEpo: membrane-associated alpha globin was significantly decreased (P less than .01), thiol group reactivity of ankyrin was significantly improved (P less than .05), spectrin alterations were reduced, and deformability of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human beta thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease.

Mouse beta thalassemia, a model for the membrane defects of erythrocytes in the human disease.

The present study describes the pathophysiology, at the cellular level, of the mouse beta thalassemia and shows the pertinence of this model for the human disease. The homozygous state of mouse beta thalassemia is characterized by a clinical syndrome similar to the human beta thalassemia intermedia, but it cannot be explained by the small deficiency in beta chain synthesis. The small pool of unpaired and soluble alpha chains present in mouse reticulocytes contrasts with the large amount of insoluble alpha chains in erythrocytes which is induced by the high instability of mouse alpha chains and the absence of significant proteolysis. The amount of insoluble alpha chains associated with red cell ghosts is similar in human and mouse disease of similar severity. The study of membrane protein defects showed a decreased amount of spectrin (alpha and beta chains) and dramatic changes in the distribution of the most reactive thiol groups of membrane proteins. These results were similar to that previously described in the human disease (Rouyer-Fessard, P., Garel, M. C., Domenget, C., Guetarni, D., Bachir, D., Colonna, P., and Beuzard, Y. (1989) J. Biol. Chem. 264, 19092-19098). Abnormal density distribution curves of erythrocytes and oxidant-induced lysis of red blood cells used as functional tests were similar in the human and mouse beta thalessemia. We conclude from the present study that 1) mouse beta thalassemia is an excellent model for the membrane defects occurring in the human disease; 2) disease expression is not the reflection of the globin chain unbalance only nor of the soluble pool of alpha hemoglobin chain but mainly is a reflection of insoluble alpha chains; and 3) the rate of proteolysis and instability of alpha chains are important factors which must be taken into consideration in the pathophysiology and the clinical heterogeneity of the disease.

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia.

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Towards a mouse model for sickle cell disease: HB SAD.

Very recently a high expression of human hemoglobin S, which causes sickle cell disease, has been obtained in transgenic mice. We have constructed a modified beta S gene, beta SAD which carries two additional mutations in order to induce polymerization of transgenic hemoglobin when diluted by endogenous mouse Hb. The transgenic SAD mice are not anemic but exhibit a low percentage of irreversible sickle cells. Sickling is induced by deoxygenation of erythrocytes in vitro. In addition, the anemia of neonates and the low incidence of SAD animals in the progeny suggest a deleterious effect of SAD Hb during development. Finally, hypoxia induces a high mortality in SAD adults suggesting the induction of vaso-occlusive events.