Beta-thalassemic Mice Research Articles

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human beta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice.

Adeno-associated virus 2 (AAV), a nonpathogenic human parvovirus, has gained attention as a potentially useful vector for human gene therapy. Here, we report successful AAV-mediated stable transduction and high-efficiency, long-term, erythroid lineage-restricted expression of a human beta-globin gene in primary murine hematopoietic stem cells in vivo. Bone marrow-derived primitive Sca-1(+), lin(-) hematopoietic stem cells from homozygous beta-thalassemic mice were transduced ex vivo with a recombinant AAV vector containing a normal human beta-globin gene followed by transplantation into low-dose-irradiated B6.c-kitW(41/41) anemic recipient mice. Six months posttransplantation, tail-vein blood samples were analyzed by PCR amplification to document the presence of the transduced human beta-globin gene sequences in the peripheral blood cells. Semiquantitative PCR analyses revealed that the transduced human beta-globin gene sequences were present at approximately 1 copy per cell. The efficiency of the human beta-globin gene expression was determined to be up to 35% compared with the murine endogenous beta-globin gene by semiquantitative RT-PCR analyses. Peripheral blood samples from several positive recipient mice obtained 10 months posttransplantation were fractionated to obtain enriched populations of granulocytes, lymphocytes, and erythroid cells. PCR analyses revealed the presence of the human beta-globin gene sequences in granulocytes and lymphocytes, indicating multilineage reconstitution. However, only the erythroid population was positive following RT-PCR analyses, suggesting lineage-restricted expression of the transduced human beta-globin gene. Southern blot analyses of total genomic DNA samples isolated from bone marrow cells from transplanted mice also documented proviral integration. These results provide further support for the potential use of recombinant AAV vectors in gene therapy of beta-thalassemia and sickle-cell disease.

Postnatal cytokines and boosts improve chimerism and hematological parameters in beta-thalassemic mice transplanted in utero.

We have developed a murine model of in utero transplantation in nonanemic, beta-thalassemic mice to study chimerism, tolerance, and changes in hematological parameters in response to cytokines and postnatal boosts with donor cells. We have documented improved survival of homozygous fetuses by 40% as compared with controls. Low-level, mixed chimerism was improved by postnatal cytokine therapy and boosts and was associated with improvement in hemoglobin levels, reticulocyte counts, and iron stores. Cytotoxicity assays demonstrated higher responses to donor cells in control mice as compared with in utero transplanted animals (at 50:1 effector to target ratios, transplanted mice showed 8.66% target lysis and control mice showed 51.85% target lysis, P=0.0003), indicating tolerance. The combination of prenatal tolerance to allogeneic cells with postnatal boosts in primed hosts may become an effective, nontoxic strategy for the improvement of hemolytic anemia in beta-thalassemic patients.

Improvement of mouse β-thalassemia by electrotransfer of erythropoietin cDNA

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. Intramuscular injection of small amounts of a plasmid encoding mouse EPO, immediately followed by controlled electric pulses, was used. 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. 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.

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.

Removal of erythrocyte membrane iron in vivo ameliorates the pathobiology of murine thalassemia.

Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.

Defective assembly of membrane proteins in erythroid precursors of beta- thalassemic mice

beta-Thalassemic mice provide a useful model for studying the pathophysiology of human beta-thalassemia in that one can perform experiments that are difficult to perform in humans. The ease of access to beta-thalassemic mouse marrow provided the opportunity to explore the cause of the ineffective erythropoiesis that characterizes severe beta-thalassemia in mouse and man. We hypothesized that the accumulation of excess alpha-globin might interfere with the normal assembly of red blood cell (RBC) membrane proteins, thus contributing to the severe intramedullary lysis. Femoral marrow was obtained from normal and beta-thalassemic mice, and RBC precursors were purified (&gt; 90%) by panning and harvesting CD45- cells. The assembly of RBC membrane proteins was assessed by observing immunofluorescence patterns obtained on fixed permeabilized precursors using rabbit polyclonal antibodies directed against human spectrin, and band 4.1, and murine band 3. The distribution of the proteins was shown with a fluorescein- tagged goat antirabbit antibody. In contrast to normal mice, about 30% of intermediate and late stage erythroblasts in beta-thalassemic mice appear abnormal. Neither spectrin nor band 4.1 formed crisp rim fluorescence in these erythroid precursors of thalassemic mice, whereas assembly of band 3 appeared normal. Therefore, the assembly of membrane skeletal proteins is abnormal in murine beta-thalassemic erythroid precursors perhaps because of the deposition of unmatched alpha-globin chains.

Defective Assembly of Membrane Proteins in Erythroid Precursors of ß-Thalassemic Mice

beta-Thalassemic mice provide a useful model for studying the pathophysiology of human beta-thalassemia in that one can perform experiments that are difficult to perform in humans. The ease of access to beta-thalassemic mouse marrow provided the opportunity to explore the cause of the ineffective erythropoiesis that characterizes severe beta-thalassemia in mouse and man. We hypothesized that the accumulation of excess alpha-globin might interfere with the normal assembly of red blood cell (RBC) membrane proteins, thus contributing to the severe intramedullary lysis. Femoral marrow was obtained from normal and beta-thalassemic mice, and RBC precursors were purified (> 90%) by panning and harvesting CD45- cells. The assembly of RBC membrane proteins was assessed by observing immunofluorescence patterns obtained on fixed permeabilized precursors using rabbit polyclonal antibodies directed against human spectrin, and band 4.1, and murine band 3. The distribution of the proteins was shown with a fluorescein-tagged goat antirabbit antibody. In contrast to normal mice, about 30% of intermediate and late stage erythroblasts in beta-thalassemic mice appear abnormal. Neither spectrin nor band 4.1 formed crisp rim fluorescence in these erythroid precursors of thalassemic mice, whereas assembly of band 3 appeared normal. Therefore, the assembly of membrane skeletal proteins is abnormal in murine beta-thalassemic erythroid precursors perhaps because of the deposition of unmatched alpha-globin chains.

Locus-Control-Region-Coupled Beta (S)(Antilles)- and Alpha(2)-Hemoglobin Genes Select for High Alpha(2)-Hemoglobin Expression in Adult Transgenic Mice.

Two transgenic lines of mice were produced which contained the beta(S)(Antilles)- and alpha(2)-hemoglobin genes tandemly coupled to the 'micro' locus control region (&mgr;LCR). The &mgr;LCRbeta(S)(Antilles)alpha(2)-hemoglobin transgenic mice expressed high levels of alpha(2)-hemoglobin while beta(S)(Antilles)-hemoglobin expression was virtually undetectable. Abundant alpha(2)-hemoglobin protein was observed in the blood of transgenic mice, while beta(S)(Antilles)-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of beta-thalassemic mice. The &mgr;LCRbeta(S)(Antilles)alpha(2) transgenic mice demonstrate that if the &mgr;LCR is coupled to the beta(S)(Antilles)- and alpha(2)-hemoglobin genes in tandem, only the distal alpha(2)-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching. Copyright 1994 S. Karger AG, Basel

Fetal hematopoietic stem cell transplantation into β-thalassemic mice

The transplantation of fetal-derived hematopoietic stem cells (HSCs) may potentially be used to treat hemoglobinopathies, immunodeficiencies, and storage diseases. The levels of donor cell engraftment needed to reconstitute the recipient's hematopoietic system are disease-dependent and remain unknown for most deficiencies. We have explored the application of fetal hematopoietic stem cell transplants for the amelioration of hemolytic disease in a murine model of beta-thalassemia. Nonirradiated neonatal homozygous beta-thalassemic mice were transplanted intraperitoneally (IP) with 10(6) fetal liver cells from syngeneic nonthalassemic murine fetal donors (14 to 16 days gestation). Donor hemoglobin was demonstrated in the peripheral blood of 9 of 14 transplant recipients at levels ranging from 8.8% to 27.1% at 30 days. The levels of engraftment in 6 of these 9 transplant chimeras remained stable or increased up to 150 days after transplantation, with levels ranging from 13.6% to 54.6% at 280 days. Three chimeras have demonstrated gradually decreasing engraftment after 200 days. The degree of engraftment correlated with clinically relevant improvement: decreased reticulocyte counts (8.4% to 15.7% in chimeras [n = 9] v 17.1% to 19.1% in controls [n = 8], P = .01), increased mean RBC deformability, and the significant reduction in extramedullary hematopoiesis and iron deposits seen on histological examination of chimeric liver and spleen. These data demonstrate that fetal HSC transplants results in significant long-term chimerism with favorable alterations in red cell characteristics, and decreased hemolytic anemia in beta-thalassemia.

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 β-Thalassemia by Recombinant Human Erythropoietin

Abstract 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.

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.

Human Sickle Hemoglobin in Transgenic Mice

DNA molecules that contain the human alpha- and beta s-globin genes inserted downstream of erythroid-specific, deoxyribonuclease I super-hypersensitive sites were coinjected into fertilized mouse eggs and a transgenic mouse line was established that synthesizes human sickle hemoglobin (Hb S). These animals were bred to beta-thalassemic mice to reduce endogenous mouse globin levels. When erythrocytes from these mice were deoxygenated, greater than 90 percent of the cells displayed the same characteristic sickled shapes as erythrocytes from humans with sickle cell disease. Compared to controls the mice have decreased hematocrits, elevated reticulocyte counts, lower hemoglobin concentrations, and splenomegaly, which are all indications of the anemia associated with human sickle cell disease.

Effect of human beta (s)-globin chains on cellular properties of red cells from beta-thalassemic mice.

The transgenic mouse system provides an in vivo setting in which to examine the effects on mouse red cells of hemoglobin genes that have been genetically introduced into the animals' genome. In this report, we have analyzed the cellular properties of red cells from homozygous beta-thalassemic mice (Hbbth-1/Hbbth-1), homozygous beta-thalassemic transgenic mice containing a human beta-sickle (beta(s)) gene (Hbb(th-1)/Hbb(th-1) + beta(s)), and normal animals. The presence of human beta(s)-globin chains in red cells from the Hbbth-1/Hbb(th-1) + beta(s) transgenic animals was noted to have a significant effect on cellular deformability and density distribution, as well as on the degree of anemia in these animals. We conclude from these studies that red cell deformability and density distribution is a sensitive means for assessing at the cellular level the effects of globin genes genetically introduced into whole organisms. In addition, these studies suggest that small decreases in the amount of excess alpha-globin chains can significantly ameliorate the severity of anemia in the beta-thalassemic mouse.

Iron homeostasis in beta-thalassemic mice

To explore the pathogenesis of nontransfusional iron overload in iron-loading anemia, we examined features of external iron exchange, internal iron kinetics, and tissue iron burden in adult mice with inherited gene-deletion beta-thalassemia. Mice homozygous for beta-thalassemia display moderate anemia, reticulocytosis, and shortened red cell survival, whereas heterozygous carriers appear hematologically normal. Quantitative iron determination revealed that iron content and concentration in liver, spleen, and kidney, but not heart, were far higher (P less than .01) in 15-to 35-week old homozygous thalassemic mice than in age-matched normal and heterozygous controls; of these tissues, iron content increased with age only in kidneys (P = .01) of homozygous affected mice. Although plasma iron levels were only minimally elevated in homozygotes, plasma iron turnover was threefold greater (P less than .001) than that seen in heterozygote controls. Nevertheless hyperabsorption of enteric radioiron, discernible among homozygous thalassemic mice as late as 6 to 8 weeks after birth, was not observed in older mice, additionally, thalassemic and control mice at 18 to 34 weeks showed comparable iron excretion after intravenous radioiron. We conclude that adult mice with beta-thalassemia regain balanced external iron exchange, despite substantial tissue iron excess and accelerated internal iron transit.

Hematology of a murine beta-thalassemia: a longitudinal study.

Mice homozygous for a spontaneous mutation, in which the beta-major globin gene is deleted, have clinical symptoms of beta-thalassemia. These mice have a hypocellular, hypochromic, microcytic anemia that becomes more severe with increasing age. The defective red cell morphology, decreased osmotic fragility of erythrocytes and shortened red cell life span found in beta-thalassemic mice are similar to those observed in human beta-thalassemia. Synthesis of beta-globin is depressed but not as much as might be expected because the expression of the beta-minor globin gene is enhanced to encode two to three times more globin than in normal mice. Splenomegaly, an enlarged pool of stem cells for erythropoiesis, and iron overloading occur in older mice. The fact that these mice remain moderately healthy makes them a very suitable animal model in which to develop and test alternative techniques of gene therapy that could be successfully applied to the treatment of human thalassemia. Homozygous beta-thalassemic mice have large deposits of iron in their tissues, which might make these mice also useful for in vivo tests of the effectiveness and possible long-term side effects of newly developed iron chelators.