Humanized mouse models of Cooley's anemia: correct fetal-to-adult hemoglobin switching, disease onset, and disease pathology

Abstract

beta thalassemia major or Cooley's Anemia (CA) has been difficult to model in mice due to their lack of a fetal hemoglobin gene equivalent. This summary describes novel preclinical humanized mouse models of CA that survive on human fetal hemoglobin at birth and are blood-transfusion dependent for life upon completion of their human fetal-to-adult hemoglobin switch after birth. These CA models are the first to recapitulate the temporal onset of the disease in human patients. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; examining the onset of disease pathology; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Figure 1.

Gene-targeting strategy in ES cells used to replace the adult mouse β globin genes with human globin genes in the mouse β globin locus. Top line represents the wild-type mouse β globin locus. A single-targeting vector is drawn below the locus illustrating homologous recombination between 5′ and 3′ mouse homology regions. After removal of the hygromycin marker gene (hyg) by recombinase (CRE), the resulting human globin gene KI alleles are shown. Locus control region (LCR) represented by blue ovals, functional mouse embryonic genes (εy and βh1), and adult genes (β^maj and β^min) represented by blue boxes, mouse pseudogenes drawn as gray boxes, human genes are red boxes, hygromycin marker gene is brown box, loxP sites are green triangles.

Figure 2.

HPFH mutations modulate the human γ globin levels in CA mice. (A) Total β-like globin chains in peripheral blood hemolysates of humanized newborn to 5-week-old mice were separated by HPLC and quantified. High fetal γ globin levels at birth are replaced by adult β globin as the hemoglobin switch is completed after birth. Genotype of each humanized KI mouse shown at the top of each figure. Fetal γ globin chains shown in red, adult β globin in blue, and adult δ globin in orange. Values represent mean ± SEM. n ≥ 9 at each time point. (B) Survival curves of humanized CA mice. The percentage of mice alive for each genotype is plotted with age. The few humanized γβ° KI CA mice that survive to birth die within the first 24 h. Incorporation of the HPFH mutation in the human γ globin gene promoter in CA mice results in 100% survival to birth and an average lifespan of 14 and 15 daysforthe γ^HPFHδβ° and γ^HPFHβ° CA mice, respectively. (C) F-cell distribution in newborn and adult heterozygous γ^HPFHβ°/γβ^A KI mice. Peripheral mouse blood was fixed and stained with anti-human fetal hemoglobin monoclonal antibody and analyzed by flow cytometry. HbF is pancellularly distributed in newborn humanized mice, whereas adult mice have a heterocellular distribution. Green line is wild-type mouse control.

Figure 3.

Hematological RBC indices and histopathology of humanized control, heterozygous, and homozygous CA mice. (A) RBC indices of peripheral blood from humanized mice showed a slight but significant anemia in heterozygous CA mice. Homozygous CA mice are severely anemic with marked reductions in RBC, hemoglobin, and hematocrit levels and an extreme reticulocytosis compared to control mice. Humanized control and heterozygous mice were analyzed eight weeks after birth. Moribund homozygous CA mice were analyzed prior to their euthanasia around two weeks of age. Values represent mean ± SEM. n ≥ 9 for each mouse line. Statistical significances were determined compared to the γβ^A/γβ^A control mice. P values were calculated by two-tailed unpaired Student’s t-test. (B) Peripheral blood smear, spleen, and liver sections of humanized control (γβ^A/γβ^A), heterozygous (γ^HPFHδβ°/γβ^A), and homozygous (γ^HPFHδβ°/γ^HPFHδβ°) CA mice. Control mice have normocytic RBCs and typical splenic and hepatic structure. Heterozygous CA mice are slightly thalassemic with frequent RBC targeting, but little erythroid hyperplasia in the spleen or extramedullary hematopoiesis in the liver. Homozygous CA mice have numerous nucleated RBCs and erythroblasts in the peripheral blood, marked erythroid hyperplasia in the spleen and extramedullary hematopoiesis, and extensive iron overload in the liver.

Figure 4.

Humanized CA mice can be rescued from lethal anemia by blood transfusion. (A) Blood transfusion scheme for CA mice. Each week fresh peripheral RBCs were collected from GFP transgenic mice, pelleted through Ficoll, washed, resuspended to 70% hematocrit in saline, and transfused into CA mice. (B) Blood chimerism (blue line) and hemoglobin levels (red line) measured over time in transfused CA mice. The donor RBC chimerism was determined by measuring the percentage of GFP⁺ RBCs in the peripheral blood by flow cytometry. Hemoglobin concentrations maintained at 14 g/dL efficiently suppressed endogenous erythropoiesis indicated by the virtual absence of GFP negative RBCs in the peripheral blood. Transfused CA mice live into adulthood free of anemia but develop severe iron overload.