Yongliang Huo

Humanized Mouse Model of Cooley's Anemia

A novel humanized mouse model of Cooleys Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human γ to β0 globin gene cassette (γβ0) was inserted directly into the murine β globin locus replacing both adult mouse β globin genes. The inserted human β0 globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human β globin polypeptide chains are produced. Heterozygous γβ0 mice suffer from microcytic anemia. Unlike previously described animal models of β thalassemia major, homozygous γβ0 mice switch from mouse embryonic globin chains to human fetal γ globin during fetal life. When bred with human α globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

Preclinical transfusion-dependent humanized mouse model of β thalassemia major

A preclinical humanized mouse model of beta thalassemia major or Cooley anemia (CA) was generated by targeted gene replacement of the mouse adult globin genes in embryonic stem cells. The mouse adult alpha and beta globin genes were replaced with adult human alpha globin genes (alpha2alpha1) and a human fetal to adult hemoglobin (Hb)-switching cassette (gamma(HPFH)deltabeta(0)), respectively. Similar to human infants with CA, fully humanized mice survived postnatally by synthesizing predominantly human fetal Hb, HbF (alpha(2)gamma(2)), with a small amount of human minor adult Hb, HbA2 (alpha(2)delta(2)). Completion of the human fetal to adult Hb switch after birth resulted in severe anemia marked by erythroid hyperplasia, ineffective erythropoiesis, hemolysis, and death. Similar to human patients, CA mice were rescued from lethal anemia by regular blood transfusion. Transfusion corrected the anemia and effectively suppressed the ineffective erythropoiesis, but led to iron overload. This preclinical humanized animal model of CA will be useful for the development of new transfusion and iron chelation regimens, the study of iron homeostasis in disease, and testing of cellular and genetic therapies for the correction of thalassemia.

Human Globin Knock-in Mice Complete Fetal-to-Adult Hemoglobin Switching in Postnatal Development

ABSTRACT Elevated levels of fetal γ-globin can cure disorders caused by mutations in the adult β-globin gene. This clinical finding has motivated studies to improve our understanding of hemoglobin switching. Unlike humans, mice do not express a distinct fetal globin. Transgenic mice that contain the human β-globin locus complete their fetal-to-adult hemoglobin switch prior to birth, with human γ-globin predominantly restricted to primitive erythroid cells. We established humanized (100% human hemoglobin) knock-in mice that demonstrate a distinct fetal hemoglobin (HbF) stage, where γ-globin is the dominant globin chain produced during mid- to late gestation. Human γ- and β-globin gene competition is evident around the time of birth, and γ-globin chain production diminishes in postnatal life, with transient production of HbF reticulocytes. Following completion of the γ- to-β-globin switch, adult erythroid cells synthesize low levels of HbF. We conclude that the knock-in globin genes are expressed in a pattern strikingly similar to that in human development, most notably with postnatal resolution of the fetal-to-adult hemoglobin switch. Our findings are consistent with the importance of BCL11A in hemoglobin switching, since removal of intergenic binding sites for BCL11A results in human γ-globin expression in mouse definitive erythroid cells.

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

β thalassemia major or Cooleys 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.

Allogeneic bone marrow transplant in the absence of cytoreductive conditioning rescues mice with β-thalassemia major

β-thalassemia is a group of inherited blood disorders that result in defects in β-globin chain production. Cooley anemia (CA), or β-thalassemia major, is the most severe form of the disease and occurs when an individual has mutations in both copies of the adult β-globin gene. Patients with CA fail to make adult hemoglobin, exhibit ineffective erythropoiesis, experience severe anemia, and are transfusion dependent for life. Currently, allogeneic bone marrow transplantation (BMT) is the only cure; however, few patients have suitable donors for this procedure, which has significant morbidity and mortality. In this study, a novel humanized murine model of CA is rescued from lethal anemia by allogeneic BMT in the absence of cytoreductive conditioning. A single intravenous postnatal injection of allogeneic bone marrow results in stable, mixed hematopoietic chimerism. Five months after transplantation, donor cells accounted for approximately 90% of circulating erythrocytes and up to 15% of hematopoietic stem and progenitor cells. Transplanted mice are transfusion independent, have marked improvement of hematological indices, exhibit no growth retardation or signs of graft-versus-host disease, and are fertile. This study describes a method for the consistent engraftment of allogeneic donor hematopoietic cells that rescues a humanized mouse model of CA from lethal anemia, all in the absence of toxic cytoreductive conditioning.