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Advisory Committee Chair

Thomas M Ryan

Advisory Committee Members

Tim M Townes

Peter J Detlaf

Anupam Agarwal

David F Crawford

Document Type

Dissertation

Date of Award

2009

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

β thalassemia major or Cooley’s anemia (CA) is a heterogeneous group of inherited disorders characterized by deficient β globin chain production. Newborns with CA are healthy because of the high fetal hemoglobin (HbF) levels present in their red blood cells (RBCs). As HbF levels decline during the first year of life, patients develop severe anemia that necessitates regular blood transfusion to maintain life. CA has been difficult to model in the mouse due to the lack of a fetal hemoglobin equivalent in the mouse. By utilizing a human fetal to adult delayed hemoglobin switching cassette, I hypothesize that a CA mouse model could be generated that more closely mimics the disease in humans. Humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse β globin genes with a delayed switching γβ0 or γδβ0 globin gene cassettes. The nonfunctional human β0 globin knock-in (KI) allele contains a G to A mutation at IVS1.1. Wild type and hereditary persistence of fetal hemoglobin (HPFH) mutations were employed in the promoter of the human γ globin allele to further increase γ globin expression levels. Heterozygous KI mice exhibit β thalassemic phenotypes. Newborn homozygous humanized CA mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, and survive from one day to several weeks after birth, but can survive into adulthood through repeated blood transfusions. These CA models are the first to recapitulate the temporal onset of the disease in human patients. i Interestingly, humanized CA mice are rescued from lethal anemia by a single intravenous postnatal injection of allogeneic bone marrow in the absence of any cytoreductive conditioning. Stable hematopoietic chimerism is reached 8 weeks post transplantation. Transplanted CA mice have a marked improvement of their anemia, exhibit no growth retardation or graft versus host disease, and are fertile. These novel humanized CA disease models are useful for the study of the regulation of globin gene expression, synthesis, and switching; 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.

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