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Advisory Committee Chair

Thomas M Ryan

Advisory Committee Members

Louise T Chow

Christopher A Klug

Rakesh P Patel

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Cooley’s Anemia (CA) is a hereditary disease which occurs when an individual inherits two null β-globin alleles. CA presents during the latter part of the first year of life due to the high levels of fetal hemoglobin (HbF) in the circulating red blood cells (RBCs) at birth. The absence of β-globin chains, to dimerize with α-globin chains in the newly formed bone marrow derived erythroblasts, results in premature destruction of the erythroid cells in the marrow and ineffective erythropoiesis. I hypothesized that increas-ing the amount of β-like globin chains for hemoglobin assembly in erythroblasts would lessen disease severity or even cure a humanized mouse model of CA. Employing gene editing techniques, we increased the levels of the fetal β-like globin chain, γ-globin, by introducing mutations that prevent or delay the completion of the switch from fetal to adult hemoglobin. This goal was first achieved by the introduction of a T to C promoter mutation at position -175 upstream of the γ-globin gene transcription start site (TSS). The animals generated from this experiment survived normally but were phenotypically thalassemic with enlarged spleens, extramedullary hematopoiesis and low levels of liver iron accumulation. The second approach employed germline gene editing of the Bcl11a erythroid specific enhancer (ESE) with CRISPR/Cas9. Several mutant founder lines were generated, but the mutations with the greatest effect on γ-globin upregulation extended at least 77 bp 3’ of the sgRNA cut site. The 77 bp deletion resulted in a heterocellular in-crease in γ-globin transcription and protein chain synthesis through adulthood at levels sufficient to prolong the survival of 84% of CA mice over 10 weeks with no RBC trans-fusions. The final approach was a proof-of-principle experiment in which we used CRISPR/Cas9 mediated homology directed repair (HDR) to repair the mutant β-globin gene in homozygous CA embryos. Of the mice born, two had repaired alleles and subse-quent homozygotes generated demonstrated no symptoms of thalassemia. These animals provide novel models for studying in vivo globin switching mech-anisms, identifying new modulators of globin switching and gene editing approaches to increasing HbF, and testing new therapeutic drugs for increasing HbF levels in erythro-blasts.

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