| Summary: | <p>The thalassaemias are the most common monogenic disorders worldwide. Both α- and β-thalassaemia are caused by genetic mutations that result in a decrease in globin gene expression and protein synthesis. The more clinically significant of the two disorders, β-thalassaemia is mainly caused by point mutations in the β-globin gene or proximal sequences. Patients develop severe anaemia early in life and are dependent on life-long blood transfusions. The relative excess free α-globin chains in developing erythroblasts causes apoptosis and necrosis, resulting in ineffective erythropoiesis and the resulting severe phenotype. Decades of careful clinical observational studies have shown that individuals with severe β-thalassaemia who co-inherit α-globin gene mutations often have a mild or asymptomatic phenotype. This is especially, but not limited to, individuals with HbE/β-thalassaemia which causes half of all severe β-thalassaemia worldwide. The α- and β-globin gene clusters are among the most finely characterised loci in the human genome, and this understanding of gene regulation in normal and pathological contexts allows the development of tailored therapeutic strategies. In this study, I have to tried to use this knowledge to develop two therapeutic strategies to cure β-thalassaemia. The first aims to tunably reduce α-globin expression by editing its main enhancer, MCS-R2, to restore globin chain balance in patients with severe β-thalassaemia. The second aims to recapitulate the asymptomatic carrier β-thalassaemia trait state by using new technology to repair the HbE mutation in-situ. </p>
<p>Initially I aimed to further characterise the role of the main enhancer of α-globin, MCS-R2. This work was enabled through the study of a patient with severe HbH disease who had an extremely rare mutation, leaving her with two α-globin genes and a deletion of MCS-R2 on the same allele. CD34+ cells were obtained and differentiated erythroid cells were studied using various methods, including ATAC-seq and Capture-C to assess gene-expression, chromatin accessibility and architecture in the absence of the main enhancer. Next a variety of techniques were used to profile the sequence of MCS-R2 to identify key regulatory sequences that are important to its function. Several putative transcription factor binding sites were identified, and these were targeted in the development of the first therapeutic strategy. Individual binding sites were mutated using CRISPR/Cas9 with high efficiency to try and tunably reduce α-globin expression. The mutation of binding sites for canonical erythroid transcription factors resulted in the reduction of α-globin expression commensurate with a single α-globin gene deletion, which has been shown previously to be of clinical benefit in individuals with severe β-thalassaemia mutations. Next base editors, a modular variant of the CRISPR/Cas9 system, were used to repair the pathogenic codon 26 mutation in the HBB gene that causes HbE/β-thalassaemia. This system was optimised and then used in patient-derived CD34+ cells, resulting in high base-deamination efficiencies and robust production of normal β-globin protein. Serial mouse xenotransplantation experiments were performed, proving that base editing is possible in long-term repopulating haematopoietic stem cells, and the off-target landscape was assessed. </p>
<p>In summary, through the development and utilisation of knowledge of how the main α-globin enhancer controls gene expression, and the adoption of new gene-editing technologies, this work outlines two new therapeutic strategies for curing HbE/β-thalassaemia. These have the potential to deliver significant benefit, both alone or in conjunction with gene-editing therapies currently being trialled in the clinic. </p>
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