| Sammanfattning: | <p>β-Thalassaemia is a disorder of haemoglobin production characterised by severe anaemia requiring life-long blood transfusions. The common genetic defects are predominantly based in and around the β-globin gene resulting in reduced or absent β-globin chain synthesis. The resultant excess of free α-globin chains, which precipitates in red blood cells and their precursors and causes ineffective erythropoiesis, is the main pathophysiological mechanism of anaemia in these patients. Clinical and genetic data accumulated over the last 30 years have indicated that reduction of α-globin expression is clinically beneficial to patients with β-thalassaemia, and in the subset of patients with HbE β-thalassaemia it could be transformational. Attention has been centred on pathways that increase γ-globin expression and hence the production of foetal haemoglobin, but I have explored avenues that down regulate the α-globin gene expression without affecting the β-like globin expression, to equalise globin chain imbalance and could have potential clinical applications.</p> <p>Initially, an <em>in vitro</em> erythroid differentiation system in serum-free medium was optimised and characterised. This generates a large number of erythroid cells from human CD34<sup>+</sup> cells with minimal non-erythroid contamination. This system appears to be a faithful recapitulation of normal erythropoiesis and was validated with newly developed assays. A library of epigenetically active small molecules and licenced drugs was then screened and this identified a number of interesting compounds including IOX1, a histone demethylase inhibitor. Secondary assays confirmed IOX1 as a selective down regulator of α-globin expression, with no significant adverse effects on β-like globin expression, erythroid cell viability, erythroid differentiation or global erythroid transcriptome.</p> <p>Next, CRISPR/Cas9 genome editing technique was used to introduce targeted deletions of multispecies conserved sequences (MCS) R2 region, the most critical distant <em>cis</em>-acting regulatory element enhancing α-globin expression in humans. High transfection and mutation efficiencies were achieved in human CD34<sup>+</sup> cells and single cell assays confirmed high frequency of homozygous and heterozygous deletion events. More importantly, and analogous to patients with an identical mutation, the deletion of MCS-R2 element resulted in selective knockdown of α-globin expression without altering β-globin expression or erythroid differentiation. In conclusion, I have demonstrated that selective down regulation of α-globin expression is plausible through pharmacological and genome engineering approaches and these findings unveil new pathways of therapy for β-thalassaemia.</p>
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