Quantitative mRNA expression profiling of primary human erythroid progenitor cells exposed to small molecules for HbF induction

Abstract

β-thalassaemia and sickle cell disease (SCD) are the most prevalent haemoglobinopathies affecting millions of patients around the word. These haemoglobinopathies display clinical heterogeneity despite both being monogenic and both affect the β-globin gene. In some individuals fetal haemoglobin remains persistent throughout their life. Such condition is known as Hereditary persistence of fetal haemoglobin (HPFH). It was observed that higher levels of HbF ameliorates the condition of SCD and β-thalassaemia. Till now the only curative therapy for such haemoglobinopathies is allogenic transplantation, usually from a matched donor. However due to availability, this treatment is only reserved to less than 15% of the patients. These patients receive palliative care instead and are regularly transfused with red blood cells together with iron chelation therapy. Such treatment greatly impinges on the quality of life and is associated with several complications. Pharmacological induction of fetal haemoglobin can be beneficial in rendering these patients transfusion independent. The specific aim of this M.Sc project was to conduct a pharmacological induction of fetal haemoglobin (HbF) in primary human erythroid progenitor stem cells (HEPs) using three novel drugs obtained from the European Molecular Biology Laboratory (EMBL). These compounds served to probe and better understand the relationship between the Krüppel like Factor 1 gene (KLF1) and the B-cell lymphoma/leukemia 11 A gene (BCL11A) and the gamma (γ) to beta (β) globin gene switch mechanism. KLF1 and BCL11A are two important transcriptional factors which are involved in globin gene switching by silencing fetal haemoglobin expression. In this project, a combination of molecular biology and cell culture tools were used in order to test the hypothesis that one or more drugs in the study will increase the levels of HbF by knocking down the expression of KLF1 and BCL11A. The three drugs under study, going by their codified names 744363, 163213 and 163435 were tested at 11 different concentrations (1-50,000nM) and an accompanying Dimethyl sulfoxide (DMSO) control for each drug. The serial dilution of each drug ensured that a very wide and broad concentration range was tested and to see at which range does HbF respond. Indeed, drugs 163213 and 163435 were maximally efficient at low drug concentrations, whilst 744363 was optimal at moderate doses. A direct relationship between repressing BCL11A alone, or in combination with KLF1 was observed in some instances, with a concomitant increase in HBG mRNA expression. The increase in gamma globin was at times accompanied with an increase also in HBB. The highest Haemoglobin subunit globin gene (HBG) expression (FC 2.69) was obtained when the differentiated HEPs were exposed to 1nM of drug 163213. At this concentration the Haemoglobin subunit beta gene (HBB) levels also increased to FC 1.81 while the levels of KLF1 remained unperturbed (FC 1.03) and BCL11A slightly repressed (FC 0.72). Thus, suggesting that HBG expression increased independently of KLF1 and BCL11A. Using such approach to uncover the molecular basis of γ-globin gene control might serve as a model for other clinically significant gene switch mechanisms. This study laid the necessary groundwork for which further research can be optimized to develop therapeutic targeting methods in the treatment of haemoglobinopathies.