Advancing lentiviral gene therapy vectors for β-thalassaemia

Student thesis: Doctoral ThesisDoctor of Philosophy


Background: β-thalassaemia is a potentially lethal hereditary anaemia, caused by reduced or absent expression of HBB polypeptide chains of adult haemoglobin (HbA: α2β2). Current curative treatment options are limited to few patients, while alternative, chronic palliative therapy consisting of frequent transfusions coupled with iron chelation therapy, is costly and reduces patients’ quality of life. Increased levels of fetal haemoglobin (HbF: α2γ2) were shown to lessen the severity of β-thalassaemia, highlighting the therapeutic potential of a gene-therapy-mediated increase in HBG1 and HBG2 (HBG) expression.
Aims: The aim of this project was to develop a new generation of lentiviral vectors (LVs) in order to deliver a combinatorial gene therapy for β-thalassaemia (and sickle cell disease). LVs were designed to enhance HbF levels by engineering the HBB-expressing GLOBE LV to co-express short-hairpin RNAs (shRNAs) that will post-transcriptionally knockdown repressors of HBG expression, such as BCL11A. Conceptually, it was envisaged that the combination of LV-derived HBB plus increased endogenous HBG chains would achieve levels of total haemoglobin that would reproducibly be in the range (~50% of normal values) to be curative for β-thalassaemia major (and sickle cell disease).
Methodology/Results: A series of modified LVs with simple shRNAs or microRNA-adapted shRNAs (shRNA-miRs) inserted within the second intron of HBB (βIVS-II) within GLOBE, were produced and functionally tested in erythroid tissue culture cell lines (K562, HEL, MEL) and in a human primary erythroid cell culture model system. Early experiments were conducted using shRNA-bearing vectors targeting mRNA of the GFP reporter to rapidly establish proof-of-principle for the vector design. Technical difficulties with the execution of experiments in GFP-expressing transgenic cell lines prompted the parallel use of BCL11A-targeting vectors aiming to induce HBG expression. The cell line work did not produce concrete findings, with every indication that these systems were unsuitable platforms for the intended analyses.
Lastly, vectors were functionally tested in primary erythroid cells derived from CD34+ haematopoietic stem cells from both normal individuals and patients with β-thalassaemia. To this end, extraction protocols of CD34+ cells from peripheral blood samples were optimised, based on density-gradient centrifugation and simple ACK lysis of red blood cells. The effect of these protocols on cell viability, proliferation and differentiation was compared in standardised small-scale assays on semi-solid methylcellulose media. Standardising liquid cultures for large-scale CD34+ cell expansion and differentiation, however, posed unexpected challenges and prompted the evaluation of three separate protocols before suitable cell numbers and differentiation levels were achieved. Using the established systems, the RNAi competency of stem-loop sequences in shRNAs was functionally validated by expression from the RNA-polymerase-III U6 promoter of control vectors. Vectors carrying the same basic shRNA cassettes in the GLOBE-encoded HBB βIVS-II showed no discernible effect on target genes. In contrast, subsequent preliminary analyses of second-generation shRNA-miR vectors demonstrated their RNAi activity, making them the subject of further investigation for combinatorial gene therapy for the haemoglobinopathies.
Date of Award2015
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMichael Antoniou (Supervisor)

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