Novel Approaches to Thalassaemia Gene Therapy

Student thesis: Doctoral ThesisDoctor of Philosophy


The haemoglobinopathies are the most prevalent genetically inherited disorders worldwide. Gene therapy for p-thalassaemia is particularly challenging given the requirement for high p-globin chain production in a lineage-specific manner. Advances in lentiviral vector (LV) technology, together with efforts to incorporate erythroid-specific regulatory elements into gene therapy vectors have yielded some promising results in both in vitro and in vivo studies with a number of clinical trials either underway or planned all using an ex vivo approach for the genetic correction of patient haematopoietic stem cells (HSCs). Despite this, current vectors are still sub-optimal: LV integration into the host-cell genome takes place near or within genes being actively transcribed, carrying a potential chance of causing insertional mutagenesis; long-term silencing of integrated transgenes with effect on reproducibility of expression; genetic correction in later developmental stages or adulthood that fails to avert phenotypic manifestations of the condition. This study forms the basis of a long-term programme to develop novel approaches to thalassaemia gene therapy, namely via an in utero delivery route that in principle could be readily applicable worldwide and thus have a positive global impact in the treatment of the haemoglobinopathies. We started with a comparative study with ubiquitous and tissue specific promoter/enhancer combinations in an integration defective lentiviral vector (ID-LV) configuration to assess their ability to express efficiently. Our results show that tissue specific expression of a human P-globin gene (HBE) regulated cassette from within an ID-LV can be obtained albeit at low levels of per vector copy number. The use of ID-LV for targeting HSC clearly awaits the development of versions of this vector system that are capable of replications and retention.
We next performed a comparative analysis of UCOE-based lentiviral vectors in a murine neonatal intravascular delivery model system in order to find the ideal marking vector for further in utero studies, hi this comparative study UCOE-eGFP-WPRE proved to be the vector less prone to positional variegation and silencing effects, showing in reproducible levels of expression. Hence, this vector was chosen for further in vivo studies. Our main objective was to address the possibility of an in utero gene therapy approach for the haemoglobinopathies. This methodology aims to take advantage of the migration of HSCs from the foetal liver to bone marrow during development. Therefore, genetic correction of HSC in foetal liver should in principle provide a life-long cure. We initially embarked on a comparative experiment with a UCOE-eGFP-WPRE LV and its twin vector containing a luciferase reporter gene (UCOE-LUC-WPRE), to determine the optimal time of delivery in a murine in utero delivery model system. Injection via the vitelline vessel at E14 and El6 showed little difference with respect to marking of erythroid precursors. However, there appeared to be a higher level of transduced myeloid cells from injections conducted at El6. Following this, we performed vitelline vessel injection at E14 in wild-type mice with a therapeutic GLOBE-based LV containing an HBB cassette consisting of PLCR elements HS2 and HS3 linked to a mini-HBB gene, and its associated transcription termination region. Analysis of recipient mice 3 months post-injection showed vector presence (average copy number 1.347) and detectable haemoglobin levels in peripheral blood.
Date of Award2013
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorMichael Antoniou (Supervisor) & Simon Waddington (Supervisor)

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