Vetharoy, Winston; (2024) Molecular characterisation of ex vivo expanded Peripheral Blood Stem Cells for the treatment of Primary Immune Deficiencies. Doctoral thesis (Ph.D), UCL (University College London).

Text Winston Vetharoy - PhD Thesis_Final.pdf - Accepted Version Access restricted to UCL open access staff until 1 September 2027. Download (12MB)

Abstract

Primary Immunodeficiency diseases (PIDs) comprises of over 430 inborn errors of immunity. For most of these PIDs, Hematopoietic stem cells transplantation remains the only curative option. However, the paucity of a compatible donor precludes this as a viable option for many paediatric patients. Nowadays, correction of patient’s own hematopoietic stem cells constitutes a valid alternative. To this end, gene therapy using viral vectors has been employed to deliver a copy of the correct gene to target cells. However gamma-retroviral or lentiviral gene therapy for PIDs have been fraught with various challenges including the risk of insertional mutagenesis and oncogene activation. Recently, gene editing of stem cells using engineered nucleases has been explored / pursued as a therapeutic approach for restoration of a functional immune system for various PIDs. However, as with gene therapy using retro or lentiviral vectors, there are key challenges being faced in terms of retaining/preserving the stemness and self-renewal capacity of the geneedited stem cells. One of the strategies used to preserve the stemness is through the use of small molecules. Most of the work in this area is focussed on the effect of small molecules for expanding Cord blood CD34+ cells for allogeneic transplantation in leukaemic patients while mobilised CD34+ from peripheral blood is the preferred source for gene therapy applications. The aim of this project is to characterise peripheral blood mobilised stem cells in order to find suitable protocols to culture them during the gene therapy procedure. A new protocol that can sustain long term correction of hematopoietic cells can overcome the limitations of the current gene therapy approaches for those disorders, like chronic granulomatous disease or Fanconi Anaemia where the HSC pool is compromised. 4 In this project, I first describe the development of a novel clinically relevant protocol to retain the stemness of ex vivo expanded mobilised PBSCs for gene therapy. Secondly, I describe the in vitro and in vivo tests performed to examine the characteristics of the expanded graft. One of the main observations in vitro has been the upregulation of the key markers of haematopoietic stem cell population (over two-fold increase compared to the control). In the in vivo studies the ability of the expanded graft to successfully engraft immunodeficient murine recipients was ascertained, and found that the human CD34+ cells cultured using the novel protocol moderately enhanced the engraftment in the analysed murine bone marrow tissue. Then, I set out the extensive molecular characterisation carried out on the expanded cells - using RNAseq and BioMark platform – in order to study the gene expression profiles that contributed to the stemness of the ex vivo expanded graft. Further to the molecular characterisation, I have attempted to characterise some of the key genes that were found to be differentially expressed upon culturing using the novel protocol, in order to understand their putative role in maintaining the stemness of the ex vivo expanded graft. In the final part of the project, I have characterised the novel protocol in terms of its ability to maintain the stemness of CD34+ cells isolated from clinical paediatric patients having X-linked agammaglobulinemia (XLA) and X-linked chronic granulomatous disease (XCGD). The novel protocol was able to enhance and sustain the stem cell phenotype in vitro, better than the current clinical protocols. This was further verified in a scale up experiment under stringent good manufacturing practice (GMP) setting to demonstrate clinical manufacturing feasibility.

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