Blundell, M.P.; (2007) Molecular investigations into Wiskott-Aldrich Syndrome. Doctoral thesis , University of London.

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Abstract

Wiskott-Aldrich Syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by eczema, thrombocytopaenia and immunodeficiency. WAS encodes for a haematopoietic restricted protein, WASp, involved in transduction of signals from the cell membrane to the actin cytoskeleton. Mutations lead to impaired actin dynamics in response to stimuli and are seen as defects of receptor capping, chemotaxis, phagocytosis and proliferation. This leads to susceptibility to pyogenic, viral and opportunistic infections and increased incidence of lymphoproliferative disease and malignancy. Due to the high morbidity and mortality associated with mismatched transplantation, WAS is considered a good target for gene therapy. We were able to reconstitute a murine model of WAS using a gamma retroviral vector, with reconstitution of specialised actin structures, podosomes, as a functional readout. Reservations concerning the safety of such vectors, following adverse events in a clinical trial, led to the development of third generation self-inactivating lentiviral vectors. Under the promotion of CMV, SFFV LTR or short sequences of the proximal endogenous Wiskott-promoter, WASp was able to restore cytoskeletal abnormalities in dendritic cells in-vitro from WASp-deficient mice. In addition we have demonstrated stable expression of WASp in T cell, B cell and myeloid lineages following transduction and engraftment of lineage negative murine bone marrow (up to 9 months) using both an SFFV LTR and endogenous promoter sequences. We have demonstrated the ability to restore cytoskeletal abnormalities and proliferative responses from the reconstituted mice. Recently a novel mutation in WAS has led to the discovery of a constitutively active WASp, with a novel monocytopaenic and neutropaenic phenotype. Here we characterised the patient phenotype of a novel constitutively active WASpn94T mutant. In addition to monocytopaenia and neutropaenia, there was abnormal cytoskeletal actin, manifesting itself as abnormal podosome distribution and an inability to phagocytose or produce an oxidative burst to physiological stimuli. There was also increased apoptosis in the bone marrow and evidence of genomic instability. Utilising lentiviral vectors, WASpI294T was expressed in a cell line to elucidate the possible mechanisms responsible for the patient phenotype. These studies demonstrate the efficacy and feasibility of a lentiviral vector-mediated gene therapy strategy for WAS using endogenous promoters where a more regulated expression level may be achieved.

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