Thrasher, Adrian James; (1995) Molecular studies towards gene therapy of chronic granulomatous disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Chronic granulomatous disease (CGD) is human disorder arising from heterogeneous molecular lesions in the genes encoding a phagocyte specific enzyme system, the NADPH-oxidase. The importance of this system to host immunity is exemplified by the clinical phenotype arising from its defective function, which is characterised by recurrent bacterial and fungal infection. Bone marrow transplantation can be curative, but the paucity of suitable donor material, and the increased risks and complications associated with use of matched unrelated donor grafts, restricts application of this procedure to a minority of cases. CGD is an excellent candidate disorder for the development of somatic gene therapy. The studies presented in this thesis investigate potential mechanisms for determination of the myeloid and differentiation-specific expression of one of the NADPH-oxidase components, p47phox, and demonstrate the applicability of a gene transfer approach using recombinant retrovirus-based systems to permanently reconstitute NADPH- oxidase activity in immortalised B cells. However, the current generation of retroviral vectors are limited by inefficient transduction of relatively quiescent cells, which include the pluripotent haematopoietic stem cell population. This greatly restricts their potential to effect lifelong correction of the clinical phenotype. One system which may be less dependent on cell cycle for successful transduction is based on the dependovirus, adeno-associated virus (AAV). These vectors are shown to stably transduce and restore NADPH-oxidase activity to patient-derived B cells, but are also shown to be difficult to propagate. A method for rescue of recombinant AAV particles from replication-defective adenoviruses is described, which facilitates production, and significantly reduces the risk of contamination with wild-type adenovirus. Adenoviral vectors are also shown to restore function to primary monocytes derived from patients, and may prove to be useful for delivery of the recombinogenic AAV vector genome to target cells.

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