Fields, Paul Andrew; (2002) Towards gene therapy for haemophilia using muscle as a target: A study of vector transduction and the implications of the immune response. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A major hope for treatment of haemophilia is via a gene based approach. But with this hope as for any new novel treatment for haemophilia, is the occurrence of an immune response. This thesis attempts to examine the role of the immune response to Gene transfer by a muscle directed approach. It characterises the humoral and cellular immune response to the secretable human transgene product human factor IX in both small and large animal models of haemophilia. The humoral responses were studied by means of Elisa, Western Blot and Clotting based assays in both normal and haemophilic animal models. The aim of these experiments was to look for the presence of an antibody to human factor IX, determine whether it was inhibitory or not, and look for factors which may influence it's occurrence. Cellular immune responses were analysed histologically, and also by devising an invitro, and later an in vivo based cytotoxic T cell assay (CTL) assay to look for the presence of specific cellular based responses to the secretable transgene product human factor IX. The results demonstrated that humoral responses were observed to the transgene product human factor IX, and that the antibody subtype IgG1 was induced, in keeping with a TH2 cellular driven immune response to adeno-associated viral gene transfer. In the case of plasmid and Adenoviral vectors, a more dominant TH1 cellular driven response was observed. In the large animal studies a humoral response was observed, but in this scenario the response was less inhibitory than observed in the mice. The development of a humoral response could be overcome by setting immunomodulatory strategies designed to suppress inhibitor formation. The cellular based responses showed that for Adenoviral based gene transfer, strong Cytotoxic T cell responses were observed, but not for AAV based muscle transduction. The conclusions from these studies showed that likely immune responses induced were influenced by vector and transgene selection. Other factors such as route of gene transfer administration and underlying mutation status in recipient animals also affected outcome. Finally it was shown that some of these immune responses could be overcome by the design of immunomodulatory strategies. These findings have significant implications for the design of human clinical studies of gene transfer treatments for haemophilia.

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