Ling, Gavin Chien Pang; (2019) Use of bispecific single chain variable fragments in haemophilia A gene therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Haemophilia A is an X-linked monogenic disorder which has showcased the significant potential of gene therapy with long term Factor VIII (FVIII) expression following a single intravenous administration of adeno-associated Virus (AAV). There remains a critical unmet need in patients who develop neutralising antibodies to Factor VIII (FVIII) (inhibitors), resulting in significant morbidity and mortality and for whom current AAV gene therapies are unsuitable. Using the sequence of emicizumab, a bispecific full-length IgG4 antibody which acts as an activated FVIII (FVIIIa) mimetic, I generated a novel bispecific structure utilising single chain variable fragments linked in tandem (‘Bit8’). In vitro testing of Bit8 confirmed functional activity as demonstrated by the conversion of Factor X (FX) into activated FX (FXa), which persisted in the presence of FVIII inhibitors. Iterative optimisation through inter-scFv linker and heavy-light chain rearrangements identified a lead candidate with enhanced FXa generation and was further characterised using thrombin generation and chromogenic assays and by SPR. The optimal Bit8 construct was inserted into an AAV expression cassette with resulting transduction in the HUH7 hepatoma cell line demonstrating both secretion and functional activity. Wild type and haemophilia A knockout mice were injected with AAV-Bit8 constructs demonstrating successful transduction into liver and protein expression in vivo. However tail clip assays failed to show significant reductions in bleeding which may be related to relatively low levels of protein expression and is further explained therein. Despite the advances in gene therapy for haemophilia A, additional strategies for making this revolutionary treatment are needed, and this work shows exciting promise as a pilot study in haemophilia in preclinical studies, with new scientific challenges to overcome.

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