Lee, Doyoung; (2017) Investigation of mechanisms for enhancing expression of human FVIII in vitro; application to therapeutic protein production. Doctoral thesis , UCL (University College London).

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Abstract

Haemophilia A is the most common bleeding disorder and is caused by deficiency or abnormality in coagulation Factor VIII (FVIII). Deletion of the B-domain dramatically improves secretion of this protein with no alteration of the pro-coagulant activity of the B-domainless FVIII. Previous work has shown that the codon optimisation of FVIII increases expression by 10-fold and the novel variant, FVIII-V3, is expressed at higher levels than B-domain deleted FVIII (BDD-FVIII) in the context of AAV mediated gene transfer in mice. However, the mechanisms underlying these enhancements to expression remain undefined. The purpose of this study was to investigate if (1) codon optimisation increases FVIII expression by increasing transcription of FVIII mRNA, (2) higher level of FVIII expression observed with V3 is due to N-linked glycosylation motifs in the B-domain linker and V3. In this thesis, I characterised a range of novel FVIII variants to demonstrate that functional characteristics were similar to native FVIII. Firstly, comparison of FVIII expression levels in lentivirally transduced HEK-293 stable cell lines showed codon optimised FVIII variants expressed higher than wild type FVIII not only improved mRNA transcription but also FVIII expression. The V3co variant expressed FVIII at the highest level in-vitro and by in-vivo AAV gene delivery to mice. Secondly, the glycosylation profile of purified FVIII variant proteins was described. We show incremental formation of N-glycans within the B-domain linker sequences as detected by mass spectrometry analysis. In addition, these novel FVIII variants showed thrombin digestion patterns, identical to the commercial rFVIII protein. Furthermore, the thrombin generation and vWF/FVIII affinity assay revealed that all variants were similar to commercially available B-DD rFVIII. Finally, the expression of N-linked glycosylation mutant V5 and through a deletion analysis, short regions from the V3 linker variants using in-vivo AAV gene transfer confirming glycosylation of the linker sequence is not an important factor but conformation changes may be the key for the improved expresssion profile. Overall, these insights will significantly increase the potential for the transition of engineered FVIII molecules to clinic.

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