1 Bioengineering of Coagulation Factor VIII for Improved Secretion

Abstract

PURPOSE: Mechanisms that limit recombinant factor VIII (rFVIII) within heterologous systems (eg. commercial rFVIII production or gene therapy applications) include: 1) inefficient expression of FVIII mRNA, 2) inefficient folding of the primary translation product within the endoplasmic reticulum (ER) with retention by chaperone proteins and 3) a requirement for facilitated transport from the ER to the Golgi apparatus. The B domain of FVIII shares no homology with any identified protein, is dispensable for functional activity and is extensively glycosylated (18 asparagine (N)-linked glycosylation attachment sites). B domain-deleted (BDD)-FVIII generates higher concentrations of mRNA compared to full-length FVIII and therefore increased primary translation product but exhibits a reduced rate of secretion and increased intracellular retention. Bioengineering of the FVIII B domain effectively improves the trafficking of rFVIII through facilitated ER-Golgi transport. METHODS: We have used a series of B domain variants to map key regions of the B domain that regulate mRNA levels and secretion efficiency. Each construct has increased B domain size (ranging from 29 to 774 amino acids (aa) and increased number of N-linked oligosaccharides (from 1 through 18). RESULTS: BDD-FVIII, FVIII wild-type (WT) and FVIII B domain variants were transfected into COS and CHO cell lines and the media harvested for analysis of FVIII activity and antigen. The addition of even a few N-linked oligosaccharides within a short B domain spacer (optimal at 226 aa and 6 N-linked oligosaccharides) improves secretion of BDD-FVIII approximately 5 to 10-fold. With additional increase in B domain size and oligosaccharide content, secretion efficiency declined in a stepwise fashion to similar levels observed for FVIII WT. An alternative construct was prepared in which the human FVIII B domain was replaced with the FVIII B domain from puffer fish (fugu rubripes). The fugu FVIII B domain is 224 aa and shares only 6% sequence identity to the human FVIII B domain, yet has a high density of N-linked oligosaccharides (11). This construct was secreted as efficiently as the 226aa/N6 human construct. CONCLUSIONS: The secretion efficiency of FVIII can be regulated by the size and oligosaccharide content of the B domain. The results suggest that it is primarily the presence of N-linked oligosaccharides that improves secretion efficiency. FVIII bioengineered for improved secretion will be an alternative for gene therapy strategies as well as recombinant FVIII production in manufacturing or transgenic strategies.