Stable expression of native Coagulation factor VIII using the 2A self-processing sequence and furin cleavage site

Abstract

Hemophilia A is an X-linked recessive bleeding disorder, widely prevalent throughout the world, for which, replacement therapy is the current treatment done by infusion of either recombinant FVIII or human plasma derived FVIII. The expression of FVIII is limited by many aspects, bioengineered FVIII with increased the activity and/or the stability can overcome some of these limitations. We demonstrate that a furin cleavage site (RKRR) and a 2A self-processing peptide derived from FMDV can efficiently and apparently facilitate the equimolar expression of full-length FVIII heavy and light chains from a single ORF, and the FVIII heavy and light chain can self-assemble to form a functional molecule in vivo and in vitro. In addition, our results shown that retaining the 6 N-linked oligosaccharides within a short B-domain spacer associated with 2A and furin cleavage site is expressed more efficiently both in vitro in traditional heterologous expression systems as well as in vivo in a mouse model of hemophilia A.