Identification of Porcine Coagulation Factor VIII Domains Responsible for High Level Expression via Enhanced Secretion

Abstract

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency. Factor VIII (fVIII)1 is a plasma protein that functions in proteolytically activated form as a cofactor within the intrinsic pathway of blood coagulation to increase the rate of proteolytic activation of factor X by activated factor IX. fVIII contains a domain structure designated A1-A2-B-ap-A3-C1-C2 that is defined based on internal sequence homology (1, 2). The fVIII A domains share homology with the copper-binding protein ceruloplasmin (3, 4), which has an A1-A2-A3 domain structure in which the three A domains are arranged along a pseudo-3-fold axis of symmetry (5). Before cell secretion, fVIII is cleaved at the B/ap-A3 domain junction into A1-A2-B (heavy chain) and ap-A3-C1-C2 (light chain) subunits. fVIII circulates in the plasma as an inactive heavy chain/light chain heterodimeric procofactor that is non-covalently bound to von Willebrand factor. Proteolytic activation of fVIII by thrombin results from cleavages at Arg-372 between the A1 and A2 domains, Arg-740 between the A2 and B domains, and Arg-1689 between the ap and A3 domains. During this process, the covalent linkage between the A1 and A2 domains is lost, and the B domain and 41-residue ap are released, producing a heterotrimeric,

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

“…1) in the mammalian expression vector ReNeo has been described previously (27,28). These constructs contain nucleotide sequence between the A2 and ap-A3 domains encoding a linker region of human or porcine origin that includes the Arg-His-Gln-Arg recognition sequence for PACE/furin processing (29,30).…”

Replacing bad (F)actors: hemophilia

“…9 For many secreted proteins, transit from the endoplasmic reticulum to the Golgi apparatus is rate limiting and the primary determinants of this limitation are believed to be specific amino acid sequences within the molecule itself. 10,11 Using this knowledge, we and others have focused on the development of biobetter FVIII molecules and their implementation into gene transfer vectors that can be used in stem cell gene therapy applications. For example, it was shown by Kaufman et al that coexpression of von Willebrand factor results in the improved accumulation of recombinant FVIII in the culture medium of heterologous cells.…”

“…For example, we and others have shown that BDD p-FVIII is expressed at 10-fold or greater levels compared with BDD h-FVIII. 11,15,16 This led to the rationale of using a recombinant murine stem cell (␥-retrovirus) viral vector encoding BDD p-FVIII in HSCT gene therapy studies. These studies were highly successful with respect to the plasma FVIII activity levels achieved in transplanted hemophilia A mice conditioned with lethal or sublethal irradiation-based conditioning.…”

“…This bioengineered FVIII transgene demonstrated equivalent transgene product expression to BDD p-FVIII in vitro and in vivo using both SIV-and HIV-based expression vectors. 11,17,18 An alternative, but also successful, preclinical HSCT gene therapy strategy incorporating nonengineered BDD h-FVIII, and more recently FIX, transgenes involves specific targeting of transgene expression to the megakaryocyte/platelet hematopoietic lineage. This approach was first demonstrated by Poncz et al using transplantation of HSCs from transgenic mice to correct the hemophilia A bleeding phenotype.…”

Hemophilia: New Protein Therapeutics

“…Further study using porcine/human hybrids has localized certain protein sequences within porcine FVIII that confer this advantage, although additional studies will be necessary to characterize the mechanism. 10 Bioengineering rFVIII for high-efficiency production could potentially reduce costs and increase the availability of concentrates for therapy.…”