Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Abstract

Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.

Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

“…Deficiency of FVIII results in bleeding disorder commonly known as hemophilia A. 9,31–33 We used a list of 27 peptides each 12 residue long selected against mAb Bo2C11 targeting the C 2 domain of FVIII. 31 Using the X-ray crystal structure of the C 2 domain of FVIII 34 (PDB id:1IQD), EpiSearch method predicted a potential epitope on the C 2 domain of FVIII that correspond to a patch centered at the residue R2220 with the highest score (0.937).…”

Delivery of Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector

“…The human B-domain deleted FVIII was chosen in this study, because the B-domain structure does not require procoagulant activity in contrast to full-length of human FVIII cDNA which contains a domain structure of A1-A2-B-A3-C1-C2. 16 The B-domain deleted hFVIII retains coagulant activity and is secreted from cultured cells more efficiently than with full length FVIII. Furthermore, it is less sensitive to degradation.…”

Prospects for the Use of Artificial Chromosomes and Minichromosome-Like Episomes in Gene Therapy

“…By contrast, the open reading frame of FVIII is 7055 bp long and it has been much harder to express in viral vectors [ 140 ]. Different strategies have been applied to make the FVIII cDNA a bit shorter, and hence easier to introduce into different vectors, including using a B-domain deleted version of FVIII cDNA [ 141 , 142 ] as this domain does not appear to be essential for coagulation [ 14 , 143 – 145 ].…”

“…Indeed, studies performed in transgenic mice have demonstrated that these “minigene constructs” are very susceptible to being switched off by neighbouring chromatin [ 13 ]. On the other hand, there are cases where the cDNA is too big to be contained in one of these vector systems and some “nonessential” regions have to be eliminated [ 14 ].…”