Development and Characterization of Recombinant Ovine Coagulation Factor VIII

• Type of Material: Article
• Authors: Philip M. Zakas, Emory University; Bagirath Gangadharan, Emory University; Graca Almeida-Porada, Wake Forest Institute for Regenerative Medicine; Christopher D. Porada, Wake Forest Institute for Regenerative Medicine; Harold Trent Spencer, Emory University; Christopher Doering, Emory University
• Language: English
• Date: 2012-11-09
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: © 2012 Zakas et al.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0049481
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 7
• Issue: 11
• Start Page: e49481
• End Page: e49481
• Grant/Funding Information: This work was supported by a grant from the National Institutes of Health, National Heart, Lung, and Blood Institute, HL092179.
• Research Categories: Health Sciences, Medicine and Surgery

Abstract

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.