Functional factor VIII made with von Willebrand factor at high levels in transgenic milk

Background—Current manufacturing methods for recombinant human Factor VIII (rFVIII) within mammalian cell cultures are inefficient which limit the abundance needed for affordable, worldwide treatment of hemophilia A. However, rFVIII has been expressed at very high levels by the transgenic mammary gland of mice, rabbits, sheep and pigs. Unfortunately, it is secreted into milk with low specific activity due in part to the labile, heterodimeric structure that results from furin processing of its B domain. Objectives—To express biologically active rFVIII in the milk of transgenic mice through targeted bioengineering. Methods—Transgenic mice were made with a mammary specific FVIII gene (226/N6) bioengineered for efficient expression and stability containing a B domain with no furin cleavage sites. 226/N6 was expressed with and without von Willebrand Factor (VWF). 226/N6 was evaluated by ELISA, SDS-PAGE, Western blot, and one- and two-stage clotting assays. Hemostatic activity of immunoaffinity enriched 226/N6 was studied in vivo by infusion into hemophilia A knockout mice. Results and conclusions—With or without co-expression of VWF, 226/N6 was secreted into milk as a biologically active single chain molecule that retained high specific activity similar to therapeutic-grade FVIII. 226/N6 had \u3e450-fold higher IU/ml than previously reported in cell culture for rFVIII. 226/N6 exhibited similar binding to plasma-derived VWF as therapeutic-grade rFVIII and intravenous infusion of transgenic 226/N6 corrected the bleeding phenotype of hemophilia A mice. This provides proof-of-principle to study expression of 226/N6 and perhaps other single chain bioengineered rFVIII in the milk of transgenic livestock

Recombinant factors for hemostasis

Trauma deaths are a result of hemorrhage in 37% of civilians and 47% military personnel and are the primary cause of death for individuals under 44 years of age. Current techniques used to treat hemorrhage are inadequate for severe bleeding. Preliminary research indicates that fibrin sealants (FS) alone or in combination with a dressing may be more effective; however, it has not been economically feasible for widespread use because of prohibitive costs related to procuring the proteins. To meet future demands for hemostatic therapies, FS will likely include recombinant human fibrinogen (rFI) and recombinant human Factor XIII (rFXIII). The underlying hypothesis of the research presented in this dissertation is that a liquid fibrin sealant (LFS) composed of recombinant FI, FXIII and FIIa in optimized proportions can assist hemostasis in the presence and absence of a bioresorbable bandage while using considerably fewer biologics than commercial products currently available. This dissertation characterized rFI produced in the milk of transgenic cows, plasma-derived thrombin (pdFIIa) activated by sodium citrate and rFXIIIa expressed in genetically engineered Pichia pastoris with respect to their capacity to serve as components in a LFS. The ratios of these factors were optimized to yield a LFS with a rapid clot formation rate and high viscoelastic strength. This optimized LFS was preliminarily tested ex vivo and in vivo. The clotting kinetics and viscoelastic strength of our optimized LFS was equivalent to those of a commercially available LFS; however, it uses approximately 75% less fibrinogen and thrombin. Our optimal LFS successfully achieved hemostasis in a significant number of the wounds that included extensive tissue and vascular damage. LFS applied without the assistance of a dressing was able to stop bleeding of oozing wounds or those with small vessels; however, a scaffold was needed when wounds contained large vasculature

Recombinant Human Fibrinogen That Produces Thick Fibrin Fibers with Increased Wound Adhesion and Clot Density

Human fibrinogen is a biomaterial used in surgical tissue sealants, scaffolding for tissue engineering, and wound healing. Here we report on the post-translational structure and functionality of recombinant human FI (rFI) made at commodity levels in the milk of transgenic dairy cows. Relative to plasma-derived fibrinogen (pdFI), rFI predominately contained a simplified, neutral carbohydrate structure and >4-fold higher levels of the γ′-chain transcriptional variant that has been reported to bind thrombin and Factor XIII. In spite of these differences, rFI and pdFI were kinetically similar with respect to the thrombin-catalyzed formation of protofibrils and Factor XIIIa-mediated formation of cross-linked fibrin polymer. However, electron microscopy showed rFI produced fibrin with much thicker fibers with less branching than pdFI. In vivo studies in a swine liver transection model showed that, relative to pdFI, rFI made a denser, more strongly wound-adherent fibrin clot that more rapidly established hemostasis