Development and characterization of recombinant ovine coagulation factor VIII.

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

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells

OBJECTIVE: CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS: Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS: All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION: Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes

Discrimination of the BDD OfVIII Heavy and Light Chains.

<p>Immunoprecipitation using domain specific MAbs was performed by incubation with ofVIII in the presence and absence of thrombin. Heavy and light chains were dissociated using M-PER lysis buffer supplemented with 150 mM NaCl prior to MAb addition. FVIII heavy chain was precipitated with 4F4 1B, an A2 domain-specific mAb, and light chain was precipitated with I14 1B, a C2 domain-specific mAb. MAbs incubated with vehicle served as negative controls.</p

Thrombin-Activated Decay Rate of OfVIIIa.

<p>Human (closed circle) and ovine (open circle) fVIIIa decay was measured by chromogenic Xase assay in which 20 nM fVIII was activated with thrombin and then stopped with desulfatohirudin. Activated fVIIIa in complex with phospholipid vesicles, activated factor IXa, and factor X was measured at 0.5, 3, 5, 8, 15, and 30 minutes to determine residual fVIIIa activity. Half-lives of 1.8±0.09 and 3.5±0.37 minutes were calculated for human and ovine fVIIIa, respectively. Data shown represents the percent of initial activity by semi-log extrapolation to time  = 0. Regression analysis revealed Pearson correlation coefficients of 0.999 for both treatments.</p

Biochemical Analysis of BDD OfVIII.

<p>Recombinant ofVIII (2 µg) ± thrombin and PNGase treatment was resolved by SDS-PAGE and visualized by Coomassie blue staining. A molecular weight ladder was used to determine the relative mobility of the polypeptides.</p