Recombinant canine B-domain–deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model

Abstract

Production of recombinant B-domaindeleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia IntroductionHemophilia A (HA) is an X-linked bleeding disease resulting from a functional factor VIII (FVIII) deficiency affecting 1 in 5000 males worldwide. For several decades, the HA dog model has been the most extensively used for preclinical studies. 1 Notably, in 2 strains of dogs, the underlying mutation consists of an inversion in intron 22 of the FVIII gene that is analogous to the most common human mutation. 2 This model faithfully replicates the human disease at both genotypic and phenotypic levels. 3,4 To date, there is no characterization of the cFVIII protein resulting from difficulties in purifying large amounts from canine plasma and to the relative poor performance in recombinant FVIII expression systems in general. Although the cFVIII cDNA sequence has a high sequence identity to human FVIII (hFVIII), 5 adult HA dogs develop immune responses on exposure to hFVIII that preclude the assessment of the efficacy and safety of potential novel therapies for HA. Notably, among humans, even small nucleotide changes in the hFVIII gene may predispose to inhibitor formation. 6 To overcome these limitations, we established a heterologous expression system for cFVIII. Our findings uncovered unforeseen enhanced biologic properties of the protein. This work fills an important void for the study of cFVIII biologic properties and immune responses in HA dogs. Methods Production and characterization of recombinant cFVIII-BDDPermission was obtained from the Institutional Animal Care and Use Committee of the University of Pennsylvania and the Children's Hospital of Philadelphia for all studies. cFVIII-BDD 7 and hFVIII-BDD 8 were expressed in baby hamster kidney cells and purified as previously described (supplemental data and supplemental Table 1, available on the Blood website; see the Supplemental Materials link at the top of the online article). [8][9][10][11] Canine and human FVIII-BDD protein concentrations were determined by absorbance at 280 nm using an extinction coefficient (E 280 , 1%) of 1.60 and molecular weight of 160 000; these values, obtained with porcine FVIIIa, were assumed to be the same as canine and human FVIII-BDD. 12 Protein specific activity was determined by activated partial thromboplastin time (aPTT) with minor modifications. 13 Decay of activated FVIII activity was monitored by purified component assay using reconstituted human factor Xase complex and plasma models as previously described. 11 N-terminal sequencing was determined in the laboratory of Dr Alexander Kurosky and Steven Smith at University of Texas Medical Branch (Galveston, TX). Enzymatic cleavage of N-...

In vivo efficacy of platelet-delivered, high specific activity factor VIII variants

“…17 Levels of hBF8 and IR8 in plasma and platelets of both transgenic and bone marrow reconstituted mice were determined using an anti-human specific enzyme linked immunosorbent assay (ELISA) kit (Affinity Biologicals) compared with recombinant full-length hF8 protein (Baxter Health Corporation). cBF8 levels were detected using a cF8-specific ELISA as described 24 compared with purified recombinant cBF8 protein. 24 Total pF8 determinations were done on platelet releasate after 3 cycles of freeze/thaw at Ϫ80°C/37°C of the platelet pellet.…”

“…cBF8 levels were detected using a cF8-specific ELISA as described 24 compared with purified recombinant cBF8 protein. 24 Total pF8 determinations were done on platelet releasate after 3 cycles of freeze/thaw at Ϫ80°C/37°C of the platelet pellet. For platelet-releasate F8 determination, platelets were incubated at 37°C with thrombin (final concentration, 1 U/mL; Sigma-Aldrich) for 10 minutes.…”

“…23 Thus, while pF8 appears an attractive strategy for hemophilia A therapy because of its potential use in the setting of inhibitors, one also needs to carefully examine whether this nontraditional delivery system would not lead to untoward clot embolization. In this study, we tested whether using 2 F8 variants with increased F8 activity, the abovementioned IR8 and canine (c) BF8, a species of F8 that has 3-to 5-fold greater specific activity than hBF8, 24,25 could decrease clot instability. We found that both F8 species were stored in murine platelets and released upon platelet activation.…”

“…This observation of low pcBF8 could be due to RNA message level, but the same cDNAs were used in the baby hamster kidney cell expression studies, where cBF8 was expressed at levels several fold higher than hBF8. 24 More likely the underlying defect in pcF8 levels is related to the well-known difficulty for F8 to undergo intracellular processing, leading to intracellular accumulation and cellular apoptosis. Both the transgenic and lentivirus/BMT pcBF8 mice had normal steady-state platelet counts (data not shown).…”

Using Factor VII in Hemophilia Gene Therapy

“…Ishiwata and colleagues have been treated hemophilia mice using AAV8 vector containing canine BDD -FVIII gene (Ishiwata et al, 2009). Sabatino and colleagues report indicated that canine BDD-FVIII dogs is stable than human BDD-FVIII, it can be considered in the hemophilia treatment (Sabatino et al, 2009). Doering et al were transferred hFVIII -sFVIII hybrid in to hematopoietic stem cells with lentiviral vector; cells expressed FVIII more than 100-8 times of cells transfected with hFVIII only (Doering et al, 2009).…”