title: Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.
abstract: Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy.
  
  
  
  Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: G N
authors.initials: GN
authors.lastname: Nguyen
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: George
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: J I
authors.initials: JI
authors.lastname: Siner
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Davidson
authors.affiliation: Division of Laboratory Medicine, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: C B
authors.initials: CB
authors.lastname: Zander
authors.affiliation: Division of Laboratory Medicine, Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: X L
authors.initials: XL
authors.lastname: Zheng
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: V R
authors.initials: VR
authors.lastname: Arruda
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Camire
authors.affiliation: The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.|Department of Pediatrics, Division of Hematology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Sabatino
mesh_terms: D000818:Animals; D001665:Binding Sites; D006224:Cricetinae; D005169:Factor VIII / Q000235:genetics*; D045683:Furin / Q000737:chemistry*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D006467:Hemophilia A / Q000235:genetics* / Q000628:therapy; D006487:Hemostasis; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D011994:Recombinant Proteins; D005169:Factor VIII; D045683:Furin
keywords: dependovirus; factor VIII; furin; genetic therapy; hemophilia A