Blood Journal
Leading the way in experimental and clinical research in hematology

Minimal modification in the factor VIII B domain sequence ameliorates the murine hemophilia A phenotype

  1. Joshua I. Siner1,
  2. Nicholas P. Iacobelli1,
  3. Denise E. Sabatino2,
  4. Lacramiora Ivanciu1,
  5. Shangzhen Zhou1,
  6. Mortimer Poncz1,
  7. Rodney M. Camire1, and
  8. Valder R. Arruda1,*
  1. 1 Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA, United States;
  2. 2 Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States
  1. * Corresponding author; email: arruda{at}email.chop.edu

Key points

  • Novel FVIII variant (FVIII-RH) with enhanced stability and procoagulant activity in both in vitro and in vivo models

  • FVIII-RH is efficacious and safe, thus it is an attractive molecule for protein replacement and as a transgene in gene therapy strategies

Abstract

Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed (>75% vs. <20% human FVIII-BDD) as a single-chain protein and exhibits greater stability after activation compared to human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the PACE/furin cleavage site within the B domain (R1645H) that mimics the canine sequence (HHQR vs. human RHQH). Compared to human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single chain form. Notably, FVIII-BDD exhibited a 2-fold increase in biological activity compared to FVIII-BDD likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and micro-circulation. These findings were successfully translated to AAV-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was ~2-fold higher compared to AAV-FVIII-BDD injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared to FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule to improve efficacy without increased immunogenicity, and may be suitable for both protein- and gene-based strategies for HA.

  • Submitted October 31, 2012.
  • Accepted January 25, 2013.