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

Joshua I. Siner, Nicholas P. Iacobelli, Denise E. Sabatino, Lacramiora Ivanciu, Shangzhen Zhou, Mortimer Poncz, Rodney M. Camire, Valder R. Arruda

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.