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.