Inhibitory immune response to exogenously infused factor VIII (FVIII) is a major complication in the treatment of hemophilia A. Generation of such inhibitors has the potential to disrupt gene therapy for hemophilia A.We explore what we believe to be a novel approach to overcome this shortcoming. Human B-domain-deleted FVIII (hBDDFVIII) was expressed under the control of the platelet-specific αIIb promoter in platelets of hemophilic (FVIII null ) mice to create 2bF8 trans mice. The FVIII transgene product was stored in platelets and released at the site of platelet activation. In spite of the lack of FVIII in the plasma of 2bF8 trans mice, the bleeding phenotype of FVIII null mice was corrected. More importantly, the bleeding phenotype was corrected in the presence of high inhibitory antibody titers introduced into the mice by infusion or by spleen cell transfer from recombinant hBDDFVIII-immunized mice. Our results demonstrate that this approach to the targeted expression of FVIII in platelets has the potential to correct hemophilia A, even in the presence of inhibitory immune responses to infused FVIII. IntroductionMonogenic diseases, characterized by the loss of a specific plasma protein, are currently treated by repetitive replacement therapy and are choice candidates amenable to gene therapy. Hemophilia A, a severe congenital bleeding disorder caused by the loss of clotting factor VIII (FVIII) (1), is a prototype of such monogenic diseases. Currently, hemophilia A is treated by infusion of recombinant or plasma-derived FVIII (2). However, 25-30% of patients develop antibodies (FVIII inhibitors) that selectively inactivate the clotting activity of FVIII and negate its therapeutic efficacy (3). Hemophilia A is considered a strong candidate for gene therapy because the therapeutic window is broad and even a minimal plasma level of plasma FVIII is clinically advantageous. The development of inhibitory antibodies to the FVIII transgene product in plasma remains a significant barrier to some patient candidates. Many groups have developed various strategies for directing FVIII synthesis (4-15), although inadequacies of gene delivery and expression and inhibitor formation remain clinical problems (7,(16)(17)(18).The approach we investigated, which we believe to be novel, is based on the hypothesis that targeting the production of FVIII to a secreting cell type that acts in the immediate vicinity of sites where FVIII is needed could overcome the presence of inhibitory antibodies. Furthermore, by sequestering the FVIII, the generation of antibodies in naive individuals might be prevented or at least rendered less relevant.

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The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of “VWF activity” by this assay and may not reflect a functional defect or true hemorrhagic risk.