Patricia A. Morateck

Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies

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 alphaIIb promoter in platelets of hemophilic (FVIIInull) mice to create 2bF8trans 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 2bF8trans mice, the bleeding phenotype of FVIIInull 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.

Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity

Although genetic induction of factor VIII (FVIII) expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of preexisting FVIII inhibitory antibodies to determine whether such antibodies would affect therapeutic engraftment. We generated a line of transgenic mice (2bF8) that express FVIII only in platelets using the platelet-specific alphaIIb promoter and bred this 2bF8 transgene into a FVIII(null) background. Bone marrow (BM) from heterozygous 2bF8 transgenic (2bF8(tg+/-)) mice was transplanted into immunized FVIII(null) mice after lethal or sublethal irradiation. After BM reconstitution, 85% of recipients survived tail clipping when the 1100-cGy (myeloablative) regimen was used, 85.7% of recipients survived when 660-cGy (nonmyeloablative) regimens were used, and 60% of recipients survived when the recipients were conditioned with 440 cGy. Our further studies showed that transplantation with 1% to 5% 2bF8(tg+/-) BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not negate engraftment of 2bF8 genetically modified hematopoietic stem cells, and transplantation of these hematopoietic stem cells can efficiently restore hemostasis to hemophilic mice with preexisting inhibitory antibodies under either myeloablative or nonmyeloablative regimens.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.