A mouse model for inhibitor development in mild hemophilia A

Abstract number: OC202

Van Helden^* P. M. W., Bril^* W. S., Zuurveld^* M. G., Hollestelle^* M. J., Fijnvandraat† K., Reitsma‡ P. H., Van Mourik^* J. A., Van Lier‡ R. A. W., Mertens^* K., Voorberg^* J.

‡AMC, Netherlands †Emma Children's Hospital AMC, Netherlands; ^*Sanquin Research at CLB, Netherlands;

Hemophilia A is a bleeding disorder caused by the functional absence of clotting factor VIII. A serious complication of replacement therapy is the formation of inhibitory antibodies to factor VIII that occurs in approximately 25% of patients with severe hemophilia A. Inhibitor formation in mild and moderate hemophilia A is rare. Several reports have described inhibitor development in patients with mild hemophilia A caused by an Arg593 to Cys mutation. At present, it is not clear why some patients with this genetic defect develop inhibitors and others do not. To mimic the immune response in these patients, a transgenic mouse expressing human factor VIII-R593C was generated. Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of mice carrying the transgene revealed that expression of human factor VIII-R593C was confined to the liver. HuFVIII-R593C mice were crossed with factor VIII-deficient E16KO mice. The immune response in huFVIII-R593C/E16KO mice was subsequently compared to that of E16KO mice. E16KO mice developed inhibitory antibodies following 5 serial intravenous injections with human factor VIII. Also significant proliferation of spleen-derived T cells was observed in E16KO mice. In contrast, huFVIII-R593C/E-16KO mice did not develop an immune response following intravenous injection of factor VIII. No factor VIII-dependent proliferation of T cells derived from the spleen of huFVIII-R593C/E16KO mice was observed. Analysis of plasma samples of these mice did not reveal the presence of antibodies reactive with human factor VIII. These results show that under these experimental conditions huFVIII-R593C/E16KO mice are tolerant for human factor VIII. Next, we evaluated antibody formation in huFVIII-R593C/E16KO and E16KO mice following subcutaneous administration of factor VIII in the presence of an adjuvant. In plasma of 50% of huFVIII-R593C/E16KO mice significant levels of antibodies were detected whereas all treated E16KO mice developed high titer antibodies against factor VIII. These data indicate that inflammatory conditions contribute to loss of tolerance in this mouse model of mild hemophilia A.