Summary

Inhibitory antibodies develop in approximately 25% of patientswith severe hemophilia A following treatment with factorVIII. InE-16KO or E-17KO mice, in which the factor VIII gene has beeninactivated by insertion of a neo cassette, inhibitors develop followingadministration of factor VIII. Here, we describe the generationof transgenic mice expressing human factor VIII-R593C(huFVIII-R593C). Human factor VIII-R593C cDNA under controlof a mouse albumin enhancer/promoter was injected intofertilized oocytes. Analysis of transgenic mice revealed thathuman factor VIII-R593C was expressed in the liver.Transgenicmice were crossed with factor VIII-deficient mice (E-16KOmice). In plasma of E-16KO mice antibodies were detected afterfive serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levelsof antibodies. No antibody secreting cells were observed ineither spleen or bone marrow of huFVIII-R593C/E-16KO mice.Also, factor VIII-specific memory B cells were not observed inthe spleen of huFVIII-R593C/E-16KO mice. Analysis of T cellresponses revealed that splenocytes derived of E-16KO micesecreted IL-10 and IFN-? following restimulation with factorVIIIin vitro . In contrast, no factor VIII-specific T cell responses wereobserved in huFVIII-R593C/E-16KO mice.These results indicatethat huFVIII-R593C/E-16KO mice are tolerant to intravenouslyadministered factor VIII. It is anticipated that this model mayprove useful for studying immune responses in the context offactor VIII gene therapy.