This project seeks to develop a comprehensive understanding of the immune response to coagulation factors using normal and hemophilic animals that are used in pre-clinical testing to assess the immunogenicity of new coagulation factor products. The animal models under study in this project include spontaneous hemophilia A and B dogs, factor VIII and factor IX knockout mice, and various normal mice that have been genetically defined in great detail. Thus far, I have demonstrated that the Chapel Hill hemophilia A dogs have a severe bleeding phenotype that is the result of a gene inversion that mimics the factor VIII gene inversion seen in ~40% of humans with severe hemophilia A. Similar factor VIII gene inversions have now been observed in other hemophilia A dogs, which also parallels the observed repeated de novo incidence of this mutation in humans. As in humans, bleeding in hemophilia A dogs is treated with factor VIII (as cryoprecipitate) and in some animals inhibitor antibodies may develop. Thus, the Chapel Hill hemophilia A dog colony is a potential model for study of the inhibitor phenomenon associated with administration of factor VIII. In related studies, I have mapped genes that influence the antibody response to coagulation factor IX in mice. The results of these studies indicate that the mouse MHC H2 locus (on chromosome 17) has a highly significant association with antibody formation (LOD score >4.5), and other genes on chromosomes 10 and 1 also contribute to the antibody response. The current focus of this project is to establish which genes in addition to the MHC H2 locus contribute to the pre-disposition for antibody development, and to begin a survey of canine histocompatibility genes in dogs with hemophilia A that have inhibitor antibodies. The genetic defect in the hemophilia A dogs is under further study so as to define the breakpoint with sufficient specificity that a rapid (PCR-based) genetic diagnostic test can be developed. Such a test will permit rapid identification of the gene defects in as many dogs as can be ascertained with hemophilia A. This work is being done in collaboration with researchers at the University of North Carolina at Chapel Hill where the hemophilia A dog colony is maintained. During the past year I have published a study that describes the gene inversion that is responsible for hemophilia A in a dog colony in Chapel Hill, North Carolina. The key features of this animal model are that the factor VIII levels are undetectable and the bleeding phenotype is identical to that of humans with severe hemophilia A (e.g., spontaneous bleeding in joints and soft tissues, prolonged bleeding with trauma and surgery, and prolonged in vitro clotting times of plasma). The key findings of my research were that the hemophilia A dog colony in Chapel Hill has an inversion that is identical in mechanism to a common mutation seen in humans with hemophilia A. Further, the local sequence of DNA is one which may be amenable to correction by mRNA splicing, and is identical to that found in humans. This animal model could therefore be used to test mRNA splicing constructs that could be used in humans to correct the common inversion that causes hemophilia A. During the past year I have published with my CBER collaborators and external collaborators the results of studies of the effects of adenovirus vectors on non-human primates. These results indicate dose-dependent toxicity that manifests as abnormalities of the coagulation system, including abnormalities of fibrinogen, prolongation of clotting times, and thrombocytopenia. In addition, there are increases in high-molecular weight von Willebrand factor multimers which may mediate the platelet consumption that takes place. The observation that these abnormalities occur with a relatively innocuous adenovirus vector (at high doses) suggests that this animal model might be studied to assess the beneficial effects of certain biologicals (particularly activated protein C) which have proven to be clinically useful for patients with bacterial sepsis and disseminated intravascular coagulation. If such agents could be shown to have a protective effect against the toxic effects seen with certain viral infections, then it may prove worthwhile to study the interactions to see if they might be applicable to therapy of certain viral infections characterized by DIC (e.g., those caused by dengue, ebola and other hemorrhage-inducing viruses). If data to this effect can be developed in the upcoming year, I may introduce a separate proposal to study these agents in the context of counter-bioterrorism efforts. During the last year I have begun a study whose aim is to map genetic loci in mice that control the immune response to human factor IX. As it turns out, there are inbred strains of mice (i.e., C57BL/6J and A/J) that have dramatically different antibody responses to human factor IX when given as a protein (without adjuvant) or in the context of gene therapy vector delivery. The value of this observation is that there are inbred mice which are derived from these parental strains and which are mapped very extensively with regard to genetic polymorphisms found in the mouse genome. These well-characterized mice have been immunized with an adenovirus vector expressing human factor IX and scored for the immune response. An interim analysis indicates that marker D17Mit62, which is immediately adjacent to the mouse MHC H2 locus, has a highly significant association with the immune response (LOD score >4.5) in both males and females. For both males and females there is suggestive linkage (LOD scores > 2.5) for markers on chromosome 10. It remains to be seen what candidate gene is near the chromosome 10 markers that have an association with the immune response. These loci can be investigated for their relevance to the immune response to factor IX in humans with hemophilia B and inhibitor antibodies. The importance of this is that inhibitor antibodies are a well-known and feared complication of conventional therapy with clotting factor concentrates and a feared and for now hypothetical complication of gene therapy of hemophilia B.
