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Our laboratory has focused on structure-function relationships of normal and mutant factors VIII and IX to identify interactive sites important for hemostasis in vivo. Dysfunctional hemophilia A & B mutations are substituted into molecular models and mutants based on homologous proteins expressed to identify functionally important surface sites on factors VIII or IX. Alloimmune and autoimmune responses are being characterized in terms of changes in B-cell and T-cell epitopes across time. A second major focus has been support for basic and clinical studies on gene therapy for the hemophilias.
Factor IX circulates as a single chain 415 aa zymogen at 90 nM. Its 34 kb gene in Xq26 has 8 exons, coding for signal and propeptides, the Gla domain, 2 EGF- domains, connecting and activation peptides and the catalytic (serine protease) domain (Bajaj & Thompson, 2005). The crystal structure of an inhibited porcine factor IXa is known. Nearly 700 distinct mutations have been reported in about 2000 families. In the Seattle series, over 120 distinct genotypes have been characterized in 99% of over 250 families with hemophilia B studied.
Factor VIII is a 2332 aa cofactor that circulates as a double chain precursor at 0.5-1 nM. Its 186 kb gene in Xq28 has 26 exons, coding for a signal peptide and a mature protein with A1-A2-B-A3-C1-C2 domains (Thompson, 2003). Cleavages by thrombin dissociate a von Willebrand factor (vWF) binding site and activate the cofactor. They crystal structure of ceruloplasmin, with homologous A1-3 domains, serves as a model as does the recent crystal structure of a partial bovine factor Va; over 800 distinct mutations have been reported (half, missense). The two C domains provide a surface for lipid and a secondary vWF binding site; the crystal structure of a recombinant C2 domain has been solved and from that, a model of the homologous C1 domain constructed (Liu et al, 2000). Allo- and auto antibodies have common epitopes, most frequently in the A2 and C domains and are under active investigation.
Nearly half of severe hemophilia A is due to recurrent gene inversions and over 150 families have been identified in the Seattle series (Liu et al, 1999); ~20% are associated with allo-antibody inhibitors in at least one affected member (Thompson, Nakaya & Johnson, 2006). In over 350 other families with hemophilia A, a variety of genotypes have been identified (Liu et al, 1998 & 2000). Baseline plasma testing for factor VIII antigen by a monoclonal ELISA allows distinction of the ~1/4th of patients with missense mutations that circulate excess, dysfunctional factor VIII antigen. These have provided a focus on the relationship between structural changes and their functional consequences. Selected mutations have been expressed in mammalian cells or in fragments. Partial gene deletions have undergone breakpoint analysis (Nakaya et al, 2004). We are scaling up expression of human C1C2 fragment (Hsu & Thompson, 2003, abstract) that, with data on C2 and the light chain of factor VIIIa will allow definition of phospholipid and von Willebrand factor binding sites, interactions with vitamin K-dependent factors (Liu & Thompson, 2000 & 2001, abstracts) and definition of inhibitory epitopes.
Little is known about which residues are involved as immunodominant epitopes for either B or T lymphocytes. We are preparing isolated domains of factor VIII with normal and surface mutant sequences to identify B cell epitope clusters (Pratt et al, 2005). Serial patient samples will be used to characterize the extent of epitope spreading and/or maturation as the response develops and may be predictive of responsiveness to therapy. We are studying T-cell epitopes in patient samples, including tetramer analysis to identify and isolate specific T-cell clones and determine the degree to which T-cell epitopes vary during an immune response to factor VIII.
A clinical trial of intramuscular delivery of an AAV-factor IX vector into hemophilia B patients was conducted in Philadelphia and Palo Alto (Manno et al, 2000) and included 2 of 8 subjects that have been followed in Seattle; safety was demonstrated but efficacy, if any, was marginal. A trial with liver delivery of a more potent construct with tissue-specific promoter developed by our collaborators in Seattle showed the first evidence of a significant response. Unfortunately, there was a delayed inflammatory response to the capsid protein that was not predicted in animal models leading to termination of the protocol. As alternatives to viral gene transfer, we have found prolonged expression of therapeutic levels of factor IX in deficient mice due to episomal liver expression of therapeutic levels of factor IX in deficient mice due to episomal liver expression (Miao et al, 2000 & 2001) although the rapid, hydrodynamic delivery method used would not be suitable for humans. This technique also works to deliver factor VIII to hemophilia A mice and that provides an animal model for evaluating the immune response to factor VIII (Ye et al, 2004).
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