MOLECULAR BASIS AND THERAPY OF HEMOPHILIA A

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The broad long-term goals of this laboratory are 1) to understand the factor Vlll gene and the manner by which mutations in this gene give rise to hemophilia A, and 2) to contribute to improved therapy for hemophilia patients by developing new DNA-based genetic tests and by producing a laboratory animal model for hemophilia A. The preliminary results germane to the proposed research include the following: the isolation of the human factor Vlll gene; the discovery of two putative genes within the factor Vlll gene; the description of two classes of hemophilia-causing mutations; and the development of sensitive methodologies for rapid genetic prediction of hemophilia A and detection of mutations. The four specific aims of this research are the following: 1) To discover and determine the base change(s) of hemophilia- causing mutations in coding, regulatory and exon-splicing sequences of the factor Vlll gene. Genomic DNA sequences from hemophiliacs will be amplified and screened for mutations by denaturing gradient gel electrophoresis. The nature of the mutation will be examined in light of the patient's factor Vlll activity, antigen, and clinical phenotype. One important outcome of this research will be to determine the origin of the mutations in the many cases of hemophilia that arise de novo. 2) To discover DNA sequence polymorphisms in and flanking the factor Vlll gene in order to improve genetic diagnosis, and to develop a rapid non-radioactive assay for these so that genetic diagnosis can be made readily available throughout the world. The sequence polymorphisms will also be discovered by denaturing gradient gel electrophoresis. 3) To determine the structure and function of the two genes that appear to lie within the factor Vlll gene, particularly regarding their relationship to factor Vlll itself. This will be achieved mainly by cDNA cloning, mapping and sequencing. 4) To create a laboratory animal model for hemophilia A by inactivating the murine factor Vlll gene. Transgenic mice will be produced from embryonic stem cells into which a defective factor Vlll gene has been introduced by homologous recombination. In the future, these mice will be used for in vivo testing of new factor Vlll products and therapies, as well as for somatic cell gene therapy experiments.