Fanconi anemia (FA) is a recessive disease characterized by nearly universal progressive bone marrow failure and a constellation of serious physical anomalies. There is a high rate of fatality in childhood, most frequently from complications of aplasia within an average of 5 years from diagnosis. Supportive care, in terms of platelet and erythrocyte transfusions, the use of androgens to stimulate hematopoiesis in vivo, and the use of HLA-identical sibling transplants (available to a minority of patients) has extended the life- expectancy of FA patients, however. However, taking into account the incidence of FA disease in subsequent siblings, the availability of potential HLA-identical sibling donors is <30%. Matched unrelated donor transplants remains highly experimental in this disease with a survival after an unrelated donor transplant as low as 30%. Identification of donors, graft failure and graft versus host disease remain significant obstacles. FA cells have a defect in DMA repair that leads to increased spontaneous chromosomal breakage. This feature increases the sensitivity of FA cells to DNA bifunctional cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). The diagnosis of FA now relies upon detecting increased chromosomal breakage after exposure in vitro to DEB. Similarly, cells cultured from patients with FA display increased susceptibility to the cytotoxicity of mitomycin C. More recently, cells from patients with FA have been demonstrated to display G2 phase prolongation arrest, increased sensitivity to oxygen, defective p53 induction and increased apoptosis. FA can be classified into at least eleven complementation groups by somatic cell hybrids. The complementation is based upon correction of the chromosomal sensitivity to cross-linking agents in hybrid cells. Nine independent genes have been cloned and characterized within these 11 complementation groups (A-C, D1, D2 and E-G, and L). Transgenic expression of these genes in the respective FA cells in vitro corrects the increased chromosomal breakage from DEB and the increased sensitivity to MMC. In addition, expression of these genes in bone marrow progenitors from patients with FA increases cell survival in in vitro assays. Recent studies have demonstrated that clinical progression of the disease may be influenced by inter- and intra-genic variations, suggesting that complementation assignment and mutation identification will be increasingly important for clinical management in the future. In this proposed project, we seek to develop a comprehensive approach to stem cell collection, complementation assignment/mutation analysis, and genetic modification of FA patients (by gene therapy) in order to effectively treat patients of complementation groups A, C, and G, which make up -90% of North American patients. These studies take advantage of our long-standing expertise in retrovirus-mediated gene transfer, an emerging large group of FA patients that are being seen in the Cincinnati Children's Hospital Medical Center (CCHMC) Fanconi Anemia Comprehensive Care Clinic (FACCC) well before the onset of severe aplasia and the potential for using complementation group ascertainment as a screening method to direct high-throughput methods for mutation analysis. A stem cell collection and a gene therapy trial have been developed, approved by the Institutional Review Board of CCHMC, the NIH Recombinant DNA Advisory Committee (RAC) and Investigational New Drug applications have been approved for these trials by the Food and Drug Administration. This grant proposal seeks funding to conduct these clinical trials and further develop retrovirus-mediated complementation analysis in a CAP/CLIA environment for use in clinical management of FA patients.
