CHILDREN'S HOSPITAL CORPORATION, THE

Fanconi anemia (FA) is a rare recessive syndrome characterized by bone marrow failure, congenital anomalies and a predisposition to malignancy. FA cells have a defect in DNA repair that leads to increased spontaneous chromosomal breakage. This feature increases the sensitivity of FA cells to DNA bifunctional cross-linking agents such as MMC and DEB. Relating directly to this application, increasing evidence including from the Principal Investigators (PI) of this proposal suggests FA cells have specific defects in non-homologous end joining (NHEJ) and homologous recombination (HR). Transgenic expression of FA genes in genetically deficient cells in vitro corrects the phenotypic abnormalities of FA cells. Bone marrow transplantation has cured some patients of their bone marrow failure or hematologic malignancies. However, there is increased toxicity due to the conditioning regimens compared to patients without the disorder. Two clinical gene transfer trials for patients with FA have been reported, including one by a PI of this proposal. In this study, we demonstrated that gene transfer per se is no longer a limitation to the effectiveness of this approach, but that even early in the disease process there are significant deficiencies in the number and/or function of hematopoietic stem cells (HSCs) that can be collected and utilized as targets in gene transfer. Thus, new therapeutic approaches are needed to treat this disease. One approach that could address this deficiency is the derivation of HSC from induced pluripotent stem cells (iPS) reprogrammed from other somatic tissues. Important advantages of iPS cells for therapy are the unlimited proliferative capacity of such cells (allowing production of large numbers of HSC) and the ability to clone such cells, which allows precise single cell correction of genetic mutations and prospective molecular characterization of manipulated cells prior to therapeutic use. The reprogramming of a panel of disease specific human iPS cells has been accomplished by a PI of this proposal, but the reprogramming of somatic Fanconi anemia cells has proven difficult. This is likely related to the defects in FA NHEJ or HR and to recent reports of involvement of the p53 DNA damage response pathway in reprogramming. Indeed, we have recently found a link between DNA damage and cytokinesis failure in FA that may play a critical role in the bone marrow failure phenotype. Thus, overall, we believe that understanding FA DNA damage response, which is intimately involved in the FA cellular phenotype and appears to be involved also in somatic cell reprogramming, is critical to development of novel cell, genetic and small molecule-based therapies in FA. The three projects in this multi-investigator proposal are linked via a common goal of acquiring increased understanding of reprogramming technology and DNA repair pathways as involved in FA cells and application of this new knowledge for rapid development of new therapeutic approaches to the aplastic anemia of FA utilizing state-of-the-art basic technology. PUBLIC HEALTH RELEVANCE: This study will develop new methodologies to correct genetic disease. We aim to gain new insights into the biology of Fanconi anemia, the most common inherited bone marrow failure syndrome. Our study will explore the potential of a very promising novel cell type, called induced pluripotent stem cells (iPS), to provide unique insights into Fanconi anemia biology and to enable the development of novel treatment modalities for this severe disorder.

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