GENE TRANSFER AND FUNCTIONAL STUDIES OF FANCONI ANEMIA

点击翻译按钮获取中文摘要

Fanconi anemia (FA) is defined by hypersensitivity to bifunctional alkylating agents (MMC, DEB), progressive pancytopenia, and cancer susceptibility. Initially thought to be strictly a disorder of DNA repair, studies on the function of FA type C (FAC) have suggested possible roles of the gene in cell cycle regulation and hematopoiesis as well. Early gene therapy experiments with type C show a survival advantage of corrected progenitor cells, suggesting that FA may provide an excellent model to study hematopoietic stem cell gene therapy. We have obtained cDNA of the recently cloned FA type A gene (FAA, 65% of FA patients). The focus of this grant proposal will be to optimize gene transfer of retroviral vectors developed in collaboration with Dr. Richard Mulligan's lab and to use the vectors and other tools to study FAA function. First, using aliquots of peripheral blood progenitor cells (PBPC) and bone marrow from FA patients enrolled in a stem cell mobilization protocol, we will optimize FAA retroviral gene transfer protocols to establish high-level transduction into long term repopulating hematopoietic stem cells (LTR-HSC) as assayed by in vitro and in vivo assays. Second, we will make FAA antiserum and use it to screen for FAA mutations in FA cell lines and patient samples. We will also use the antiserum to identify the cellular localization of the FAA polypeptide, assay for cell-cycle specific expression, and assess for co-imunoprecipitation with FAC and other cellular proteins. Finally, we will create a series of truncation mutants of the FAA protein and test their function in FA cell lines, attempting to correlate different functional mutations with varied FA phenotypes. Basic understanding of FAA function and creation of retroviral vectors for gene therapy may lead toward the development of FAA clinical gene therapy trials. This proposal blends well with a strong institutional commitment to the field of gene therapy, guided by Dr. Nathan, a co-sponsor and PI of a hematopoietic gene therapy SCOR grant. The mentors chosen to supervise this grant have an outstanding record of training skilled scientists. Dr. Sieff, Dr. Mulligan, and Dr. D'Andrea are acclaimed researchers in the fields of human hematopoiesis, gene therapy, and Fanconi anemia respectively, and the strengths of each lab will be combined with this project. My clinical background in pediatric hematology and previous experience studying human hematopoiesis and gene transfer have prepared me to focus on FA gene transfer and function. The proposed grant period, combines rigorous didactic courses with close oversight of an intensive lab experience, allowing me to develop into a skilled, independent researcher.