GENE TRANSFER AND FUNCTIONAL STUDIES OF FANCONI ANEMIA

范可尼贫血（FA）的定义是对双功能抗体超敏反应。 烷化剂（MMC、DEB）、进行性全血细胞减少症，以及 癌症易感性最初被认为是严格意义上的 DNA修复，对FA C型（FAC）功能的研究 提示该基因在细胞周期调控中可能的作用， 造血也是。C型糖尿病的早期基因治疗实验 显示了纠正的祖细胞的生存优势，表明 FA可能为研究造血功能提供一个极好模型 干细胞基因治疗我们已经获得了最近的cDNA 克隆的FA A型基因（FAA，65%的FA患者）。的焦点 这项拨款提案将用于优化逆转录病毒的基因转移， 与Richard Mulligan博士的实验室合作开发的载体 利用向量等工具研究FAA功能。第一、 使用外周血祖细胞（PBPC）和骨的等分试样 来自参加干细胞动员的FA患者的骨髓 我们将优化FAA逆转录病毒基因转移方案， 建立高水平转导进入长期再增殖 造血干细胞（LTR-HSC），如通过体外和体内测定的 体内测定。第二，我们将制造FAA抗血清， 筛选FA细胞系和患者样品中的FAA突变。我们 也将使用抗血清来确定细胞定位的 FAA多肽，细胞周期特异性表达的测定，和 评估与FAC和其他细胞的免疫共沉淀 proteins.最后，我们将创建一系列的截短突变体， FAA蛋白，并在FA细胞系中测试它们的功能， 将不同的功能突变与不同的FA 表型基本了解FAA的功能和创建 用于基因治疗的逆转录病毒载体可能会导致 FAA临床基因治疗试验的发展。 这一建议与一项强有力的机构承诺很好地结合在一起， 基因治疗领域，由Nathan博士指导，共同赞助商和 造血基因治疗SCOR基金的PI。导师 被选中监督这项补助金的人都有出色的培训记录 熟练的科学家。Sieff医生Mulligan医生和D 'Andrea医生 在人类造血、基因、 治疗，范可尼贫血分别，和每一个强度 实验室将与该项目结合起来。我的临床背景是 儿科血液学和既往研究人类的经验 造血和基因转移使我准备专注于FA 基因转移和功能。拟议的赠款期限，结合 严格的教学课程与密集的实验室密切监督 经验，让我发展成为一个熟练的，独立的 研究员

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.