Gene Transfer of Hematopoietic Stem Cells

描述(摘自申请者的摘要)：为了高效的基因 造血干细胞的转移是要实现的，我们相信检测 需要新的载体和更好地定义干细胞种群。我们有 选择Fanconi贫血(FA)作为促进基因改良的模型疾病 造血细胞的移植方案。FA是一种罕见的常染色体隐性遗传 以骨髓衰竭为主要特征的疾病及其发展 白血病的症状。FA的血液学表现是由于干的紊乱 细胞功能。FANC蛋白(FANC互补基团)的功能 A-H)尚不清楚，但所有FANC细胞对DNA都表现出超敏反应 交联剂和建议在维持DNA稳定性方面的作用。虽然我们 证明了经基因校正的FA具有选择性生长优势 基因敲除模型中的造血细胞，校正后的干细胞群体 需要进一步的定性。在这里，我们建议对基因进行鉴定和测试 原始人造血干细胞分离部分的转移 来自小鼠和人类的生理学方法而不是免疫学方法。这 纯化方案分离了前面描述的新的侧群 小鼠和人造血细胞中的组份(SP)。我们的战略是 分离和转导范可尼贫血互补组A(FANCA)和C (FANCC)敲除小鼠原始干细胞。将对接受动物进行检查 它们对DNA损伤剂和细胞因子的反应以及它们 用Moloney-小鼠基因转移重建造血功能 逆转录病毒和艾滋病毒、马和猫为基础的慢病毒载体。隔离 将对FA患者的人CD34+、CD34+/CD38-和SP组分进行检测 NOD/SCID免疫缺陷小鼠逆转录病毒载体的转导 系统。目前，我们正在进行一项针对FANCA患者的试验。信息 从计划中获得的研究将提供更好的理解 FA中的异常造血和更好地确定治疗策略的重要性 用于设计未来的人类临床试验。

DESCRIPTION(adapted from applicant's abstract): In order for efficient gene transfer of hematopoietic stem cells to be achieved we believe that the testing of new vectors and better defined stem cell populations are required. We have chosen Fanconi anemia (FA) as a model disease to facilitate improved gene transfer protocols of hematopoietic cells. FA is a rare autosomal recessive disorder characterized principally by bone marrow failure and the development of leukemia. The hematologic manifestations of FA are due to a disorder of stem cell function. The functions of the FANC proteins (FANC complementation groups A-H) are not understood but all FANC cells exhibit hypersensitivity to DNA crosslinkers and suggest a role in maintaining DNA stability. Although we demonstrated that a selective growth advantage exists in gene-corrected FA hematopoietic cells in a knockout model, the corrected stem cell population requires further characterization. Here we propose to identify and test gene transfer on isolated fractions of primitive hematopoietic stem cells based on physiologic rather than immunologic methods from both mouse and human. This purification scheme isolates the previously described novel side population fraction (SP) in both mouse and human hematopoietic cells. Our strategy is to isolate and transduce Fanconi anemia complementation group A (FANCA) and C (FANCC) knockout mice primitive stem cells. Recipient animals will be examined for their response to DNA damaging agents and cytokines and their ability to reconstitute hematopoiesis following gene transfer using moloney-murine retroviral and HIV, equine and feline-base lentiviral vectors. Isolation of human CD34+, CD34+/CD38- and SP fractions from FA patients will be tested for transduction by retroviral vectors using the NOD/scid immunodeficient mouse system. Currently we have an ongoing trial for FANCA patients. Information obtained from the planned studies will provide a better understanding of abnormal hematopoiesis in FA and better define therapeutic strategies important for designing future human clinical trials.