Gene Transfer of Hematopoietic Stem Cells

描述（改编自申请人的摘要）：为了获得有效的基因 造血干细胞移植的实现，我们认为， 需要新的载体和更好定义的干细胞群。我们有 选择范可尼贫血（FA）作为模型疾病，以促进基因改良， 造血细胞的转移方案。FA是一种罕见的常染色体隐性遗传病 以骨髓衰竭为主要特征的疾病， 白血病FA的血液学表现是由于干细胞异常引起的， 细胞功能FANC蛋白的功能（FANC互补基团 A-H）不清楚，但所有FANC细胞都表现出对DNA的超敏性 交联剂，并建议在维持DNA稳定性的作用。虽然我们 证明了基因校正的FA存在选择性生长优势， 在敲除模型中，校正的干细胞群体 需要进一步的表征。在这里，我们建议识别和测试基因 转移分离的原始造血干细胞组分， 生理学方法而不是免疫学方法。这 纯化方案分离了先前描述的新的侧群 在小鼠和人类造血细胞中，我们的策略是 分离和纯化范可尼贫血互补群A（FANCA）和C （FANCC）敲除小鼠原始干细胞。将对动物进行检查 对DNA损伤剂和细胞因子的反应， 使用莫洛尼鼠基因转移后重建造血 逆转录病毒和HIV、马和猫慢病毒载体。分离 将检测来自FA患者的人CD 34+、CD 34 +/CD 38-和SP组分， 使用NOD/scid免疫缺陷小鼠通过逆转录病毒载体转导 系统目前，我们正在对FANCA患者进行试验。信息 从计划的研究中获得的信息将使人们更好地了解 FA中的异常造血和更好地确定治疗策略很重要 设计未来的人体临床试验。

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.