PERIPHERAL BLOOD STEM CELLS AS TARGETS FOR GENE TRANSFER

不能转导足够比例的干细胞库是 镰状细胞病基因治疗的一个主要限制。在这 项目我们试图通过以下方式克服这一障碍：a)确定 体外转导人造血干细胞的最佳条件 体外培养。新型细胞因子，如Flk-2配体，将在我们的 转导方案，以及底物(CS-1、VCAM-1或 纤维连接蛋白)，它可以提供逆转录病毒颗粒的集中病灶。 由于人类和狒狒的造血、转导具有相似性。 对人类最有利的条件将适用于我们的灵长类动物 模型；b)将确定灵长类动物的最佳动员条件 以使尽可能多的干细胞能够被采集用于EX 活体操作；c)反复动员和回输将 使用包含显性可选标记的向量来执行。在……里面 体内选择将使用一种药物应用于包含 标记物都是抗性的。将使用药物管理时间表， 旨在同时提供温和的条件反射和体内 选择；d)作为c)中的条件反射和选择的控制，重复 动员、转导和回输的循环，没有 将进行清髓或选择；e)相对效率 MLV与AAV为基础的载体转导长期再填充 细胞将在单个动物身上进行比较；以及f)疗效和 一类新型人泡沫病毒和慢病毒载体的安全性 将会受到考验。

The inability to transduce a sufficient fraction of the stem cell pool is a major limitation to gene therapy for sickle cell disease. In this project we attempt to overcome this obstacle by: a) identifying the optimal conditions for the transduction of human hemopoietic stem cells in vitro. Novel cytokines, such as flk-2 ligand, will be tested in our transduction protocols, as will the effects of substrata (CS-1, VCAM-1, or fibronectin), which may provide concentrated foci of retroviral particles. Due to the similarity between human and baboon hemopoiesis, transduction conditions that are optimal in humans will be applied to our primate model; b) Optimal mobilization conditions will be identified in primates so that the greatest possible number of stem cells can be harvested for ex vivo manipulation; c) Repeated cycles of mobilization and reinfusion will be performed using a vector containing a dominant selectable marker. In vivo selection will be applied using a drug to which cells containing the marker are resistant. A schedule of drug administration will be used that is intended to simultaneously provide mild conditioning and in vivo selection; d) As a control for conditioning and selection in c), repeated cycles of mobilization, transduction, and reinfusion, without myeloablation or selection will be performed; e) The relative efficiencies of MLV- versus AAV- based vectors to transduce long-term repopulating cells will be compared in individual animals; and f) the efficacy and safety of a new class of vectors based on human foamy virus and lentivirus will be tested.