PERIPHERAL BLOOD STEM CELLS AS TARGETS FOR GENE TRANSFER

不能使足够部分的干细胞池再生是一个问题。 这是镰状细胞病基因治疗的一个主要限制。在这 我们试图通过以下方式克服这一障碍：a）确定 人造血干细胞转导的最佳条件 体外新的细胞因子，如flk-2配体，将在我们的 转导方案，如底物（CS-1，VCAM-1，或 纤连蛋白），其可以提供逆转录病毒颗粒的集中病灶。 由于人类和狒狒造血的相似性， 对人类最有利的条件将被应用到我们的灵长类动物身上 模型; B）将在灵长类动物中确定最佳动员条件 这样就可以收获尽可能多的干细胞， 体内操作; c）反复循环的动员和再输注将 使用含有显性选择标记的载体进行。在 将使用药物进行体内选择， 标记是耐药的。将使用药物给药时间表， 旨在同时提供温和的调理和体内 d）作为c）中调节和选择的对照，重复 动员、转导和再输注的循环， 将进行骨髓消融或选择; e）相对效率 基于MLV和AAV的载体的比较， 将在个体动物中比较细胞;和f）功效和 基于人泡沫病毒和慢病毒的一类新载体的安全性 会得到考验

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.