TRANSDUCTION OF BABOON HEMATOPOIETIC STEM CELLS

该项目的目标是开发策略，以达到临床相关水平的基因转移到造血干细胞，目的是治疗遗传性疾病的造血干细胞及其后代。到目前为止，单独的体外刺激，即使有多种生长因子，也不能增强对造血干细胞的基因转移。我们假设，就像在小鼠身上一样，有必要诱导干细胞增殖以达到基因转移的治疗水平。在小鼠中，在采集骨髓之前用5-FU治疗动物是必要的，以实现高效的基因转移到骨髓再生干细胞。我们假设，用细胞因子组合和/或细胞毒性药物干扰骨髓的治疗将引起干细胞的增殖，从而使它们更容易受到逆转录病毒或AAV介导的基因转移的影响。在这些研究过程中，我们将比较不同来源的CD34+干细胞对不同载体转导的敏感性。我们将确定在采集骨髓和血液CD34+干细胞之前用造血生长因子治疗动物是否会改变向骨髓再生细胞的基因转移。我们将检测来自不同来源(骨髓、血液、脐带血)的转基因CD34+细胞在体内重建淋巴造血的能力。这些研究确定了将基因有效转移到狒狒造血干细胞所需的条件，这将提供进一步的见解，直接适用于人类的基因治疗。

The goal of this project is to develop strategies for achieving clinically relevant levels of gene transfer into hematopoietic stem cells for the purpose of treating inherited disorders of hematopoietic stem cells and their progeny. To date, in vitro stimulation alone, even with multiple growth factors, has not resulted in enhanced gene transfer into hematopoietic stem cells. We hypothesize that, as in mice, it will be necessary to induce stem cell proliferation to achieve therapeutic levels of gene transfer. In mice it is essential to treat animals with 5-FU prior to harvesting marrow to achieve efficient gene transfer into marrow repopulating stem cells. We hypothesize that treatment with cytokine combinations and/or cytotoxic drugs to perturb marrow will elicit proliferation of stem cells, thereby rendering them more susceptible to retroviral or AAV mediated gene transfer. In the course of these studies we will compare CD34+ stem cells from different sources for susceptibility to transduction by different vectors. We will determine if treating animals with hematopoietic growth factors prior to harvesting marrow and blood CD34+ stem cells will alter gene transfer into marrow repopulating cells. We will determine the ability of genetically altered CD34+ cells from different sources (marrow, blood, cord blood) to reconstitute lymphohematopoiesis in vivo. These studies to define the conditions necessary to achieve effective gene transfer into baboon hematopoietic stem cells will provide further insights that will be directly applicable to gene therapy in humans.