IN VIVO SELECTION OF TRANSDUCED HEMATOPOIETIC STEM CELLS

虽然造血干细胞基因治疗提供了一种潜在的手段来治疗各种各样的遗传性疾病，影响血细胞，其临床效用已受到低效的基因转移到再生干细胞。为了解决这个重要的限制，我们已经开发了一个系统，在体内选择的遗传修饰的造血干细胞。该策略需要使用突变的二氢叶酸还原酶（DHFR）基因作为显性选择标记。然后通过在移植后向动物施用基于新型抗叶酸的药物组合来选择含有表达DHFR基因的逆转录病毒载体的干细胞。我们已经表明，该系统能够在移植小鼠中显著富集转导的干细胞。我们现在建议通过追求两个具体目标来进一步开发用于临床基因治疗的该系统。在第一个具体目标中，我们将确定转导的干细胞在选择后是否经历自我更新分裂，以及强制自我更新是否会增加选择的效率。还将分析诱导的干细胞循环对选择效率的影响。这些新的信息将在一个可能改进的选择方案中进行测试，以确定转导的干细胞是否可以在非骨髓清除的宿主中扩增。第二个具体目标是开发用于非人类灵长类动物的系统。一个有效的方案转导干细胞将系统地在移植恒河猴。将进行一组单独的实验，以确定适当的药物选择方案。然后，这些结果将被纳入实验，以测试是否可以在这种临床前模型中获得干细胞选择。我们预计，这些研究将充分确定这种方法的临床可行性，并将产生广泛相关的干细胞基因治疗的数据。

Although hematopoietic stem cell gene therapy offers a potential means to treat a wide variety of genetic diseases that affect blood cells, its clinical utility has been hampered by inefficient gene transfer into repopulating stem cells. To address this important limitation, we have developed a system for the in vivo selection of genetically modified hematopoietic stem cells. This strategy entails using a mutated dihydrofolate reductase (DHFR) gene as a dominant selectable marker. Stem cells containing a retroviral vector expressing the DHFR gene are then selected by administered a novel anti-folate-based drug combination to animals after transplant. We have shown that this system enables significant enrichment of transduced stem cells in transplanted mice. We now propose to further develop this system for clinical gene therapy by pursuing two specific aims. In the first specific aim, we will determine if transduced stem cells undergo self-renewal divisions after selection, and if enforced self-renewal will increase the efficiency of selection. The effects of induced stem cell cycling on selection efficiency will also be analyzed. This new information will then be tested in a potentially improved selection protocol to determine if transduced stem cells can then be amplified in non-myeloablated hosts. The second specific aim will develop this system for use in non-human primates. An effective protocol for transducing stem cells will be systematically derived in transplanted Rhesus monkeys. A separate set of experiments will be undertaken to define an appropriate drug selection scheme. These results will then be incorporated into experiments that will test if stem cell selection can be obtained in this preclinical model. We anticipate that these studies will fully determine the clinical feasibility of this approach, and will yield data broadly relevant to stem cell gene therapy.