TRANSDUCTION OF BABOON HEMATOPOIETIC STEM CELLS

基因治疗在造血系统遗传性疾病中的有效应用在很大程度上仍未实现。临床研究表明，移植异基因干细胞可以纠正许多此类疾病。因此，该项目的目标是加强基因转移到狒狒的骨髓再生细胞中，作为一种相关的、非人类灵长类的干细胞移植的临床前模型。利用竞争性再繁殖策略研究了与基因转移相关的不同变量，我们证明了1)长臂猿白血病病毒-伪型载体；2)通过从重组人纤维连接蛋白片段CH-296上培养细胞可以实现骨髓再生细胞的高效转导；3)逆转录病毒介导的基因转移在G-CSF+SCF启动(动员)的外周血和骨髓祖细胞中得到增强。然而，尽管移植后最初阶段血液中的标记水平在10%到20%之间，但对于大多数遗传病来说，长期的基因转移水平仍然不够高。因此，在长期再生干细胞的转导和植入方面需要进一步的改进。我们建议建立动员可有效转导的再生细胞的最佳方法，并确定是否可以在不抑制转导细胞体内再生的情况下减少条件反射。此外，由于非人类灵长类动物模型在逻辑上是有限的，我们将确定是否使用免疫缺陷小鼠模型提供相同的信息。最后，该项目将扩展其他项目的研究成果。研究将集中在新型杂交载体转导骨髓再生细胞的潜力，以及RAR和Notch受体家族的配体加强基因转移到这些再生细胞中的潜力。非人灵长类动物提供了一个高度相关的模型，在该模型中，这些策略可以在引入人类临床试验之前进行直接测试。

The effective application of gene therapy to heritable diseases of the hematopoietic system remains largely unrealized. Clinical studies demonstrate that the transplantation of allogeneic stem cells can correct many of these diseases. Therefore the goal of this project is to enhance gene transfer into marrow repopulating cells in baboons as a relevant, non-human primate, pre-clinical model for stem cell transplantation. Using a competitive repopulation strategy to study different variables related to gene transfer in baboons we have demonstrated 1) that Gibbon Ape Leukemia Virus-pseudotype vectors; 2) that efficient transduction of marrow repopulating cells can be accomplished by culturing cells from over the recombinant human fibronectin fragment CH-296; 3) that retrovirus mediated gene transfer is enhanced in G-CSF plus SCF primed (mobilized) peripheral blood and marrow progenitors. However, in spite of transient levels of marking in blood of between 10% and 20% during the initial post-transplant period, long-term gene transfer levels still are not high enough for most genetic diseases. Therefore, further improvements are needed in the transduction and then the engraftment of long-term repopulating stem cells. We propose to establish optimal methods for mobilization of repopulating cells that can be efficiently transduced and determine whether conditioning can be reduced without inhibiting in vivo repopulation by transduced cells. In addition, as the non-human primate model is logistically limited, we will determine whether identical information is provided using an immune-deficient mouse model. Finally, this project will extend findings from other projects. Studies will focus on the potential of novel hybrid vectors to transduce marrow repopulating cells and for ligands for the RAR and Notch families of receptors to enhance gene transfer into these repopulating cells. Non-human primates provide a highly relevant model in which these strategies can be directly tested before being introduced into human clinical trials.