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，异体干细胞移植可以纠正许多这些疾病。因此，该项目的目标是增强基因转移到狒狒骨髓再生细胞中，作为干细胞移植的相关非人类灵长类动物临床前模型。利用竞争性再种群策略研究与狒狒基因转移相关的不同变量，我们已经证明：1)长臂猿白血病病毒-伪型载体；2)利用重组人纤维连接蛋白片段CH-296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引物（动员）外周血和骨髓祖细胞中增强。然而，尽管在移植后最初阶段血液中的瞬时标记水平在10%到20%之间，但对于大多数遗传疾病，长期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移植方面需要进一步的改进。我们建议建立可有效转导的再生细胞动员的最佳方法，并确定是否可以在不抑制转导细胞在体内再生的情况下减少条件作用。此外，由于非人类灵长类动物模型在后勤上受到限制，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，本项目将扩展其他项目的研究成果。研究将集中在新的杂交载体转导骨髓再生细胞的潜力，以及RAR和Notch受体家族的配体增强基因转移到这些再生细胞中的潜力。非人类灵长类动物提供了一个高度相关的模型，这些策略可以在引入人体临床试验之前直接进行测试。

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.