GENE THERAPY IN HEMATOPOIETIC CELLS

该项目的目标是开发造血干细胞基因治疗影响造血系统的疾病。最近的一项研究表明，造血干细胞基因治疗在X连锁严重联合免疫缺陷儿童中的应用是成功的。虽然这些结果对于基因治疗领域来说是非常令人鼓舞的，但大多数遗传疾病对于基因校正的细胞没有选择性优势，因此不太可能通过目前可用的技术治愈。需要进一步提高基因转移效率和使用毒性较小的非清髓性预处理方案植入转导细胞。我们已经使用狗模型来研究基因转移到造血再生细胞中，因为我们在该模型中进行造血干细胞移植的长期经验，并且因为疾病模型的可用性。在之前的资助期间，我们通过以下方式改进了向造血再生细胞中的基因转移：（1）使用巨猿白血病病毒（GALV）包膜，（2）在包被有人纤连蛋白片段CH-296的烧瓶中转导富含CD 34的细胞，以及（3）使用包括犬干细胞因子（cSCF）的生长因子组合，犬粒细胞集落刺激因子（cG-CSF）和人FLT 3-L。虽然基因标记，特别是在非清髓性设置。因此，我们对这个项目的目标有三个方面。首先，在具体目标1-3中，我们建议研究进一步提高造血干细胞中基因转移率的技术。第二，具体目标4和5将探索毒性较小的预处理方案与免疫抑制剂的组合，并研究一种新的体内选择系统。第三，具体目标6和7将基因转移技术应用于疾病模型。范可尼贫血已被选为造血干细胞基因治疗的第一个目标，因为在这种疾病中，基因校正的干细胞被认为具有选择性优势，超过未校正的干细胞。

The goal of this project is to develop hematopoietic stem cell gene therapy for diseases affecting the hematopoietic system. A recent study in children with X-linked serve combined immunodeficiency has shown the successful application of hematopoietic stem cell gene therapy. While these results were very encouraging for the gene therapy field, most genetic diseases do not have selective advantages for gene-corrected cells, and are therefore not likely to be cured by currently available techniques. Further improvements in gene transfer efficiency and in the engraftment of transduced cells using less toxic non-myeloablative conditioning regimens will be required. We have used the dog model to study gene transfer into hematopoietic repopulating cells because of our long-standing experience with hematopoietic stem cell transplantation in this model and because of the availability of disease models. During the previous funding period, we have improved gene transfer into hematopoietic repopulating cells by (1) using a gibbon ape leukemia virus (GALV) envelope, (2) transducing CD34-enriched cells in flasks coated with the human fibronectin fragments CH-296, and (3) using a growth factor combination which included canine stem cell factor (cSCF), canine granulocyte-colony stimulating factor (cG-CSF) and human FLT3-L. Although gene marking, especially in a non-myeloablative setting. Thus our objectives for this project are threefold. First, in Specific Aims 1-3, we propose to study techniques to further improve gene transfer rates in hematopoietic stem cells. Second, Specific Aims 4 and 5 will explore less toxic conditioning regimens in combination with immunosuppression and also investigate a novel in vivo selection system. Third, Specific Aims 6 and 7 will apply gene transfer techniques to disease models. Fanconi anemia has been chosen as the first target for hematopoietic stem cell gene therapy since gene-corrected stem cells in this disorder are thought to have a selective advantage over uncorrected stem cells.