GENE THERAPY IN HEMATOPOIETIC CELLS

该项目的目标是开发针对影响造血系统的疾病的造血干细胞基因疗法。最近在X连锁SERE联合免疫缺陷儿童中的一项研究表明，造血干细胞基因治疗的应用是成功的。虽然这些结果对基因治疗领域非常鼓舞人心，但大多数遗传病对经过基因校正的细胞没有选择优势，因此不太可能通过现有的技术治愈。使用毒性较低的非清髓性调节方案，在基因转移效率和转导细胞植入方面将需要进一步改进。由于我们在该模型中进行造血干细胞移植的长期经验，以及疾病模型的可用性，我们使用了狗模型来研究基因转移到造血再填充细胞中。在之前的资助期间，我们通过(1)使用长臂猿白血病病毒(GALV)外膜，(2)在涂有人纤维连接蛋白片段CH-296的培养瓶中转导CD34丰富的细胞，以及(3)使用包括犬干细胞因子(CSCF)、犬粒细胞集落刺激因子(CG-CSF)和人Flt3-L在内的生长因子组合，改进了向造血再生细胞的基因转移。尽管基因标记，尤其是在非清髓性环境中。因此，我们对这个项目的目标有三个。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基因转移率的技术。其次，特定的AIMS 4和5将探索结合免疫抑制的毒性较低的调节方案，并研究一种新的体内选择系统。第三，特定目标6和7将把基因转移技术应用于疾病模型。Fanconi贫血已被选为造血干细胞基因治疗的第一靶点，因为这种疾病中经过基因校正的干细胞被认为比未校正的干细胞具有选择性优势。

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.