IN VITRO MODELS OF SICKLE CELL GENE THERAPY

造血干细胞基因治疗在镰状细胞治疗中的应用 疾病(SCD)有一天可能会为患有这种疾病的患者提供治愈的机会。 无序。虽然在实现这一目标方面取得了重大进展， 许多障碍依然存在。SCD基因治疗的主要障碍是 这一阶段是基因转移到造血系统的效率非常低的阶段。 干细胞。目前SCD的基因治疗方法以插入为目标 在造血干细胞基因组中的一个正常的珠蛋白基因 而不是用正常基因替换突变基因。这样的基因“加法” 会导致细胞中正常珠蛋白链的过度合成 完整的突变体链。这种操控对 红细胞的总体血红蛋白组成及其倾向 镰刀是未知的。这是一个非常困难的情况，然后是一间镰刀形牢房 胎儿血红蛋白水平高或镰刀状β-地中海贫血患者 患者的高水平血红蛋白a在哪里正常或β-珠蛋白 链取代了红细胞中一些突变的镰刀状珠蛋白链。 由于基因转移到造血干细胞的效率较低 细胞，不可能确定这种基因转移的效果。 在活体环境中。我们建议开发一种体外系统来评估 正常珠蛋白基因转入红系的效果 SCD患者的祖细胞对转导细胞表型的影响。 体外扩增和扩增方法的研究进展 转导细胞的表型分化。最近的 体外扩增和分化方法的研究进展 两相液体培养系统中的红系是成功的关键 对于这样一个系统来说。这使得逆转录病毒转导和 选择整合了转移的珠蛋白基因的红系祖细胞 并在体外培养系统中对它们进行扩增和分化。这个 基因转移对血红蛋白表型和镰刀状的影响 镰状障碍患者转导细胞的潜力可以 然后通过建立红血球的体外检测方法进行分析。我们 将分析基因转移对细胞表型的影响 患有各种镰状障碍的患者，包括SS、SC、Sbeta- 地中海贫血，全部存在和不存在相互作用的α- 地中海贫血，这个实验系统的设计是为了提供 这种类型的基因转移可能有效地防止 镰刀和它的并发症。我们也希望它能成为一个有用的 比较不同基因治疗效果的临床前试验 在安全和相对便宜的体外环境中转移载体。

The application of hematopoietic stem cell gene therapy to sickle cell disease (SCD) may one day offer the chance of cure to patients with this disorder. Although significant progress has been made towards this goal, many obstacles remain. The major obstacle to the gene therapy of SCD at this stage is the very low efficiency of gene transfer into hematopoietic stem cells. Current approaches to the gene therapy of SCD aim at inserting a normal globin gene in the genome of the hematopoietic stem cell rather than replacing the mutant gene with a normal gene. Such gene "addition" would result in excess synthesis of normal globin chains in a cell with a full complement of mutant chains. The effects of this manipulation on the overall hemoglobin composition of the red cell and its propensity to sickle are unknown. This is a very difficult situation then a sickle cell patient with a high level of fetal hemoglobin or a sickle beta-thalassemia patient with a high level of hemoglobin a where the normal or beta-globin chains replace some of the mutant sickle globin chains in the red cell. As a result of the low efficiency of gene transfer to hematopoietic stem cells, it is not possible to determine the effects of such gene transfer in an in vivo setting. We propose to develop an in vitro system to assess the effects of the transfer of normal globin genes into erythroid progenitors of patients with SCD on the phenotype of the transduced cells. The recent development of in vitro methods for the expansion and differentiation of phenotype of the transduced cells. The recent development of in vitro methods for the expansion and differentiation of erythroid in a two-phase liquid culture system is crucial for the success for such a system. This has made it possible to retrovirally transduce and select erythroid progenitors that incorporate the transferred globin genes and expand and differentiate them, all in an in vitro culture system. The effects of gene transfer on the hemoglobin phenotype and the sickling potential of transduced cells from patients with sickling disorders can then be analyzed by established in vitro assays of red blood sickling. We will analyze the effects of gene transfer on the phenotype of cells from patients with a variety of sickling disorders including SS, SC, Sbeta- thalassemia, all in the presence and absence of interacting alpha- thalassemia, this experimental system is designed to provide the proof of principle that this type of gene transfer may be effective in preventing sickling and its complications. We also hope it will serve as a useful pre-clinical test for comparing the effectiveness of different gene transfer vector in a safe and relatively inexpensive in vitro setting.