Kiem H P, Andrews R G, Morris J, Peterson L, Heyward S, Allen J M, Rasko J E, Potter J, Miller A D
Clinical Research and Molecular Medicine Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Blood. 1998 Sep 15;92(6):1878-86.
We have used a competitive repopulation assay in baboons to develop improved methods for hematopoietic stem cell transduction and have previously shown increased gene transfer into baboon marrow repopulating cells using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vector (Kiem et al, Blood 90:4638, 1997). In this study using GALV-pseudotype vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in culture for their ability to increase gene transfer into baboon hematopoietic repopulating cells. Baboon marrow was harvested after in vivo administration (priming) of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). CD34-enriched marrow cells were divided into two equal fractions to directly compare transduction efficiencies under different gene transfer conditions. Transduction by either incubation with retroviral vectors on CH-296-coated flasks or by cocultivation on vector-producing cells was studied in five animals; in one animal, transduction on CH-296 was compared with transduction on bovine serum albumin (BSA)-coated flasks. The highest level of gene transfer was obtained after 24 hours of prestimulation followed by 48 hours of incubation on CH-296 in vector-containing medium in the presence of multiple hematopoietic growth factors (interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor). Using these conditions, up to 20% of peripheral blood and marrow cells contained vector sequences for more than 20 weeks, as determined by both polymerase chain reaction and Southern blot analysis. Gene transfer rates were higher for cells transduced on CH-296 as compared with BSA or cocultivation. In one animal, we have used a vector expressing a cell surface protein (human placental alkaline phosphatase) and have detected 10% and 5% of peripheral blood cells expressing the transduced gene 2 and 4 weeks after transplantation as measured by flow cytometry. In conclusion, the conditions described here have resulted in gene transfer rates that will allow detection of transduced cells by flow cytometry to facilitate the evaluation of gene expression. The levels of gene transfer obtained with these conditions suggest the potential for therapeutic efficacy in diseases affecting the hematopoietic system.
我们在狒狒中使用竞争性再增殖试验来开发改进的造血干细胞转导方法，并且之前已经表明，使用长臂猿白血病病毒（GALV）假型逆转录病毒载体可增加基因导入狒狒骨髓再增殖细胞（Kiem等人，《血液》90:4638，1997）。在这项使用GALV假型载体的研究中，我们检查了其他一些据报道能增加培养的造血祖细胞基因转导的变量，看它们是否有能力增加基因导入狒狒造血再增殖细胞。在体内给予干细胞因子（SCF）和粒细胞集落刺激因子（G-CSF）（预刺激）后采集狒狒骨髓。将富集CD34的骨髓细胞分成两个相等的部分，以直接比较不同基因转导条件下的转导效率。在五只动物中研究了通过在CH-296包被的培养瓶上与逆转录病毒载体孵育或通过在载体产生细胞上共培养进行转导；在一只动物中，将在CH-296上的转导与在牛血清白蛋白（BSA）包被的培养瓶上的转导进行了比较。在多种造血生长因子（白细胞介素-6、干细胞因子、FLT-3配体和巨核细胞生长与发育因子）存在的情况下，先进行24小时预刺激，然后在含载体的培养基中在CH-296上孵育48小时后，获得了最高水平的基因转导。使用这些条件，通过聚合酶链反应和Southern印迹分析确定，高达20%的外周血和骨髓细胞在20周以上都含有载体序列。与在BSA上或共培养转导的细胞相比，在CH-296上转导的细胞基因转导率更高。在一只动物中，我们使用了一种表达细胞表面蛋白（人胎盘碱性磷酸酶）的载体，通过流式细胞术检测到移植后2周和4周分别有10%和5%的外周血细胞表达转导基因。总之，这里描述的条件所产生的基因转导率将允许通过流式细胞术检测转导细胞，以促进基因表达的评估。在这些条件下获得的基因转导水平表明在影响造血系统的疾病中具有治疗效果的潜力。
