Sakurai F, Kawabata K, Yamaguchi T, Hayakawa T, Mizuguchi H
Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Osaka, Japan.
Gene Ther. 2005 Oct;12(19):1424-33. doi: 10.1038/sj.gt.3302562.
Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.
腺病毒基因转移至造血干细胞（HSCs）/祖细胞将为造血系统疾病的治疗及造血系统的研究提供一种新方法。我们之前报道过，一种由属于B亚组的全血清型35腺病毒（Ad35）组成的腺病毒载体，可有效地将基因转移至人骨髓CD34+细胞。然而，Ad35载体介导的转导进入人HSCs/祖细胞尚未得到充分优化。在本研究中，我们系统地检测了Ad35载体在人骨髓CD34+细胞和原始CD34+亚群中的启动子活性，以优化其在人造血干/祖细胞中的转导效率。在首次转导实验中，改良的体外连接方法被应用于Ad35载体构建，以实现简单高效地生产E1/E3缺失的Ad35载体。使用该方法，我们构建了一系列在多种强大的病毒和细胞启动子控制下编码增强型绿色荧光蛋白（GFP）的Ad35载体。在所测试的六种启动子类型中，人延伸因子1α启动子（EF1α启动子）、带有β-肌动蛋白内含子的人巨细胞病毒（CMV）立即早期1基因增强子/β-肌动蛋白启动子（CA启动子）以及带有CMV最大内含子（内含子A）的CMV启动子/增强子（CMVi启动子）在人CD34+细胞及未成熟亚群（CD34+ CD38（低/-）和CD34+ AC133+亚群）中实现了显著更高的转导效率。特别是，发现CA启动子在整个人CD34+细胞和未成熟造血亚群中均能实现最高的转导效率。此外，CA启动子介导的GFP表达细胞可分化为所有谱系的祖细胞。这些结果表明构建了一种优化的Ad35载体骨架，用于高效转导至HSCs/祖细胞。
