Mitani Kohnosuke
Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama, Japan.
Methods Mol Biol. 2014;1114:163-7. doi: 10.1007/978-1-62703-761-7_10.
Helper-dependent adenoviral vector (HDAdV), which is also called gutless AdV, has been used to deliver donor DNA for gene targeting in human pluripotent stem cells. Surprisingly, the targeting efficacies, both per chromosomal integration (drug-resistant colony) and per treated cells, are much higher than those by standard electroporation and equivalent to those by utilizing artificial nucleases, such as TAL effector nucleases (Aizawa et al., Mol Ther 20:424-431, 2012; Suzuki, Proc Natl Acad Sci U S A 105:13781-13786, 2008). Importantly, gene targeting with HDAdVs was equally efficient in transcriptionally inactive loci in human ES/iPS cells. Therefore, multiple gene-targeted clones can be obtained from human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) cultured in one 100-mm dish. For virus-mediated gene targeting, it is not required to introduce artificial double-strand breaks. By using electroporation for gene targeting, target cells should be expanded to 10⁶-10⁷ cells. In contrast, as an advantage of virus-mediated method, DNA delivery efficiency is high even in a smaller number of cells, resulting in minimizing the number of passages/cell divisions before performing gene targeting. The characteristics suggest that HDAdV-mediated gene targeting has potential advantages for manipulation of chromosomes of pluripotent stem cells for therapeutic applications.
辅助依赖型腺病毒载体（HDAdV），也被称为无基因组腺病毒载体，已被用于在人类多能干细胞中递送供体DNA以进行基因靶向。令人惊讶的是，无论是按染色体整合（耐药菌落）还是按处理细胞计算，其靶向效率都远高于标准电穿孔法，并且与使用人工核酸酶（如转录激活样效应因子核酸酶）的效率相当（相泽等人，《分子治疗》20:424 - 431，2012；铃木，《美国国家科学院院刊》105:13781 - 13786，2008）。重要的是，在人类胚胎干细胞/诱导多能干细胞中转录失活位点处，HDAdV介导的基因靶向同样高效。因此，从培养在一个100毫米培养皿中的人类胚胎干细胞（hESC）和人类诱导多能干细胞（hiPSC）中可以获得多个基因靶向克隆。对于病毒介导的基因靶向，无需引入人工双链断裂。通过电穿孔进行基因靶向时，靶细胞需扩增至10⁶ - 10⁷个细胞。相比之下，作为病毒介导方法的一个优势，即使细胞数量较少，DNA递送效率也很高，从而在进行基因靶向之前将传代次数/细胞分裂次数降至最低。这些特性表明，HDAdV介导的基因靶向在用于治疗应用的多能干细胞染色体操作方面具有潜在优势。
