Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Ave., St-Petersburg 194064, Russia.
Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Cells. 2020 Apr 3;9(4):879. doi: 10.3390/cells9040879.
Human artificial chromosomes (HACs), including the de novo synthesized alphoid-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoid-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoid-HAC in hamster CHO cells. Subsequently, the therapeutic alphoid-HAC-FVIII was transferred into the iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoid-HAC.
人类人工染色体（HACs），包括从头合成的α-卫星 HAC，是将目的基因导入真核细胞的有力工具。HAC 是有丝分裂稳定的、非整合的附加体单元，具有较大的转基因插入容量，并允许高效和稳定的转基因表达。以前，我们已经表明，α-卫星 HAC 载体不会干扰多能状态，并在人诱导多能干细胞（iPSCs）和小鼠胚胎干细胞（ESCs）中提供稳定的转基因表达。在这项研究中，我们详细研究了基于 iPSC 和 HAC 的外生性治疗血友病 A 单基因疾病的小鼠模型。iPSCs 是从 突变型小鼠成纤维细胞中开发出来的，由普遍启动子驱动的 FVIII cDNA 被引入仓鼠 CHO 细胞中的α-卫星 HAC。随后，通过 retro-microcell 介导的染色体转移方法将治疗性的α-卫星 HAC-FVIII 转移到 iPSCs 中。治疗性的 HAC 作为一个非整合的附加体在小鼠 iPSCs 中保持稳定，表现出持续的 FVIII 表达。这项研究是使用新一代合成染色体载体——α-卫星 HAC 治疗血友病 A 单基因疾病的第一步。
