School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Molecular Engineering Lab, Biomedical Sciences Institute, Agency for Science Technology and Research, 61 Biopolis Drive, Singapore 138673.
Nucleic Acids Res. 2018 Sep 19;46(16):e99. doi: 10.1093/nar/gky500.
Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.
干细胞工程、基因治疗和分子医学的进展常常涉及细胞水平的基因组工程。然而，将功能较大或多转基因盒插入人类基因组仍然是一个挑战。目前的方法，如随机转基因整合或基于靶向内切酶的基因组编辑并不理想，并且可能存在安全隐患。考虑到这一点，我们之前开发了一种源自噬菌体 λ 整合酶 (Int) 的转基因工具，该工具可将大质粒 DNA 精确地重组到人类长散布元件-1 (LINE-1) 中的一个内源性序列中。尽管取得了这一进展，但与质粒的细菌成分相关的生物安全问题、增强的摄取和有效的转基因表达仍然是问题。因此，我们进一步改进并在此报告了一种更优越的基于 Int 的转基因工具。这个新型的 Int 平台允许通过分子内重组从常规质粒中高效且轻松地衍生出大量无接头的超螺旋转基因载体，并且随后将其进行分子间的特异性基因组整合到 LINE-1 中。此外，我们鉴定了某些 LINE-1 作为 Int 介导的无缝载体转基因的优选插入位点，并表明靶向抗 CD19 嵌合抗原受体基因整合可在人胚胎干细胞克隆中实现高水平持续的转基因表达，用于潜在的下游治疗应用。
