Thornton Christopher D, Fielding Stuart, Karbowniczek Kinga, Roig-Merino Alicia, Burrows Alysha E, FitzPatrick Lorna M, Sharaireh Aseel, Tite John P, Mole Sara E, Harbottle Richard P, Caproni Lisa J, McKay Tristan R
Centre for Bioscience, Manchester Metropolitan University, Manchester M1 5GD, UK.
Medicines Discovery Catapult, Alderley Park, Cheshire, UK.
Mol Ther Methods Clin Dev. 2021 Oct 5;23:348-358. doi: 10.1016/j.omtm.2021.09.018. eCollection 2021 Dec 10.
The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy.
诱导多能干细胞（iPSC）在先进疗法中的应用正在迅速增加，但人们对其临床安全性仍存在担忧。我们报告了首次应用狗骨DNA（dbDNA）载体来生成人类iPSC。dbDNA载体是封闭 capped 的线性双链DNA基因表达盒，不包含细菌DNA，并通过化学定义的、符合现行良好生产规范（cGMP）的方法进行扩增。我们使用瞬时表达的dbDNA载体实现了与使用具有与最先进的OriP/EBNA1附加型载体相同的iPSC重编程编码序列相当的iPSC重编程效率，但关键的是，在没有p53 shRNA抑制的情况下。此外，细菌来源附加体中EBNA1的持续表达导致干扰素反应的刺激、DNA损伤的增加和自发分化的增加。这些细胞活性在dbDNA-iPSC中减少或不存在，从而产生了具有更高稳定性和细胞治疗安全潜力的细胞系。
