Radukic Marco T, Le Dinh To, Krassuski Timo, Borchert Philipp, Leach David R F, Müller Kristian M
Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, Universitätsstraße 25, 33615 Bielefeld, North Rhine-Westphalia, Germany.
Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh, EH9 3BF, UK.
Nucleic Acids Res. 2025 Jan 11;53(2). doi: 10.1093/nar/gkae1170.
Current plasmid propagation in E. coli compromises large inverted repeats, such as inverted terminal repeats (ITRs) of adeno-associated virus (AAV). Direct long-read sequencing analyses upon varying strains and culture conditions revealed ITR instability caused by a slipped misalignment mechanism, although other mechanism probably contribute. ITRs stabilized in absence of SbcC, which is part of the SbcCD nuclease complex, a human Mre11-Rad50 homolog, or at elevated growth temperatures (e.g. 42°C), with a combination being optimal. Resulting full ITR transgene plasmids improved rAAV yield and purity in HEK-293 productions. The findings advance plasmid biology, cloneable sequences and therapeutic AAV manufacturing.
目前在大肠杆菌中进行的质粒扩增会损害大型反向重复序列，如腺相关病毒（AAV）的反向末端重复序列（ITR）。对不同菌株和培养条件进行的直接长读长测序分析表明，ITR的不稳定性是由滑动错配机制引起的，不过可能还有其他机制起作用。在缺乏SbcC（SbcCD核酸酶复合体的一部分，一种人类Mre11-Rad50同源物）的情况下，或者在较高生长温度（如42°C）下，ITR会得到稳定，两者结合效果最佳。由此产生的完整ITR转基因质粒提高了在HEK-293细胞中生产重组腺相关病毒（rAAV）的产量和纯度。这些发现推动了质粒生物学、可克隆序列以及治疗性腺相关病毒制造技术的发展。
