Degradation and stable maintenance of adeno-associated virus inverted terminal repeats in E. coli

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Abstract
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.
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Funding Information
  • Bielefeld University
  • Deutscher Akademischer Austauschdienst (91651068)
  • Bielefeld University
  • Deutsche Forschungsgemeinschaft

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