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

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.

SUBMITTER: Radukic MT 

PROVIDER: S-EPMC11754738 | biostudies-literature | 2025 Jan

REPOSITORIES: biostudies-literature