Preferential Encapsulation of DNA Impurities Derived from pAAV Plasmid Backbone into Recombinant Adeno-Associated Virus Capsids

Recombinant adeno-associated viruses (rAAVs) are useful vectors for clinical gene therapy. It is crucial to examine DNA impurities, such as plasmid DNA, for quality control of rAAV products. In this study, we examined highly purified rAAV1, rAAV2, rAAV5, and rAAV6 samples produced on a three-plasmid platform, using a high-throughput sequencer. These samples contained 0.49–3.80% detectable DNA impurities derived from the three plasmids, as estimated by the ampicillin resistance gene ( amp R ). The plasmid impurities consisted of 90.62–95.84% pAAV, 3.21–6.83% pRC, and 0.95–2.55% pHelper DNA. These trends were consistent with those of DNA impurities determined by droplet digital PCR (ddPCR), indicating that cleaved pAAV backbone DNA was the primary source of DNA impurities encapsulated into the capsids. To examine the preferential encapsulation of pAAV backbone DNA into capsids, short sequencing reads were mapped to the entire pAAV backbone sequence, and we found that the reads were relatively evenly distributed across the backbone sequence, with occasional sharp drops. Furthermore, the read length distribution containing the pAAV backbone sequence showed a main peak at 3.2 kb in Oxford Nanopore Technologies sequencing. This length was consistent with that of backbone DNA nicked at two terminal resolution sites by Rep78/68. Analysis of the terminal sequences of the long reads containing backbone sequences revealed that 92.7–98.4% of them contained Rep-binding elements. These results indicate that the pAAV backbone in linear DNA form was cleaved from pAAV-ZsGreen1 by Rep78/68 nicking at trs in the nucleus, and that the single-stranded DNA was efficiently translocated into the capsids through the encapsulating machinery, similar to the rAAV genome.