Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (£0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

Adeno‐associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV‐based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co‐transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high‐throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV‐Seq 2.0, a PCR‐free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR‐free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS‐based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real‐time PCR and are useful methods to improve the safety and efficacy of these viral vectors.