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Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

Abstract : Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.
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https://www.hal.inserm.fr/inserm-01799968
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Submitted on : Friday, May 25, 2018 - 2:10:58 PM
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Emilie Lecomte, Benoit Tournaire, Benjamin Cogné, Jean-Baptiste Dupont, Pierre Lindenbaum, et al.. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing. Molecular Therapy - Nucleic Acids, Nature Publishing Group, 2015, 4, pp.e260. ⟨10.1038/mtna.2015.32⟩. ⟨inserm-01799968⟩

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