| Autor: |
Emilie Lecomte, Benoît Tournaire, Benjamin Cogné, Jean-Baptiste Dupont, Pierre Lindenbaum, Mélanie Martin-Fontaine, Frédéric Broucque, Cécile Robin, Matthias Hebben, Otto-Wilhelm Merten, Véronique Blouin, Achille François, Richard Redon, Philippe Moullier, Adrien Léger |
| Jazyk: |
angličtina |
| Rok vydání: |
2015 |
| Předmět: |
|
| Zdroj: |
Molecular Therapy: Nucleic Acids, Vol 4, Iss C (2015) |
| Druh dokumentu: |
article |
| ISSN: |
2162-2531 |
| DOI: |
10.1038/mtna.2015.32 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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