Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.
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.
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Emilie
E
Lecomte
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Benoît
B
Tournaire
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Benjamin
B
Cogné
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Jean-Baptiste
JB
Dupont
University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.|INSERM UMR 1087/CNRS UMR 6291/L'Institut du Thorax, Nantes, France.
Pierre
P
Lindenbaum
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Mélanie
M
Martin-Fontaine
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Frédéric
F
Broucque
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Cécile
C
Robin
GENETHON, Evry, France.
Matthias
M
Hebben
GENETHON, Evry, France.
Otto-Wilhelm
OW
Merten
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Véronique
V
Blouin
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Achille
A
François
University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.|INSERM UMR 1087/CNRS UMR 6291/L'Institut du Thorax, Nantes, France.
Richard
R
Redon
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.|Molecular Genetics and Microbiology Department, College of Medicine, University of Florida, Gainesville, Florida, USA.
Philippe
P
Moullier
INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.|University of Nantes, Nantes, France.|Nantes University Hospital, Nantes, France.
Adrien
A
Léger