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.

Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells

“…Our laboratory has pioneered the development of an exhaustive NGS-based approach to quantify levels of DNA impurities relative to the rAAV genome in rAAV preparations produced by transfection of mammalian HEK293 cells. 11 The present study describes our development of a novel tool for the analysis of DNA impurities originating from the baculovirus-Sf9 production system. Using our SSV-Seq protocol, we determined that the predominant DNA contaminant is derived from the baculoviral genome, although levels of this contaminant did not exceed 2.1% in the seven batches tested.…”

“…11 To facilitate sample assignment from mixed clusters, we improved on our previously published methodology 11 by (1) developing a new bioinformatics program for sample demultiplexing and (2) preparing dual-indexed Illumina libraries instead of single-indexed libraries. First, we developed a new demultiplexing program, named Quade (http:// a-slide.github.io/Quade/), to remove low index quality reads.…”

“…Samples were treated or not with the PS-DNase and Baseline Zero cocktail, as described in Lecomte and colleagues. 11 No baculoviral DNA was detected by qPCR after DNase treatment (Bac qPCR limit of quantification [LOQ], 3.3 · 10 3 copies), indicating that the percentage contamination determined by SSV-Seq did not correspond to free baculoviral DNA. At this stage, contamination of the final stocks by baculoviral particles copurified with rAAV particles could not be ruled out.…”

“…Using SSV-Seq combined with Illumina (San Diego, CA) highthroughput sequencing, we can exhaustively quantify and characterize DNA species copurified and encapsidated in rAAV particles produced by plasmid transfection of mammalian cells. 11 As an alternative to HEK293 cells, the baculovirusSf9 platform has been established as a scalable, Good Manufacturing Practice-compatible system for the production of rAAV vectors. 12 The production of rAAV vectors by infection of the Spodoptera frugiperda insect cell line (Sf9) with three baculovirus expression vectors (BEVs) was first described in 2002 by Kotin's group.…”

“…By contrast, next-generation sequencing (NGS) allows exhaustive characterization and distribution of all contaminant DNA sequences. In the present study, we adapted our previously described SSV-Seq protocol 11 to quantify DNA species in rAAV stocks produced in the Sf9 insect cell line. We developed a specific bioinformatics pipeline for the baculovirus-Sf9 production system, and single-molecule real-time (SMRT) sequencing was used to generate an accurate reference sequence for the recombinant baculovirus genome.…”