Unlocking Insights: Exploring Genetic Profiles Of rAAVs Through Orthogonal DNA Profiling Methods

By F. Dunker, K. Breunig, M. Magerl, S. Geiger, SM. Schermann, M. Hoerer, and RC. Feiner

The majority of capsids in an rAAV vector preparation contain the intended transgene cassette, both in full-length form and as incomplete fragments. Additionally, a significant portion of the packaged DNA consists of unwanted sequences from residual plasmid DNA or host cell genomic DNA. To assess safety and efficacy, regulatory authorities mandate comprehensive vector batch characterization using various analytical methods, including PCR-based techniques such as qPCR and ddPCR, alkaline gel electrophoresis, short- and long-read NGS, full/empty capsid analysis, and transcriptional profiling of both the transgene and impurities.

This study focuses on analyzing encapsidated DNA size distribution and kanamycin resistance gene (Kan) impurities using alkaline gel electrophoresis, ddPCR, and NGS. While multiplexed ddPCR has proven effective in profiling impurity lengths, PCR-based methods are limited by their reliance on sequence-specific primers. In contrast, long-read NGS provides extensive sequencing without prior selection, offering a powerful approach to characterize single-stranded AAV genomes and all associated packaged impurities.