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In Vivo and in Vitro Assessment of Residual DNA Impurity-Derived Transcriptions [II]

ABSTRACT: Gene therapy products, such as rAAV, are typically produced by DNA plasmid transfected to mammalian cell lines. Host cell DNA and plasmid DNA can be inadvertently packaged inside the rAAV capsids during production. It is important to characterize these residual DNA impurities in gene therapy products due to the theoretical risk of immunogenicity, infectivity, and oncogenicity. Previous studies showed the level of residual DNA impurity is around 1-2% of the expected rAAV genome. However, it remains unclear whether these DNAs undergo transcription and translation. To address this question, RNA was isolated from mouse liver various days after rAAV transduction, followed by reverse transcription for cDNA synthesis and library preparation. RNA-seq and dPCR were used to quantify the level of the gene of interest and the impurity genes. Results showed a low but detectable level of AAVhu37 Cap, AAV2 Rep, and KanR transcripts. The result from these studies enables targeted risk assessment and better understanding of the potential adverse effects associated with AAV gene therapy. It also points to the importance of improving vector design to reduce the level of DNA impurities.

ORGANISM(S): synthetic construct

PROVIDER: GSE280789 | GEO | 2025/05/09

REPOSITORIES: GEO

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