(M1030-09-56) Investigating In Vitro Transcription of Host Cell and Plasmid DNA Impurities in Recombinant AAV Drug Substances

Purpose: Residual DNA impurities are DNA from production cells (host cell DNA) and plasmids, associated with transfection and production of Adeno Associated Virus (AAV) gene therapy vectors, that are incorporated into the purified drug product (1). The presence of these residual DNA impurities can pose genotoxic, oncogenic, immunogenic and infectivity risks to recipients of AAV therapies (1,2). While purification process and plasmid optimization can minimize the amount of DNA impurities outside of the viral capsid (3), it can be challenging to remove low levels of encapsidated DNA contaminants. To reduce risks, regulatory agencies recommend decreasing the quantity of DNA impurities associated with AAV vectors, as well as reducing the size of DNA impurities to below the size of a functional gene (2). In this study we aimed to investigate the presence of functional residual DNA gene expression by quantifying the in-vitro transcription of host cell (18S), viral derived (E1A), and plasmid (Kan^R) genes after transduction with a recombinant AAV drug substance.

Methods: Cultured Huh7 Cells were incubated with recombinant AAV drug substance, Product A, at three different multiplicities of infection (MOI). Cells were incubated with formulation buffer as no transduction control. After 70-72 hours of incubation, cells were lysed and RNA was isolated, purified, and quantified with a NanoDrop Spectrophotometer. cDNA was synthesized from total cellular RNA by reverse transcription using random hexamer primers and subsequently diluted to 5X and 10X concentrations. Expression of the AAV transgene, 18S, Kan^R and E1A were quantified by Real-time Quantitative PCR using qualified primer and probe assays. For each assay, standard curve and positive controls were quantified to monitor assay performance. Expression of the housekeeping gene, ACTB was quantified to control for RNA input. Gene expression was normalized to ACTB and values were expressed as Fold-increase when normalized to cells that were not transduced with AAV vector.

Results: Transgene expression was observed at high (1.11e6), medium (1.06e5) and low (1.01e4), MOIs in a dose dependent manner, indicating successful transduction of Huh7 cells with the drug substance, compared to no transduction controls (Table 1). Expression of the plasmid gene, Kan^R, was below the limit of quantification at high, medium, and low MOIs (Table 1). The presence of E1A expression was undetectable at all MOIs (Table 1) while the standard curve and positive controls passed assay validity criteria demonstrating assay robustness. 18S expression at high, medium, and low MOIs were 1.01, 1.05 and 0.99 folds respectively when normalized to cells that were not transduced with AAV vector. (Table 1).

Conclusion: An investigation of in-vitro transcription of residual DNA impurities after transduction with recombinant AAV drug substances demonstrated no increase in host cell (18S), viral- derived (E1A) and plasmid (Kan^R) gene expression compared to cells that were not transduced with AAV vector. Thus, while low levels of DNA contaminants may exist in purified AAV drug substances the risk of residual DNA transcription remains low.

References: (1) J.F. Wright (2014). Product-related impurities in clinical-grade recombinant AAV vectors: characterization and risk assessment. Biomedicines, 2: 80-97
(2) Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications. FDA Guidance Documents (2020)
(3) B. Hauck, S. L. Murphy, P. H. Smith, G. Qu, X. Liu, O. Zelenaia, F. Mingozzi, J.M. Sommer, K.A. High, and J.F. Wright (2009). Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses. Molecular Therapy. 12: 744-152

Figure 1. Method workflow for quantification of residual DNA transcript. NC= Negative Control. High MOI- 1.11e6, Medium MOI- 1.06e5, and Low MOI- 1.01e4.

Table 1. Expression of residual DNA genes after transduction with recombinant AAV drug substance. Values are represented as fold-increase compared to negative control cells. Results shown are mean values from three independent experiments. LOQ- Limit of Quantification. LOQ for KanR assay is 64 copies/10µL.