Residual DNA impurities in AAV vectors-nature and transcription

Abstract

Recombinant adeno-associated viruses (rAAVs) produced by transfecting DNA plasmids into mammalian cells can inadvertently package host cell DNA (hcDNA) and plasmid DNA inside their capsids. Although the percentage of these DNA impurities is low compared to the rAAV genome in vector preparations, it is essential to characterize the DNA impurities in gene therapy products due to the theoretical risks associated with unwanted gene expression and potential immunogenicity and oncogenicity in treated patients. We performed long-read sequencing in rAAV vector, with a focus on analyzing residual, non-transgene DNA within the capsids. Although we detected host cell and residual plasmid DNA impurities, they were predominantly incomplete sequences without coding potential. This indicated that while DNA impurities may be present in rAAV preparations, host cell and residual plasmid genes were unlikely to be expressed. This was supported by RNA sequencing (RNA-seq) analyses that showed minimal plasmid RNA transcripts and host cell RNA transcripts in the livers of mice dosed with rAAV. Overall, the results from these studies enable data-based risk assessment of co-packaged DNA impurities and a better understanding of potential adverse effects associated with rAAV gene therapy.

Keywords: AAV product quality assessment; AAV safety assessment; DNA impurities; PacBio sequencing; gene therapy; host cell DNA transcription; rAAV; residual DNA.

Graphical abstract

Figure 1

rAAVhu37-EGFP vector diagram and analytics (A) Plasmids used in triple transfection for viral production. (B) rAAV products and capsid variants that contain different DNA species. (C) Analytical data for rAAVhu37-EGFP product.

Figure 2

Quantity and size distribution of residual plasmid DNA (A) Size distribution for residual plasmid DNA impurities. (B) Number of residual plasmid DNA genes and their complete open reading frames.

Figure 3

Distribution of residual HcDNA (A) Genome distribution of hcDNA sequences. (B) Distribution of residual hcDNA in different chromosomes. (C) Motif enrichment analysis of the hcDNA.

Figure 4

Expression of EGFP in the rAAVhu37-EGFP infected mouse liver (A) EGFP protein measured by ELISA (n = 3 controls, n = 5 per experimental group). (B) EGFP mRNA calculated from RNA-seq (n = 3). Data are shown as means (SD), p values were determined by non-paired student t test. (C) Mapping results of RNA-seq reads from livers of control (n = 3) and rAAVhu37-EGFP-dosed mice (n = 3 per group).

Figure 5

RNA expression of genes from residual plasmid DNA in mice dosed with rAAVhu37-EGFP (A) Abundance of plasmid DNA-derived mRNA in control and rAAVhu37-EGFP-dosed mice by RNA-seq (n = 3). (B) Average read depth for the residual plasmid DNA impurities. Data are shown as means (SD). (C) Normalized quantification of plasmid DNA impurities by dPCR (n = 3). (D) Correlation between percentage of residual DNA transcripts (in mice livers) and percentage of residual DNA (in AAV vectors) with or without open reading frame.