Residual DNA Impurities in AAV Vectors – Nature and Transcription

 

Gene therapies offer a transformative approach to treating genetic diseases by delivering therapeutic genes directly to target tissues. Among delivery platforms, recombinant adeno-associated virus (rAAV) has emerged as a leading vector due to its capacity for long-term gene expression and its favorable safety profile. However, concerns remain about residual DNA impurities that co-purify with rAAV vectors. A recent study by Ultragenyx Pharmaceutical, published in Molecular Therapy: Methods & Clinical Development, provides a comprehensive analysis of these impurities—examining their composition, transcriptional potential, and possible safety implications.

Regulatory Challenges for DNA Impurities in Gene Therapy

Current regulatory guidelines from the FDA and WHO cap residual host cell DNA (hcDNA) in biologics at less than 10 ng per dose and limit fragment sizes to under 200 base pairs. These standards were originally developed for conventional biologics, not gene therapy products in which DNA is an integral component. Studies, including the one under discussion, show that rAAV preparations often exceed these thresholds, highlighting the need for gene therapy-specific risk assessment frameworks.

Types and Composition of Residual DNA in rAAV Vectors

This study analyzed rAAVhu37-EGFP vectors produced via triple transfection in HEK293 cells and identified three main impurity types:

  • Host cell DNA (hcDNA) comprised approximately 2.01% of the packaged genome, mostly originating from intronic (41.3%) and intergenic (42.7%) regions, with minimal exon content and no full transcription units, indicating low transcriptional risk.
  • Plasmid backbone DNA made up about 2.26%, with fragmented sequences and a minority containing intact open reading frames (ORFs): 22% for AAVhu37 Cap, 12% for AAV2 Rep, and 63% for Kanamycin resistance (KanR), suggesting limited but possible transcriptional activity.
  • Inverted terminal repeat (ITR)-containing fragments appeared in 32% of plasmid-derived sequences and 1.15% of hcDNA. Given that ITRs can facilitate second-strand synthesis and act as weak promoters, their presence may modestly increase the transcriptional potential of these impurities.

Mechanisms Driving Impurity Packaging

Several mechanisms appear to influence the encapsidation of non-target DNA into AAV particles. Notably, there was significant enrichment of the Rep Binding Element (RBE) sequence,GAGCGAGCGAGCGCGC,within packaged hcDNA (p = 1.03 × 10⁻¹⁴⁶), particularly in non-coding regions. This suggests a preferential binding and packaging facilitated by AAV Rep proteins. Additionally, transcription factor binding sites were overrepresented in hcDNA, implicating possible Rep–transcription factor interactions. The predominance of non-coding DNA likely results from production-associated fragmentation and the vector’s limited packaging capacity (~4.7 kb), which favors shorter, non-coding sequences.

Transcriptional Potential of DNA Impurities In Vivo

To evaluate whether residual DNA impurities were transcriptionally active in vivo, the researchers performed PacBio long-read sequencing and RNA-seq on mouse livers following rAAV administration. While over 98% of mapped sequences corresponded to the intended transgene cassette, approximately 2% derived from plasmid backbone or human genome DNA. Most of these were fragmented and lacked complete ORFs, minimizing their transcriptional capacity. However, a small fraction, particularly KanR sequences containing intact ORFs, did show transcriptional activity, albeit at levels <0.01% of the EGFP transgene.

There was a clear correlation between transcriptional activity and the presence of intact ORFs, suggesting that long-read sequencing could serve as a predictive tool for assessing transcriptional risk. Importantly, plasmid-derived transcripts did not accumulate over time, unlike the sustained expression of the EGFP cassette, reflecting differences in stability and replication competence.

Drivers of Impurity Transcription

Several features may contribute to low-level transcription of residual DNA. Approximately 32% of plasmid impurities contained ITRs, which can function as weak mammalian promoters and likely explain the limited KanR expression observed. For Cap and Rep genes, transcription may originate from regulatory elements such as the P5 promoter found in the helper plasmid, which includes active sites like YY1 and TATA boxes. Additionally, single-stranded DNA impurities may be less transcriptionally active due to their lower abundance and reduced ability to form stable episomes compared to the double-stranded therapeutic cassette.

Transcriptional Silence of Host Cell DNA

In contrast to plasmid impurities, hcDNA fragments demonstrated no transcriptional activity in vivo. PacBio sequencing confirmed that most hcDNA was intronic or intergenic and lacked full ORFs, while RNA-seq analysis showed no detectable human gene transcripts in treated mouse livers. Although promoter regions and RBEs were enriched within hcDNA, they did not result in functional transcription, reinforcing the view that hcDNA carries minimal transcriptional risk.

Safety Considerations and Future Directions

Despite the absence of transcription from hcDNA and only minor activity from plasmid impurities, the presence of residual DNA still raises concerns. Unassessed risks, including insertional mutagenesis, immune activation, or oncogenesis, remain relevant. Moreover, impurity profiles vary across production methods, emphasizing the need for case-by-case evaluations.

Given that clinical rAAV doses often exceed 1.0 × 10¹⁴ GC/kg, reducing DNA impurities remains critical. Future efforts should focus on vector engineering to minimize unintended promoter activity, alongside improvements in purification processes to enhance product safety and consistency.

Conclusion

This study provides a detailed investigation into the composition and behavior of DNA impurities in rAAV gene therapy vectors. While low-level transcription from plasmid DNA was observed and host cell DNA remained transcriptionally silent, potential safety concerns still warrant attention. Ongoing monitoring, improved manufacturing practices, and long-term clinical follow-up will be essential to ensure the continued safety and efficacy of rAAV-based therapies.

Reference

Jen, Hsin-I et al. Residual DNA Impurities in AAV Vectors – Nature and Transcription. Molecular Therapy Methods & Clinical Development, Volume 0, Issue 0, 101503. DOI: 10.1016/j.omtm.2025.101503

 

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Our Recent Publication/Meeting Presentation on Gene Therapy

1. Development of VNX-101, an Adeno-Associated Virus with Less Immunogenicity and Efficient Long-Term Expression of a CD19 T-Cell Engager. Molecular Therapy Methods & Clinical Development, published online July 24, 2025.

2. Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217.

3. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1.

4.  American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity  Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

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