Precision DNA Impurity Reduction Approaches for Ultra-Pure rAAV Manufacturing

A. Introduction

Recombinant Adeno-associated virus (rAAV) vectors have become one of the most widely used platforms for gene delivery in both research and clinical applications. Their favorable safety profile, broad tissue tropism, and long-term transgene expression have enabled landmark therapies such as Luxturna and Zolgensma. However, despite these advances, large-scale AAV manufacturing continues to face significant challenges, particularly in achieving consistent product quality and purity.

One of the most critical quality concerns in rAAV production is the presence of residual DNA impurities, including plasmid backbone sequences and host cell DNA (hcDNA). These contaminants may be unintentionally packaged into AAV capsids during production and pose risks such as immunogenicity, off-target gene expression, and variability in therapeutic efficacy. Current approaches to mitigate these impurities—such as plasmid redesign, minicircle DNA, or intensified downstream purification—often introduce additional process complexity, cost, or yield trade-offs.

In our recent preprint publication, “Precision DNA Impurity Reduction Approaches for Ultra-Pure rAAV Manufacturing”, we systematically investigated the sources of DNA impurities in rAAV and developed a series of upstream engineering strategies to minimize contamination at its origin. By combining advanced analytical tools with molecular and process innovations, the study establishes a scalable framework for producing ultra-pure AAV vectors aligned with GMP and regulatory expectations.

B. Method Overview

To understand and reduce DNA impurities in recombinant rAAV, our scientists used a widely adopted AAV production system and combined it with advanced analytics and innovative upstream engineering strategies.

AAV Production System

rAAV vectors were generated using a standard triple-plasmid system, consisting of:

• A gene-of-interest (GOI) plasmid containing the therapeutic cassette flanked by AAV inverted terminal repeats (ITRs)
• A Rep/Cap plasmid encoding AAV replication and capsid proteins
• A helper plasmid providing adenoviral functions (E2A, E4, VA RNA) required for viral replication

In selected experiments, additional plasmid was used to introduce new functional elements (e.g., nuclease or recombinase expression). Cells were transfected using PEI-based reagents, and viral particles were harvested ~72 hours post-transfection. Crude lysates were treated with benzonase to remove free nucleic acids, followed by purification using CsCl gradient ultracentrifugation and dialysis to obtain high-quality rAAV preparations.

Analytical Characterization of DNA Impurities

To comprehensively profile encapsidated DNA species, the study integrated multiple orthogonal analytical methods:

• Next-Generation Sequencing (NGS):
  Viral genomes extracted from purified AAV particles were sequenced and aligned against production plasmids and the human reference genome. This enabled quantitative mapping of on-target genomes versus contaminating DNA species (plasmid backbone, Rep/Cap, helper sequences, and host cell DNA).
• Quantitative PCR (qPCR):
  Targeted assays were used to quantify specific DNA components, including:

• • WPRE (vector genome proxy)
  • ori (plasmid backbone marker)
  • Rep/Cap and helper-derived sequences
  • Host cell DNA (hcDNA)

• Capsid and genome titration:
  Capsid titers were measured by ELISA, while genome titers were determined via qPCR, allowing direct comparison of yield versus impurity levels across different strategies.

Upstream Engineering Strategies Evaluated

To reduce DNA impurities at the source, the study systematically evaluated multiple upstream molecular strategies:

• TelN/TelROL protelomerase system:
  Introduced TelROL recognition sites flanking the ITR cassette to enable enzymatic excision of plasmid backbone, generating miniDNA structures during production.
• I-SceI meganuclease cleavage:
  Incorporated rare-cutting I-SceI recognition sites into plasmids to induce targeted DNA fragmentation and reduce packaging of backbone sequences.
• CRISPR/Cas9-mediated cleavage:
  Designed guide RNAs targeting plasmid backbone regions, enabling programmable degradation of contaminating DNA during AAV production.
• Cre/LoxP recombination system:
  Flanked the transgene cassette with loxP sites and expressed Cre recombinase to recombine plasmids into separate circular DNA molecules, effectively isolating the therapeutic genome from the backbone.

These strategies were implemented through plasmid engineering, transient expression systems, or stable knock-in cell lines, allowing evaluation of both performance and scalability.

Host Cell DNA Mitigation Strategy and Comparative Evaluation Framework

To address host-derived DNA contamination, the study introduced caspase inhibitors (e.g., Emricasan, Q-VD-OPh) during the transfection phase. This approach aimed to suppress apoptosis-induced genomic fragmentation, thereby reducing the availability of host DNA fragments for unintended packaging into AAV capsids.

All strategies were systematically benchmarked based on:

• Reduction of plasmid backbone DNA (ori/WPRE ratio)
• Reduction of host cell DNA (hcDNA levels)
• Impact on vector genome yield and capsid production

This integrated framework enabled identification of strategies that achieve optimal balance between purity, productivity, and scalability, providing direct relevance for GMP manufacturing.

C. Key Results

Baseline contaminant profiling

The study began by establishing a quantitative baseline of DNA impurities in rAAV preparations using NGS. As shown in Table 1, the vast majority of encapsidated DNA in full rAAV particles (rAAV‑eGFP group) corresponded to the intended ITR-flanked transgene cassette (~98.7%), confirming efficient packaging of the target genome. However, a measurable fraction of contaminating DNA was consistently detected, including plasmid backbone sequences (~0.675%) and host cell DNA (~0.548%). Notably, empty capsid (rAAV‑Em) preparations exhibited dramatically higher levels of non-target DNA, with plasmid-derived sequences accounting for over 20% of total reads and host DNA reaching ~3.5%. These findings demonstrate that DNA impurities are not negligible and are significantly enriched in non-productive particles, suggesting that mispackaging is influenced by both vector biology and production conditions. Importantly, the data confirm that plasmid backbone DNA and host cell DNA are the dominant impurity species in AAV manufacturing.

TelN/TelROL excision

Building on this baseline, we evaluated the TelN/TelROL protelomerase system (Figure 1) as an upstream strategy to reduce plasmid-derived contamination. By introducing TelROL recognition sites flanking the transgene cassette, the system enabled excision of plasmid backbone DNA to generate miniDNA structures. Experimental results showed that TelN-mediated processing reduced plasmid backbone contamination to approximately 20–30% of baseline levels. While in vitro digestion led to a decrease in vector genome titers, intracellular expression of TelN during AAV production improved the balance between purity and yield, in some cases restoring or even enhancing genome output. Additional experiments revealed that the GOI plasmid is the primary contributor to residual plasmid DNA, whereas the helper plasmid contributes minimally. These findings highlight that targeted excision of backbone DNA from key plasmids can significantly reduce impurities, though optimization is required to preserve productivity.

I‑SceI meganuclease

The I-SceI meganuclease strategy (Figure 2) provided an alternative approach based on targeted DNA cleavage. Incorporation of I-SceI recognition sites flanking the ITR regions enabled fragmentation of plasmid DNA during AAV production. This approach consistently reduced plasmid backbone contamination to approximately 20–40% of baseline levels across multiple configurations, including transient expression and stable knock-in systems. However, these reductions were accompanied by modest decreases in vector genome titers, indicating a trade-off between impurity removal and production efficiency. Compared with TelN/TelROL, the I-SceI system exhibited slightly lower efficiency, likely due to incomplete cleavage or the persistence of linear DNA fragments that remain susceptible to packaging.

CRISPR/Cas9-mediated DNA cleavage

We next evaluated CRISPR/Cas9-mediated cleavage (Figure 3) as a programmable strategy to degrade plasmid DNA during AAV production. By targeting backbone regions with guide RNAs, Cas9 reduced plasmid contamination to approximately 10–20% of baseline levels, representing a substantial improvement over earlier methods. In certain configurations, genome titers were increased, suggesting that reducing competing DNA substrates may enhance packaging efficiency. However, the effectiveness of CRISPR/Cas9 was highly dependent on expression timing and delivery format. Stable Cas9-expressing cell lines showed only limited improvement, indicating that continuous nuclease activity is less effective than transient expression. Additionally, concerns regarding off-target activity and regulatory complexity limit the practicality of this approach for large-scale manufacturing.

 Cre/LoxP recombination strategy

Among all strategies tested, the Cre/LoxP recombination system (Figure 4) demonstrated the most significant improvement in AAV purity. By flanking the transgene cassette with loxP sites and expressing Cre recombinase during production, the plasmid was recombined into two separate circular DNA molecules, effectively isolating the therapeutic genome from the plasmid backbone. This strategy reduced plasmid backbone contamination to near-undetectable levels, with NGS analysis (Table 2) showing a decrease from ~0.186% in control samples to as low as ~0.004–0.006%. Importantly, this dramatic improvement in purity was achieved without compromising vector genome titers; in some cases, yields were even enhanced, likely due to reduced competition during packaging. These results establish Cre/LoxP recombination as a highly effective and scalable solution for minimizing plasmid-derived impurities in AAV manufacturing.

Reduction of host cell DNA via caspase inhibitors

Finally, we addressed host cell DNA contamination (Figure 5), which represents a distinct and complementary challenge. By supplementing AAV production cultures with caspase inhibitors, the study demonstrated that suppression of apoptosis significantly reduces genomic DNA fragmentation and subsequent packaging into AAV capsids. Treatment with compounds such as Emricasan and Q-VD-OPh reduced hcDNA levels to approximately 1–5% of baseline without affecting vector genome titers. These findings indicate that host-derived impurities can be effectively controlled through process-level interventions, providing a complementary strategy to plasmid engineering approaches.

D. Conclusion

This study provides a systematic evaluation of DNA impurity sources and mitigation strategies in AAV manufacturing. The results confirm that plasmid backbone DNA and host cell DNA are the primary contaminants and that their encapsidation is driven by both structural and biological mechanisms, including ITR-mediated packaging and apoptosis-induced DNA fragmentation.

Among the strategies tested, while nuclease-based approaches such as TelN, I-SceI, and CRISPR/Cas9 provide incremental reductions in plasmid-derived DNA, the Cre/LoxP system achieves near-complete elimination of backbone contamination with minimal trade-offs. When combined with caspase inhibitor supplementation to reduce hcDNA, the study establishes a robust, scalable, and GMP-compatible framework for high-purity AAV production.

Importantly, this work shifts the paradigm of AAV manufacturing from reliance on downstream purification to upstream process engineering, emphasizing prevention rather than removal of impurities. By integrating plasmid design, enzymatic processing, and cellular control mechanisms, it is possible to produce ultra-pure AAV vectors without compromising yield or scalability. As the AAV field moves toward commercialization, such innovations will be critical for meeting regulatory expectations and enabling broader patient access to gene therapies.

Reference:

https://www.biorxiv.org/content/10.64898/2026.04.07.716878v1