A recent clinical case report (Buddle et al., Nature Medicine) described prolonged, severe hepatitis following systemic AAV therapy. Beyond known vector-related risks, investigators identified plasmid backbone DNA in ~5–6% of hepatocytes.
These sequences originated from bacterial plasmids used during AAV manufacturing and may include:
• Origins of replication
• Antibiotic resistance genes
• Other non-therapeutic backbone elements
Traditionally classified as process-related impurities, such DNA fragments may have biological relevance, as liver toxcity and hepatitis, particularly in high-dose systemic applications where immune activation is a key safety concern. The key question is shifting from “Are impurities within specification?” to “What biological impact might they have?”
How Backbone-free Minicircles Shift the Paradigm
Schnoedt et al. (2017) showed that bacterial backbone sequences are difficult to fully remove during plasmid-based AAV manufacturing and that residual fragments can persist through purification and be unintentionally packaged into AAV capsids, reducing yield and efficiency. This highlights the limits of downstream purification in conventional transfection workflows.
Minicircle DNA addresses this challenge at the source. As backbone-free, circular supercoiled DNA molecules containing only the expression cassette, minicircles provide:
• No bacterial backbone sequences in the starting material
• Elimination of backbone-packaging into AAV particles
• Impurity reduction by design rather than purification alone
This shifts the strategy from downstream removal to upstream prevention, lowering back-packaging risk and enabling cleaner AAV preparations. This is particularly relevant for high-dose systemic applications and late-stage programs with strict safety and regulatory expectations.
Performance and Scalability
The small, backbone-free, supercoiled Minicircle DNA format is optimized for AAV production and translates structural DNA advantages into higher functional yields and improved vector safety.
• Improved producer cell transfection and downstream transduction
Due to their compact, supercoiled structure, minicircles support more efficient transfection of AAV producer cells, leading to improved virus quality. This translates into up to ~30% higher transduction efficiency in target cells, reflecting improved functionality of the produced vectors rather than changes in the target cells.• Higher functional titers and full capsid rates
The absence of bacterial backbone sequences enables more efficient genome processing and encapsidation during AAV assembly. This results in a higher proportion of correctly packaged, full capsids and a strong increase in functional AAV particle output. Minicircle-based AAV production has been reported to deliver 3–4× higher functional titers compared to conventional plasmid formats• Enhanced safety and reduced impurity burden
Because minicircles are backbone-free by design, they eliminate a major source of plasmid-derived DNA impurities and reduce the risk of unintended backbone packaging into AAV particles. This supports cleaner virus preparations and may simplify downstream purification and control strategies.
Together, these properties make DNA format selection a strategic factor for balancing safety, manufacturability, and scalability in advanced AAV programs.
How Can Minicircle DNA Upgrade AAV Manufacturing Without Platform Disruption?
Minicircle DNA can be integrated into established AAV production workflows with minimal changes to existing processes — supported by PlasmidFactory’s EU-based GMP manufacturing capabilities from early development to large-scale production.
Flexible implementation models
Depending on platform strategy, serotype, and development stage, different DNA format and manufacturing approaches can be applied — with or without Minicircle integration.
Option 1 — Optimized system approach
Adopt PlasmidFactory's fully optimized proprietary 2-plasmid or 2-Minicircle-based production systems.
Option 2 — Incremental upgrade
Integrate Minicircles into existing 2- or 3-plasmid AAV workflows by replacing only the transfer vector.
Option 3 — Design conversion
Convert existing AAV plasmid constructs into backbone-free Minicircle formats.
Option 4 — Amplification of established plasmid systems
Continue with your conventional AAV plasmid-based setup and scale production from research to clinical and commercial supply under GMP conditions.
All options can be combined with our innovative ITRRESCUE® & ITRPROTECT® technologies to optimize vector performance.
This flexibility allows developers to align DNA format choice, risk profile, and GMP pathways, enabling both incremental improvements and fully optimized next-generation manufacturing strategies — supported by PlasmidFactory’s DNA manufacturing and GMP expertise.
Why Partner with PlasmidFactory?
With more than 25 years of DNA manufacturing experience and EU-based GMP capabilities, PlasmidFactory supports AAV developers from early research to clinical production with:
• Expertise in backbone-free vector design
Learn More
Explore how Minicircle DNA can strengthen vector quality, safety, and manufacturing robustness in your AAV program.
Contact us at contact@plasmidfactory.com
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