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Touchlight’s dbDNA can enable significant cost savings for AAV gene therapy developers, accelerating the drive towards more cost-effective AAV gene therapies. An independent analysis conducted by Decisional Points Limited revealed that switching from pDNA to dbDNA without transfection process optimisation, reduced CoGs per dose by 14%. Further CoGs savings beyond those modelled in this study could be achieved when switching to dbDNA due to the elimination of the need for bacterial MCB manufacture and efficiencies in downstream processing due to improvements in % full capsids.​

Download the poster to see the full cost savings analysis.

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Recombinant adeno-associated virus (rAAV) clinical translation remains constrained by significant manufacturing and safety challenges. One approach to address these limitations is to substitute the E. coli derived plasmid DNA with cell-free DNA. Touchlight’s linear dbDNA, an enzymatically amplified DNA construct, offers rapid, scalable generation of GMP-grade material in a fraction of the time of conventional plasmid DNA. dbDNA lacks bacterial sequences, making it an ideal DNA template for AAV production. To characterise the purity and quality of dbDNA-derived AAV vectors and plasmid-derived AAV vectors, nanopore sequencing was employed to profile the encapsidated DNA. Successful packaging of the ITR-to-ITR AAV genome using dbDNA was confirmed. Furthermore, the dbDNA-derived AAV population contained significantly fewer process- and product-related DNA impurities. While the reverse packaged plasmid sequences contained the antibiotic resistance and bacterial replication cassette, reverse packaged dbDNA comprised short inert stuffer sequences. Thus, dbDNA offers safety advantages over plasmid as an AAV template. Illumina sequencing confirmed full length ITRs are maintained during the production of dbDNA. Nanopore ITR characterisation of AAV genomes revealed high levels of intact ITRs in both dbDNA- and plasmid-derived AAV preparations. The dbDNA-derived AAV vectors contained lower levels of partial genomes compared to plasmid-derived AAV vectors. Thus, the high vector batch quality and low DNA impurity levels observed in dbDNA-produced AAVs further support their enhanced safety profile.

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Recombinant adeno-associated virus (AAV) is one of the most promising delivery vehicles for genetic medicines, offering potential to treat a range of previously untreatable diseases. With hundreds of AAV-based therapies progressing through clinical trials, the demand for high-quality DNA starting material is high. But today’s AAV production still relies on plasmid DNA (pDNA), which brings with it challenges that slow progress and limit scalability.

In this blog, we take a closer look at the limitations of pDNA and explain how our dbDNA provides a promising alternative. 

The challenges of plasmid-based AAV production

Today, the most common approach to manufacturing AAV is the co-transfection of HEK293 cells with three separate plasmids: one carrying the Rep and Cap genes for viral replication and capsid proteins, another providing adenoviral helper functions, and a third containing the therapeutic transgene flanked by inverted terminal repeats (ITRs). This system has been the gold standard of AAV research for years, but when it comes to clinical and commercial manufacturing, using pDNA introduces limitations. 

Limited scalability

pDNA is produced in E. coli through bacterial fermentation, a process that is slow, expensive, resource-intensive and difficult to scale. Producing GMP-grade pDNA can take 10-12 months, as complex fermentation, purification and quality control steps all add time. With demand for AAV vectors rising rapidly, this lengthy production cycle is a serious bottleneck for the gene therapy field. 

Poor fidelity

ITRs form cruciform secondary structures that are difficult to maintain in E. coli. During plasmid propagation, this structural instability can lead to deletions or rearrangements within the ITR regions, generating heterogeneous plasmid preparations. Compromised ITR integrity reduces the efficiency of viral rescue and packaging, resulting in lower and less consistent AAV yields. 

Risk of antibiotic resistance

Because plasmids rely on antibiotic resistance markers for selection in bacterial systems, transfected plasmids can contain backbone sequences encoding antibiotic resistance genes. Plasmid-derived bacterial sequences are known to be inadvertently packaged into AAV capsids at frequencies ranging from 1-5%, and in some cases as high as 26%. This poses obvious safety concerns, as it risks transferring resistance traits. It also raises serious regulatory red flags, creating more hurdles for developers trying to move studies forward. 

High manufacturing costs

On top of these issues, plasmid production in E. coli is expensive – most of the cost comes from time in GMP suites, specialist equipment, and the labour of highly trained staff. This reliance on resource-heavy facilities makes it difficult to expand capacity and to meet the growing demand for AAV vectors. 

The solution: dbDNA

At Touchlight, we’ve developed an advanced cell-free DNA technology platform that addresses the bottlenecks of plasmid-based AAV production. Our process is entirely in vitro and produces high-fidelity, covalently closed, linear DNA constructs, known as dbDNA.  

Unlike pDNA, dbDNA is generated without bacteria and relies solely on enzymes. The process begins with a circular, double-stranded DNA molecule containing the sequence of interest, flanked by short protelomerase recognition sites. This template is amplified by Phi29 DNA polymerase through rolling circle amplification, producing concatemeric repeats of the construct with high fidelity. A protelomerase then cleaves and covalently closes the ends to generate monomeric, linear dbDNA. Residual bacterial backbone is removed by restriction digest and exonuclease, leaving only the dbDNA sequence of interest. 

The resulting dbDNA offers several advantages for AAV production. 

Superior purity and safety

dbDNA contains no bacterial backbone, meaning no antibiotic resistance genes and no risk of them being carried into AAV particles. The result is a high-purity DNA construct, free from unwanted genetic sequences that raise regulatory concerns and delay clinical development. 

Faster timelines and scalable manufacturing

Because dbDNA is made entirely in vitro, it does not require large fermentation facilities or complex bacterial processes, which makes scaling simpler and less expensive. And with GMP-quality dbDNA produced in 2 weeks instead of the 10–12 months often needed for plasmids, it offers a faster, more economical way to meet growing demand. 

Stable and consistent

dbDNA can amplify complex sequences with high fidelity, ensuring that ITRs remain stable. This eliminates the heterogeneity often seen in plasmid preparations, and stable ITRs in turn support more efficient replication and reliable packaging into viral particles. 

Greater efficiency

dbDNA requires significantly less starting material to achieve equivalent or greater AAV titres compared with plasmids. In fact, dbDNA uses around 40% less DNA per transfection while delivering a more than two-fold increase in the proportion of full capsids. This “copy advantage” reduces input costs while supporting higher overall efficiency in vector production. 

Summary

As gene therapy continues to advance, the limitations of plasmid-based AAV production threaten to slow progress and restrict patient access to life-changing treatments. Our dbDNA offers a way forward. By providing a faster, safer and more scalable source of DNA, dbDNA has the potential to resolve many of the major challenges facing AAV manufacturing. For the field of genetic medicine, this technology has the potential to transform how therapies are made and bring life-changing treatments to patients sooner.

Ready to bring your therapy to patients faster? Download our whitepaper and see how dbDNA AAV production streamlines your journey to the clinic.

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Discover how synthetic, backbone-free DNA offers a safer, scalable alternative to traditional plasmid DNA for AAV production, addressing key challenges in transfection efficiency, cost, and impurity control.

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