Unlocking the Potential of Minicircle DNA

There are a range of potential limitations when using pDNA for cell and gene therapy applications.

The first and most obvious is that pDNA contains the bacterial backbone – including the origin of replication and the antibiotic resistance genes – which is required for amplification via microbial fermentation. This puts limits on the size of the genes that can be delivered, preventing the use of longer sequences that may be preferable for their therapeutic effect. In addition, transfection efficiency is reduced due to the larger size and bacterial elements, reducing overall expression of any therapeutic protein(s).

The second is the increased risk of contamination – including endotoxin, host cell proteins and host cell genomic DNA – that can occur as part of microbial fermentation. The purification of the plasmid has to remove any traces that could be immunogenic, to ensure patient safety.

Minicircles, on the other hand, are much smaller, meaning they can incorporate larger therapeutic genes, have enhanced transfection and also enhanced expression which is longer-lasting. Finally, the safety profile is much improved due to the elimination of bacterial sequences.

The traditional method for producing minicircles involves the generation of a custom plasmid and the use of recombination to eliminate the bacterial backbone. It still requires the use of microbial fermentation to amplify the pDNA, which comes with its own challenges.

Our process completely eliminates the need for microbial fermentation, which means we can produce pure minicircles without any risk of contamination from the process itself. It is also significantly simpler than traditional methods meaning our process is faster, potentially cheaper and producing a cleaner product.

It was a significant milestone and the first of many, we hope!

We have demonstrated that it is possible to produce fully synthetic minicircle at significant scale with full characterization. It shows there is now an alternative to traditional methods – and also proves that the technology works. With further development, we can move on to democratizing minicircles for a wider range of modalities, including monoclonal antibody production. This will allow manufacturers to bypass the need for microbial fermentation moving directly to mammalian culture, saving time and cost.

There are a wide range of potential benefits to a fully synthetic approach to minicircle production. Cell-free systems bypass plasmid amplification bottlenecks and recombination inefficiencies, with direct synthesis avoiding the complex multi-step process of bacterial transformation, culture, induction and recombination. This can lead to reductions in production cycles from weeks/months to hours/days, dramatically accelerating manufacturing timelines.

The elimination of microbial hosts from the process removes concerns surrounding endotoxins, host cell proteins or residual bacterial genomic DNA. Moreover, high-fidelity polymerases in cell-free systems can reduce error rates compared to bacterial replication machinery, leading to higher-quality synthetic DNA.

Cell-free reactions are more easily scaled through parallelization or volume increases without the complexities of bioreactor scale-up. In addition, precise control over reaction components allows for reproducible, consistent production batches.

Without cellular debris and bacterial contaminants, downstream purification becomes significantly simplified, with elimination of antibiotic selection markers and bacterial sequences from the entire production process addressing key regulatory concerns.

With an enzymatic approach, new minicircle designs can be quickly produced without bacterial cloning steps. Enzymatic systems can also accommodate a wider range of minicircle sizes without the constraints imposed by bacterial maintenance. Furthermore, enzymatic approaches allow for site-specific incorporation of modified bases or attachments that might be toxic to bacterial hosts, opening up new opportunities downstream.

The regulatory burden is expected to be light, compared to other novel forms of DNA that are being developed. Minicircle DNA is well understood and the introduction of a physically and functionally identical synthetic version should be relatively straightforward.

Issues to explore are most likely to involve comparisons between a cell-free synthetic process with the traditional methods – for example, determining if there are any unique process-related impurities, demonstrating sequence fidelity and structural integrity, and ensuring a similar stability profile. The standard controls, such as process validation, control of raw materials and testing for toxicity/immunogenicity, will need to be included.

NunaBio is investing heavily in a range of characterization methods, as well as implementing the highest quality standards, to potentially deliver material to GMP. Again, regulatory considerations will likely be lower as the final product is a raw material, rather than a therapeutic, and the format is well understood with a minimal level of novelty.

We are also investing heavily in the development of a range of prototype instruments based on our proprietary process. We are looking at methods to both scale-up production to produce larger single batches, but also scale-out to allow for the development of hundreds to thousands of sequences in parallel. Finally, we are exploring the development of a continuous process, to produce DNA 24 hours a day.

Our eventual aim is to be able to move from sequence to manufacturing scale in seven days.