No bacteria, no bottlenecks: The future of DNA IVT templates is synthetic

The remarkable success of mRNA therapies and vaccines, highlighted during the COVID-19 pandemic, is revolutionizing modern medicine. Central to the production of these mRNA-based interventions is the DNA template used in in vitro transcription (IVT) reactions. Traditionally, plasmid DNA (pDNA) has been the foundation for such therapies. However, as the demand for mRNA therapies surges, the limitations of pDNA are becoming more apparent, pushing manufacturers and researchers to look for alternative solutions.  A promising alternative is synthetic DNA, paving the way for more efficient, scalable and safe manufacturing of mRNA therapeutics and vaccines. 

Challenges with plasmid DNA and the need for alternatives 

Despite its widespread use, pDNA production is not without its challenges and the reliance on bacterial fermentation introduces a range of complexities. For example, there is risk of contamination with bacterial components, such as endotoxins, which pose safety risks if present in the final product. Additionally, bacterial fermentation is time-consuming and resource-intensive, requiring careful optimisation to achieve high yields. This can hinder scalability, making it difficult to meet the growing demand for mRNA therapies. 

Several bioprocessing steps also introduce process bottlenecks and increased production costs. Plasmid purification involves multiple steps, including cell lysis, filtration, and chromatographic separation, each of which adds complexity to the process. Furthermore, pDNA requires linearization before using in the IVT reaction, adding another processing step, which is both difficult to monitor and necessitates a further purification step to remove the enzymes. As demand for mRNA-based therapies and vaccines continues to rise, the inherent inefficiencies of pDNA production highlight the urgent need for alternative DNA production methods.  

Finally, maintaining polyA tail length of IVT templates is a major challenge in pDNA production since recombination of these homopolymeric sequences is a common occurrence in bacteria. This leads to multiple populations of differing polyA tail length, necessitating master cell bank production and careful monitoring during scale-up and between batches. Maintaining polyA tail length is vital for mRNA stability and biological function. 

Enter Synthetic DNA: A Novel Solution for mRNA Manufacturing 

Synthetic DNA has emerged as a viable alternative to pDNA, streamlining manufacturing whilst ensuring quality, safety and scalability. 4basebio’s synthetic DNA is produced through using proprietary enzymes, rather than relying on bacterial fermentation. This bacteria-free approach amplifies DNA sequences with high fidelity, whilst eliminating the risk of bacterial contamination and increasing the overall safety and compliance. 

4Basebio’s opDNA® is a partially opened, linear, double-stranded DNA template, that has been specifically designed as an IVT template. Each molecule features a hairpin at the 5’ end, with an opposite open 3’ end, allowing it to be fed directly into the IVT reaction without a linearisation or de-ending step. 

Benefits of opDNA® in mRNA Manufacturing

1. No need for linearization
   The 3’ open-end facilitates use directly in IVT reactions, eliminating the costly enzymatic linearisation step which is required when using circular pDNA templates.  

2. Enhanced PolyA stability
   Synthesising DNA enzymatically avoids the issue of recombination in bacterial hosts. Consequently, synthetic DNA templates can accommodate a stable polyA tail (>180 PolyA length). 

3. Enhanced Safety and Compliance
   prepared by enzymatic amplification of an engineered circular DNA template, which uniquely, is devoid of bacterial backbone sequences, including antibiotic resistance genes. Combined with the reduced risk of endotoxin and bacterial genomic DNA contamination, this improves the safety profile of the DNA template, compliant with regulatory standards for clinical use.  

4. Increased mRNA yields
   Bacterial backbones can often be 2 kb or greater in length. This is completely absent in opDNA® templates meaning only the relevant gene of interest including UTRs and poly A sequence are present downstream of a bacteriophage (T7 or SP6) promoter. Consequently, a higher copy number of desired IVT template per mass of synthetic DNA, results in greater IVT yields. This translates to lower production costs and offering a more economically viable alternative to pDNA. 

5. Sequence Flexibility
   Accommodates a variety of DNA sequences, ranging from 140 bp to 20 kb. The process is optimised to handle long and complex sequences, including regions of high GC content and viral derived sequences that are often a bottleneck for pDNA manufacturing. For example, self-amplifying mRNAs (SAMs) can be synthesised from opDNA® with improved mRNA integrity to pDNA templates.   

6. Flexibility of scale
   The enzymatic approach facilitates GMP-grade production at a range of scales, flexible to produce large-scale batches to meet the demands of a public health crises or, small-scale batches for personalised cancer vaccine applications. 

The inherent challenges associated with bacterial-based production of pDNA are evident and efficient, more scalable production methods must be adopted to meet the increasing demand of mRNA products. Synthetic DNA offers an innovative alternative, providing enhanced safety, scalability, and flexibility in mRNA manufacturing.

As the field of mRNA therapeutics continues to evolve, embracing these cutting-edge techniques will play an essential role in the production of next-generation therapies, meeting global demand whilst ensuring safety, cost-efficiency, and regulatory compliance.

To explore more about our opDNA® construct and how it compares to pDNA, click the button below.

References

1. Dhir, A., Walker, A. (2023, July 11). Cell-free synthetic DNA in mRNA manufacturing. Labiotech.eu. https://www.labiotech.eu/in-depth/enzymatically-produced-cell-free-synthetic-dna-mrna-manufacturing/  

2. Thalhamer, J., Weiss, R., & Scheiblhofer, S. (2011). Gene vaccines. https://doi.org/10.1007/978-3-7091-0439-2  

3. Challener, C.A. Synthetic DNA as an Alternative to Plasmids. BioPharm International 2024 37 (10)