By Ashish Dhir, PhD and Amy Walker, PhD, 4basebio
The pace, efficacy and scalability with which the success of mRNA vaccines against COVID-19 was demonstrated opened endless possibilities for mRNA medicine in broad areas of infectious diseases, cancers, protein-encoding replacement therapies.
The advantages of RNA medicine are obvious, with the ease and speed of design and testing, rapid scale up and manufacturing, and the negligible risk of insertional mutagenesis often seen as an Achilles heel for DNA-based therapy. Nevertheless, the requirement for intracellular delivery of the mRNA to target cells while preserving its stability remains an important challenge in the field.
RNA is intrinsically an unstable molecule with inherent immunogenicity, and much of the early work on turning the concept of mRNA medicines into a reality focused on stabilization and the prevention of RNA mediated unwanted immune responses. Various approaches based on our fundamental knowledge of RNA have been exploited, including optimizing the 5′ cap structure and the 3′ poly(A) tail length, as well as regulatory elements within the 5′ and 3′ untranslated regions that in the presence of certain RNA modifications (most commonly N1-methylpseudouridine) enhance stability while restricting the innate immune response to synergistically boost mRNA-derived protein production.
The ingenuity lies in mRNA harnessing human cells as its own vaccine-production facility, with several accompanying advantages as it allows human post-translational modification (PTM) of protein products with the potential for less immunogenicity and full functionality. Moreover, it allows for an efficient way of dealing with multimeric proteins including cocktail vaccines and the localization of transmembrane and intracellular proteins to increase the scope of treatment. This promise is reflected in the number of mRNA-based clinical trials currently under progress.
In mRNA-based clinical trials, including protein replacement and oncology-based precision medicine, various vaccination platforms are being evaluated in the clinic that primarily rely on plasmid DNA as template for RNA synthesis. However, there are fundamental issues with the use of plasmid DNA that are not only limited to the presence of a bacterial backbone, antibiotic resistance genes and endotoxins, but also long lead times to produce large-scale (several grams) clinical grade material that is GMP compliant.
Plasmid DNA production involves bacterial fermentation in large-scale bioreactors that are often at risk of batch failures. Also, plasmid DNA encoded homopolymeric sequences (poly(A) tails and ITRs) recombine during bacterial amplification of the plasmid DNA, resulting in high sequence instability and heterogeneity. These prevalent issues, combined with a large footprint, and often accompanied with low yields, can add extreme cost and time to this upstream step.
Synthetic DNA can overcome challenges in mRNA manufacturing
The use of enzymatically-produced synthetic DNA templates can greatly overcome these issues.
From a manufacturing standpoint, enzymatic DNA synthesis has a significantly reduced manufacturing footprint; typically DNA is amplified at greater than 1g/L yields. This allows not only for scale up and scale out – a 10g batch of DNA can be manufactured using simple, single use, bench top equipment – but also enables rapid tech transfer. For example, the process could be easily installed in difficult to access areas in remote parts of the world, for rapid pandemic response.
The use of defined enzymes and components for in vitro DNA synthesis also results in a simplified down-stream purification processes; unlike conventional plasmid fermentation.
Synthetic DNA reduces error rate
One concern with enzymatically-produced DNA is the fidelity of amplification; conventional polymerases have high error rates when compared to bacterial amplification systems, which poses a hindrance to the PCR approach in both the cost of large-scale production and high risk of mutations.
The use of engineered enzymes with proofreading capabilities can lead to a significantly reduced error rate more in line with plasmid systems. Our 4bb Trueprime polymerase allows a high yielding, accurate, primer free DNA amplification with a resulting fidelity = 3.75*10-9 (PCR has an error rate about 200,000 times higher) with no sequence bias.
Additionally, the use of a primase, rather than sequence specific primers, can further enhance fidelity when combined with an engineered DNA polymerase. An input DNA template, required for rolling circle amplification (RCA), can be a plasmid DNA template, however, this results in the amplification of the plasmid backbone, which then needs to be enzymatically digested downstream. This plasmid amplification increases the chance of antibiotic resistance genes being incorporated into the final product. Alternatively, the use of a G-block template, which can then be circularized using standard molecular biology techniques, means that the entire process can be conducted using synthetic technologies, and be entirely devoid of bacterial fermentation.
A key benefit of synthetic DNA templates over plasmid DNA for mRNA production is the fact that the template does not need to be enzymatically linearized prior to in vitro transcription (IVT), a step that is often limited by choice of restriction sites, efficiency and quality of digestion, hence reducing the time and cost effectiveness of this critical upstream step.
Another benefit is the efficient/reliable inclusion of longer poly(A) tails that is well established in the industry to enhance stability and translational capacity of the mRNA. Poly(A) tracts are prone to recombination in conventional plasmid fermentation, and often researchers compromise by including a linker to break up the poly(A) tract, or by accepting a shorter poly(A) with a degree of heterogeneity within the mRNA transcript.
Alternatively, mRNA can be polyadenylated post-transcriptionally, which is a costly enzymatic process that is difficult to control and can lead to a heterogeneous poly(A) length within the final mRNA product. The strand displacement capabilities of the polymerases used in RCA reactions enable the incorporation of long homopolymeric tracts within the DNA template, enabling a clean, homogenous polyA tail. IVT yields per mass of DNA are likely to be higher for synthetic DNA as, unlike plasmid DNA, it lacks any non-transcribing DNA backbone.
With the emergence of the RNA-based therapeutics era, there is an unmet demand for readily compatible and “ready to use” DNA templates for RNA manufacturing at all scales. We envision that synthetic DNA will be an enabler for the commercialization and scale-up of mRNA therapeutics and vaccines with improved safety benefits and fast turnaround.
Ashish Dhir is RNA development lead, and Amy Walker is the director of discovery at 4basebio