Addressing the Plasmid Production Bottleneck: Isothermal Amplification in IVT 

Can we ditch the plasmids? Template-free preparation is gaining momentum in mRNA therapeutics for rare diseases and beyond.

 

The intermediate between the originating genetic sequence and the final functional protein, the messenger RNA (mRNA), has been put into the spotlight thanks to several inherent advantages. The global COVID-19 pandemic highlighted the abbreviated mRNA development cycle and a simplified manufacturing process to boot. Further, mRNAs represent a therapeutic modality with high platform potential, seen as a critical enabler to address rare disease since the cost of development of one-off treatments for smaller patient populations simply isn’t feasible.¹

 Together, mRNAs offer a rapid, cost-effective, and platform-ready method to target previously undruggable targets. As an industry, we have witnessed a logarithmic expansion of clinic-ready traditional nonreplicating and self-amplifying mRNAs intended to be used as prophylactic vaccines, cancer immunotherapy, and other indications.^2,3

While the mRNA space continues to grow and progress, one bottleneck threatening the exciting breakneck pace involves the plasmid production step. Researchers and industry leaders are developing creative alternatives to traditional DNA template preparation and the plasmids it requires. Novel isothermal amplification-based approaches show promise in breaking the bottleneck in therapeutic mRNA production at scale for rare diseases and beyond.

Current Trends in mRNA Manufacturing

With a significant front-loading of the mRNA clinical pipeline, a renewed focus on manufacturing has emerged with a keen emphasis on quality and consistency of the mRNA.⁴ Input reagents, and their quality level, have been the focus as selection of these key materials is crucial as mRNA continues to scale.⁵  “There are obviously going to be advantages of using GMP materials from the start”, said Daniel Dixon, a Field Applications Scientist with Thermo Fisher Scientific. “Transfer from process development to manufacturing requires a transfer process that is simplified by using the same materials”, stated Dixon. However, complications around GMP-grade raw material supply as well as an inflationary market has certainly gained the attention of developers. “A good supply of material is crucial”, said Dixon.

RNA therapeutics are mostly developed and manufactured using in vitro transcription (IVT).⁶ The source sequence of the mRNA is encoded in a DNA plasmid (pDNA) template. This critical IVT raw material at a quality level suitable for large scale pharmaceutical production is a particular challenge. ⁷  Since the production of template pDNA is done with fermentation, significant cost and time is added to the mRNA manufacturing process. Although the pDNA is purified, leftover contaminants, such as proteins and nucleic acids from the E. coli host as well as endotoxins, can also be of concern. ⁷ Though well-controlled in GMP-grade pDNA, these contaminants do present a source of variability depending on the method and scale of pDNA purification. Finally, flux in market demands for pDNA, as seen during the COVID-19 pandemic, can translate to long lead times to acquire suitable GMP plasmid and represents a glaring weak spot in supply chain continuity.

Alternate IVT Template Amplification Methods

Isothermal amplification methods present an attractive alternative strategy for DNA template manufacturing intended for therapeutic mRNA IVT considering some of the potential setbacks with pDNA. Collectively, these methods consist of techniques such as rolling circle amplification (RCA), nucleic acid sequence-based amplification (NSABA), Strand displacement amplification (SDA), and Loop mediated isothermal amplification (LAMP). ⁸ These methods present mRNA developers with a simplified solution for template DNA preparation. “Isothermal methods have the advantage of reducing complexities. RCA is isothermal and it operates at one temperature, which negates the need for complex equipment and simplifies workflows”, noted Dixon. These methods utilize polymerases with the unique ability for strand displacement, such as Phi29. The amplification potential, and really the true advantage of RCA, is gained through the continuous addition of nucleotides to a primer annealed to the circular template while the reaction is held at a constant temperature. This results in long sequences with tens to hundreds of tandem repeats and the potential to identify a single molecule with the target sequence, all with minimal equipment investment.⁹

Translating this advantage to IVT, RCA could be utilized first through synthesizing circular DNA templates with the specific required elements added.⁹ Given the single molecule sensitivity of RCA, these synthetic templates could be efficiently and easily amplified via RCA to levels required for large scale IVT. This synthetic process-based approach would eliminate potential contaminants observed with E. coli-based plasmid amplification as well as providing additional timeline flexibility and less raw material variability.

How We Are Resolving pDNA Bottlenecks for IVT

The convenience and sensitivity of isothermal-based DNA amplification methods is an attractive strategy for pDNA replacement in the IVT reaction. Driving innovation in the mRNA space through the support of these isothermal amplification methods, like RCA, we are leveraging high quality and ICH Q7-compliant reagents, robust supply chains, and comprehensive technical support. Through the support of novel applications of isothermal amplification methods, Thermo Fisher Scientific aims to facilitate process scale-up through the removal of process and technology bottlenecks that will ultimately enhance mRNA success as a modality in therapeutics for rare diseases and beyond.

 

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References

1. Fang, E. et al. Advances in COVID-19 mRNA vaccine development. Signal Transduction and Targeted Therapy vol. 7 Preprint at https://doi.org/10.1038/s41392-022-00950-y (2022).
2. Wang, Y. et al. mRNA vaccine: a potential therapeutic strategy. Molecular Cancer vol. 20 Preprint at https://doi.org/10.1186/s12943-021-01311-z (2021).
3. Miao, L., Zhang, Y. & Huang, L. mRNA vaccine for cancer immunotherapy. Molecular Cancer vol. 20 Preprint at https://doi.org/10.1186/s12943-021-01335-5 (2021).
4. Kate Goodwin. Medicine’s Hot New Modality, mRNA, Faces Unclear Regulatory Landscape. https://www.biospace.com/article/medicine-s-hot-new-modality-faces-unclear-regulatory-landscape/.
5. Whitley, J. et al. Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform requirements and development of a scalable production process to support early phase clinical trials. Translational Research vol. 242 38–55 Preprint at https://doi.org/10.1016/j.trsl.2021.11.009 (2022).
6. Karikó, K., Muramatsu, H., Ludwig, J. & Weissman, D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 39, (2011).
7. lahijani1998.
8. Mohsen, M. G. & Kool, E. T. The Discovery of Rolling Circle Amplification and Rolling Circle Transcription. Acc Chem Res 49, 2540–2550 (2016).
9. Ali, M. M. et al. Rolling circle amplification: A versatile tool for chemical biology, materials science and medicine. Chemical Society Reviews vol. 43 3324–3341 Preprint at https://doi.org/10.1039/c3cs60439j (2014).

 

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