Rok Sekirnik

The poly(A) tail of mRNA plays a vital role in mRNA transcript stability, translational efficiency, and immunogenicity. Co-transcriptionally polyadenylated in vitro transcribed (IVT) mRNAs typically contain poly(A) tails of 50–120 nucleotide tail length due to limitations in production of template pDNA with longer poly(A) sequences. In contrast, post-transcriptional enzymatic polyadenylation of mRNA with poly(A) polymerase (PAP) presents a modular alternative to increase the tail length. However, the lack of real-time control strategies for PAP-mediated tailing has limited its broader applicability in mRNA production. Here, we develop a methodology for controlling poly(A) tail length in post-transcriptional polyadenylation of mRNA that uses adenosine triphosphate (ATP) consumption measured at-line to predict the poly(A) tail length. We establish a novel analytical method based on monolith reverse-phase chromatography to validate the poly(A) predictions. We were able to produce longer poly(A) tails and accurately determine their length in 300–700 nt range. The resulting longer poly(A) tailed reporter mRNAs outperformed the encoded and shorter poly(A) tailed mRNAs in cell-based assays. This work presents a new strategy for controlled post-transcriptional polyadenylation using ATP consumption as a process control metric, an approach which may in future be expanded to other NTP-dependent enzymatic conversions.

The so-called in vitro transcription (IVT) reaction, the enzymatic process converting a DNA template into RNA using bacteriophage RNA polymerases, has become a defining gateway to a new class of medicines. By enabling the scalable production of synthetic mRNA, IVT laid the foundation for the rise of mRNA vaccines, which received an incredible amount of public and scientific attention due to their role in mitigating the risks of the Covid-19 pandemic. Since then, mRNA technology has entered a renaissance, reflected in the steep increase in clinical trialsrising from fewer than 200 in 2021 to approximately 450 by the summer of 2025 -and in the approval of the first non-Covid-19 vaccine (RSV vaccine). Therapeutic areas have broadened to individualised cancer treatment (neoantigen therapies, Rojas et al., 2023) and gene therapies targeting genetic diseases such as cystic fibrosis (Bai et al., 2024) and sickle cell anemia (Breda et al., 2024). mRNA manufacturing involves mRNA sequence design, synthesis and purification for therapeutic applications. The synthesis step is performed through in vitro transcription of a linear DNA template, typically a linearized plasmid DNA (pDNA). This process requires the preparation of pure linearized pDNA which -in addition to the promoter required to initiate the transcription reaction -contains key sequence elements required for functional mRNA: 5' and 3' untranslated regions (UTRs), coding region, and poly(A) tail. Contrary to plasmid DNA, which is generally produced in E. coli, the subsequent IVT reaction is carried out in a completely cell-free environment. This reaction minimally requires the above-described DNA template, an RNA polymerase, nucleoside triphosphates (NTPs), and Mg 2+ . A 5' cap structure on the mRNA can improve mRNA stability by protection against exonucleases and recognition by initiation factors to promote translation. Therefore, a 5' cap can be added to the mRNA during the in vitro transcription either co-transcriptionally ('co-capping') by adding a suitable cap-analogue to be incorporated as the initiating nucleotide building block, or post-transcriptionally ('post-capping') using a capping enzyme. Improvements in capping strategies can enhance translation efficiency and mRNA stability. Chemical modifications of the cap structure, e.g., within the triphosphate linkage, can further improve mRNA stability and enhance ribosome recruitment. The poly(A) tail length also affects both mRNA stability and translation efficiency. Increasing the tail length increases protein expression, but beyond an optimal range, translation efficiency plateaus as additional adenosine residues provide no further benefit. UTRs from various genes, including globin and genes from tobacco etch virus, are often used to enhance translation and stability.The Research Topic 'In Vitro Transcription (IVT) Reaction -The Gateway to New Therapeutic Modalities' brings together eight complementary contributions that collectively advance our molecular, analytical, and process-engineering understanding of IVT. These works span topics from enzyme and substrate design to real-time analytics and impurity control, reflecting the maturation of IVT from a bench-top reaction to an industrially relevant, quality-by-designenabled manufacturing process for diverse RNA modalities, including mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA), and transfer RNA (tRNA) therapeutics.As editors, we were amazed to observe the progress reported across most of these fronts, and beyond. Contributions to the collection include not only the science of mRNA, but also studies on DNA as a critical raw material for IVT. For example, the Research topic published the first report of the impact of DNA template purity on the quality of IVT product (Martinez et al., 2023) which demonstrated that impurities in linearised DNA template can lead to production of aberrant RNA, including dsRNA, if impurities are recognised as template by RNA polymerase. The latter received much attention: Nair and Kis (Nair and Kis, 2024) examined the phylogenetic and structurefunction relationship of T7 RNA polymerase and its engineered mutants designed to reduce immunogenic impurities, linking enzyme activity to the quality attributes of the mRNA product. This topic was further explored by Lenk et al., 2024, who provided a comprehensive analysis of product-and process-related contaminants arising from IVT, systematically categorising nucleotide-based impurities (such as dsRNA, abortive transcripts, and RNA:DNA hybrids) and non-nucleotide contaminants (including RNAse, endotoxins, and metal ions). Their work highlights how these impurities can activate pattern-recognition receptors and underscores the interplay between reaction design, template engineering, and purification strategy in achieving high-purity, clinically efficacious mRNA. mRNA capping received due attention in this collection, reflecting its tremendous impact on the cost-of-goods for mRNA synthesis. Kurpijewski et al. (2024) reported the synthesis of cap analogues modified at the N2 position of 7-methylguanosine and demonstrated their dual application as translation inhibitors and as capping reagent. This work is likely to spur further research into improved capping structures.To be optimally utilised in IVT reaction, reagents including capping analogues, NTPs and mRNA can be monitored at-line or in-line. The minireview by Lee et al. (2023) explored available analytical approaches for monitoring IVT, including light-up RNA aptamer and fluorescent dye pairs, fluorophore-labelled antisense probes, and HPLC methods. Welbourne et al. (2024) further advanced this area by developing an HPLC-based method for at-line monitoring of IVT progression, providing near-real-time information on the concentration of both building blocks (NTPs) and product (mRNA). A related analytical technique was used by Megušar et al. (2024) to investigate factors affecting...

MRNA synthesis by in vitro transcription reaction (IVT) is a critical step in the production of mRNA therapeutics. IVT is a complex, multi-component enzymatic reaction where mRNA is synthesized from a DNA template using RNA polymerase, typically T7 RNA polymerase, and nucleoside triphosphates (NTPs) in a buffered solution containing magnesium ions. The concentrations and ratios between reaction components need to be carefully balanced to maximize mRNA yield while minimizing the formation of product-related impurities and the cost of the reaction. This protocol describes the use of a chromatographic analytical method for separation of NTPs, pDNA, and mRNA with a 4-minute read-out. At-line analysis which allows to monitor mRNA production and NTP depletion can be used to study the influence of reaction parameters on reaction kinetics and yield as well as for reaction optimization and transfer to fed-batch mode.

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents, IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA polymerase, e.g. T7. Due to the high cost of IVT reagents, IVT is a critical step in the mRNA drug substance production process and has been the focus of intense optimization in the field. To decrease the cost of mRNA production, reagents must be utilized optimally. Effective optimization necessitates a comprehensive understanding of the impact of individual reagents on reaction kinetics, i.e., the consumption of nucleoside triphosphates (NTPs) and the production of mRNA. Traditionally, low-throughput, end-point analytical techniques have been used for the analysis of mRNA. Though such methods give valuable information on mRNA content, at-line, near real-time analytics is needed to fully understand IVT reaction. We demonstrate how a liquid chromatography analytical method that separates NTPs, as well as pDNA and mRNA, can be used in near real-time to study IVT reaction kinetics. With the knowledge of the influence of different IVT components on kinetics and yield, rapid chromatographic analysis can be used to convert batch IVT reaction into a fed-batch mode that further increases productivity and reduces the overall cost of the reaction.

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Engineered transfer RNA is an emerging therapeutic modality, particularly suited to treatment of diseases caused by genetic disorders based on premature termination codons, frameshifts, or missense mutations. It is also extensively used in reprogramming of in vitro translation systems to generate non-canonical amino acid-containing proteins and peptides, such as in mRNA display. Due to its length, chemical synthesis of tRNA is challenging and production of engineered tRNA at scale is currently limited to in vitro transcription from a DNA template. Previously, the highest reported in vitro transcription yield was 2.5 g/L, significantly below the industry standard for mRNA production of 7–10 g/L. To improve this process, we implemented monitoring of nucleoside triphosphate consumption and tRNA production during in vitro transcription, using at-line high-performance liquid chromatography, with a monolithic solid phase. This allowed for optimization of nucleoside triphosphate concentration, reduction of the in vitro transcription time to 90% step yield was then developed. These improvements in the production and purification of tRNA represent an important step in facilitating production of tRNA for research purposes, and provide a method for purification of therapeutic tRNAs that is scalable and compatible with Good Manufacturing Practice requirements for clinical production.