An Overview Of In Vitro Transcription-Based mRNA Production

Messenger RNA (mRNA) therapeutics have dominated the news cycle since the COVID-19 vaccines were first created. mRNA vaccines and therapeutics have the potential to prevent or treat many life-threatening conditions. mRNA is delivered to a cell's ribosomes, instructing the body to create copies of a protein that either treats a specific disease or, in the case of vaccines, instructs the body’s immune system to fight a disease. Unlike cell or gene-based therapeutics, mRNA drugs do not affect DNA and break down once the protein is produced. Due to its effectiveness and potential to combat many previously incurable and/or untreated diseases, mRNA has rapidly become an attractive avenue for drug development.

However, manufacturing mRNA drugs is more complex than it may seem on the surface. The first step to manufacturing mRNA therapeutics is to create the drug substance that forms the basis of the drug product. This manufacturing process is known as the in vitro transcription (IVT) process. Creating a drug substance suitable for mRNA delivery via IVT is a complex journey with many stops along the way.

Understanding mRNA Structure And Function

mRNA is a single-stranded molecule that carries genetic information from the DNA in the cell's nucleus to the cytoplasm, which creates the proteins that govern bodily processes. mRNA is necessary for protein synthesis or translation and contains a 5′ cap, 5′ UTR, coding region, 3′ UTR, and poly(A) tail. During translation, ribosomes use the instructions conveyed by mRNA as a template for assembling amino acids to create the desired peptides or proteins.

IVT, also known as enzymatic synthesis, creates a synthetic mRNA product designed to mimic natural eukaryotic endogenous mRNA. Like naturally occurring mRNA, synthetic mRNA typically also consists of a 5' cap, 5' and 3' untranslated regions (UTRs), an open reading frame (ORF), and a poly(A) tail structure. The 5' cap is fundamental for mRNA stability and efficient translation, while the UTRs and poly(A) tail contribute to translational regulation and mRNA stability.

The structural features of IVT mRNA are carefully designed to ensure its stability, translational efficiency, and reduced immunogenicity. Modifications such as the incorporation of pseudouridine or N1-methyl-pseudouridine during the IVT process can further enhance IVT mRNA's stability and translational properties. Therefore, the structure of IVT mRNA is optimized to simulate endogenous mRNA and achieve specific therapeutic goals.

Fundamentals Of The IVT Process

For decades, scientists have understood that DNA functions as an architect's blueprint in all living cells, while RNA acts like a general contractor bringing the architect's vision to life. To create the mRNA drug substance, manufacturers rely on IVT. IVT uses bacteriophage-derived modified RNA polymerases to create mRNA based on synthetic DNA templates. Next, the mRNA is purified to remove contaminating molecules that might trigger undesirable immune responses.

During IVT, RNA polymerases, such as T7, T3, and SP6, assist in initiating and elongating RNA synthesis. The resulting RNA transcripts can be used in analytical techniques, structural studies, biochemical and genetic studies, and as functional molecules. The IVT process involves template design, RNA polymerase-assisted transcription, template removal, capping, and adding a poly(A) tail. High-quality IVT enzymes and NTPs optimize the reaction and ensure the production of pure RNA transcripts. The use of in vitro-synthesized RNA is essential for creating RNA-based therapeutics.

The most famous example of successful IVT mRNA application is the mRNA-based COVID-19 vaccines. These vaccines’ unprecedented success pushed IVT mRNA production into the limelight and paved the way for future vaccines and therapeutics, encouraging scientists to continue to refine and perfect the IVT process.

The Role Of Enzymes In mRNA Synthesis

Synthesizing mRNA via IVT involves several enzymes to ensure that the mRNA is efficiently and accurately generated. IVT involves several key enzymatic steps, including:

RNA Synthesis: RNA polymerases instigate RNA synthesis from a DNA template. During the IVT reaction, RNA polymerases are used to initiate and elongate RNA synthesis in the presence of a DNA template containing a specific promoter sequence. Several RNA polymerases used in IVT include T7, T3, and SP6. Advancements in IVT technology have led to the engineering of RNA polymerases to minimize the production of immunostimulatory byproducts, thereby improving the quality and safety of the synthesized mRNA products.

RNA Capping: In IVT, capping enzymes add a protective cap structure to the manufactured mRNA. The cap provides stability and translational efficiency. The process adds a 7-methylguanylate cap structure (Cap 0) to the 5' end of the RNA. The capping enzymes used in IVT, such as Vaccinia Capping Enzyme (VCE), are derived from eukaryotic organisms or DNA viruses and are added to the IVT reaction to facilitate the capping process.

Tailing of RNA: Adding a poly(A) tail, a stretch of adenosine nucleotides, to the 3' end of an RNA molecule is carried out by enzymes called poly(A) polymerases (PAPs).

Poly(A) polymerases catalyze the addition of multiple adenosine monophosphates to the 3' end of an RNA transcript, forming the poly(A) tail. The poly(A) tail is an important feature of most mature messenger RNAs (mRNAs) in eukaryotes and is crucial to mRNA stability, export from the nucleus, and translation efficiency. Other commonly used enzymes include the following:

Pyrophosphatases: remove unwanted byproducts from RNA synthesis
RNase inhibitors: prevent RNA degradation that may occur during the manufacturing process
DNase: removes the template DNA
RNA modification enzymes: used for post-transcriptional modifications of mRNA to enhance stability and translational properties
Ligases: used in IVT to stabilize the fluorescence resonance energy transfer (FRET) signal during the detection of mRNA production, which allows for the quantification of mRNA production.

In this discussion, John Stubenrauch, Ph.D., COO, Nutcracker Therapeutics, and Hari Pujar, Ph.D., COO, Tessera, speak with Anna Rose Welch, Editorial & Community Director, Advancing RNA, about mitigating issues posed by raw material variability in the IVT process, particularly with enzymes.

IVT Workflow For mRNA Therapeutics

The IVT workflow for mRNA therapeutics begins with template selection, optimization, and enzyme selection and validation.

Template Selection And Optimization

First, researchers choose the precise DNA template to produce the desired mRNA construct. The template is a plasmid DNA (pDNA) engineered to contain the gene of interest. This template contains the sequence to be transcribed with an upstream RNA polymerase promoter site. Before IVT, the DNA template undergoes purification to ensure high-quality mRNA while protecting transcription yield and mRNA integrity. Various methods, such as UV absorption, agarose gel electrophoresis, and sequencing, are used to check the integrity and quality of the DNA template. Optimizing the template design is crucial to simplifying downstream processing and ensuring the appropriate balance of the mRNA structure.

Enzyme Selection And Validation

The next step is choosing which enzymes will optimize the template design and the IVT workflow and validating them through mechanistic, computational, and laboratory screenings. First, researchers assess an enzyme's ability to produce high-quality mRNA with minimal impurities, particularly immunostimulatory byproducts. For example, in the case of T7 RNA polymerase (T7 RNAP), which is commonly used in IVT, researchers have engineered mutant variants to reduce the production of immunostimulatory impurities during mRNA synthesis. This process entails evaluating the mutant's impact on impurity profiles, RNA yields, and the potential for downstream purification. The goal is to identify and optimize enzymes that can streamline mRNA manufacturing processes by minimizing the generation of undesirable byproducts, thereby enhancing the safety and efficacy of the resulting therapeutics.

mRNA Synthesis Steps

Synthesizing RNA from DNA involves initiation, elongation, and termination. These steps are fundamental to mRNA synthesis and are carefully regulated to ensure mRNA molecules' accurate and timely production.

Initiation: occurs when RNA polymerase binds to the promoter region of the DNA. The DNA unwinds, and the RNA polymerase uses the DNA template to create synthetic RNA.
Elongation: the RNA polymerase moves along the DNA template, synthesizing an RNA molecule complementary to the DNA sequence. The RNA molecule lengthens as the RNA polymerase continues to add nucleotides to the growing mRNA chain.
Termination: the RNA polymerase reaches a specific termination sequence in the DNA. The RNA polymerase and the newly synthesized mRNA are released from the DNA, and the mRNA undergoes further processing to become a mature, functional molecule.

Post-synthesis Modifications

The 5' cap and 3' poly(A) tail are critical modifications added to mRNA molecules during their synthesis and processing. The 5' cap and 3' poly(A) tail function synergistically - the cap enhances poly(A) tail function, and vice versa, for optimal mRNA translation in vivo. Proper incorporation of these modifications is crucial for producing functional mRNA drug substances. Methods for creating these modifications include the following:

Adding the 5' Cap

The 5' cap is added co-transcriptionally during mRNA synthesis by the capping enzyme complex. This enzyme has RNA triphosphatase, guanylyltransferase, and methyltransferase activities to form the cap-0 structure (m7GpppN). An additional 2'O-methyltransferase can modify the cap-0 to the cap-1 structure (m7GpppNm), which helps evade immune detection. Capping enzymes from viruses like Vaccinia or Faustovirus are commonly used for in vitro capping of synthetic mRNAs.

Adding the 3' Poly(A) Tail

The poly(A) tail is typically encoded in the DNA template used for in vitro transcription (IVT) of mRNA. Alternatively, it can be enzymatically added post-IVT using poly(A) polymerases like E. coli PAP. The length of the poly(A) tail (usually >100 nt) is optimized for mRNA stability and translation efficiency. Kits like the HiScribe T7 ARCA mRNA kit enable co-transcriptional capping and post-IVT poly(A) tailing.

Purification And Quality Control

After capping and polyadenylation, the synthesized mRNA undergoes purification to remove any remaining impurities, template DNA, or byproducts. Quality control measures are essential to ensure that the mRNA meets the required further development and therapeutic use standards.

Advantages And Challenges Of IVT

IVT enables the design of tailored mRNA molecules for specific therapeutic applications. It is also highly scalable, allowing for the production of large quantities of mRNA with high reproducibility, making it suitable for commercial manufacturing.

Despite these advantages, IVT also comprises unique challenges, such as enzyme specificity and efficiency, cost considerations, and process optimization, as well as managing the stability and shelf life of synthesized mRNA. First, enzyme specificity and efficiency are essential to synthesize high yields of mRNA accurately. Validating enzymes, such as RNA polymerases and capping enzymes, is necessary to ensure the precision and effectiveness of the mRNA synthesis process. However, although IVT can be cost-effective compared to other manufacturing methods, it can still be expensive. Companies must optimize their processes to minimize production costs while creating high yields and consistent quality. Raw material usage, enzyme efficiency, and purification processes can all be optimized to reduce expenditures.

Meanwhile, stability and shelf life continue to complicate mRNA therapeutics' manufacturing and distribution processes, especially when strategizing formulation, storage, and transportation options. Safeguarding the stability of mRNA products is crucial to ensuring efficacy.

John Stubenrauch, Ph.D. (Nutcracker Therapeutics), Hari Pujar, Ph.D. (Tessera), and Anna Rose Welch (Advancing RNA) discuss innovations that are being made — or still need to be made — in the IVT process.

Emerging Technologies And Innovations

Innovations and emerging technologies are being developed globally due to the potential of mRNA therapeutics to prevent or cure life-threatening diseases. Scientists are developing novel enzymes and improving enzyme engineering to enhance the efficiency, scalability, and rapid manufacturing of mRNA-based drugs.

Novel Enzymes And Enzyme Engineering

Emerging science is developing novel enzyme biocatalysts as well as improving existing enzymes to enhance their activity and specificity. Additionally, the development of mutant variants of RNA polymerases aims to reduce the generation of immunostimulatory impurities during mRNA synthesis. These developments are anticipated to advance the field of IVT, opening the door for more efficient production of mRNA drug substances.

Automation And Robotics In IVT

Automation is expected to radically improve many processes in drug manufacturing, including developing mRNA drug substances. Automating IVT, in particular, can increase efficiency, quality, and safety. Reducing human involvement through automation creates higher throughput and reproducibility. Additionally, automation can help overcome sample-handling problems, create exhaustive experimentation capabilities, and provide a more rigorous environment for controlled synthesis.

AI And Machine Learning In Experimental Design & Process Optimization

AI and machine learning (ML) can predict and overcome potential mRNA manufacturing challenges, ensure quality control, and continuously improve the process by learning from real-time data.

By analyzing vast amounts of experimental data, AI models can identify fundamental physicochemical properties and molecular features that influence the behavior of mRNA-based therapeutics, thereby facilitating the design of more effective drug delivery systems. They perform real-time monitoring and analysis, enabling prompt issue detection and resolution. AI is also used for predictive maintenance, reducing equipment downtime.

ML algorithms assist in experimental design for mRNA substance manufacturing by enabling the prediction of RNA secondary structures, which is crucial for understanding the functional properties of mRNA. These algorithms can learn from training data that include RNA sequences and their structures, thus constructing more accurate models by increasing the number of parameters. ML algorithms optimize drug candidates by considering various factors, including efficacy, safety, and pharmacokinetics, thus aiding in developing innovative mRNA-based therapeutics. Finally, ML can optimize process parameters, improving product quality and reducing resource consumption — including labor.

Future Prospects And Potential Advancements In IVT

mRNA substance manufacturing will continue to evolve rapidly as the popularity of mRNA therapeutics grows. Researchers continue to pioneer more efficient, scalable, and cost-effective manufacturing processes for mRNA-based therapeutics, contributing to their broader accessibility and potential for revolutionizing drug development.

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