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5 Things to Know About mRNA Capping for Next-Generation Vaccines and Therapies

Scientist on May 23, 2024

This installment of Tech Snapshot was written by TriLink BioTechnologies, a global leader in nucleic acid and mRNA solutions. TriLink delivers unrivaled chemical and biological experience, CDMO services and high-quality readymade and custom materials, including its patented CleanCap® mRNA capping technology.

The field of messenger RNA (mRNA) vaccines and therapeutics is rapidly evolving, offering new hope for treating and preventing a wide range of diseases. But behind the scenes, a crucial process ensures these therapies function correctly: mRNA capping. This essential step adds a specialized structure to the beginning of the mRNA molecule, acting like a protective shield, enabling it to generate the desired therapeutic effect. Here are five key takeaways researchers in this exciting field should know about mRNA capping.

1. Understanding the importance of mRNA capping

mRNAs are messengers carrying vital instructions for our cells. They are fragile and susceptible to degradation within the cellular environment. Capping at their 5’ end acts as a defensive shield, safeguarding the mRNA molecule from premature breakdown. This ensures the message reaches its intended destination – the ribosomes, cellular machinery responsible for protein production. Without proper capping, the mRNA would degrade rapidly, rendering its intended purpose as a vaccine or therapeutic useless.

Figure 1. Components of a mature mRNA.

2. Exploring the capping strategies and their considerations

Outside the cells, mRNA is made from a DNA template through a process known as in vitro transcription for its biological applications. The cap is not part of the DNA template and must be added to the mRNA. Several mRNA capping methods exist, each with its own set of considerations. Here’s a closer look at three main approaches:

  • ARCA method: Introduced in 2001, anti-reverse cap analog (ARCA) offers a simple approach since capping and mRNA synthesis take place in one step — commonly known as co-transcriptional capping. However, it generates a basic cap structure (cap 0) that might not be recognized as “self” by the human immune system. This can trigger unintended immune responses, reducing the vaccine’s effectiveness or causing side effects. Additionally, ARCA capping exhibits lower efficiency, producing less capped mRNAs compared to newer methods.
Figure 2. mRNA cap structures: cap 0, cap 1, and cap 2. Methylation on the first and second nucleotides of mRNA following N7-methylguanosine (m7G) determines their structures.
  • Enzymatic capping: This approach utilizes enzymes to add a cap-0 or cap-1 structure to the mRNA molecule. It provides more control over the capping process and can achieve higher efficiency than ARCA. However, enzymatic capping takes place after transcription (i.e., post-transcriptional capping) and involves multiple reaction steps and purification procedures, making it more complex and time-consuming.
  • CleanCap® technology: This innovative method stands out for its efficiency and streamlined process. It utilizes a patented cap analog to introduce a natural cap-1 structure, mimicking the structure found in mature human mRNAs. This approach not only enhances the stability of the mRNA molecule but also reduces the risk of unwanted immune response. Additionally, CleanCap technology operates as a one-pot solution, significantly reducing processing steps and simplifying manufacturing compared to enzymatic capping.
Figure 3. The steps of three common mRNA capping strategies involved in the production of capped mRNAs.

3. Capping for stability and efficiency

Effective mRNA vaccines and therapeutics rely on two key factors: the stability of the mRNA molecule and its ability to efficiently generate the desired protein product. mRNA capping addresses both these concerns. By protecting the mRNA from degradation, capping ensures it has a longer lifespan inside the cell, increasing the chances of successful protein production. Furthermore, certain cap structures, like the natural cap-1 generated by CleanCap technology, are recognized by the cellular machinery as being responsible for protein translation. This recognition enhances the overall efficiency of protein production, maximizing the potential of the mRNA vaccine or therapeutic.

4. Capping for a desired immune response

Vaccines work by stimulating the immune system to recognize and combat a specific pathogen. However, the vaccine itself shouldn’t trigger an immune response. Uncapped mRNA molecules, with their exposed 5’ phosphate group, can activate the immune system in an unintended way. This can not only reduce the vaccine’s efficacy, but also potentially lead to side effects. Proper mRNA capping with a cap-1 structure prevents this unwanted immune response. By adding a structure recognized as “self” by the human body, capping ensures the immune system focuses its attack on the target antigen associated with the specific pathogen encoded by the vaccine’s mRNA, leading to a more specific and effective response.

5. Choosing the right capping method: a balancing act

Selecting a suitable mRNA capping method is crucial for vaccine and therapeutic development. Each method comes with its own set of advantages and disadvantages, impacting factors like:

  • Efficiency: How effectively does the method add the cap to the mRNA molecule?
  • Immunogenicity: Does the generated cap structure trigger unwanted immune response?
  • Manufacturing complexity: How many processing steps are involved, affecting cost and production time?

For instance, while ARCA offers a simple approach, its lower efficiency and potential for immunogenicity might not be ideal. Enzymatic capping provides more control but comes with increased complexity. CleanCap technology emerges as a compelling option due to its high efficiency, natural cap-1 structure and streamlined process, offering a balance between effectiveness and manufacturing ease.

In conclusion, mRNA capping plays a critical role in the success of mRNA vaccines and therapeutics. Understanding the different capping methods and their impact on stability, efficiency and immunogenicity is essential for researchers and developers working in this rapidly advancing field. As we unlock the full potential of mRNA capping, we pave the way for a future filled with innovative therapies and a healthier tomorrow.

Visit TriLink’s website for technical resources supporting CleanCap technology.