Nature in-depth review: the development strategy and future of mRNA technology

The success of mRNA technology in the development of new crown vaccines has not only accumulated a wealth of positive efficacy and safety data in the vast population, but also demonstrated the way to promote mRNA technology from research and development to regulatory approval

Recently, Nature Reviews Drug Discovery published two articles.

Principles of mRNA design and synthesis

One of the main parts of mRNA vaccines is synthetic mRNA molecules, which instruct cells to produce antigens that stimulate immune responses

One of the functions of the 5'end cap is to prevent mRNA from being detected by sensors that recognize viral RNA in cells, thereby preventing unnecessary immune responses

The length of the 3'polyadenine tail indirectly regulates the translation and half-life of mRNA

The 5'and 3'UTRs regulate the translation, half-life and intracellular localization of mRNA

▲The 5 parts of mRNA structure (picture source: reference [1])

The open reading frame encoding the antigen is the most important part

Another improvement measure to improve translation is the introduction of modified nucleosides, such as pseudouracil, N1-methyl pseudouracil and other nucleosides

The use of modified nucleosides can prevent recognition by pattern recognition receptors, thereby allowing mRNA to generate sufficient levels of protein antigens to stimulate an immune response

CureVac's strategy to prevent mRNA from being discovered by pattern recognition receptors is to eliminate uracil in vaccine mRNA through sequence engineering and codon optimization, while increasing GC levels

In addition to improving the mRNA sequence, the mRNA production process has also been simplified in the past few decades

Adding the polyadenine tail sequence to the plasmid DNA simultaneously solves the problem of different synthetic lengths of the polyadenine tail

The combination of these various innovations overcomes many challenges in mRNA production and promotes the birth of a simple, effective, and scalable mRNA synthesis process

Vectors for delivery of mRNA vaccines

Because mRNA is a large molecule with negative charges, it cannot pass through the cell membrane composed of anionic lipids, and in the body, it will be swallowed by cells of the innate immune system or degraded by nucleases.

Lipid nanoparticles

Lipid nanoparticles (LNP) are currently the fastest clinically progressing mRNA delivery technology.

Usually LNP contains four components, ionizable lipids, cholesterol, auxiliary phospholipids and PEG-modified lipid molecules

▲The main lipid components of lipid nanoparticles (picture source: reference [1])

Ionizable lipid molecules are arguably the most important component of LNP.
They carry a positive charge in an acidic environment, allowing them to combine with negatively charged mRNA to form LNP
.
They are neutral at physiological pH, which improves their safety and prolongs their residence time in the blood circulation
.
After being engulfed by cells into the endosome, the acidic environment of the endosome will allow them to carry a positive charge again, thereby promoting fusion with the endosomal cell membrane and releasing mRNA into the cytoplasm
.

Cholesterol improves the stability of nanoparticles and helps the fusion of liposomes with endosomal cell membranes
.

The auxiliary lipid molecules regulate the fluidity of the nanoparticles and assist in fusion with the cell membrane of the endosome
.

PEG-modified lipid components increase the stability of LNP, regulate the size of nanoparticles by limiting lipid fusion, and increase the half-life of nanoparticles by reducing non-specific interactions with macrophages
.

Composite and polymer nanoparticles

Although the clinical progress is not as rapid as LNP, polymers and lipids have similar advantages and can effectively deliver mRNA
.
Cationic polymers can form complexes of different sizes with mRNA
.
There are already a variety of biodegradable polymer materials for effective mRNA delivery
.

Similar to ionizable lipid molecules, pH-sensitive polymers have also been used to deliver mRNA
.
These polymers will be protonated at the acidic pH of the endosome to promote the release of RNA
.

▲Polymer nanoparticles for mRNA delivery (picture source: reference [1])

Other delivery systems

In addition to lipid and polymer-based delivery methods, polypeptides can also be used to deliver mRNA, because some amino acids carry cationic or amphiphilic amino groups and can bind to mRNA
.

Finally, squalene-based cationic nanoemulsions have also been used to deliver mRNA
.
These nanoemulsions consist of a squalene-based core and a lipid shell
.
mRNA is adsorbed on the surface of the nanoemulsion
.
Some squalene formulations can act as an adjuvant
.
For example, Novartis's MF59 is used as an adjuvant in FDA-approved influenza vaccines
.
MF59 causes cells at the injection site to secrete chemokines, recruits antigen-presenting cells, promotes the differentiation of monocytes into dendritic cells, and enhances antigen uptake by antigen-presenting cells
.

▲Cation nanoemulsion for mRNA delivery (picture source: reference [1])

Progress of mRNA vaccines for infectious diseases

In terms of the clinical application of mRNA technology, the development of vaccines against infectious diseases is the fastest-moving direction
.
The review pointed out that as of June 18 this year, of the 102 new coronavirus vaccines currently in clinical development, 19 are mRNA vaccines
.
Among them, the new crown vaccine developed by Pfizer/BioNTech and Moderna has obtained emergency or temporary authorization from many regulatory agencies
.
A few days ago, the US FDA has also formally approved the new crown vaccine developed by Pfizer/BioNTech
.

In addition to the new coronavirus, a variety of mRNA vaccines have also entered the clinical development stage to prevent infection of cytomegalovirus (CMV), Zika virus, respiratory syncytial virus (RSV), influenza virus, rabies virus and other pathogens
.

For different pathogens, the development of mRNA vaccines will also encounter different challenges
.
Including the rapid mutation of the virus (HIV virus), high lethality (rabies virus), the production of new virus strains and mutants (influenza virus and new coronavirus) and so on
.
Therefore, the design of mRNA vaccines needs to be adjusted accordingly, including targeting conserved regions in antigens, carrying antigens of multiple mutants or virus strains, and so on
.

▲Challenges and countermeasures in developing mRNA vaccines against different pathogens (picture source: reference [1])

Key issues in the development of mRNA vaccines

Persistence of antibody response

After vaccination, the translated antigen is taken up by antigen-presenting cells and transported to the lymph nodes
.
There, the interaction between antigen-presenting cells, B cells, and follicular helper T cells promotes the formation of germinal centers
.
In the germinal center, B cells proliferate, differentiate, and produce mutations in antibody genes, thereby producing high-affinity neutralizing antibodies against the antigen
.
The germinal center response and the induction of follicular helper T cells are essential for long-term protection
.

In order to enhance this immune response process, some mRNA delivery systems actively target antigen-presenting cells, making them "cell factories" for the production of antigens
.
By coupling monoclonal antibodies or ligands that specifically bind to dendritic cells on the surface of LNP, gratifying effects have been obtained in actively delivering mRNA into antigen-presenting cells
.

In addition, extending the time for mRNA to translate protein can change the pharmacokinetic characteristics of the vaccine and can also enhance the antibody response
.

Vaccine development for emerging virus variants

Viral genomes often undergo mutations during replication
.
Although most mutations have no effect on the function of the virus, some mutations may enhance immune escape and limit the effectiveness of the vaccine
.
For example, the rapid mutation of the HIV virus has caused vaccine development efforts over the past 30 years to still not produce an effective vaccine
.
Mutations in influenza vaccines require vaccine developers to modify vaccine formulations for major virus strains every year
.

At present, innovative strategies for virus mutations include the development of vaccines that target conserved areas of the virus, such as the "stalk" of influenza virus hemagglutinin; or the vaccines to encode broad-spectrum neutralizing antibodies
.

The recent mutations of the new coronavirus have also attracted attention to the effect of mRNA vaccines on mutants
.
At present, many companies have begun to develop enhanced vaccines against mutant strains
.
In the long run, it is very important to develop a pan-coronavirus vaccine that can prevent the new coronavirus and other future coronaviruses
.
The current research has provided a proof of concept
.
Drawing on the experience of HIV and influenza viruses, new insights into the structure of the new coronavirus will facilitate the discovery of sites that are conserved in different coronaviruses, speeding up antigen discovery and vaccine design
.

safety

In general, the safety characteristics of mRNA vaccines are good, and only mild or moderate adverse events have been found in clinical trials
.

The review pointed out that the proportion of allergic reactions caused by the vaccine in the population vaccinated with Pfizer/BioNTech's new crown vaccine is about 4.
7 cases per million vaccination, and the value of Moderna vaccine is 2.
5 cases per million vaccination
.
This value is 2-4 times that of more traditional vaccines
.
One hypothesis explaining allergic reactions is related to the PEG-modified lipid molecules used in LNP
.
As people have used many daily products containing PEG (such as toothpaste, shampoo, etc.
), antibodies against PEG already exist in the body
.
These antibodies may increase the likelihood of allergic reactions and limit the effectiveness of vaccines
.

Obviously, the field of mRNA vaccine development needs to have a deeper understanding of the mechanism of vaccine formulations that cause allergic reactions, so as to improve the formulation and improve the safety characteristics
.

Availability of vaccines

The accessibility of vaccines is the biggest challenge in achieving widespread protection, especially in low-income countries
.
For the licensed mRNA COVID-19 vaccine, its accessibility is further restricted by cold storage conditions
.

If billions of people are to be vaccinated globally, vaccines with better heat tolerance are needed
.
In preclinical studies, CureVac demonstrated that the sequence-optimized rabies virus vaccine RABV-G can remain stable in the region between -80°C and 70°C for several months
.
Moreover, there are currently reports showing that the two new mRNA vaccines under development can remain stable at room temperature
.
If these heat-resistant vaccine candidates obtain positive results in clinical trials, it is expected to increase the global availability of mRNA vaccines
.

Look to the future

While mRNA technology has made breakthroughs in the field of infectious disease vaccines, the application of mRNA technology in other fields is also being carried out
.
An article published on Nature Reviews Drug Discovery counted 180 pipeline projects of 31 mRNA companies as of July this year
.
This analysis found that in addition to preventive vaccines that mainly target infectious diseases, many companies are also developing therapeutic vaccines (mainly cancer vaccines) and innovative mRNA therapies
.

▲List of pipelines for research and development of mRNA vaccines and therapeutics (picture source: reference [2])

We expect that with the advancement of science and the development of technology, mRNA technology can be more widely used, benefiting the majority of patients!

Reference materials:

[1] Chaudhary et al.
, (2021).
mRNA vaccines for infectious diseases: principles, delivery and clinical translation.
Nature Reviews Drug Discovery, https://doi.
org/10.
1038/s41573-021-00283-5

[2] Xie et al.
, (2021).
Evolution of the market for mRNA technology.
Nature Reviews Drug Discovery, https://doi.
org/10.
1038/d41573-021-00147-y

Note: The original text is slightly deleted