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mRNA vaccines: current trends and prospects

 Last Update: 2021-09-19
 Source: Internet
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With the rapid release of highly effective vaccines (94-95%) by Pfizer-BioNTech and Moderna, the epidemic of COVID-19 has made mRNA vaccines the focus of attention
.

Once the SARS-CoV-2 sequence was published, Moderna obtained the first batch of vaccine candidates in just 28 days

In addition to shortened development time and efficiency (at least for COVID), there are other advantages to using mRNA for preventive and therapeutic vaccines
.

The first is the safety feature, which includes that the antigen is usually only expressed for a few days and can be regulated by the design of the mRNA

Second, unlike DNA-based methods, mRNA vaccines do not need to enter the nucleus, so the risk of genome integration and mutagenesis is small
.

Finally, mRNA vaccines provide stable development of cellular and antibody immune responses, which can be changed between the two through the design of mRNA, the choice of delivery methods, or other methods

The production method of mRNA vaccine also has advantages, because mRNA synthesis is based on a mature in vitro transcription process in a cell-free system
.

Cell-free systems help reduce costs, time and simplify manufacturing processes

Nevertheless, there are still some areas that need to be improved
.

Because mRNA is negatively charged, it is difficult for mRNA to enter the cell and will be rapidly degraded by nucleases (such as RNase)
.

This can be alleviated to a certain extent by lipid nanoparticle (LNP) encapsulation (which has been used in current mRNA-based COVID-19 vaccines), modification, base substitution, mRNA design, or other methods

In addition, the distribution of vaccines is a problem, and currently mRNA vaccines need to be stored frozen
.

Alternative methods such as freeze-drying are under investigation

From a manufacturing perspective, there are many challenges
.

Compared with traditional recombinant proteins, one of the main challenges of mRNA processing is the lack of specialized equipment and consumables suitable for relatively small and large-sized mRNA

Trends in RNA-based therapy

In 1990, after injecting naked mRNA into mouse skeletal muscle and observing the expression of a protein in vivo, the potential of mRNA vaccines attracted the attention of the scientific community
.

Since then, the industry has grown rapidly

Two forms of mRNA structures are currently under development: conventional non-replicating mRNA and self-amplifying mRNA
.
The non-replicating mRNA vaccine has a traditional mRNA form and does not have the replication ability built into the mRNA sequence
.

The antigen sequence is flanked by untranslated (UTR) regions, 3'poly (A) tails and 5'caps
.
The cap, UTR, ORF and tail can be customized to up-regulate or down-regulate the expression level, or to regulate the immune response
.
Modified nucleotides, such as pseudouridine and 5-methylcytidine, can be used to reduce adverse innate immune system responses and improve translation efficiency
.
Therefore, many aspects of clinical response can be adjusted simply through the design of mRNA
.

Non-replicating mRNA vaccines are short-lived in nature, usually expressing antigens for hours or days (the cell half-life of Pfizer and modern vaccines is estimated to be 8-10 hours)
.
For some applications, this may be beneficial, but for other applications, such as systemic protein therapy, extended expression of proteins will be beneficial
.

Self-amplified mRNA (saRNA) methods are under development to enable mRNA replication
.
In turn, this can extend the expression window to several weeks
.
Generally, saRNA is based on adding viral replicase genes in cis or trans configuration, these genes are derived from alphavirus, flavivirus or picornavirus
.

These strategies can increase expression levels or reduce mRNA dosage requirements by 10-100 times
.
Self-replicating mRNA may expand mRNA technology in many applications while reducing manufacturing requirements
.
There are many areas of mRNA technology being developed and optimized, and mRNA design and optimization are important aspects of current efforts
.

In addition to mRNA, there are other RNA therapies under development or have been approved
.
These include antisense oligonucleotides that change gene expression; small interfering RNA (siRNA), which also changes gene expression through different mechanisms; aptamers can bind to other ligands, including RNA and guide RNA, for CRISPR targeting Design and other functional RNA
.

These RNA therapies share overlapping technologies with mRNA vaccines
.
One example is Onpattro, an approved siRNA therapeutic using LNP technology
.
Therefore, in addition to mRNA vaccines, the entire field of RNA therapy is developing rapidly
.

Based on different types of mRNA therapies

COVID vaccine is a preventive vaccine for infectious diseases.
In addition to bacterial infections such as staphylococcus and tuberculosis, there are many other preventive vaccines under development, including influenza, Zika virus, dengue fever, rabies, and Venezuelan equine encephalitis
.
The unique method involves the expression of chikungunya virus neutralizing monoclonal antibodies
.

mRNA vaccines have also gained attention as a treatment method for cancer
.
mRNA can be used to trigger an immune response to mutated oncogenes or regulatory cancer genes (such as p53), which are shared in many cancers in the treatment of widespread cancer treatments
.

Other ways to treat cancer include personalized treatment, which is the development of vaccines for individual cancer mutations
.
In this regard, the patient’s mutant group will be identified by the next-generation sequence, and some customized mRNA vaccines for individual specific neoantigens will be developed
.
The development of therapeutic cancer vaccines is progressing rapidly, more than 70 clinical trials have been completed, and more results are expected in the next 2-3 years
.

Many technologies are under evaluation, including direct stimulation of antigen-presenting cells (APC) by in vitro electroporation of mRNA
.
Other methods include direct intratumoral injection, systemic methods, and targeted organ methods
.
Currently, more than 50% of clinical trials using mRNA focus on the treatment of melanoma, prostate cancer and brain cancer
.
Therefore, although LNP targeting specific organs, tissues and cells to specific organs, tissues and cells is still under research, many applications of mRNA vaccines are in different stages of development from concept to clinical trials
.

mRNA production scale and manufacturing bottlenecks

The scale of mRNA required for manufacturing varies depending on the indication, the effectiveness of the method, market demand, and other factors
.
A customized and personalized process may only require the production of milligrams of mRNA
.
Global demand may require more mRNA production capacity
.

For example, the current Pfizer and Moderna COVID vaccines contain 30 and 100 micrograms of non-replicating mRNA, respectively
.
In this case, the production of 1 billion doses of vaccine will require the production of 30-100 kg of highly purified cGMP mRNA, which can best be solved in a production batch of at least a few grams
.

One of the most common bottlenecks in current mRNA manufacturing is scale
.
Since COVID products have reached the scale of billions of doses, a larger-scale production technology is definitely needed
.
We see that the supplier can customize the complete solution and deliver it to the customer's solution
.
These solutions have been developed and delivered for mAb applications as well as plasmid and viral vectors
.

However, small-scale cGMP production also needs improvement, because most of the current equipment is modified from the biotechnology industry and is designed for a production scale much larger than the production scale required for mRNA
.
The industry can benefit from equipment designed for mRNA cGMP manufacturing, including smaller-scale equipment
.

The upstream manufacturing process of mRNA is quite mature
.
The cGMP plasmids, polymerases, and enzymes needed to synthesize mRNA in vitro are available, but can be expensive
.
The Poly A tail can be created by being included in a template or by using enzymes
.
There are high-efficiency, co-synthesis, capping options, or it can be processed by high-efficiency enzyme treatment
.
Due to the cell-free nature, mRNA vaccines may be cheaper than other vaccine methods, but their production costs are currently higher
.

In order to improve the overall cost situation, it is necessary to reduce the cost of GMP reagents, capping reagents, proprietary LNP components and other proprietary components
.
The capacity limitation of the plasmid used as the starting template in the mRNA process is also a challenge faced by this application field and the growing field of viral vectors
.

In addition, manufacturing companies are looking for ways to eliminate this bottleneck, and new technologies surrounding the cell-free process of plasmid production may improve this initial process step
.

However, downstream manufacturing process fluids need to be improved
.
High-purity mRNA is required for efficient translation and reduction of adverse immune reactions
.
There are many impurities, including enzymes, nucleotides, plasmid templates, abnormal RNA species, and other impurities that currently require a multi-step purification process
.

These multi-step processes are diverse and in a state of development
.
Techniques such as precipitation, affinity oligomeric dT, ion pair chromatography (IPC) with or without cellulose, ion exchange, tangential flow filtration (TFF), etc.
can be used
.
Therefore, alternative purification ligands and refined purification methods will greatly benefit the industry
.

Although mRNA production is certainly suitable for standardization and platformization, as has happened in the monoclonal therapy industry, most of the current production is carried out in multiple steps using suitable equipment
.
Other aspects, such as disposable items and continuous processing strategies, will benefit this emerging industry
.

The speed and potential cost benefits of mRNA technology make it a technology for personalized medicine
.
Vaccines are developed for individual cancer mutations in a person
.
Many companies are developing integrated system mRNA processing solutions for this
.

Although many of the steps in the process are the same, there are many additional challenges due to scale and cost
.
But we are monitoring and contributing to the development of this field, which can reach the commercial stage within a few years
.

Finally, a deeper understanding of process science is required
.
For example, LNP is usually formed in a rapid mixing process using microfluidic devices, which is more of an art than an established method
.
A better understanding of the impact of LNP components and their impact on LNP stability, delivery, efficiency, immune response, and final patient outcome will benefit the industry
.
The optimization of LNP and other delivery technologies is a key attribute that determines the ultimate success or failure of treatment
.

Packaging and delivery technology

The use of nanostructures, such as LNP, is common in mRNA therapy because they generally provide higher efficiency than naked mRNA and allow multiple routes of administration
.
One challenge of nanostructure technology is that it is inherently complex, it involves many potential components, and has many possible clinical outcomes
.

The understanding of this aspect is not complete
.
Nanostructural properties are critical to clinical outcomes, including: nucleic acid protection, controlled release of intracellular RNA, cell and tissue selectivity, translation efficiency, toxicity, and long-term stability
.

Nanostructures are complex in structure, and they may be composed of multiple components, such as common lipids, polymers (PEG, PEI, polylysine, etc.
), proteins, cholesterol, or customized proprietary components, such as ionizable lipids
.
Conjugates such as PEG-lipids are usually used
.

Each of these will have a huge impact on its attributes
.
For example, polymer content can control particle size and affect efficiency and cell tropism
.
Structural lipids, such as cholesterol, can affect particle stability
.
If mixed incorrectly, empty nanoparticles with no payload may be formed
.

Therefore, the composition and formation of nanostructures are critical to the desired clinical effect
.
Currently, LNP is the landing non-viral delivery system for many systems, including gene therapy
.

There are other delivery methods under research and development
.
Exosomes are believed to use receptors and can provide more efficient uptake, higher specificity, and fewer side effects
.
This is a promising area of early research
.

Other areas include conjugated RNA, such as GalNac-siRNA, which has been shown to target liver cells
.
Similarly, GALA peptide-conjugated mRNA has been shown to increase APC uptake
.
There are other methods under evaluation to increase target specificity or improve cellular uptake
.

Naked mRNA has been evaluated for cancer treatment by direct injection into tumors or other methods
.
It is generally considered that naked RNA is less efficient than other methods, but its advantage is that it is easy to prepare because it only requires buffers
.
In certain applications, the inherent high immunogenicity of naked mRNA can provide benefits by enhancing adjuvant activity
.

mRNA industry prospects

With the advent of the COVID-19 epidemic, the mRNA of preventive vaccines has become the focus of public attention due to its urgent need
.
These vaccines prove the promise of mRNA therapy through their rapid development time and high efficiency
.

Although the COVID vaccine is compelling, so far, most mRNA vaccines have focused on cancer treatment, and there have been dozens of complete or ongoing clinical trials to date
.
Many of these trials should be completed in the next two to four years
.
Many of these are personalized therapeutic cancer vaccines
.
The promising results in this field can further promote the development of the mRNA industry
.

In addition, there are many treatments in the early development of different fields, and if successful, they will have a significant impact
.
The success of mRNA therapy may replace less effective treatments in the future, such as influenza vaccines, tuberculosis vaccines or other applications
.

Summarize

mRNA therapy is an emerging field that is rapidly developing and expanding
.
There are many applications in development that are too large to be covered in detail in this short article
.
Due to its advantages in flexibility, cost and speed of development, this technology offers great benefits and potential for infectious diseases and personalized medicine
.
Of course, to fully realize the potential of this technology, challenges still need to be overcome, including the lack of experience and knowledge of expanding the mRNA process, perceived regulatory uncertainty, and targeted delivery technology
.
The COVID-19 epidemic has proved the promise of mRNA therapy, and the prospects of this emerging industry are bright
.

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