RNA Viruses, such as the SARS-CoV-2HIV and Influenza, tend to pick up mutations rather quickly, once they are copied inside the cells of our bodies. This happens because the little cell workers that copy the RNA (called enzymes) are sloppy workers and prone to make errors. 

The SARS-CoV-2 virus genetic code has just 30,000 nucleotides blocks of RNA, or letters that can spell at least 29 genes1; and, the most common mutations are single-nucleotide changes between viruses from different people, that have little effect on the overall performance of the virus infection rate, but that allow researchers to track the spread by linking closely-related viruses2

In fact, sequencing data actually suggests that coronaviruses change more slowly than most other RNA viruses, because they have “proofreading” mechanisms that correct potentially fatal copying mistakes, accumulating only two single-letter mutations per month in its genome — a rate of change about half that of Influenza and one-quarter that of HIV2.

The problem is, that scientists can spot mutations faster than they can make sense of them, or what problems they may cause. Many mutations will have no consequence for the virus’s ability to spread or cause disease, because they do not alter the shape of a protein; whereas those mutations that do change proteins are more likely to harm the virus than improve it2. For example, in the beginning of the pandemic in Singapore, the ∆382 deletion made the infection of the virus milder3. On the contrary, the G614 variant showed a fitness advantage of infection, where individuals had a higher concentration of pseudotyped virions, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity4

If we think about it, the virus just wants to spread, but not actually kill – because, if it kills, it can no longer spread.

More recently, a new SARS-CoV-2 virus variant showed up in the UK, where multiple spike protein mutations are seen (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H)5. Changes are also seen in other genomic regions, inclusively in the receptor binding domain (N501Y, “Nelly”). 

The unusually high number of spike protein mutations suggests that the variant has not emerged through gradual accumulation of mutations6. Instead, one possible explanation for the emergence of the variant, is prolonged SARS-CoV-2 infection in a single patient, potentially with reduced immunocompetence, similar to what has previously been described7,8. Such prolonged infection in immunocompromised patients can lead to accumulation of immune-escape mutations at a higher rate. 

This explanation is also suggested to the current SARS-CoV-2 virus variant found in Brazil (P.1)9, and in South Africa (501Y.V2)10

Unfortunately, this shows that the emergence of successful variants with similar properties is not so rare6; and, just like the need to develop new influenza vaccines every season, that also might be the case for SARS-CoV-2. The persistence of the pandemic, may enable accumulation of immunologically relevant mutations in the population, even as vaccines are developed. 

But, because science tries to be one step ahead, this is exactly what manufacturers are already working on, specifically the ones using mRNA technology that can quickly adapt their platforms to the current mutations seen.

Mutation luck
Mutation of luck


1          Naqvi, A. A. T. et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim Biophys Acta Mol Basis Dis 1866, 165878-165878, doi:10.1016/j.bbadis.2020.165878 (2020).

2          Callaway, E. The coronavirus is mutating — does it matter? Nature 585, 174-177, doi: (2020).

3          Young, B. E. et al. Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study. The Lancet 396, 603-611, doi:10.1016/S0140-6736(20)31757-8 (2020).

4          Korber, B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 182, 812-827.e819, doi:10.1016/j.cell.2020.06.043 (2020).

5          Andrew Rambaut, N. L., Oliver Pybus, Wendy Barclay4, Jeff Barrett5, Alesandro Carabelli6, et al. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations: COVID-19 genomics UK consortium. December 2020 (2020).

6          ECDC. Rapid increase of a SARS-CoV-2 variant with multiple spike protein mutations observed in the United Kingdom. December 2020 (2020).

7          Choi, B. et al. Persistence and Evolution of SARS-CoV-2 in an Immunocompromised Host. N Engl J Med 383, 2291-2293, doi:10.1056/NEJMc2031364 (2020).

8          McCarthy, K. R. et al. Natural deletions in the SARS-CoV-2 spike glycoprotein drive antibody escape. bioRxiv, 2020.2011.2019.389916, doi:10.1101/2020.11.19.389916 (2020).

9          Kupferschmidt, K. New mutations raise specter of ‘immune escape’. Science 371, 329-330, doi:10.1126/science.371.6527.329 (2021).

10        Karim, S. S. A. The 2nd Covid-19 wave in South Africa. December 2020 (2020).

Moderna mRNA COVID-19 vaccine: how did they do it?

Moderna uses an efficient method to make large quantities of individual mRNAs with high purity, based on a technique developed in 1984 by D. Melton and colleagues at Harvard University and published in Nucleic Acids Research. In here bacteriophage polymerases were used to transcribe plasmid DNA and make purified RNAs. D. Melton went further and showed that he could transcribe a synthetic mRNA injected into frog oocytes, and get expression of b-globin (Krieg & Melton, Nucleic Acids Research, 1984). With this report, the protocol to make pure mRNAs was settled.

Today, at Moderna, they moved on from this idea to a GMP drug-product inside of a vial in under two months. In the case of the COVID-19 vaccine, they went from a sequence of the virus to having the material ready for a Phase I clinical trial in 45 days. 

This automated process starts with back-translation using Moderna’s proprietary computer algorithm, that has been defined over the years to pick the best mRNA sequence for the target in question. 

Then, the therapeutic mRNA is engineered to avoid the innate immune sensors that defend against RNA viruses and might destroy the vaccine. These defences come in to main types: Toll-like receptors (TLR) in the endoplasmic reticulum, and RIG-I/MDA-5-like receptors in the cytoplasm. 

To avoid TLRs, Moderna places modified nucleotides. This technique is based on a paper by Diebold and colleagues published in Science (2004), where the researchers took macrophages from mice and incubated them with different polymers (polyA, polyC, polyG, polyI, polyU), then checking for an IFN-alpha response. What they have seen was that the only polymer that created the IFN-alpha response was the one that contain Uridine (polyU), which seems to be the only nucleotide that is being recognized by the TLR-driven innate immune response. As such, at Moderna, mRNAs are engineered to replace all U’s with N1-methyl-pseudiuridine without affecting base-pairing, but avoiding TLR recognition. N1-methyl-pseudiuridine is a naturally occurring nucleotide that our bodies recognize as native.

Another problem though, is avoiding the creation of double-stranded RNA (dsRNA) to escape the RIG-I/MDA-5-like receptors. The state-of-the-art used in Moderna to avoid this trap, is HPLC purification to reduce dsRNA content. This is a technique based on Karikó & Weismann (2011, Nucleic Acids Research), that results in very little dsRNA in the end-product, and an avoidance of the innate immune response. The developed technique at Moderna has recently been published in Science Advances (June 2020), where Moderna scientists describe a highly sensitive assay to detect dsRNA and purify therapeutic mRNAs.

(It seems Moderna scientists are now going even further, and have reengineer T7 RNA polymerase not to produce dsRNA at all – although this has not been published yet)

The third step to make a good mRNA-drug, and a successful vaccine, is to find an efficient delivery method.  With that in mind, Moderna researchers looked back at work done by Wolff and colleagues in the 90s (Science, 1990, vol.247, 1465-1468), where they were able to directly inject RNA in the mouse skeletal mouse and have protein expression, with no special delivery system involved. 

In 2014, researchers at Moderna were able to go further, and measured with a Luciferase system, the in vivo expression of biologically active proteins in a dose-dependent manner, by injecting mice with Moderna’s naked mRNA. Nowadays, Moderna targets different tissues via multiple Routes of Administration with specific delivery vehicles (ROAs: direct injection in the heart or tumours, subcutaneous, intravenous, intramuscular). 

Most ROAs require mRNA encapsulation with Lipid nanoparticles (LNPs, 80-100nm). Each encased particle contains 2-6 molecules of mRNA, phospholipid cholesterol, a PEG lipid that keeps the lipid nanoparticles stable avoiding aggregation, and an ionizable lipid that at low pH interacts with the mRNA. These mRNA-LNPs are akin to endogenous lipid transport complexes, slightly bigger than Very-Low Density Lipoproteins (VLDLs), and seen as “friendly bubbly characters” by our bodies.

At Moderna, they empower a rational-structure LNP design by analysing and engineering each individual step of the component mixture, to enhance chemical/physical stability of mRNA-LNPs to allow an optimal biodistribution, cellular uptake, endosomal escape and protein expression. In comparison to the competitor LNPs in the field, which have a 1-2% delivery rate, Moderna’s LNPs can deliver 30% mRNA into cells. To date, Moderna has published two peer-revied articles describing these efforts and their applications in different ROAs (Sabis … Benenato, Mol Ther, 2018Hassett … Britto, Mol Ther Nuc Acids, 2019). 

Finally, there needs to be the right knowledge to engineer the best mRNA sequence for a particular purpose in order to make a good mRNA-drug or vaccine. 

So, there needs to be translation initiation fidelity, so that it always starts in the right place and there’s faithful decoding; and, also, the ribosome needs to stop in the right place. 

The mRNA needs to have a functional half-life, to function before it gets destroyed. 

Also, the therapeutics or vaccine needs to hit the correct cell type, which is achieved by putting “off-logic gates”, like microRNAs target sites in the 3’prime UTR, that cause the mRNA to be degraded in case it hits an undesired cell type.

At Moderna, they have all steps in place for a rapid development of mRNA as medicines:

  1. Efficient methods to make large quantities of individual mRNAs at high purity;
  2. Ability to avoid the innate immune sensors that defend against RNA viruses;
  3. Efficient delivery methods,
  4. Knowledge of how to engineer the best mRNA sequence for a particular purpose.

Besides proprietary algorithm to define the mRNA sequence and unique LNP design; one of the big differences between BioNTech/Pfizer vaccine and Moderna, is that Moderna does not use self-amplifying RNA in their vaccines. According to Dr Melissa Moore, Chief Scientific Officer at Moderna Therapeutics Inc., self-amplifying RNA first starts by building dsRNA which pushes-up the immune response against the vaccine, as such Moderna hasn’t gone down that road to avoid such road-blocks.

The important take-out message is not which vaccine we take.

With a mRNA vaccine, the cell is doing the protein itself, and the antigen-presenting cells are exhibiting it on their surface, which causes an activation of B and T cells.

This protects us and others from developing this devastating disease.

COVID-19 mRNA vaccine, the BNT162b2 candidate

The principle of mRNA therapeutics is to introduce therapeutic messenger (m) RNAs encoding the genetic information for a protein, into a cell of interest. The mRNA structure is designed to increase half-life, translation and protein functionality. 

The mRNA synthesis is made by in vitro transcription using a linear DNA template, which provides more than 500 copies of mRNA per template. They promote a transient expression of the encoded protein/antigen, and are degraded into nucleotides, without the formation of toxic metabolites. Since mRNA is highly sensitive, it gets degraded after a short-time, with no risk of genomic integration. 

This one process can be used to manufacture essentially any mRNA sequence. 

The raw reaction mixture has mRNA and all types of impurities, both process-related (T7 RNA polymerase, remaining building blocks, hydrolyzed DNA…), and product-related impurities (break-off transcripts, side products…), that need to be further purified with high affinity chromatographic methods.

Once purified RNA is obtained, the therapeutic is ready to deliver to the patient.

BioNTech together with their partner Pfizer and FosunPharma, managed to do all this in a record time of 84 days to develop a COVID-19 vaccine. 

Their scientists designed multiple antigen variants using the SARS-CoV-2 Spike (S) protein.

Next, they ran preclinical and toxicology evaluation tests in vitro and in vivo (e.g., in vitro expression data, antibody titers in animal models, pseudo-virus neutralization (pVN) assay in animal models), so they could discover the most active antigen and bring it into the clinic. Also, using their Good Manufacturing Practice (GMP) facilities they produced small scale batches for fast entry into the clinic to demonstrate safety, tolerability (i.e., low reactogenicity), and immunogenicity. 

Afterwards, they initiated clinical trials, where not one but four vaccine candidates were simultaneously studied, in order identify the safest and most effective candidate for further development. In parallel, there was already preparation of larger manufacturing batches of the candidate with the best output in the clinical studies; and, the identification of collaboration partners to develop and provide the vaccine worldwide (Pfizer, FosunPharma).

BioNTech and Pfizer studied three mRNA types during all Phase I/II studies, to evaluate safety and efficacy. The basis for such studies was that different types of mRNA vaccines lead to different responses. 

As such, vaccine prototype (1) was a Uridine mRNA (uRNA) with strong intrinsic adjuvant effect, strong antibody response, that stimulates the production of more CD8+T cells then CD4+T cells.

Vaccine prototype (2) was a Nucleoside-modified mRNA (modRNA), with a moderate intrinsic adjuvant effect, very strong antibody response, lower cytokine induction, that stimulates more CD4+T cells then CD8+T cells.

In the case of vaccine prototype (3), this was a Self-amplifying mRNA (saRNA) with long duration, and a higher likelihood for good efficacy antibody response with lower dosage. 

Early and constant interaction with the regulatory agencies (e.g.EMA) has allowed that BNT162b2 vaccine candidate raced to the finish line, being now the leading candidate from BioNTech & Pfizer; and, currently in review for conditional marketing authorization approval.

It is outstanding that if a new variant of the virus is required (e.g., due to a mutated virus sequence), a new GMP batch of a possible vaccine could be available within half of the time (around 42 days).

If there is a positive message out of these dark days, is that Science is there to save us from our doom. 

Let’s embrace Science.

mRNA vaccine

mRNA: the therapeutics of the future! (or, 2021 the way research is going…)

Today’s top selling drugs are mostly biologics, which means proteins (e.g., monoclonal antibodies) being used as medicine. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018, and is expected to generate revenue of $150 billion by the end of 2019, and $300 billion by 20251. Thus, the market for therapeutic antibody drugs has experienced an explosive growth with new drugs being approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases, which have increasingly fewer adverse effects due to their high specificity. 

But, these types of treatments carry their own limitations. Besides of a long period of development, they can only access the extracellular space, the serum. Yet, ~2/3 of the human proteome are intracellular or transmembrane proteins; which means that biologics cannot treat disorders that are caused by these types of proteins. 

As such, an alternative mechanism to make these proteins is needed in order to go further in the advancement of therapeutics. 

One such mechanism, is to supply the mRNA that encodes that protein. As such, any protein can become a drug. And, since only the coding region varies from mRNA-drug to mRNA-drug; once the delivery vehicle problem is resolved (e.g., using lipid nanoparticles), the development of new mRNA drugs should be quite fast in comparison with other types of medicine.

The other positive aspect of mRNA as therapeutics, is that it can be used in many different types of modalities: prophylactic vaccines, cancer vaccines, intertumoral immune-oncologicals, localized regenerative therapeutics, systemic secreted medicine, and cell surface therapeutics…

Another positive aspect of mRNA-drugs is that several mRNAs can be delivered at once, like in the case of prophylactic vaccines (e.g., Moderna Therapeutics Cytomegalovirus (CMV) mRNA vaccine delivers 6 different mRNAs to encode a multiprotein complex). 

Or, another possibility, is the personalization of cancer vaccines, where different patient-specific neoepitopes are sequenced, and mRNA is created that encodes those 20-35 specific tumour-neoepitopes. 

Those mRNAs can also encode cytokines, that are able to boost the immune system to attack a tumour, like intertumoral immune-oncologicals therapeutics. 

In the field of localized regenerative therapeutics, there is currently a project between Moderna Therapeuticsand AstraZeneca in Phase II clinical trials, where Vascular Endothelial Growth Factor-A (VEGFA) mRNA is injected NAKED, without lipid nanoparticles, directly into the heart muscle of patients with a recent Myocardial Infarction (MI) to help rebuild blood vessels. 

Of course, many challenges still exist. 

mRNA needs to be rightly sequenced and be made at high purity. It also needs to have the ability to avoid the innate immune sensors, like Toll-like receptors (TLR) in the endoplasmic reticulum, and RIG-I/MDA-5-like receptors in the cytoplasm. What researchers do to avoid the first ones (TLRs) is to use a Pseudouridine, which looks like a Uridine and still base-pairs, but escapes TLR recognition. To avoid the RIG-I/MDA-5-like receptors, researchers need to highly purify the therapeutic mRNA, so that no double-stranded RNA exists (or, reengineer T7 RNA polymerase not to produce dsRNA at all).

The next step on making a good mRNA-drug is to find an efficient delivery method. For that, most therapeutics will need some sort of mRNA encapsulation with Lipid nanoparticles, which are akin to endogenous lipid transport complexes, similar to Very-Low Density Lipoproteins (VLDLs).

Finally, in order to make a good mRNA-drug, there needs to be knowledge on how to engineer the best mRNA sequence for a particular purpose. As such, there needs to be translation initiation fidelity, so that it always starts in the right place and there’s faithful decoding; and, also, the ribosome needs to stop in the right place. There needs to be high translation efficiency, a functional mRNA half-life, and hit the correct cell type, by putting off-logic gates, like microRNAs target sites in the 3’prime UTR and causing the mRNA to be degraded in case it hits an undesired cell type. Also, the mRNAs need to be tailored to the protein type (e.g., secreted, mitochondrial).

So, let’s see what the future brings from mRNA therapeutics…


1          Lu, R.-M. et al. Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science 27, 1, doi:10.1186/s12929-019-0592-z (2020).