Messenger RNA (mRNA)

Definition

Found in all cells, messenger ribonucleic acid, or mRNA, is a single-stranded molecule. It is responsible for transferring genetic information from DNA, found in the nucleus of the cell, to ribosomes floating in the cell's cytoplasm which carry out the synthesis of proteins. Each mRNA molecule provides a copy of the DNA blueprint for a chain of amino acids. Further configuration of this chain results in a protein.

This diagram depicts the role of mRNA in the production of proteins.

Importance

mRNA is pivotal to the translation of DNA instructions into proteins which are involved in every bodily function and are a key component in every cell of the body. Proteins play multiple roles, including helping to copy DNA, carry messages, break down chemicals and facilitate digestion, blood clotting, respiration and metabolism. They also help the body to repair and make new cells.

The mRNA molecule plays a central role in protein synthesis. This process involves several steps. To start with, enzymes, called RNA polymerases, build mRNA molecules. Made in the cell's nucleus, these molecules are complementary to a portion of one strand of the DNA double helix. This process is called transcription. Once made, the mRNA moves from the cell nucleus to the cytoplasm where it gets read by the ribosome, the machinery responsible for making proteins. The genetic information contained in mRNA is read in a specific direction, from five prime (5') to the three prime (3') end of the molecule. This gets translated into a chain of amino acids, known as a peptide chain, with three nucleotides coding for one amino acid. The peptide chain then naturally folds into its secondary and then tertiary structure to form a protein. Sometimes a protein can then undergo further modifications before it reaches its functional form.

Explored for many years by researchers due to its central position in the flow of genetic information within a cell, and as such providing a novel therapeutic target, mRNA has recently grabbed public attention because of its successful development as vaccines against COVID-19. Rather than traditional vaccines, which rely on administering weakened or inactive pathogens or just parts of the pathogens, known as an antigen, mRNA can be used to provide the information to the body's cells to create a pathogenic protein themselves. Like traditional vaccines, this can then be recognised by the immune system as foreign and thus elicit an immune response and subsequent immune memory. mRNA vaccines have been shown to have a greater specificity and efficacy than traditional vaccines (Maruggi, Chiarot, Giovani). By January 2021 two mRNA vaccines, encoding for a specific protein found on the surface of the SARS-CoV-2 virus, one developed by Moderna and another by BioNTech with Pfizer, had been approved for COVID-19 and several others are now in the pipeline.

Innate and adaptive immunity

Figure 1: Different mechanisms involved in innate and adaptive immunity.

The innate response is set off (figure 1) as soon as the body encounters an antigen. After physical and chemical barriers such as the skin and mucous membranes, the innate immune system is the first line of defence and involves macrophages and other innate immune cells rushing to respond to foreign intruders. These cells also activate the production of T and B lymphocytes (T and B cells). Known as adaptive immune cells, these cells take longer to develop. Their job is to destroy any foreign invaders that survive the first line of attack and to remember them so they can swiftly eliminate them next time they enter the body (Lewis).

Differences between innate and adaptive immunity

Characteristics Innate immunity Adaptive immunity
Presence Innate immune cells reside in most tissues in the body Adaptive immune cells reside within specific tissues until they are triggered by the innate cells to respond to an infection
Defence system First line of defence (skin, gut, mucous membranes) carried out by a group of cells that patrol the body to help to raise the alarm and kill an invader Second layer of defence (lymphatic system: lymph nodes, spleen, thymus etc)
Target Broad acting and non-targeted - deals with any foreign substance Fights only specific pathogens
Response Immediate Delayed
Potency Limited and lower potency High potency
Inheritance Generally inherited from parents and passed onto offspring Not passed on from parents to offspring
Memory Not retained Memory cells develop following an infection and reside in the body for some time so that upon reinfection the adaptive immune system can elicit a faster response
Diversity Limited High
Composition Phagocytic leukocytes, dendritic cells, natural killer cells and plasma proteins B and T cells
Example White blood cells fighting bacteria as seen in redness and swelling when you have a cut Vaccination creating memory to fight specific pathogens

Differences between innate and adaptive immunity

Characteristics

Presence

Innate immunity

Innate immune cells reside in most tissues in the body

Adaptive immunity

Adaptive immune cells reside within specific tissues until they are triggered by the innate cells to respond to an infection

Characteristics

Defence system

Innate immunity

First line of defence (skin, gut, mucous membranes) carried out by a group of cells that patrol the body to help to raise the alarm and kill an invader

Adaptive immunity

Second layer of defence (lymphatic system: lymph nodes, spleen, thymus etc)

Characteristics

Target

Innate immunity

Broad acting and non-targeted - deals with any foreign substance

Adaptive immunity

Fights only specific pathogens

Characteristics

Response

Innate immunity

Immediate

Adaptive immunity

Delayed

Characteristics

Potency

Innate immunity

Limited and lower potency

Adaptive immunity

High potency

Characteristics

Inheritance

Innate immunity

Generally inherited from parents and passed onto offspring

Adaptive immunity

Not passed on from parents to offspring

Characteristics

Memory

Innate immunity

Not retained

Adaptive immunity

Memory cells develop following an infection and reside in the body for some time so that upon reinfection the adaptive immune system can elicit a faster response.

Characteristics

Diversity

Innate immunity

Limited

Adaptive immunity

High

Characteristics

Composition

Innate immunity

Phagocytic leukocytes, dendritic cells, natural killer cells, and plasma proteins

Adaptive immunity

B and T cells

Characteristics

Example

Innate immunity

White blood cells fighting bacteria as seen in redness and swelling when you have a cut

Adaptive immunity

Vaccination creating memory to fight specific pathogens

One of the attractions of mRNA vaccines is that they can be designed very quickly for different targets (Maruggi, Chiarot, Giovani). Once the genomic sequence of a particular pathogen is known, an mRNA vaccine template can be produced within a few weeks (Mascola, Fauci). Such speed is particularly important for responding to new disease outbreaks. Some idea of how fast this process can be is seen from the fact that Moderna managed to create a template for its mRNA COVID vaccine within just five days after the SARS-CoV-2 genome was first sequenced (Corbett, Edwards, Leist).

The main advantage of mRNA vaccines is that they are easier and quicker to manufacture than traditional vaccines. Most influenza vaccines, for example, are grown in chicken embryos present in fertilised eggs. Used for decades to produce influenza vaccines, this method requires millions of eggs for production and can take six months which impedes the ability to respond to new outbreaks (Bender). Other vaccines, such the one for the hepatitis B virus or the human papillomavirus are reliant on the production of proteins by genetically modified yeast and insect cell lines. Importantly, mRNA vaccines can be synthesised using enzymes and template DNA molecules. Such a process is also much easier to update, scale-up and mass produce than traditional vaccines, and the same manufacturing facility can also be used to make mRNA vaccines for different diseases, which reduces the amount of investment required by companies (Ball).

Another attraction with mRNA is that it is non-infectious. This means it is safe both for the technicians involved in the manufacture of the vaccine and for patients who receive it. In addition, once mRNA has done its work it naturally dissolves within the body so there is no risk that it can integrate with the recipient's DNA (Pardi, Hogan, Porter, Weissman; Maruggi, Chiarot, Giovani).

Many regard the quick breakthrough with the COVID-19 vaccines as just the start of the potential revolution mRNA can bring to medicine. Well before the pandemic hit, mRNA was already attracting considerable investment. Between 2015 and 2018 three mRNA therapeutic companies (Moderna Therapeutics, BioNtech, and CureVac) had gained US$2.8 billion of private investment. In 2018 Moderna set a record for the biggest biotech IPO with an approximate value of $7.6 billion (Wang, Travis, Watts). Just how fast the market is now expected to grow can be gauged by the fact that at the end of 2019 the combined market capitalisation of five publicly listed mRNA companies was ~US$15 billion. By August 2021 their capitalisation was more than $300 billion (Xie Chen Wong).

A search of the ClinicalTrials.gov database in May 2021, using the words 'mRNA', 'messenger RNA', 'messenger ribonucleic acid' showed that 177 trials have been launched to assess various mRNA therapies since 2002. These trials only include those that directly used a mRNA therapeutic agent. They do not cover drugs that use RNA interference designed to silence particular genes. Out of the total trials, 68 began in the first five months of 2021. This is four times the number of trials started for the whole of 2020, which totaled 17 (figure 2). As might be expected, a large proportion of the trials started in 2021 concern mRNA vaccines for COVID-19. By May 2021 62 trials had started for COVID-19 vaccines.

Innate and adaptive immunity

Figure 2: Number of trials listed in Clinicaltrials.gov for mRNA therapies started each year.

Innate and adaptive immunity

Figure 3: Number of trials listed by condition in Clinicaltrials.gov for mRNA therapies started each year.

Well before the outbreak of COVID-19, mRNA vaccines were being explored for other infectious diseases like rabies, HIV and influenza. mRNA vaccines have also been developed against the Ebola, Zika, hepatitis C and rabies viruses and the cytomegalovirus, a common herpes virus which although usually harmless can cause serious complications in pregnancy and in people with impaired immunity (Pardi, Hogan, Porter, Weissman; Meyer, Huang, Yuchakov). Work is also underway to create a mRNA vaccine against Streptococci, a group of bacteria linked to many disorders including pneumonia, wound and skin infections, sepsis and endocarditis (Maruggi, Chiarot, Giovani). A search of the Clinicaltrials.gov database in May 2021 listed 22 trials started with mRNA vaccines for various infectious diseases since 2002 (figure 1).

Overall cancer was by far the largest group of conditions for which mRNA therapies were started before 2021. At least 74 of the trials listed in the Clinicaltrials.gov database launched since 2002 were for this category. Many of these trials involve mRNA based cancer vaccines. Seen as a form of immunotherapy, such vaccines aim to help train the immune system to recognise tumour-associated or tumour-specific antigens (receptors) found on the surface of malignant cancer cells to mount an attack and destroy the tumour.

mRNA also offers a promising platform for more personalised cancer vaccines because it can be specifically designed for patients' unique tumour genetics. This is important because one of the major challenges with cancer is that tumours are very heterogeneous and differ between patients (Albert). One of the ways it is possible to identify a tumour's mutations is to extract DNA from an individual patient's tumour cells and then compare it with germline DNA from normal cells. Once this is done the mutations are evaluated to determine their immunogenic potential and the neo-antigens identified as the development targets for the mRNA vaccines (Huang, Huang).

Many of the mRNA cancer vaccines target dendritic cells. First discovered in 1973 by Ralph Steinman and Zanvil Cohn, dendritic cells are a type of immune cell found in the bloodstream that helps present antigens to the immune system to activate the T cell response to destroy it. Scientists have been exploring the use of dendritic cells since the 1990s to make cancer cells more visible to the immune system. In 2010 the US Food and Drugs Administration approved sipuleucel-T to treat late-stage prostate cancer. It is a personalised treatment that involves optimising white blood cells, primarily dendritic cells, harvested from the patient which helps to enhance the immune response to the cancer (Marte).

Clinical responses to dendritic cell vaccines have so far been disappointing. But new methods are now being developed to improve their efficacy. This includes introducing mRNA to dendritic cells to code for desired antigens. Several techniques have been developed for this purpose over the past decade. The simplest method is to incubate the dendritic cells with mRNA. Another uses an eclectic pulse to transfer mRNA molecules through the cell membrane, known as mRNA electroporation. Alternatively mRNA can be delivered into the cell by encasing it in a lipid shell. One of the advantages with mRNA is that it circumvents the need to grow or isolate patient-specific antigens. It also offers more standardised antigen preparation and reduces the risk of prompting an auto-immune response (McNamara, Nair, Holl).

A number of large pharmaceutical companies are already partnering in the mRNA cancer vaccine space. In 2016, for example, Merck expanded a collaboration with Moderna, first struck in 2015, to develop and commercialise personalised mRNA cancer vaccines. The vaccines are designed to target neoantigens selected from a patient's tumour. Neoantigens are new proteins that form on cancer cells when their DNA accumulates certain mutations which can play an important role in how the immune system responds to cancer. By the beginning of 2021 there was one vaccine (mRNA-4157) in phase II trials as an adjuvant treatment for melanoma. Preliminary studies indicate the same vaccine could also enhance the immune response in patients given immune checkpoint inhibitors (Tong). Cancer treatment with mRNA vaccines still has some way to go, but some trials have reported promising results for their use in treating solid tumours (Milao, Zhang, Huang).

Both the mRNA vaccines for infectious diseases and cancer are designed to prime an immune response to a particular antigen. The same technology, however, also provides a means to dampen the immune system, which is useful for treating auto-immune disorders, allergies and preventing the rejection of donor (allogenic) transplants (Dolgin 2019).

Discovery

The use of mRNA for medical applications rests on the gradual coming together of knowledge from different disciplines over many years. Its development was not a smooth path and involved many different players, a number of whom struggled to get recognition for their work. The emergence of mRNA is rooted not only in new understandings of the central dogma of molecular biology and the vital role mRNA plays in it but also the development of new lipid-based delivery systems which are pivotal to preventing the molecule's disintegration and allow it to reach the right target in the body. Due to their importance, these lipid-based systems have been the subject of patent disputes over time leaving a bitter taste for many involved and hindering progress with the technology (Vardi).

The mRNA molecule was originally discovered as a result of scientists' search to understand the molecular mechanism by which DNA directs the formation of proteins within a cell. This began as soon as the double-helix, the twisted-ladder structure, of deoxyribonucleic acid (DNA) was cracked in 1953. A key question was how this genetic information resulted in the synthesis of a protein.

The first clue came from Elliot Volkin and Lazarus Astrachan, two American scientists based at Oak Ridge National Laboratory (ONL) in Tennessee. In 1956 they discovered a 'DNA-like RNA' substance that did not resemble previously found types of RNA. They noticed it when they infected Escherichia coli with a T2 bacteriophage, a virus, and then exposed the culture to radioactive 32P for a few minutes. Importantly the substance appeared to help the bacteria's cell machinery switch from making its own proteins to those that were characteristic of the virus. While greeted with great interest, the actual function of the newly found substance was not fully grasped at the time. Both Volkin and Astrachan speculated that it acted as a precursor to the synthesis of DNA (Volkin, Astrachan; Volkin; Editorial). The turning point came in 1961 (Cobb). That year a group of scientists managed to isolate mRNA and determine its key role in protein synthesis (Brenner, Jacob, Meselson; Gros, Hiatt, Gilbert, Kurland; Jacob, Monod).

Scientists continued to investigate the properties of mRNA and its role in the synthesis of proteins throughout the 1960s. This included Raymond Lockard and Jerry Lingrel, two scientists based at the University of Cincinnati, Ohio. In 1969 they showed that it was possible to get mouse lymphocytes to start producing globin, a protein that helps transport oxygen, by introducing mRNA, isolated from a rabbit, using a cell-free system developed from immature red blood cells (Lockard, Lingrel).

Over the next decade scientists began to wonder whether they could exploit the cellular messaging system provided by mRNA to allow patients' cells themselves to produce therapeutic proteins. This idea was given a boost in August 1978 by the work of Georges Dimitriadis at the National Institute for Medical Research in London and a team led by Marc Ostro at the University of Illinois. Independently they managed to produce rabbit globin respectively in mouse and human cells. They achieved this by delivering mRNA using a liposome. First described in 1965 by Alex Bangham, a British biophysicist and haematologist, a liposome is a minute hollow bubble composed of the same bilipid layer that composes cellular membranes (Dimitriadis, Feb 1978; Ostro Giacomoni Lavelle; Felgner).

New drug delivery platform for nucleic acids

Liposomes had become a popular device by the late 1970s for delivering biologically active compounds for therapeutic purposes. But the technology had some teething problems and was difficult to scale up. Additionally, liposomes were too small to accommodate DNA which made them unsuitable for gene therapy which many scientists were now focusing their energies on. Furthermore the negative charge of the nucleic acids made it hard to contain them within the similarly charged lipids (Felgner interview 1997).

In 1984, however, a team led by Philip Felgner, an American biophysicist at Syntex Research, a Californian company, managed to synthesise a cationic, positively charged, lipids that could form positively charged bilayer membranes and liposomes. This was a major breakthrough because until now scientists only had access to natural lipids which were either neutral or negatively charged. By creating a positively charged lipid, the Syntex group hoped to improve the efficiency of drug delivery. Their idea was to incorporate or encapsulate a drug in a liposome composed of cationic lipids which could fuse easily with negatively charged cell membranes to deliver the drug directly into a cell (Felgner interview 2021).

Philip Felgner

Photograph of Philip Felgner, credit Felgner. The grandson of German immigrants, Philip Felgner grew up in Frankenmuth, a farming town in Michigan known for its Bavarian-style architecture and as a popular vacation destination. The youngest of three brothers, Felgner's father was president of an automobile insurance company and his mother was a housewife. Born just at a time when television was becoming popular, Felgner first became interested in science from watching Walt Disney shows about scientists like Albert Einstein. His curiosity was further piqued in science after he visited the laboratory of Thomas Edison, commonly associated with the invention of the light bulb. Originally torn between becoming a classical guitar player or a scientist, Felgner landed up completing a BSc in biochemistry in 1972 at Michigan State University and then stayed on to do a Master's and doctorate there. In 1978 he became a post doc at the University of Virginia with the biophysicist Tom Thomspon during which time he began working on liposomes. Felgner then became a staff scientist at Syntex in 1982. For him Syntex provided a good opportunity to learn more about chemistry and the pharmaceutical industry. In 1988 Felgner joined Vical as a founder and the director of product development where he and his team helped develop DNA vaccines for infectious diseases. A decade later he took up a faculty position at the University of California, Irvine, where he now leads the Vaccine Research and Development Center and Protein Microarray Laboratory (Felgner interview 1997; Felgner interview 2021; Sforza).

At this time molecular biology genetic engineering and gene therapy was just dawning. One of the first tests that Felgner and his colleagues carried out was to see how the new lipids would react with a plasmid, a small flexible loop of DNA that replicates in bacteria. The plasmid had been genetically modified by Hardy Chan, a molecular biologist at Syntex. Chan had engineered the plasmid to carry a eukaryotic promoter and beta-galactosidase, a reporter gene. Expecting to see something on mixing the liposome with the plasmid, Felgner was very surprised to see no visible change. Unable to believe that nothing had happened, Felgner decided to run some tests on the solution, including sucrose density gradient ultracentrifugation. This revealed that, even though it was not obvious to the naked eye, the liposome had captured all of the DNA in the plasmid to form a complex. One of the reasons the plasmid and the liposome bound so well together like magnets was because they each had opposing charges. The team also found that the new complex, which they called a lipoplex, fused easily with tissue culture cells and proved efficient at delivering functional DNA into the cell (Felgner interview 2021; Felgner, Gadek, Holm).

Felgner was very excited by the results as it opened up a world of possibilities for gene therapy. Importantly, as he says, it solved a 'tremendous problem ...because everybody was trying to encapsulate the nucleic acid into the traditional liposomes and it just was not practical to do that. But here, suddenly everything became practical.' The beauty of the new method, later called lipofection, was that it was very simple and highly replicable. All that was needed was to mix the plasmid with cationic lipids and they self-assembled into a lipolex (Felgner interview 2021).

For Felgner the discovery was 'like magic' because scientists could now 'introduce any gene into a cell' and get them to produce any desired protein. As he explained, 'only nature and evolution' could do this before. The method not only held promise for gene therapy. It also had many research applications, such as investigating gene expression, control of cell growth and cell lineage (Felgner interview 2021; Felgner n.d.). But Albert Bowers, Syntex's president, failed to grasp the significance of the new technique and was unwilling to fund its development. Part of the problem was that Bowers was only interested in things that would benefit the company's bottom-line profits the next year and gene therapy was still considered a long shot (Felgner n.d.; Wollaeger interview).

Frustrated by Bowers' reaction, Felgner managed to get Syntex to patent the technique (US-7250404-B2) and began hunting for ways to make lipofectin widely available to other scientists. In the end he persuaded Bethesda Research Labs, subsequently acquired by Invitrogen and then Thermo Fisher, to licence the cationic lipid preparations from Syntex. This resulted in their wide distribution across the world (Felgner n.d.).

Lipofectin reagent

Lipofectin reagent, credit: Felgner.

Before Bethesda Research Labs had begun its commercialisation operation, Felgner informally shared some of his cationic lipids with Robert Malone. Based at the Salk Institute for Biological Sciences in La Jolla, California, Malone was well versed in developing functional mRNA and was a graduate student in the laboratory of Inder Verma, an Indian American molecular biologist and recognised leader in the field of gene therapy (Felgner interview 2021). His development of functional mRNA was helped by the work of Paul Krieg and Douglas Melton, two developmental biologists based at Harvard, who in 1984 showed it was possible to produce large amounts of biologically active mRNA in the laboratory using an RNA-synthesis enzyme extracted from the vaccinia virus and other tools (Krieg, Melton). Krieg and Melton's breakthrough was driven by a desire to find a useful research tool for investigating gene function and activity. Unable to persuade Harvard's technology development of the virtue of patenting their technique, Krieg and Melton made it possible for other researchers to gain access to their reagents by handing them over to Promega Corporation, a lab-supplies company in Madison, Wisconsin (Dolgin Sept 2021).

Early experiments with mRNA and DNA

In late 1987 Malone conducted an experiment which showed it was possible to get cells to start producing luciferase, a protein that helps produce light. He achieved this by injecting the cells with some mRNA he had prepared using Krieg and Melton's technique mixed with the cationic lipids supplied by Felgner (Malone, Felgner, Verma; Dolgin Sept 2021). What was important about Malone's experiment was he demonstrated the feasibility of carrying out gene therapy without the use of retroviruses which until now had been the standard procedure (Felgner, Rhodes; Felgner interview 2021). Malone then went one step further to show that he could get xenopus and frog embryos to absorb mRNA using the same method. It was the first time that anyone had shown the possibility of delivering mRNA into a living organism (Felgner interview 2021; Malone; Dolgin Sept 2021).

Felgner continued to look for other ways to further experiment with lipofectin. The perfect opportunity came after he joined Vical Corporation in February 1988. Set up the year before in San Diego, Vical was a small start-up that had been founded by some former executives from Hybritech, an American biotechnology company that had been acquired in 1986 by Eli Lilly for US$485 million on the back of its monoclonal antibody products (Wollaeger interview; Roach, Monroe; Felgner n.d.).

Ostensibly, Vical hired Felgner to help with its development of liposomes for the delivery of AZT treatment for HIV patients for which it had secured $5 million through a partnership with Burroughs Wellcome (Wollaeger interview; Felgner interview 1997). But Vical also agreed to sponsor his work on lipofectin that Syntex had discouraged him from pursuing. This gave him the means to start a collaboration with John Wolff, a physician-scientist with an interest in gene therapy for neurological conditions who had just taken up a faculty appointment at the University of Wisconsin. One of the attractions of working with Wolff was he had the facilities for running animal studies (Felgner interview 1997; Felgner interview 2021).

Photograph of Vical being built in 1988

Photograph of Vical being built in 1988, courtesy of Philip Felgner.

In 1989 Felgner and Wolff reported together with others that the lipofection method could successfully be used to deliver functional DNA into the skeletal muscle of live mice, which enabled the cells to start producing proteins that they could not otherwise make. Soon after this Felgner and his Vical colleagues unexpectedly found that naked DNA and naked mRNA directly injected into the muscle of an animal, without mixing it with cationic lipids, could also stimulate cells to start producing proteins they could not make otherwise (Wolff, Malone, Williams; Felgner, Rhodes).

What was especially exciting was 'the only thing' that Vical's scientists had done was inject 'a certain volume of fluid' into the muscle' (Felgner interview 2021). Recalling the moment in 1989, Felgner says 'that was so wacky. If you put DNA on cultured cells nothing happens. But in an animal, you could inject DNA and get expression. That's because, when you inject into muscle, it makes transient breaks in the membrane that allow the DNA to get in' (Sforza). Astonished at the result, Felgner and his collaborators spent several months repeating the experiment and double-checking what had happened (Roach Monroe). As Felgner explains, they discovered 'that it was a pressure mediated effect, not any kind of delivery system effect that everybody was striving for' (Felgner interview 2021).

Activation of beta-galactosidase activity in muscle cells after the injection of naked DNA

This photograph shows the activation of beta-galactosidase activity in muscle cells after the injection of naked DNA. Source: Figure 2, Wolff, Malone, Williams.

Vical's founders were thrilled with the finding. Importantly they believed it would help 'convert the company from a bare-bones start-up to a major player in the ranks of San Diego's biotechnology community' (Roach Monroe). They immediately filed for a patent with the Wisconsin Alumni Research Foundation on the gene transfer technique (Felgner interview 1997; Felgner interview 2021). Since naked DNA did not involve the use of cationic lipids like those claimed in the Syntex patent, the naked DNA patent gave Vical a very dominant intellectual property position. Consequently, Vical built its business model around this very exclusive naked DNA IP. The patent made it clear that the new technique could be used for 'gene therapy, vaccination and any therapeutic situation in which a polypeptide should be administered to cells in vivo' (Felgner, Wolff, Rhodes).

Nucelic acid vaccination

For proof of concept of nucleic acid vaccination, Felgner reached out to Nancy Haigwood who was with Chiron Pharmaceuticals in Alameda, California. She created a plasmid producing high levels of secreted HIV gp120 in Chinese hamster ovary cells intending to use the purified protein to develop a recombinant protein vaccine for HIV. When they injected that same plasmid into mice anti-gp120 specific antibody and T-cell responses were induced. Based on these results they were surprised that Chiron did not exercise their option to license the technology. Later the Vical team learned that Chiron's executives were preoccupied in merger negotiations with Cetus (Felgner interview 2021).

The simplicity of the new method quickly attracted the attention of Maurice Hilleman at Merck. Having pioneered the development of more than 40 vaccines between 1960 and 1996, including those for measles, mumps, rubella, hepatitis A, hepatitis B, influenza, Japanese encephalitis, pneumococcus, meningococcus and Haemophilus influenzae B, Hilleman immediately recognised the potential of Vical's gene delivery technique for improving the manufacturing of vaccines. He envisaged the possibility of creating new vaccines by using plasmid DNA constructs. This promised to be much less time-consuming to prepare and less difficult to scale-up than the method then used for vaccine production which relied either on weakening or inactivating a pathogen or genetically engineering sub-particles of a virus so that it could be injected safely to stimulate an immune response. Now all that would be needed to create a new vaccine would be to change the DNA sequence in a plasmid (Felgner interview 2021).

Merck quickly established a multi-million dollar partnership with Vical to explore its gene transfer technique further. In 1993 the two companies reported that they had managed to evoke a strong immune response in mice by injecting them with a plasmid containing the gene for an influenza strain (Ulmer, Donnelly, Parker). This was just the start of an explosion of research into DNA vaccines (McCarthy). But progress was slow. In part this was because most of the focus was on the development of DNA vaccines for chronic infections like HIV, tuberculosis and malaria which proved difficult targets for vaccination. DNA vaccines also tended to elicit low immune responses (Fynan, Lu, Robinson).

The mRNA challenge

Few considered exploring mRNA for vaccine or drug development in the early years. This was despite the fact that in 1990 Felgner and his colleagues had demonstrated that naked mRNA worked just as well as naked DNA when directly injected into the muscle of mice (Wolff, Malone, Williams; Felgner, Rhodes). In February 1992 Gustav Jirkowski and colleagues at The Scripps Research Institute in California also demonstrated that mRNA encoding for the hormone vasopressin directly injected into the brains of rats could temporarily relieve diabetes for up to 5 days (Jirikowski, Sanna, Maciejewski). The following year a French team, which included Frederic Martinon and Pierre Meulien based at Transgène, a small biotech company based in Strasbourg, reported that they had managed to induce an immune response in mice by injecting them with a liposome formulated with mRNA encoding for the influenza virus nucleoprotein (Martinon, Krishnan, Lenzen).

One of the reasons mRNA attracted so little interest at first was because it was considered less stable than DNA and its effect less durable. Many researchers were also concerned that it could stimulate unwanted immune responses (Liu; Pardi, Hogan, Porter, Weissman). Companies were also reluctant to embrace the technology for vaccines because the cost and feasibility of manufacturing was seen as too challenging (Dolgin Sept 2021).

Early mRNA pioneers

Such limitations, however, did not put off Katalin Karikó, a biochemist based at the University of Szeged's Biological Research Centre in Hungary. She first developed an interest in the therapeutic potential of mRNA in 1978 when she joined the RNA team of Jenő Tomasz. A couple of years before Tomasz had synthesised reference material for Aaron Shatkin, a scientist working on unravelling the genetic structure of reovirus mRNAs at the Roche Institute of Molecular Biology in Nutley, New Jersey (Banerjee). Shatkin was doing this work alongside Yasuhiro Furuichi, a Japanese researcher visiting his laboratory, who back in 1973 had observed, on the basis of work with the cytoplasmic polyhedrosis virus (CPV) of silkworms, that mRNA synthesis was activated by methylation of a specific nucleotide during the initial stage of transcription of the double-stranded RNA genome. Furuichi originally tried to publish his findings in Nature in 1973, but the editors replied they were too busy to publish it. The paper was subsequently published in Nucleic Acids Research in 1974 (Furuichi; Furuichi emails). One year later Furuichi discovered, together with Kin'ichiro Miura at the National Institute of Genetics in Japan, that the mRNA synthesis correlated with a blocked structure at the 5' terminus of the CPV mRNA (Furuichi, Miura). This paved the way for his discovery with Shatkin of a similar cap structure in eukaryotic cell and viral mRNAs which they published in 1975 Furuichi, Muthukrishnan, Tomasz, Shatkin; Hanawa; Anon; Furuichi 2021). They were helped in this work by the reference material supplied by Tomasz (Karikó email).

Like other nucleic acids, mRNA has two specific ends known as 3' and 5', each of which have different nucleotide structures. These ends act a bit like the front and back cover of a book. mRNA translation into a protein starts at the 5' end, which contains the cap structure and ends at the 3' end, which contains a sequence of adenine bases, referred to as a poly-A tail (figure 4). The cap and the polyA tails are now known to help protect the mRNA from degradation and increase the efficiency of protein synthesis (Hornblower, Robb, Tzertzinis; Karikó email).

Aaron Shatkin

Photograph of Aaron Shatkin, credit Vincent Racaniello. Shatkin completed a degree in chemistry at Bowdoin College in 1956 and in 1961 finished a doctorate under the Nobel Laureate Professor Edward Tatum. Thereafter he joined Norman Salzman at the National Institutes of Health with whom he conducted research on the mRNA biogenesis of vaccinia virus and independently began studying reoviruses, animal viruses that contain double-stranded RNA. He first made his mark in 1968 when he showed that purified virions of reovirus contain an RNA polymerase able to transcribe RNA in vitro 'that are complementary to the viral genome RNA (Banerjee).

Yasuhiro Furuichi

Photograph of Yasuhiro Furuichi, credit Furuichi. Born in 1940 Joshin in the north of Korea, when Japan occupied the country, Furuichi graduated in pharmaceutical sciences from the University of Tokyama in 1964 and then completed a doctorate at the University of Tokyo in 1969. Following this he joined the National Institute of Genetics in Japan where he began his work with the cytoplasmic polyhedrosis virus (CPV) of silkworms.

mRNA is translated by a ribosome into proteins

Figure 4: Diagram showing how mRNA is translated by a ribosome into proteins.

When Karikó joined Tomasz's laboratory he asked her to try to create a synthetic form of RNA called 2-5A (Frangou). This RNA had just been found to be produced in response to interferon, a protein that enables the body to defend itself against viruses and cancer (Wiliams, Kerr, Gilbert; Kerr, Brown). Synthesising RNA was not an easy task because the enzyme RNA polymerase was not yet available. So far scientists had only managed to chemically synthesise very short pieces of RNA that were 3-4 nucleotides long. Generating mRNA was even more challenging because it contains hundreds of thousands of nucleotides. Due to these limitations, Karikó focused her efforts on synthesising a trimer of 2'-5'-linked cordycepin, which is a nucleoside analogue of adenosine and tested it for its antiviral activity. She also produced phosphatase-resistant 2-5A analogs and published her findings in 1985 (Karikó Ludwig; Szte).

Katalin Karikó taken in 1957

Photograph of Katalin Karikó taken in 1957 with her older sister Zsuzsanna and parents Zsuzsanna and János, credit Karikó. Karikó grew up in a small Hungarian farming town called Kisújszállás, where her father worked as a butcher and her mother as a bookkeeper. Like all their neighbours their home did not have any running water, a refrigerator or a television. Karikó gained a fascination for science through watching her father dismember pig carcasses and displayed a talent for biology early on. At school she came third in the country for the biology competition. She went on to study biochemistry at the University of Szeged, which she did after participating in the university's preparatory summer courses for underprivileged students. Funded by a scholarship from the Hungarian Academy of Sciences, Karikó completed a doctorate in 1982. Her research involved synthesising short pieces of RNA and testing them for antiviral activity (Szte; Hargittai, Hargittai). As an undergraduate student, Karikó became involved in using liposomes to deliver DNA into cells which she published with colleagues in 1985 (Somlyai, Kondorosi, Karikó, Duda). In order to make the liposomes Karikó needed phospholipids and turned to a recipe from the 1940s which required cow brains, which was a very familiar ingredient to her from the days she had spent with her father's butcher shop (Borrell).

Despite her progress, Karikó could not continue with her work on mRNA because the Biological Research Centre ran out of money to support her. Made redundant in 1985, Karikó decided to start a new academic life in the United States. This was not an easy decision because Hungary was still behind the Iron Curtain and limited how much money its citizens could take abroad. In the end Karikó managed to raise enough money with Bela Francia, her engineer husband by selling their second-hand family car and illegally swapping the cash for some dollars with some foreign students. With this cash they purchased a one-way ticket and sewed up the remainder in the back of their daughter's teddy bear. The family flew to Philadelphia where Karikó had secured a postdoctoral position at Temple University (Crow; Kolata; Bendix; Trouillard).

Karikó continued working on cordycepin analogues of 2-5A molecules at Temple University where she remained for some time (Karikó Reichenback Suhadolnik; Karikó, Li, Sobol). But when her boss learned that she was planning to leave Temple University, he threatened her with deportation which made her life very difficult. Karikó's daughter had only just started school, so she was forced to take a research position in Bethesda. This entailed her commuting from Philadelphia every Monday morning at 3 a.m where she slept on the floor of her office until she returned home on Fridays (Frangou; Karikó email.

Katalin Karikó working in the laboratory

Photograph of Katalin Karikó working in the laboratory, 1980. Credit: Karikó.

In 1990 Karikó became a research assistant professor at the University of Pennsylvania with the financial support of assistant professor Elliot Barnathan, a cardiologist whose research was focused on blood vessels. Her enthusiasm for mRNA quickly got Barnathan interested in seeing if it could be used to help newly transplanted blood vessels function better. To this end they launched a series of experiments to determine if mRNA could be used to get cells to produce the urokinase receptor, a protein that binds urokinase, which inhibits blood clot formation. To their delight it did. For Karikó this was a pivotal moment. Immediately she saw data beginning to spew out from the gamma counter indication that the new receptor protein is functional; she understood mRNA could be used to get any cell to produce any desired protein. As she puts it, 'I felt like a god' (Kolata).

Keeping the mRNA dream alive against the odds

Encouraged by this success, Karikó continued to look for ways to work on the therapeutic applications of mRNA. Between 1993 and 2001 she published several papers with colleagues confirming that mRNA could be successfully introduced into cultured cells or rat brain in vivo to produce the encoded proteins (Karikó, Kuo, Boyd; Karikó, Megyeri, Xiao, Barnathan ; Karikó, Kuo, Barnthan, Langer; Karikó, Kuo, Barnthan; Karikó, Keller, Harris). Despite the exciting results, Karikó struggled to secure funding. As she recollected, 'Every night I was working: grant, grant, grant. And it came back always no, no, no'. Her dream was to deploy mRNA to treat cystic fibrosis and strokes (Kollewe). Few, however, were willing to back her idea because synthetic mRNA degraded too easily in the body for use as a drug and too little amount of protein was produced to have a biological effect (Garde, Saltzman).

The attitude at the time is captured by Matt Winkler, who in 1989 founded Ambion, one of the first RNA-focused lab supplies companies in Texas. As he put it, 'If you had asked me back [then] if you could inject RNA into somebody for a vaccine, I would have laughed in your face'. David Curiel, an oncologist based at the University of Alabama at Birmingham, encountered the same phenomenon in the early 1990s when he approached Ambion after he established in mice that mRNA could be used as a cancer vaccine (Conry, LoBuglio, Wright). He recalled that Ambion said they did not see any 'economic potential in the technology' (Dolgin Sept 2021).

In 1995 Karikó suffered another setback when the University of Pennsylvania decided to demote her from a faculty position because she had failed to secure sufficient research funding. This decision could not have come at a worse time for Karikó as she had just been diagnosed with cancer and was facing an operation. In addition, her husband had just become stranded for six months in Hungary where he had returned to pick up his green card (Cox).

Left in a precarious position by the university, Karikó was rescued by David Langer. He had worked next to her in the Cardiology lab while he was a medical student. By 1995, he was a neurosurgeon resident and convinced chairman Eugene Flamm to give Karikó a laboratory space and salary. Now on the same salary as a technician, Karikó began to wonder if she was 'not good enough, not smart enough'. With no other options open to her, Karikó decided to remain at the university. Having always dreamed of developing mRNA encoding for therapeutic proteins, she began to make mRNA for the purposes of treating a stroke. Determined not to be defeated by her knockback, whenever Karikó went to meetings or sat next to someone she took the opportunity to 'ask what they were researching' and 'always offered to make RNA' for them. This earned her the reputation as 'the RNA hassler' both within the university and beyond (Cox; Garde, Saltzman; De George; Kollewe; Karikó email).

Karikó finally found a sympathetic ear in 1998 when she began chatting to Drew Weissman at the department's photocopier while both of them were jockeying to copy the latest scientific publications. An immunologist and physician, Weissman had joined the University of Pennsylvania the year before. He was developing vaccines for infectious diseases and struggling to get immune cells to produce proteins for the vaccines. Their discussion led to the beginning of a very productive partnership, supported with NIH funding (Keener).

In 2000 Karikó and Weissman published one of their first papers together. They reported that they had managed to stimulate a potent immune response in human dendritic cells loaded with mRNA coding for the HIV gag protein (Weissman, Ni, Scales). Based on this it seemed that mRNA was ideal for a vaccine. Critically it produced a lot of antigen needed to trigger an immune response and proved capable of enhancing the immune response like an adjuvant. But for Karikó, who wanted to develop mRNA for therapeutic applications, the immune activation by the mRNA was very bad news. For this reason, she and Weissman launched efforts to understand why the mRNA was so immunogenic so that they could find a way of changing it (Karikó email).

The modified mRNA breakthrough

By 2004 the two scientists had worked out that the problem might be due to the mRNA activating a series of immune sensors, called Toll-like receptors, which act as the first line of defence against pathogens (Karikó, Ni, Capodici). Further work indicated that this was triggered by uridine, one of the nucleosides found in RNA. Based on this, they set out to create a modified form of mRNA by replacing uridine with a suitable analogue known as pseudouridine, which is the fifth most abundant nucleoside in the mammalian RNA. To their delight the human dendritic cells treated with pseudouridine-containing mRNA induced no inflammatory molecules and produced very high levels of the protein encoded by the mRNA. Karikó could not at first believe what they had achieved. It was only after they got the same result on repeating the experiment that she could truly believe it. Both she and Weissman immediately grasped the significance of their breakthrough for creating safe mRNA that could be used for many different applications (Karikó email).

Drew Weissman and Katalin Karikó

Photograph of Drew Weissman and Katalin Karikó in their laboratory at the University of Pennsylvania, 2015, credit Karikó. Born in Lexington, Massachusetts, Weissman majored in biochemistry and enzymology at Brandeis University and then completed a doctorate in immunology and microbiology and M.D. at Boston University in 1987. He then did a residency at Beth Israel Deaconess Medical Center and was a postdoctoral fellow in the laboratory of Anthony Fauci at the National Institutes of Institute for seven years. During this time he explored the role of dendritic cells in the immune response to infectious diseases like HIV infection (Weissman, Rubbert, Comadiere, Murphy, Fauci).

Karikó and Weissman initially struggled, however, to get a journal to publish their discovery (Barbaro). It was finally published in 2005 (Karikó, Buckstein, Ni, Weissman). They also had a hard time persuading the intellectual property officer at the University of Pennsylvania to see the value of patenting their work. He only became enthusiastic, Karikó recalls, after she spotted he was bald and remarked 'mRNA could be good for growing hair' (Crow).

In 2006 Karikó and Weissman founded a company, called RNARx, with a small business grant of $900,000 from the US government. Their aim was to develop pseudouridine-containing mRNA encoding erythropoietin for treatment for anaemia (Johnson). But the company never really made much headway because it was hampered by disagreements between the university and the two scientists over the licensing of their technology. The university continued to own 50 per cent of the patent despite all the efforts by Karikó and Weissman to obtain the patent for RNARx. In the end the University of Pennsylvania sold the license in 2010 to Cellscript, a company founded and headed up by Gary Dahl based in Madison, Wisconsin, which it mostly used to market kits to produce modified mRNA (Dolgin 2015; Frangou; Borrell; Karikó email).

The wrangling over the patent did not stop the two scientists making progress at a scientific level. In 2008 they confirmed that the incorporation of pseudouridine not only significantly reduced the chances of triggering an unwanted immune response but the modified mRNA had a higher translational capacity when tested in mammalian cells and mice (Karikó, Muramatsu, Welsh). Four years later they reported that regular injections with nucleoside modified mRNA in mice and monkeys helped boost the production of erythropoietin, a protein essential to the production of red blood cells, providing a new therapeutic pathway for anaemia (Karikó, Muramatsu, Keller, Weissman).

A new chapter for mRNA

Karikó and Weissman's work notably demonstrated that mRNA could be rendered invisible to a cell's defence mechanism by replacing its nucleoside uridine with pseudouridine. While largely unnoticed by the scientific community, one person who was inspired by their breakthrough was Luigi Warren. He had first developed an interest in mRNA during his graduate studies and in 2007 applied to the Agency for Science, Technology and Research (A*STAR), a biomedical research hub in Singapore, to pursue a project which envisaged the use of mRNA to reprogram cells. Unsuccessful in this bid, Warren decided instead to join Derrick Rossi, a Canadian biologist who had just set up his own laboratory focusing on stem cell research at the Immune Disease Institute at Harvard Medical School. He had first collaborated with Rossi on a single-cell gene expression analysis project at Stanford. Warren was finishing up his PhD in the lab of Stephan Quake and Rossi was nearing the end of a postdoc in Irvine Weissman's lab. They warmed to one another, sharing tastes for seventies glam rock and roundabout career paths (Warren emails).

In November 2007 Warren joined Rossi's laboratory as his first postdoctoral researcher. Six months after he started working in the laboratory, in June 2008, Warren attended the fouth annual meeting held in Philadelphia by the International Society for Stem Cell Research (ISSCR) (Warren emails). At this meeting a call was put out for scientists to find a new approach to generate pluripotent stem cells. These cells have similar characteristics to embryonic stem cells in that they can turn into any type of cell in the body. The first such cells had been announced to a stunned audience of scientists gathered at the annual meeting of the ISSCR in Toronto in 2006. They were created by Shinya Yamanaka, a Japanese scientist, and his colleagues at the University of Kyoto. Together they had managed to reprogram mature, specialised cells into 'induced pluripotent cells' (iPSCs) by using a retrovirus to artificially add four genes to skin cells from a mouse (Takahashi, Yamanaka). Crucially the new technique provided a way to side step the ethical controversies that plagued embryonic stem cells which many believed had major therapeutic potential for various conditions.

While Yamanaka went on to win the Nobel Prize in 2012, scientists quickly realised that using retroviruses to create iPCs posed a significant risk because of their potential to alter a cell's DNA and cause dangerous mutations. This presented a major stumbling block to using iPSCs for therapeutic purposes. Warren realised that mRNA offered a much more efficient and safer approach. The advantage of using mRNA was that it could carry genetic instructions into a cell but would not integrate with the cell's nucleus (Warren emails; Warren Manos Ahfeldt; Zuckerman).

Originally trained as a software engineer, Warren optimistically viewed the process of synthesising mRNA as 'a lot like computer coding'. All he needed to do was juggle with the four nucleotides that make up the molecule. To test out the feasibility of the approach he started by synthesising a modified form of mRNA encoding for a green fluorescent protein (GFP) found in jellyfish. To his delight when he delivered this mRNA into human skin cells they started to produce a green glow. This confirmed that the mRNA could be programmed to produce a desired protein. But to his consternation many of the cells died within a day or so and appeared to self-destruct whenever he gave them repeated doses of mRNA (Zuckerman).

Wondering if the problem was due to mRNA activating an unwanted immune response, Warren turned for advice to Sun Hur, a Korean-born immunologist at Harvard Medical School, and her American post-doctoral researcher Alys Peisley. In October 2008 Hur alerted Warren to Karikó and Weissman's success in modifying mRNA so that it did not trigger inflammation and other immune responses. Based on this Warren experimented with creating a new draft of mRNA encoding for the GFP with different combinations of modified nucleotides. This proved highly successful at getting human skin cells to produce large quantities of the protein. Furthermore, the cells continued to survive even when given repeated doses of the mRNA (Warren emails; Zuckerman).

Having solved the problem, Warren began experimenting with different forms of modified mRNA to see if he could convert skin cells into pluripotent stem cells. As 2009 drew to a close, he was excited to show Rossi that he had succeeded. Repeated experiments over the next year by Warren and colleagues showed that the result was not a fluke. Despite the achievement, Warren and Rossi faced major challenges getting it published (Zuckerman). Eventually appearing in the journal Cell Stem Cell in 2010, Warren's work helped publicise the earlier discovery by Karikó and Weissman and put mRNA firmly on the map (Karikó letter).

By the time the article appeared, Rossi and Warren had filed a patent and Rossi was focused on launching a spin-out company on the back of it. Called Moderna Therapeutics, the company aimed to commercialise chemically modified mRNA to create vaccines and therapeutics. Rossi founded the start-up with Kenneth Chien, a physician-scientist specialising in cardiovascular research with whom he had shown in mice that modified mRNA could help improve heart function thereby providing a promising therapeutic tool for repairing the damage caused by heart attacks (Zangi, Lui, von Gise). They were joined in the venture by Robert Langer, a prolific biochemical engineer at Massachusetts Institute and successful founder of several spin-out companies from his laboratory. The company was backed with funding from Noubar Afeyan, a Lebanese bioengineer who was chief executive of Cambridge biotech investment firm Flagship Ventures, and Timothy Springer, an immunologist and founder of several high-profile biotechnology companies (Garde, Saltzman; Dolgin 2015; Cox; Borrell).

Luigi Warren

Photograph of Luigi Warren, credit Warren. The son of an Italian mother and British father Luigi Warren grew up in Bromley, a suburb on the outskirts of London renowned as the place where David Bowie grew up. In 1982 he completed a degree in electronic engineering with computer science at University College London and then spent fifteen years in software development. Eventually bored with computer coding, in 1998 Warren enrolled for a biology degree at Columbia University which he completed in 2001. After this, he did a doctorate in biology at California Institute of Technology, completing his thesis work at Stanford University under Stephen Quake. Warren was awarded his PhD in 2008. After spending some time in academia, Warren founded and managed two service companies, Stemiotics and Cellular Reprogramming, dedicated to producing iPSCs for academic and industrial researchers using enhanced mRNA reprogramming technology.

Derrick Rossi

Photograph of Derrick Rossi, 2008, credit Tracy Rolling, Wikipedia. Born in Toronto, Canada, to Maltese immigrant parents, Rossi is the youngest of five children. His father worked at an auto body shop and his mother co-owned a Maltese bakery. Rossi first developed a passion for molecular biology when at school and completed undergraduate and master's degrees in molecular genetics at the University of Toronto before doing a doctorate at the University of Helsinki. Following a four-year postdoctoral research fellowship at Stanford University, Rossi became a professor in the department of Stem Cell and Regenerative Biology at Harvard University in 2008 (Rossi interview).

Using mRNA to combat cancer

Warren was not the only one inspired by Karikó and Weissman's mRNA advances. So too were the married couple Uğur Şahin and Özlem Türeci, two physicians and immunologists working at the medical schools of Homburg/Saar and Mainz in Germany who began researching therapies that could harness the patient's immune system to fight cancer in the early 1990s. Initially their focus was on the development of monoclonal antibodies for this purpose, for which they founded a spin-out company, called Ganymed, in 2001 (Garde, Saltzman; Miller, Cookson).

Following this work, they subsequently began to explore mRNA for creating cancer immunotherapies because mRNA could be easily and quickly tailored to target the unique tumour of each patient (Anderson; Pietzsch). Their interest in mRNA was ignited by the work of Eli Gilboa at Duke University Medical Center. In 1996 Gilboa and his colleagues showed that it was possible to induce very strong immune responses against tumours in mice using dendritic cells modified with mRNA coding for surface receptors found on the tumours (Pietzsch; Boczkowski, Nair, Snyder, Gilboa). On the back of this work Gilboa launched a spin-out company called Merix Bioscience (subsequently Argos Therapeutics and then ColImmune) with the aim of leveraging mRNA to develop cancer vaccines (Dolgin Sept 2021).

Uğur Şahin

Photograph of Uğur Şahin, 12 Aug 2019, credit BioNtech, Wikipedia. Şahin was born in Iskenderun, Turkey, in 1965, and moved to Germany with his parents when he was four years old after his father took a job in the Ford car factory in Cologne. After studying medicine at the University of Cologne, Şahin worked for several years at Saarland University Medical Center, before landing up at Mainz University.

Özlem Türeci

Photograph of Özlem Türeci, 12 Aug 2019, courtesy of BioNTech, Wikipedia. Born in 1967 in Lastrup, Lower Saxony, West Germany, Türeci's parents arrived in Germany from Turkey as immigrants. Her father was a physician and her mother a biologist. Türeci originally had dreams of becoming a nun but ended up studying medicine. She met Şahin in the early 1990s when the two of them were working as trainee doctors on a blood cancer ward in a hospital in south-west Germany.

Encouraged by Gilboa's results, Şahin and Türeci began investigating ways to optimise the manufacturing process for mRNA so that multiple versions of the molecule could be created within days for use as cancer vaccines. In 2006 they published a seminal paper with colleagues outlining a method to produce mRNA with increased stability and translational efficiency (Holtkamp, Kreiter, Seimi). As a result of this work, the German Federal Ministry of Education and Research gave them the GoBio Award which helped pave the way to their founding of a new company, in 2008, to develop mRNA as personalised cancer immunotherapies. They founded the company together with Christoph Huber, an Austrian oncologist. Originally called TRON, an abbreviation of 'translational oncology', the company was subsequently renamed Biopharmaceutical New Technologies (BioNTech).

In 2013 Şahin and Türeci invited Karikó to lecture on her technique for making modified mRNA which by now had been patented. Following this, Karikó agreed to become Senior Vice President at BioNTech to help oversee its mRNA work. A year later the three scientists published a landmark paper which provided a comprehensive overview of the progress made so far with mRNA based therapeutics and the opportunities and challenges that lay ahead. Cancer immunotherapy, vaccines for infectious diseases and protein replacement were just some of the key areas they identified where they believed mRNA had therapeutic potential (Şahin, Karikó, Türeci).

Şahin, Türeci and Karikó

Photograph of Şahin, Türeci and Karikó, 2013, credit Karikó.

The challenge of delivering mRNA into the body

Despite its appeal for therapeutics, scientists faced a major obstacle in delivering mRNA into the body because it is quickly degraded by the enzyme ribonuclease (RNase), found in all the cells in the body. Another problem was the fact that the molecule is very large and is negatively charged which makes it difficult to pass through negatively charged cell membranes (Zeng, Zhang, Walker, Dong).

One potential solution lay in wrapping the molecule in the lipids devised by Felgner and his colleagues in the 1990s. But this method had certain drawbacks; these positively charged lipids accumulated in the liver and other organs, and were inherently toxic. As early as 2006, the year RNARx was founded, Karikó approached Ian MacLachlan, a Canadian biochemist, to see if a four-lipid delivery system, known as the lipid nanoparticle system (LNP) he had pioneered, might provide a solution. He had developed this in collaboration with Pieter Cullis, a biophysicist at the University of British Columbia and scientists at two small spin-out companies, Protiva Biotherapeutics and Tekmira Pharmaceuticals, initially to improve the delivery of small molecule drugs to combat cancer and subsequently to deliver nucleic acid based drugs which offered a new approach for gene therapy (Tam, Madden, Hope). They did this by creating ionizable cationic lipids which were much less toxic and more efficient at delivering their load through the cell membrane (Delaye). In March 2006 MacLachlan and his colleagues published a landmark study showing that the new platform had proven effective in monkeys at delivering RNA to silence disease-causing genes (Zimmermann, Lee, Akinc).

Ian MacLachlan

Photograph of Ian MacLachlan, credit: MacLachlan. Born in Canada, MacLachlan completed a bachelor's degree and doctorate in biochemistry at the University of Alberta and then spent two years as a postdoctoral fellow in a gene laboratory at the University of Michigan. In 1996 he joined Inex Pharmaceuticals, a small biotechnology company co-founded in Vancouver by Cullis, where he began working on the lipid platform for delivering gene therapy. Four years later, MacLachlan left Inex when the company shifted its focus to chemotherapy. Encouraged by Cullis, MacLachlan formed a new company called Protiva Biotherapeutics where he continued improving lipid formulations for the delivery of nucleic acids with Lorne Palmer and Lloyd Jeffs. Together they found a way of creating a new lipid nanoparticle with four specific kinds of lipids. Proving highly stable and easy to manufacture, the platform marked a significant breakthrough and was duly patented (Vardi). In 2008 MacLachlan was appointed chief technical officer at Tekmira Pharmaceuticals when it merged with Protiva, He led the company's efforts to use the LNP to develop a drug against Ebola (Bacal). MacLachlan remained with the company until 2014 after which he became an independent consultant for different pharmaceutical companies (Favaro). Following his departure, Tekmira changed its name to Arbutus Biopharma.

Pieter Cullis

Photograph of Pieter Cullis, 1986, credit Cullis. Born in the UK, Cullis completed an undergraduate degree and then a doctorate in physics at the University of British Columbia, Vancouver, before undertaking postdoctoral training in biochemistry first at the University of Oxford as an MRC postdoctoral fellow and then at the University of Utrecht as a fellow of the European Molecular Biology Organisation. In 1978 he returned to the University of British Columbia to join the Department of Biochemistry and Molecular Biology. Cullis first became interested in lipid biophysics while at the University of Utrecht which he continued exploring when he returned to the University of British Columbia. His research was directed towards improving the formulation of liposomes for drug delivery, first of all for cancer and later for gene therapy (Semeniuk). Alongside his lipid work Cullis also helped to found a succession of spin-out companies, starting with Lipex Biomembranes in 1985 and subsequently Inex Pharmaceuticals in 1992. Such companies were pivotal to Cullis' research because drug development requires far more financial resources than is available in an academic setting (Horejs).

When Karikó first approached MacLachlan, he was embroiled in a complex bruising litigation case so failed to engage with her proposal. The legal battle was being fought over who owned the intellectual property rights to the LNP system. MacLachlan's initial lack of engagement however did not put Karikó off continuing to persevere with him. In 2013 she offered to relocate to Vancouver to work with him. By now the patent dispute, which lasted six years, had been settled, but the saga had left MacLachlan totally exhausted and demoralised so he did not bite (Vardi).According to MacLachlan, he was also reluctant because of the unworkable conditions the University of Pennsylvania wanted to impose on his collaboration with Karikó (MachLachlan emails).

In part the legal wrangling over the LNP system reflected the fact that it was a major game changer for drug delivery. A major milestone was reached in 2012 when the first LNP based drug was launched in human trials. This was helped by some improvements made to the LNP system. The drug was designed to deliver small interfering RNA (siRNA) to silence the action of a faulty gene produced in liver cells which causes a rare and severely debilitating and potentially life-threatening disease known as transthyretin-induced amyloidosis in which proteins building up in the body's tissues and organ can cause nerve and heart damage. The resulting drug was Patisiran (Kulkami, Cullis, van der Meel; Cross; Dolgin Sept 2021).

Subsequently approved in 2018, Patisiran marked a major turning point because researchers had spent the previous two decades struggling to deploy different forms of RNA, including siRNA and mRNA, for therapy. Just how difficult the process was is exemplified by the case of Moderna which was one of the best funded companies in the space. By 2015 it had raised more than $1 billion on the back of its potential to develop mRNA therapeutics (Dolgin 2015). In late December 2016 Moderna informed its investors that, because of safety concerns, it was unable to proceed with human trials with its mRNA therapy for Crigler–Najjar syndrome, a rare and debilitating disorder caused by a faulty gene which makes it difficult for the body to effectively convert and clear bilirubin, a byproduct of old or worn out blood cells (Servick).

Leveraging mRNA for vaccines

More progress was made on the vaccine front. In 2014 on the suggestion of Weismann, Karikó who was by that time at BioNTech, reached out to Thomas Madden, who had helped to optimise the LNP system for the development of Patisiran and was the chief executive officer of Acuitas. She wanted to see if it was possible to leverage LNPs for mRNA. By the autumn of 2014 they had completed a set of animal experiments which indicated that the Acuitas-formulated pseudouridine-containing mRNA was exceptionally potent (Karikó email).

In 2018 Weissman and the Acuitas scientists reported that LNP-formulated vaccines targeting rabies, influenza and Zika viruses could be a promising new vaccine technology. They pointed out that it was highly competitive against vaccines based on inactivated virus and elicited superior antibody and T cell responses and that these responses could be boosted and remained stable for up to 1 year (Pardi, Hogan, Porter, Weissman). This led to a collaboration between Acuitas and BioNTech to develop a flu vaccine. Recent work by a team led by Norbert Pardi, a biochemist at the University of Pennsylvania, has shown that the ionizable lipid in the LNP can enhance the efficacy of mRNA and protein subunit vaccines by inducing T follicular helper cell and humoral responses (Alameh, Tombacz, Bettini).

By the time the first case of COVID-19 struck in Wuhan, in November 2019, several mRNA companies had licensed the LNP platform from Acuitas for vaccine development (Horejs; Semeniuk). This included BioNTech and CureVac, a German company set up in 2000 on the back of work by Ingmar Hoerr. In 1998, as part of his doctoral work, Hoerr had demonstrated that a simple injection of naked mRNA could generate a strong specific immune response (Koester). Each one looked to use some variation of the LNP platform and manufacturing system and different forms of modified or codon-optimised mRNA to develop the vaccines.

Within hours of the genome of SARS-CoV-2 virus being uploaded online, on 10 January 2020, scientists around the world had managed to pinpoint the genes that carry the instructions for the spike protein, the part of the virus they needed to target for preventing its entry into human cells. This provided a crucial foundation to begin designing mRNA vaccines.

By 15 January 2020 a team of scientists led by Kizzmekia Corbett, an immunologist, at the National Institutes of Health, had managed to produce a suitable mRNA (mRNA-1273) for use as a vaccine, which they passed over to Moderna to develop with the help of the LNP platform. Moderna started testing its vaccine in animals on 4 February 2020 and the first human trials were launched just six weeks later, on 16 March 2020. Overall it had taken just 66 days from when the sequence of the SARS-CoV-2 genome was released to when clinical trials began (Corbett, Edwards, Leist).

Another mRNA vaccine was developed equally fast by the scientists at BioNTech. Back in 2018 Şahin had predicted to a room full of infectious disease experts that BioNtech had the capacity to rapidly deploy its mRNA technology to develop a vaccine if a global pandemic struck. Şahin immediately got working on the vaccine after he read of the outbreak of strange pneumonia in Wuhan reported in The Lancet on January 24 2020. On reading the article, what was most concerning to Şahin was 'that one of the family members had the virus and was virus-positive but did not have symptoms'. He realised that this meant the virus had probably already spread well beyond China, so had the capacity to become a pandemic. Within a weekend Şahin had designed 10 potential vaccine candidates on his computer to help with the problem. But BioNTech lacked the resources and capacity to develop the vaccine on its own. By February Şahin had reached out to Pfizer to help BioNtech develop it. The two companies had already been working for two years together to develop a flu vaccine and immediately switched their focus to develop and test the COVID-19 vaccine (Gelles; Nellson).

BioNTech's vaccine was approved for emergency use by the US Food and Drug Administration on December 11 2020 and the Moderna vaccine was approved 7 days later. Their approval marked a major milestone. Developed in record time, the vaccines proved the value of mRNA technology and what it could achieve. For many scientists, their approval is just the beginning of the next chapter in the application of mRNA to many other medical issues. Getting to this point required many decades of working out how best to modify mRNA and engineer a delivery mechanism so that it could be effectively used within the body using the LNP system.

Application

One of the advantages of developing mRNA for the COVID-19 vaccine is it has given companies scope to improve and streamline the full development process for advancing mRNA technology into other areas, including cancer (Albert). The fact that mRNA can encode any sequence of amino acids makes it a highly attractive platform for both the development of vaccines as well as protein-replacement therapeutics which aim to help with genetic diseases caused by missing or defective proteins (Schlake, Thess, Fotin-Mlecek, Kallen).

Issues

Despite the success with the COVID-19 vaccines, future applications with mRNA remain uncertain and the technology still has room for improvement. From the perspective of vaccines there remains the challenge that the raw materials are expensive. Additionally mRNA vaccines need to be stored at extremely cold temperatures to remain stable which makes their distribution costly (Dolgin Jan 2021; Hancock).

Billions of patients have now received the mRNA vaccines and so far they have been found to be generally safe. Where side effects have occurred, these have been confined to a handful of patients. This includes severe allergic reactions and immune thrombocytopenia, a bleeding disorder which is caused by the immune system attacking platelets after vaccination. The condition can be easily treated. For regulators, the overall benefits of the vaccines far outweigh their potential side effects. But they expect recipients to be closely monitored for a minimum of 15 minutes after being given the vaccines (UK HSA).

While mRNA vaccines have been found to stimulate a strong immune response to SARS-CoV-2, they are still too new to know exactly how durable their effect is. Current evidence suggests individuals retain strong immunity against SARS-CoV-2 and variants for at least 6 months post vaccination and helps prevent severe disease, hospitalisation and death (Goel, Painter, Apostolidis).

Nonetheless, going forward waning immunity poses a major issue, especially given the emergence of new variants of concern. For this reason health experts now recommend people get boosters to enhance their immune response. This approach is commonly used with other diseases where either an additional dose of the original vaccine is given or it has been modified to enhance protection against new variants. Boosters are given for example every year in the case of flu.

Work on the vaccines for COVID-19 has been enormously important for advancing mRNA technology. Importantly it has taught industry how to ramp up production at scale. This could have enormous benefits for cancer in the future. Yet, this will not happen overnight. One of the major challenges is that the target tissue in cancer shares many similarities with healthy tissue and contains cells with a range of mutations which makes it difficult to work out what target to go for. In addition, many tumours actively suppress immune responses. The best strategy would be to tailor a vaccine to be specific to each patient's tumour, but this type of treatment is very challenging and not financially viable. A better option is to identify common mutations so that mRNA cancer vaccines can be developed for a substantial subset of patients. Many companies are already active on this front (Albert).

Whatever happens in the future, the story of mRNA is unlikely to escape yet more patent disputes. Already the largest players in the space are actively trying to invalidate competitor patents. A lot of the contest is focused on challenging the patents concerning the LNP technology. With a lot of money at stake this issue is unlikely to be resolved any time soon (Rothwell, Figg, Ernest).The matter is not helped by the secrecy that has shrouded proprietary licensing deals and the degree to which companies have limited the information they are willing to share publicly to support vaccine manufacturing. Add to this the complication of the Superior Court of British Columbia which in 2012 granted an injunction against the use of the lipid technology by Acuitas or anyone else including Moderna, BioNtech and Curevac (MacLachlan emails; Rowland).

Acknowledgements

This piece was written in January 2022 by Lara Marks. She could not have written this piece without the invaluable feedback from Pieter Cullis, Philip Felgner, Yasuhiro Furuichi, Katalin Karikó, Ian MacLachlan and Luigi Warren. Many thanks also go to Daniel Power for his scientific help with the images and reading of the final draft.

Messenger RNA (mRNA): timeline of key events

Monod was a biochemist who, together with Francois Jacob, worked out the genetic control of enzyme and virus synthesis based on their experiments with Escherichia coli in the early 1960s. They proposed that a messenger molecule in cells carries codes from the DNA in the cell nucleus to the site of protein synthesis in the cell's cytoplasm. This molecule was later called messenger RNA which is now the basis of COVID-19 vaccines. Based on his work Monrod was awarded a Nobel Prize for Medicine in 1965. 1920-06-07T00:00:00+0000The molecule was first observed by the American scientists Elliot Volkin and Lazarus Astrachan in experiments conducted with bacteriophage-infected Escherichia coli. Calling the new molecule 'DNA-like RNA', Volkin and Astrachan published their finding in 'Phosphorus incorporation in Escherichia coli ribonucleic acid after infection with bacteriophage T2', Virology, 2 (1956), 149-61. 1956-01-17T00:00:00+0000The experiment was conducted by Sidney Brenner, Francois Jacob, and Matt Meselson and published as 'An unstable intermediate carrying information from genes to ribosomes for protein synthesis', Nature, 190 (1961), 576-81. They established the mRNA was responsible for transporting genetic information from the nucleus to the protein-making machinery in a cell. 1961-05-13T00:00:00+0000The prize was awarded to François Jacob, André Lwoff and Jacques Monod for their 'discoveries concerning genetic control of enzyme and virus synthesis'. 1965-10-14T00:00:00+0000Raymond Lockard and Jerry Lingrel conducted the experiment to understand the role of mRNA in the synthesis of proteins. They published their results in ‘The synthesis of mouse hemoglobin beta-chains in a rabbit reticulocyte cell-free system programmed with mouse reticulocyte 9S RNA’, Biochemical and Biophysical Research Communications, 37/2, (1969) 204-12.1969-10-08T00:00:00+0000Y Furuichi, '"Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA', Nucleic Acids Research, 1, (1974), 809-22.1974-07-01T00:00:00+0000Y Furuichi, K Miura (1975) 'A blocked structure at the 5' terminus of mRNA from cytoplasmic polyhedrosis virus', Nature, 253, 374-75.1975-01-31T00:00:00+0000Y Furuichi, S Muthukrishnan, J Tomasz, AJ Shatkin (1976)'Caps in eukaryotic mRNAs: mechanism of formation of reovirus mRNA 5'-terminal m7GpppGm-C', Progress in Nucleic Acid Research and Molecular Biology, 19, 3-20.1976-01-01T00:00:00+0000Monod was a French biochemist who, together with Francois Jacob, worked out the genetic control of enzyme and virus synthesis based on their experiments with Escherichia coli in the early 1960s. They proposed that a messenger molecule in cells carries codes from the DNA in the cell nucleus to the site of protein synthesis in the cell's cytoplasm. This molecule was later called messenger RNA and is now the basis of COVID-19 vaccines. Based on his work Monrod was awarded a Nobel Prize for Medicine in 1965. 1976-05-31T00:00:00+0000Two different scientific teams independently showed it was possible to get cells to produce rabbit globin using mRNA packaged in a liposome. The first was shown in mice cells by Giorgios J Dimtriadis at the National Institute for Medical Research. He published his achievement in 'Translation of rabbit globin mRNA introduced by Iiposomes into mouse lymphocytes', Nature, 274, 923-4. The second team, led by Marco Ostro at the University of Illinois, showed it was possible to get the protein produced in a human cell line by a team. This was published as Ostro, MJ, Giacomoni, D, Velle, et al (31 Aug 1978) ‘Evidence for translation of rabbit globin mRNA after Iiposome-mediated insertion into a human cell line’, Nature, 274, 921-23. 1978-08-31T00:00:00+0000Synthesised by a team led by Philip Felgner at Syntex Research the lipid was a major breakthrough because natural lipids are usually negatively or neutrally charged. The new lipid formed into positively charged liposome which fused more easily with negatively charged cell membranes to deliver drugs directly into a cell. 1984-01-01T00:00:00+0000PA Krieg, DA, Melton, (1984) 'Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs', Nucleic Acids Research, 12/18, 7057-70.1984-09-25T00:00:00+0000The result was published in RW Malone, PL Felgner, IM Verma (1 Aug 1989) 'Cationic liposome-mediated RNA transfection', Proceedings of the National Academy of Sciences USA, 86/16, 6077-6081.1987-01-01T00:00:00+0000Started by former Hybritech executives, Vical built its business around using naked DNA for gene therapy.1987-01-01T00:00:00+0000RW Malone (Fall 1989) 'mRNA Transfection of cultured eukaryotic cells and embryos using cationic liposomes', Focus, 11/4.1987-10-01T00:00:00+00001990-01-01T00:00:00+0000The discovery suggested the therapeutic possibility of using mRNA to transiently replace faulty or missing proteins. JA Wolff, RW Malone, P Williams, W Chong, G Acsadi, A Jani, et al.,'Direct gene transfer into mouse muscle in vivo', Science, 247 (1990) 1465–68.1990-03-23T00:00:00+0000GF Jirikowski, PP Sanna, D Maciejewski-Lenoir, FE Bloom, FE (1992) 'Reversal of diabetes Insipidus in Brattleboro Rats: Intrahypothalamic Injection of Vasopressin mRNA', Science, 255, 996-98.1992-02-21T00:00:00+0000Lipsomes carrying mRNA encoding the influenza virus nucleoprotein reported to induce T cells in mice. F Martinon, S Krishnan, G Lenzen, R Magne, E Gomard, JG Guillet, et al., European Journal Immunolology, 23 (1993) 1719–22.1993-07-01T00:00:00+0000RM Conry, AF LoBuglio, M Wright et al, 'Characterization of a messenger RNA polynucleotide vaccine vector', Cancer Research, 55 (1995), 1397-1400. 1995-04-01T00:00:00+0000D Boczkowski, SK Nair, D Snyder, E Gilboa, 'Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo', The Journal of Experimental Medicine, 184/2 (1996), 465-72.1996-08-01T00:00:00+0000The company was founded by Eli Gilboa on the back of his research at Duke University Medical School. It subsequently changed its name to Argos Therapeutics and then ColImmune. 1997-01-01T00:00:00+0000I Herr, R Obst, HG Rammensee, G Jung, 'In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies', European Journal Immunology, 30/1 (2000), 1-7. 2000-01-01T00:00:00+0000The company was founded on the back of work conducted by Ingmar Hoerr.2000-01-01T00:00:00+0000D Weissman, H Ni, D Scales, D et al, 'HIV Gag mRNA Transfection of Dendritic Cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response', The Journal of Immunology, 165 (2000), 4710-17.2000-10-15T00:00:00+0000K Karikó, H Ni, J Capodici, J, et al (2004) 'mRNA is an endogenous ligand for Toll-like receptor 3', Journal Biological Chemistry, 279, 12542–50.2004-03-01T00:00:00+0000K Karikó, M Buckstein, H Ni, D Weissman, D (Aug 2005) 'Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA', Immunity, 23/2, 165-75.2005-08-01T00:00:00+0000The system was devised by Ian MacLachlan in collaboration with Pieter Cullis. They first published the technique as 'Diffusible-PEG-Lipid Stabilized Plasmid Lipid Particles', Advanced Genetics (2005), 53PA:157-188. doi: 10.1016/S0065-2660(05)53006-2.2005-10-19T00:00:00+0000The company was founded by Drew Weissmann and Katalin Karikó.2006-01-01T00:00:00+0000TS Zimmerman, ACH Lee, A Akinc, et al (26 March 2006) 'RNAi-mediated gene silencing in non-human primates', Nature, 441, 111-14.2006-03-26T00:00:00+0000S Holtkamp, S, Kreiter, A Seimi, A, et al (15 Dec 2006) 'Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells', Blood, 108/13, 4009-17.2006-12-15T00:00:00+0000The company, originally called TRON, was founded by Ozlem Tureci, Ugur Sahin and Christoph Huber.2008-01-01T00:00:00+00002010-11-01T00:00:00+0000L Warren, PD Manos, T Ahfeldt, T et al (5 Nov 2010) 'Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA', Cell Stem Cell, 7/5, 618-30.2010-11-05T00:00:00+0000The team who did the research was led by Kenneth Chien. L Zangi, KO Lui, A von Gise, et al, 'Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction', Nature Biotechnology, 31/10 (Oct 2013), 898-907.2013-10-01T00:00:00+0000N Pardi, MJ Hogan, FW Porter, D Weismann, 'mRNA vaccines — a new era in vaccinology', Nature Reviews: Drug Discovery, 17 (2018), 261-79.2018-01-12T00:00:00+0000The vaccine was developed by BioNTech in partnership with Pfizer.2020-12-11T00:00:00+0000The vaccine was developed by Moderna. 2020-12-18T00:00:00+0000
Date Event People Places
7 Jun 1920Jacques Monod was born in Nancy, FranceMonodPasteur Institute
1956First observation of messenger RNA, or mRNAAstrachan, VolkinOak Ridge National Laboratory
13 May 1961Experiment confirms existence of mRNABrenner, Jacob, Meselson University of Cambridge, Pasteur Institute, California Institute of Technology
14 Oct 1965Nobel Prize awarded for the elucidation of the nature of mRNAJacob, Monod, LwoffPasteur Institute
8 Oct 1969mRNA isolated from a rabbit introduced into mouse lymphocytes shown to stimulate production of a proteinLockard, LingrelUniversity of Cincinnati
July 1974mRNA synthesis reported to be activated by a specific nucleotide during the initial stage of transcription of the double-stranded RNA genome.FuruichiNational Institute of Genetics
31 Jan 1975Unique 'cap' structure discovered at the tip end of mRNAFuruichi, Miura, ShatkinNational Institute of Genetics
1976Unique 'cap' structure discovered at the tip end of mRNA Furuichi, Muthukrishnan, Tomasz, ShatkinRoche Institute of Molecular Biology
31 May 1976Jacques Monod diedMonodPasteur Institute
31 Aug 1978First proteins produced in mouse and human cells by delivering mRNA packaged in a liposome Dimitriadis, Ostro, Giacomoni, Lavelle, Paxton, DrayNational Institute for Medical Research, University of Illinois
1984First cationic (positively charged) lipid synthesised, opening up new possibilities to deliver drugs and gene therapyFelgnerSyntex Research
25 Sep 1984Large amounts of biological active mRNA produced in the laboratory using RNA-synthesis vaccine Krieg, MeltonHarvard University
1987mRNA encapsulated into liposome made with cationic lipids injected into mouse cells shown to produce proteinsMalone, Felgner, VernaSalk Institute for Biological Sciences, Syntex
1987Vical Corporation foundedFelgner, Vical
October 1987First time mRNA encapsulated in liposome delivered into living organism demonstrated to produce proteins MaloneSalk Institute for Biological Sciences
1 Jan 1990Experiment shows mRNA can be used to get cells to produce protein that inhibits blood clot formationKariko, BarnathanUniversity of Pennsylvania
23 Mar 1990Naked mRNA and naked DNA injected directly into the skeletal muscle of mice reported to produce proteinsWolff, Malone, Williams, Chong, Acsadi, JaniUniversity of Wisconsin, Salk Institute for Biological Sciences, Vical
21 Feb 1992Diabetes reported to be temporarily relieved by injecting mRNA into rat brainsJirikowski, Sanna, Maciejewski-Lenoir, BloomScripps Research Institute
July 1993First evidence that mRNA could provide a means for vaccinesMartinon, Krishnan, Lenzen, Magne, Gomard, GuilletINSERM
1 Apr 1995mRNA vaccine vector shown to stimulate immune response against human cancer antigenConry, LoBuglio, Wright, Sumerel, Pike, Johanning, Benjamin, Lu, CurielUniversity of Alabama at Birmingham
August 1996Dendritic cells modified with mRNA shown to elicit strong immune response against tumours in miceBoczkowski, Nair, Snyder, D, GilboaDuke University
1997Merix Bioscience founded as spin-out to develop mRNA for cancer vaccinesGilboaDuke University
January 2000Freshly synthesised naked RNA and protamine-protected RNA shown to be suitable tool for vaccinationHoerr, Obst, Ramemenseee, JungUniversity of Tübingen
2000CureVac, a spin out company, set up to develop mRNA for vaccinesIngmar HoerrCureVac
October 2000mRNA encoding for HIV reported to activate potent T cell immune response Drew Weissman, H Ni, D Scales, Dude, Capodici, McGibney, Abdool, SN Isaacs, Cannon, KarikoUniversity of Pennsylvania
March 2004mRNA reported to activate series of Toll-like receptors, signalling receptors of the innate immune systemKariko, Houping Ni, Capodici, Lamphier, Drew WeissmanUniversity of Pennsylvania
August 2005mRNA rendered invisible to immune system by replacing its nucleoside uridine with pseudouridineKariko, Buckstein, Houping Ni, Drew WeissmanUniversity of Pennsylvania
19 Oct 2005Lipid nanoparticle system published for delivering drugs and gene therapyMacLachlan, CullisProtiva Biotherapeutics, Inex Pharmaceuticals
2006Spin-out company, RNARx, founded to commercialise modified mRNA for anaemia treatmentKariko, Drew Weissman 
26 Mar 2006Monkey studies show lipid nanoparticle system successfully delivered RNA to silence disease causing genesZimmerman, ACH Lee, Akinc, Bramlage, Bumcrot, Fedorik, Harborth, James Heyes, Lloyd Jeffs, Matthias John, Adam Judge, Kieu Lam, Kevin McClintock, Nechev, Lorne Palmer, Racie, Ingo Rohl, Seiffert, Shannmugam, Sood, Soutschek, Toudjarska, Wheat, Yaworski, ZProtiva Biotherapeutics
15 Dec 2006Method published to produce mRNA with increased stability and translational efficiencyHoltkamp, Sebastian Kreiter, Abderraouf Selmi, Petra Simon, Koslowski, Christoph Huber, Tureci, SahinJohannes-Gutenberg University
2008Biopharmaceutical New Technologies (BioNTech) founded to develop mRNA as personalised cancer immunotherapiesSahin, Tureci, HuberBioNTech
November 2010Moderna Therapeutics founded to commercialise modified mRNA vaccines and therapeuticsRossi, Kernneth Chien, Robert LangerModerna
5 Nov 2010Modified mRNA reported to transform skin cells into pluripotent stem cellsLuigi Warren, Philip Manos, Ahfeldt, Ahfeldt, Yuin-Han Loh, Hu Li, Frank Lau, Wataru Ebina, Pankaj Mandal, Zachary Smith, Meissner, George Daley, Brack, James Collins, Chad Cowan, Schlaeger, RossiHarvard University
October 2013Modified mRNA shown to help improve heart function in miceLior Zangi, Kathy Lui, von Gise, Qing Ma, Ebina,Ptaszek, Spater, Huansheng Xu, Tabebordbar,Gorbatov, Sena, Nahrendorf, David Briscoe,Ronald Li, Amy Wagers, Rossi, William Pu, Kenneth ChienHarvard University, Massachusetts General Hospital, Children's Hospital Boston, Mount Sinai School of Medicine, Karolinska Institute
12 Jan 2018mRNA flagged up as promising new vaccine technology for combating infectious diseasesPardi, Michael Hogan, Frederick Porter, Drew WeismannUniversity of Pennsylvania
11 Dec 2020FDA approved first mRNA vaccine for COVID-19 for emergency useBioNTech, Pfizer
18 Dec 2020FDA approved second mRNA vaccine for COVID-19Moderna

7 Jun 1920

Jacques Monod was born in Nancy, France

1956

First observation of messenger RNA, or mRNA

13 May 1961

Experiment confirms existence of mRNA

14 Oct 1965

Nobel Prize awarded for the elucidation of the nature of mRNA

8 Oct 1969

mRNA isolated from a rabbit introduced into mouse lymphocytes shown to stimulate production of a protein

Jul 1974

mRNA synthesis reported to be activated by a specific nucleotide during the initial stage of transcription of the double-stranded RNA genome.

31 Jan 1975

Unique 'cap' structure discovered at the tip end of mRNA

1976

Unique 'cap' structure discovered at the tip end of mRNA

31 May 1976

Jacques Monod died

31 Aug 1978

First proteins produced in mouse and human cells by delivering mRNA packaged in a liposome

1984

First cationic (positively charged) lipid synthesised, opening up new possibilities to deliver drugs and gene therapy

25 Sep 1984

Large amounts of biological active mRNA produced in the laboratory using RNA-synthesis vaccine

1987

mRNA encapsulated into liposome made with cationic lipids injected into mouse cells shown to produce proteins

1987

Vical Corporation founded

Oct 1987

First time mRNA encapsulated in liposome delivered into living organism demonstrated to produce proteins

1 Jan 1990

Experiment shows mRNA can be used to get cells to produce protein that inhibits blood clot formation

23 Mar 1990

Naked mRNA and naked DNA injected directly into the skeletal muscle of mice reported to produce proteins

21 Feb 1992

Diabetes reported to be temporarily relieved by injecting mRNA into rat brains

Jul 1993

First evidence that mRNA could provide a means for vaccines

1 Apr 1995

mRNA vaccine vector shown to stimulate immune response against human cancer antigen

Aug 1996

Dendritic cells modified with mRNA shown to elicit strong immune response against tumours in mice

1997

Merix Bioscience founded as spin-out to develop mRNA for cancer vaccines

Jan 2000

Freshly synthesised naked RNA and protamine-protected RNA shown to be suitable tool for vaccination

2000

CureVac, a spin out company, set up to develop mRNA for vaccines

Oct 2000

mRNA encoding for HIV reported to activate potent T cell immune response

Mar 2004

mRNA reported to activate series of Toll-like receptors, signalling receptors of the innate immune system

Aug 2005

mRNA rendered invisible to immune system by replacing its nucleoside uridine with pseudouridine

19 Oct 2005

Lipid nanoparticle system published for delivering drugs and gene therapy

2006

Spin-out company, RNARx, founded to commercialise modified mRNA for anaemia treatment

26 Mar 2006

Monkey studies show lipid nanoparticle system successfully delivered RNA to silence disease causing genes

15 Dec 2006

Method published to produce mRNA with increased stability and translational efficiency

2008

Biopharmaceutical New Technologies (BioNTech) founded to develop mRNA as personalised cancer immunotherapies

Nov 2010

Moderna Therapeutics founded to commercialise modified mRNA vaccines and therapeutics

5 Nov 2010

Modified mRNA reported to transform skin cells into pluripotent stem cells

Oct 2013

Modified mRNA shown to help improve heart function in mice

12 Jan 2018

mRNA flagged up as promising new vaccine technology for combating infectious diseases

11 Dec 2020

FDA approved first mRNA vaccine for COVID-19 for emergency use

18 Dec 2020

FDA approved second mRNA vaccine for COVID-19

References

Alameh, MG, Tombacz, I, Bettini, E, et al (2021, in press) 'Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses', Immunity.Back

Albert, H (26 March 2021) 'Will COVID-19 mRNA advances help progress cancer vaccines to the clinic?', Clinical Omics.Back

Anderson, C (3 Aug 2021), 'Uğur Şahin and Özlem Türeci: Meet the scientist couple driving an mRNA vaccine revolution', Youtube video.Back

Anon (2020) 'Cover legend: Yasuhiro Furuichi, International Journal of Oncology, 56, 865.Back

Bacal, D (19 Dec 2014) 'Tekmira says chief technical officer resigns', Proactive.Back

Ball, P (18 Dec 2020) 'The lightning-fast quest for COVID vaccines — and what it means for other diseases', Nature, 589, 16-18.Back

Banerjee, AK (13 Nov 2012) 'Aaron J. Shatkin (1934–2012)', Proceedings of the National Academy of Sciences USA, 109/46, 18629-30.Back

Barbaro, M (10 July 2021) 'The unlikely pioneer behind mRNA vaccines', New York Times, The Daily Podcast, transcript.Back

Bender, E (18 Sept 2019) 'Accelerating flu protection', Nature, 573, S60-61.Back

Bendix, A (12 Dec 2020) 'BioNTech scientist Katalin Karikó risked her career to develop mRNA vaccines', Business Insider.Back

Borrell, B (26 Oct 2021) 'On the intellectual property battle behind the COVID-19 vaccine', Literary Hub.Back

Brenner, S, Jacob, F, Meselson, M (1961) 'An unstable intermediate carrying information from genes to ribosomes for protein synthesis', Nature, 190, 576–81.Back

Boczkowski, D, Nair, SK, Snyder, D, Gilboa, E (1 Aug 1996) 'Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo', The Journal of Experimental Medicine, 184/2, 465-72.Back

Cobb M (29 June 2015) 'Who discovered messenger RNA?', Current Biology, 25, R523–48.Back

Conry, RM, LoBuglio, AF, Wright, M et al (April 1995) 'Characterization of a messenger RNA polynucleotide vaccine vector', Cancer Research, 55/7, 1397-1400.Back

Corbett, KS, Edwards DK, Leist, SR et al (5 Aug 2020) 'SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness', Nature, 586, 561-71.Back

Cox, D (2 Dec 2020) 'How mRNA went from a scientific backwater to a pandemic crusher', Wired.Back

Crow, D (14 May 2021) 'How mRNA became a vaccine game-changer', Financial Times.Back

Cross, R (6 March 2021) 'Without these lipid shells, there would be no mRNA vaccines for COVID-19', Chemical & Engineering News, 99/8.Back

De George, M (20 April 2021) 'The vaccine trenches', The Penn Gazette.Back

de la Giroday, M (Aug 2021) 'Getting erased from the mRNA/COVID-19 story', FrogHeart.Back

Delaye, F (Sept 2021) 'Messenger RNA: The outsiders strike back', Explorations.Back

Dimitriadis, GJ (15 Feb 1978) 'Entrapment of ribonucleic acids in liposomes', FEBS Letters, 86/2.Back

Dimitriadis, GF (31 Aug 1978) 'Translation of rabbit globin mRN A introduced by liposomes into mouse lymphocytes', Nature, 274, 923-24.Back

Dolgin, E (June 2015) 'The billion dollar biotech', Nature, 522, 26-28.Back

Dolgin, E (16 Oct 2019) 'Unlocking the potential of vaccines built on messenger RNA', Nature, 574, S10-S12.Back

Dolgin , E (12 Jan 2021) 'How COVID unlocked the power of RNA vaccines', Nature,589, 189-91.Back

Dolgin, E (14 Sept 2021) 'The tangled history of mRNA vaccines', Nature, 597,318-24.Back

Editorial (2004) 'ORNL's Unsung Discovery', Oak Ridge National Laboratory Review, 37/3.Back

Favaro, A (27 Sept 2021) 'How the early work of a Canadian scientist and his team made the covid-19 vaccines possible', CTV News.Back

Felgner, PL (June 1997) 'Nonviral strategies for gene therapy', Scientific American, 276/6, 102-06.Back

Felgner, PL Philip Felgner interviewed by Mark Jones (22 July 1997), UCSD library.Back

Felgner, PL Felgner, Philip interviewed by Lara Marks (24 June 2021).Back

Felgner, PL (n.d.) 'Nucleic acid vaccine origin', unpublished paper shared by Philip Felgner with Lara Marks.Back

Felgner, PL, Gadek, TR, Holm, M, et al (Nov 1987) 'Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure', Proceedings of the National Academy of Sciences USA, 84, 7413-17.Back

Felgner, PL, Rhodes, G (24 Jan 1991) 'Gene therapeutics', Nature, 349, 351-52.Back

Felgner, PL, Wolff, J, Rhodes, G et al (1989), 'Lipid-mediated polynucleotide administration to deliver a biologically active peptide and to induce a cellular immune response', US Patent 7,250,404.Back

Frangou, C (18 Feb 2021) 'Researchers looking for mRNA were ridiculed by colleagues. Luckily, that didn't stop them', Macleans.Back

Furuichi, Y (1974) '"Methylation-coupled" transcription by virus-associated transcriptase of cytoplasmic polyhedrosis virus containing double-stranded RNA', Nucleic Acids Research, 1, 809-22.Back

Furuichi, Y (30 Oct 2021) 'Exciting memories in the discovery of mRNA cap'.Back

Furuichi, Y, emails to Lara Marks, 13 Dec 2021, 8 Jan 2022, unpublished.Back

Furuichi, F, Miura, K (31 Jan 1975) 'A blocked structure at the 5' terminus of mRNA from cytoplasmic polyhedrosis virus', Nature, 253/5490, 374.Back

Furuichi, Y, Muthukrishnan, S, Tomasz, J, Shatkin, AJ (1976)'Caps in eukaryotic mRNAs: mechanism of formation of reovirus mRNA 5'-terminal m7GpppGm-C', Progress in Nucleic Acid Research and Molecular Biology, 19, 3-20.Back

Fynan, EF, Lu, S, Robinson, HL (2018) 'One group's historical reflections on DNA vaccine development', Human Gene Therapy, 29/9, 966-70.Back

Garde, D, Saltzman, J (10 Nov 2020) 'The story of mRNA: How a once-dismissed idea became a leading technology in the Covid vaccine race', STAT News, Boston Globe.Back

GAVI, 'What are nucleic acid vaccines and how could they be turned against COVID-19?'.Back

Gelles, D (10 Nov 2020) 'The husband-and-wife team behind the leading vaccine to solve Covid-19', The New York Times.Back

Goel, RR, Painter, MM, Apostolidis, SA, et al (14 Oct 2021) 'mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern', Science, 374, 6572..Back

Gros, F, Hiatt, H, Gilbert, W, Kurland, et al (1961) 'Unstable ribonucleic acid revealed by pulse labelling of Escherichia coli', Nature, 190, 581–85.Back

Hanawa, T (4 Nov 2021) 'Decades ago, this Japanese discovered what would become essential to mRNA vaccines', Japan Forward.Back

Hancock, S (4 Oct 2021) 'Difficulties of stability and storage of mRNA COVID-19 vaccines', News Medical Life Sciences.Back

Hargittai, I, Hargittai, M (2021) 'Our science and the Covid-19 pandemic—Katalin Karikó's research idea and her perseverance', Structural Chemistry, 32, 1353-56.Back

Haussecker, D (26 July 2020), 'Strong presumption Moderna Covid vaccine infringes on Arbutus IP', The RNAi therapeutics Blog.Back

Heath, D, Garcia-Roberts, G (27 Jan 2021) 'Luck, foresight and science: How an unheralded team developed a COVID-19 vaccine in record time', USA Today.Back

Helton, C (2 June 2013) 'Does Moderna Therapeutics have the next big thing?', Boston Globe.Back

Hopkins Tanne, K (26 Jan 2021) 'Covid-19: Moderna plans booster doses to counter variants', British Medical Journal, 372.Back

Holtkamp, S, Kreiter, S, Seimi, A, et al (15 Dec 2006) 'Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells', Blood, 108/13, 4009-17.Back

Horejs, C (21 Sept 2021) 'From lipids to lipid nanoparticles to mRNA vaccines', Nature, 6, 1078-76.Back

Hornblower, B, Robb, B, Tzertzinis, G (n.d.) 'Minding your caps and tails', New England Biolabs.Back

Huang, S, Huang, J (Oct 2019) 'Cancer vaccines - a new promise?', Pharma Times.Back

Jacob, F, Monod, J (1961) 'Genetic regulatory mechanisms in the synthesis of proteins', Journal Molecular Biology, 3, 318–56.Back

Jeffs, LB, Palmer, LR, Ambegia, EG (March 2005) 'A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA', Pharmaceutical Research, 22/4, 362-72.Back

Jeng, P (30 Oct 2018) 'Expert Insights: Moderna Therapeutics barrels ahead towards historic IPO', Clinical Trials Arena.Back

Jirikowski, GF, Sanna, PP, Maciejewski-Lenoir, D, Bloom, FE (21 Feb 1992) 'Reversal of diabetes Insipidus in Brattleboro Rats: Intrahypothalamic Injection of Vasopressin mRNA', Science, 255, 996-98, DOI: 10.1126/science.1546298.Back

Johnson,CY (1 Oct 2021) 'A one-way ticket. A cash-stuffed teddy bear. A dream decades in the making', The Washington Post.Back

Karikó, K, email to L. Marks (8 Dec 2021), unpublished.Back

Kariko, K to Warren, L, letter (June 2021), Twitter.Back

Karikó, K, Keller, JM, Harris, VA et al (2001), 'In vivo protein expression from mRNA delivered into adult rat brain', Journal Neuroscience Methods, 105/1, 77-86.Back

Karikó, K, Kuo, A, Boyd D, et al (1993) 'Overexpression of urokinase receptor increases matrix invasion without altering cell migration in a human osteosarcoma cell line', Cancer Research, 53, 3109–17.Back

Karikó, K, Kuo, A, Barnthan ES, Langer DS (1998) 'Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA', Biochim et Biophys Acta, 1369, 320–34.Back

Karikó, K, Buckstein, M, Ni, H, Weissman, D (Aug 2005) 'Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA', Immunity, 23/2, 165-75.Back

Karikó, K, Kuo, A, Barnathan, ES (1999) 'Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA', Gene Therapy, 6, 1092-1100.Back

Karikó, K, Li, SW, Sobol, RW et al (1987) 'Phosphorothioate analogs of 2–5A: Elucidation of the stereochemical course of the enzymes of the 2–5A synthetase/RNase L system', Nucleosides and Nucleotides, 10.1080/07328318708056190, 6, 1-2, 173-84.Back

Karikó, K, Ludwig, J (30 April 1985) 'n-Decyl-NHpppA2′p5′A2′p5′A a phosphatase-resistant, active pppA2′p5′A2′p5′A analog', Biochemical and Biophysical Research Communications, 128/2, 695-98.Back

Karikó, K, Megyeri, K, Xiao, Q, Barnathan, ES (1994) 'Lipofectin-aided cell delivery of ribozyme targeted to human urokinase receptor mRNA'm FEBS Letters, 352, 41–44.Back

Karikó, K, Muramatsu, H, Keller, JM, Weissman (May 2012) 'Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin', 20/5, 948–53.Back

Karikó, K, Muramatsu, H, Welsh, F (Nov 2008) 'Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability', Molecular Therapy, 16/11, 1833-40.Back

Karikó, K, Ni, H, Capodici, J, et al (2004) 'mRNA is an endogenous ligand for Toll-like receptor 3', Journal Biological Chemistry, 279, 12542–50.Back

Karikó, K, Reichenbach, NL, Suhadolnik, RJ et al (1987) 'Cordycepin analogs of 2–5 a as activators of RNase L: Study of the structural requirements for RNase L activation, Nucleosides and Nucleotides, 6, 1-2, 497-500, 10.1080/07328318708056268.Back

Keener, AB (Sept 2018) 'Just the messenger', Nature, 24, 1294-1300.Back

Kerr, IM, Brown, RE (Jan 1978) 'pppA2'p5'A2'p5'A: An inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells', Proceedings of the National Academy of Sciences USA, 75/1, 256-60.Back

Koester, V (4 May 2021) 'Interview with Ingmar Hoerr, pioneer of mRNA technology', Chemistry Views.Back

Kolata, G (8 April 2021) 'Kati Kariko helped shield the world from the coronavirus', The New York TimesBack

Kollewe, J (21 Nov 2020), 'Covid vaccine technology pioneer: "I never doubted it would work"', The Guardian.Back

Krieg, PA, Melton, DA (1984) 'Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs', Nucleic Acids Research, 12/18, 7057-70.Back

Kulkami, JA, Cullis, PR, van der Meel, R (2018) 'Lipid nanoparticles enabling gene therapies: From concepts to clinical utility', Nucleic Acid Therapeutics, 28/3, 147-57.Back

Lewis, L (17 Dec 2010) 'The first COVID-19 vaccines: What's mRNA got to do with it?', PLOS Blogs.Back

Liu, MA (June 2019) 'A comparison of plasmid DNA and mRNA as vaccine technologies', Vaccines (Basel), 7/2, 37.Back

Lo, C (31 March 2021) 'MRNA vaccines: The post-pandemic outlook after a breakthrough year', Pharmaceutical Technology.Back

Lockard, RE, Lingrel, JB (8 Oct 1969) 'The synthesis of mouse hemoglobin beta-chains in a rabbit reticulocyte cell-free system programmed with mouse reticulocyte 9S RNA', Biochemical and Biophysical Research Communications, 37/2, 204-12, doi: 10.1016/0006-291x(69)90720-7.Back

Lutz, J, Lazzaro, S, Habbeddine, M, et al (2017) 'Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines', Nature/ MPJ Vaccines, 29.Back

MacLachlan, I emails to L Marks 10 Dec 2021, 31 Dec 2021, unpublished.Back

Malone, RW (Fall 1989) 'mRNA Transfection of cultured eukaryotic cells and embryos using cationic liposomes', Focus, 11/4.Back

Malone, RW, Felgner, PL, Verma, IM (1 Aug 1989) 'Cationic liposome-mediated RNA transfection', Proceedings of the National Academy of Sciences USA, 86/16, 6077-6081.Back

Marte, B (Nov 2020) 'A dendritic cell cancer vaccine', Nature Milestones.Back

Martinon, F, Krishnan, S, Lenzen, G, et al (July 1993) 'Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA', European Journal of Immunology, 23, 1719-22.Back

Maruggi, G, Chiarot, E, Giovani C, et al (5 Jan 2017) 'Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens', Vaccine, 35/2, 361-68.Back

Mascola, JR, Fauci, AS (Feb 2020) 'Novel vaccine technologies for the 21st century', Nature Reviews, 20, 87-88.Back

McCarthy, M (2 Nov 1996) 'DNA vaccination: a direct line to the immune system', The Lancet, 348, 1232.Back

McNamara, MA Nair, SK, Holl, EK (2015) 'RNA-based vaccines in cancer immunotherapy', Journal of Immunology Research, ID 794528.Back

Meyer, M, Huang, E, Yuchakov, O, et al (1 Feb 2018) 'Modified mRNA-based vaccines elicit robust immune responses and protect guinea pigs from ebola virus disease', The Journal of Infectious Diseases, 217/3, 451–55.Back

Milao, L, Zhang, Z, Huang, L (25 Feb 2021) 'mRNA vaccine for cancer immunotherapy', Molecular Cancer, 20/1.Back

Miller, J, Cookson, C (16 Defc 2020) 'FT People of the Year: BioNTech's Ugur Şahin and Özlem Türeci', Financial Times.Back

Nellson, S (12 Dec 2020) 'The co-founder of BioNTech designed the coronavirus vaccine it made with Pfizer in just a few hours over a single day', Business Insider.Back

Orton, T (13 May 2021) 'The history behind the B.C. biotech that helped make the Pfizer vaccine work', Business in Vancouver.Back

Ostro, MJ, Giacomoni, D, Lavelle, et al (31 Aug 1978) 'Evidence for translation of rabbit globin mRNA after Iiposome-mediated insertion into a human cell line', Nature, 274, 921-23.Back

Pardi, N, Hogan, MJ, Porter, FW, Weissman, D (2 Jan 2018) 'mRNA vaccines — a new era in vaccinology', Nature, 17, 261-79.Back

Parikh, S (8 April 2021) 'COVID-19: A draft of history, a template for the future', Science.Back

Pietzsch, J (21 Sept 2021) 'The Paul Ehrlich and Ludwig Darmstaedter Prize 2022 will be awarded to Katalin Karikó, Özlem Türeci and Uğur Şahin', IDW.Back

Roach Monroe, L (23 March 1990) 'Biotech Firm Takes the Simple Route to Gene Therapy Success', Los Angeles Times.Back

Rossi, D (1 Oct 2014) 'Interview with Dr. Derrick Rossi by Maria Zagorulya', Journal of Young Investigation.Back

Rothwell, Figg, Ernst et al (10 May 2021) 'The mRNA patent and competitive landscape: Pioneers, litigation outlook and big pharma's next moves (Part III)', JDSUPRA.Back

Rowland, C (18 Feb 2021)'Why grandparents can't find vaccines: Scarcity of niche biotech ingredients', The Washington Post,Back

Şahin, U, Karikó, K, Türeci, O (Oct 2014) 'mRNA-based therapeutics - developing a new class of drugs', Nature Reviews: Drug Discovery, 13, 759-80.Back

Schlake, T, Thess, A, Fotin-Mlecek, M, Kallen, K-J (2012) 'Developing mRNA-vaccine technologies', RNA Biology, 9/1, 1319–30.Back

Semeniuk, I (Nov 2021) 'UBC professor Pieter Cullis awarded for role in COVID-19 vaccines', The Globe and Mail.Back

Servick, K (1 Feb 2017) 'This mysterious $2 billion biotech is revealing the secrets behind its new drugs and vaccines', Science.Back

Sforza, T (2 July 2021) 'UCI professor wins international prize for work that led to COVID-19 vaccines', The Orange County Register.Back

Somlyai, G, Kondorosi, E, Karikó, K, Duda, EG (1985), 'Liposome mediated DNA-transfer into mammalian cells', Acta Biochimica et Biophysica; Academiae Scientiarum Hungaricae, 20/3-4, 203-11.Back

Szte Info (9 April 2020) 'Katalin Karikó, an alumna of the University of Szeged, is the founder of the most promising vaccine development against the coronavirus'.Back

Takahashi, K, Yamanaka, S (Aug 2006) 'Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors', Cell, 126/4, 663-73.Back

Tam, YK, Madden, TD, Hope MJ (13 May 2016) 'Pieter Cullis' quest for a lipid-based, fusogenic delivery system for nucleic acid therapeutics: success with siRNA so what about mRNA?', Journal of Drug Targeting, 24/9.Back

Tong, A (11 Nov 2020) 'A cancer vaccine that might work? Moderna posts 'promising' early snapshot for Merck-partnered program and raids Bristol Myers for a new cancer R&D chief', Endpoint NewsBack

Trouillard, S (18 Dec 2020) 'Katalin Kariko, the scientist behind the Pfizer Covid-19 vaccine', France 24.Back

UK HSA (21 Dec 2021) 'COVID-19 vaccination programme Information for healthcare practitioners'.Back

Ulmer, JB, Donnelly, JJ, Parker, SE, et al (19 March 1993) 'Heterologous protection against influenza by injection of DNA encoding a viral protein', Science, 259/5102, 1745-9. doi: 10.1126/science.8456302.Back

Unger Baillie, K (15 Nov 2020) 'Five things to know about the promising COVID-19 vaccine news', Penn Today.Back

Warren, L, Manos, PD, Ahfeldt, T et al (5 Nov 2010) 'Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA', Cell Stem Cell, 7/5, 618-30.Back

Vardi, N (17 Aug 2021) 'Covid's forgotten hero: The untold story of the scientist whose breakthrough made the vaccines possible', Forbes.Back

Verbeke, R, Lentacker, I, De Smedt, SC, Dewitte, H (Oct 2019) 'Three decades of messenger RNA vaccine development', Nano Today, 28, 1q00766.Back

Verbeke, R, Lentacker, I, De Smedt, SC, Dewitte, H (5 Feb 2021) 'The dawn of mRNA vaccines: The COVID-19 case', Journal of Controlled Release, 333, 511-20.Back

Volkin, E, Astrachan L (April 1956), 'Phosphorus incorporation in Escherichia coli ribonucleic acid after infection with bacteriophage T2', Virology, 2/2, 149-61.Back

Volkin, E (May 1995, 'What was the message', TIBS, 20, 206-09.Back

Wang F, Travis, Z, Watts, JK (July 2020) 'RNA therapeutics on the rise', Nature Reviews: Drug Discovery, 19.Back

Warren, L (2007) 'Single-cell gene expression analysis by quantitative RT-PCR', PhD, California Institute of Technology.Back

Warren, L, emails to L Marks 11 Dec 2021, 20 Dec 2021, unpublished.Back

Weissman, D, Rubbert, A, Combadiere, C, Murphy, PM, Fauci, AS (1997) 'Dendritic cells express and use multiple HIV coreceptors', in Ricciardi-Castagnoli P (eds) Dendritic Cells in Fundamental and Clinical Immunology. Advances in Experimental Medicine and Biology, vol 417.Back

Weissman, D, Ni, H, Scales, D et al (2000) 'HIV Gag mRNA Transfection of Dendritic Cells (DC) delivers encoded antigen to MHC class I and II molecules, causes DC maturation, and induces a potent human in vitro primary immune response', The Journal of Immunology, 165, 4710-17.Back

Williams, BRG, Kerr, I, Gilbert, CS, White, CN, Ball, AL (10 July 1978) 'Synthesis and breakdown of pppA2'pSA2'pSA and transient inhibition of protein synthesis in extracts from interferon-treated and control cells', European Journal of Biochemistry, 92, 455-62.Back

Whipple, T (8 Feb 2021) 'More medical breakthroughs on the way thanks to BioNTech coronavirus vaccine', The Times.Back

Wolff, J, Malone, RW, Williams, P et al (23 March 1990) 'Direct gene transfer into mouse muscle in vivo', Science, 247/4949. Back

Wollaeger, T (2 May 1997) Wollaeger Tim interviewed by Mark Jones, transcript, UCSD library.Back

Xie, W, Chen, B, Wong, J (Oct 2021) 'Evolution of the market for mRNA technology', Nature Reviews: Drug Discovery, 735-36.Back

Xiong, F, Mi, Z, Gu, N (2011) 'Cationic liposomes as gene delivery system: transfection efficiency and new application', Phamazie, 66, 158-64.Back

Zangi, L, Lui, KO, von Gise, A, et al (Oct 2013) 'Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction', Nature Biotechnology, 31/10, 898-907.Back

Zhang, S (14 April 2021) 'The mRNA vaccines are looking better and better', The Atlantic.Back

Zeng, C, Zhang, C, Walker, PG, Dong, Y (2 June 2020) 'Formulation and delivery technologies for mRNA vaccines', Current Topics in Microbiology and Immunology.Back

Zimmerman, TS, Lee, ACH, Akinc, A et al (26 March 2006) 'RNAi-mediated gene silencing in non-human primates', Nature, 441, 111-14.Back

Zuckerman, G (2021) A Shot to Save the World: The remarkable race and ground-breaking science behind the Covid-19 vaccines.Back

Respond to or comment on this page on our feeds on Facebook, Instagram or Twitter.