The Remarkable, Unconventional Career of mRNA Researcher Katalin Karikó, the 2023 Nobel Prize Co-Winner in Physiology or Medicine
This month, the Nobel committee in Sweden awarded the 2023 Nobel Prize in Physiology or Medicine to Katalin Karikó and her colleague Drew Weissman in recognition of their long-standing contributions toward the development of mRNA as a useful tool for clinical therapy and its application toward the successful creation of the Pfizer/BioNTech coronavirus vaccine in 2021.
As we’ll see, not only was this a major achievement, it also represented a personal triumph for Karikó, whose groundbreaking work at the University of Pennsylvania was, to put it delicately, not well appreciated at the time.
During her years at Penn, Karikó never received an R01 grant to fund her research; instead, she was eventually demoted from the tenured professor track and later forced to retire.
(Ironically, Penn subsequently made millions of dollars from the patents it filed based on Karikó and Weissman’s mRNA research.)
Despite these setbacks, Karikó was able to make many significant advancements in mRNA research at Penn (along with her colleague and co-Nobel Prize winner David Weissman) – ultimately finding recognition for her work as Senior Vice President at BioNTech in Germany, which developed the Covid-19 vaccine in partnership with Pfizer.
Growing up in rural Hungary, Katalin Karikó Wanted to Train as a Scientist from a Young Age
Speaking at a 2022 conference in Canada, where she was a recipient of the Canada Gairdner International Award, Karikó recounted her childhood dream of becoming a scientist:
“I grew up the daughter of a butcher and a bookkeeper in a small town; 10,000 people lived there. And I learned from my parents that hard work is part of life. And I learned how to make sausage.
How did I go from a simple life of a singular room with no running water, no television set, no refrigerator, and (now) I’m here on the stage, accepting the Gardner International Award?
It certainly was not my intention. I was just a curious girl who watched with fascination all of the plants and animals in our yard and wanted to learn more about the internal mechanism of all of these living things.
I didn’t know a single scientist, but I was 16 years old, and I wanted and decided that I would be one.”
Karikó kept the promise she made to herself as a little girl and received her Ph.D. in biochemistry from the University of Szeged, Hungary, in 1982. Unfortunately, the biochemistry lab where she was working lost the funding to pay for her postdoc studies (which will become a recurring theme for Karikó).
So in 1985, she left communist Hungary, accompanied by her husband and daughter, along with a little over $1,000 in British pounds smuggled in the inseam of her daughter’s teddy bear, to become a postdoc at Temple University in Philadelphia.
The Past, Present, and Future of mRNA Research
What was known about RNA when Karikó arrived in the US in 1985?
Speaking at the annual AAAS conference in 2022, Karikó recounted the early breakthroughs in ribonucleic acid (RNA) research – starting with its discovery in 1961 (Brenner et al., Gros et al.), which uncovered the process of how DNA (contained in the cell nucleus) can transcribe its genetic information into messenger RNA (mRNA), which then allows it to be translated into proteins.
Katalin Karikó presents the past, present, and future of mRNA research to the 2022 AAAS Conference attendees.
By the late sixties, researchers were experimenting with mRNA isolates, injecting them into cell-free rabbit reticulocyte systems (Lockard and Lingrel, 1969), and living cells (frog oocytes) for the first time (Gurdon, et al., Lane, et al., 1971.)
According to Karikó, 1975 was the “year of the cap,” when multiple research groups around the world discovered the details of the mRNA cap at the 5 prime terminus, which one paper called ‘bizarre.’ (Both et al., Adams and Corey, Muthukrishnan, Martin et al., 1975, and Furuichi et al., 1976)
(Karikó noted that this style of mRNA terminus cap was most recently used in Moderna’s version of the Covid-19 vaccine.)
RNA Experiments that Inspired a Research Career
1978 was a pivotal year for Karikó.
This was the year that betaglobin mRNA (extracted from rodent reticulocytes) was successfully introduced into living mammalian cells (both rabbit and human cell lines) by first wrapping it in a liposome (e.g. a lipid droplet of fat, discovered by Bangham in the 1960s), which allowed the injected mRNA to diffuse into the primary cells and produce detectable amounts of betaglobin proteins.
Karikó was a biology student in Hungary when she read the two papers publishing these results (Dimitriadis and Ostro et al, 1978), and from this, she understood (as did many others in the field) that it might be possible one day to use RNA as a clinical therapy delivery mechanism.
“It’s important for students to start with something — for the rest of my life, I was remembering this experiment and thinking about it.”
(Karikó also pointed out that it may surprise a lot of people that the first RNA delivery in mammalian cells dates back to 1978 – and not during the recent lead-up to the development of the mRNA Covid-19 vaccines as many non-scientists assume.)
Working in the lipid laboratory at the Biological Research Center, Hungarian Academy of Sciences in Szeged, Hungary, Karikó performed experiments with encapsulating DNA (not RNA) in a liposome and transferring it into mammalian cells (Somlyai, Karikó, et al., 1984).
This was Karikó’s first experiment, where she worked under the guidance of mentors, a very important step in her career.
Later, she learned how to synthesize the so-called “anti-viral molecule” – 2’ 5’ linked oligoadenylate – recently identified by her colleague Ian Kerr in London (Kerr et al. 1978) that was found to be responsible for the interferon-induced anti-viral effect.
Karikó was experimenting with this 2’ 5’ linked oligoadenylate, hoping to create a workable anti-viral therapy, but (as we mentioned earlier) funding at her laboratory was cut at this critical juncture, so she made plans to move to the US to continue her research. (Karikó returned to researching this molecule in the mid-1980s, hoping to create an anti-viral clinical therapy for HIV.)
Harvard Researchers Synthesize mRNA in a Test Tube
While Karikó was working in the lab in Hungary, researchers Douglas Melton and Paul Krieg at Harvard made a critical discovery: they were able to synthesize mRNA in vitro (e.g. in a test tube) using bacteriophage RNA polymerase. (Krieg, Melton, et al.). Following this, they were able to “cap” the synthetic mRNA (using the capping enzyme from Bernhardt Moss’ lab) and inject it into frog oocytes to create human interferon (Krieg, Melton, et al., 1984).
In other words, this was the first time it was demonstrated that mRNA made by design could be injected into cells to code for a general function protein.
Karikó’s mRNA Research at Temple University and the University of Pennsylvania
At Temple University, Karikó joined the Nucleoside Laboratory, where they used mismatched double-stranded RNA to try to induce the patient’s interferon system. (Carter, et al., 1987)
Disappointingly, these efforts were not as effective for HIV patients as they hoped due to their compromised interferon mechanism.
In 1989, Karikó joined the University of Pennsylvania, becoming an adjunct professor at Penn’s Perelman School of Medicine, working with cardiologist Elliot Barnathan. Here, she submitted her initial grant applications to study mRNA gene therapy applications.
At this time, there were a lot of developments in the field of mRNA research, starting with in vivo delivery of mRNA into living cells.
John Wolff’s lab team had successfully injected luciferase mRNA directly into living mouse muscle – a process now known as IVT, short for In Vitro transcription/translation. (Wolff, et al., 1990)
Throughout the mid-nineties, researchers jumped onto the mRNA bandwagon, performing numerous clinical therapy IVT experiments, including:
- Reversing diabetes in Brattleboro rats (Jirikowsi et al., 1992)
- Creating an influenza vaccine using nanoparticle (NP) mRNA (Martinon, et al., 1993)
- Creating an influenza vaccine using self-replicative recombinant SFV RNA (Zhou, et al., 1994)
- Creating an mRNA cancer vaccine in mice (Conry, et al., 1995)
- Creating an mRNA cancer vaccine in human dendritic cells (Boczhowski, et al. 1996)
Yet, despite all these efforts, these mRNA experiments conducted during the early to mid-1990s produced disappointing results.
Karikó explains that, broadly speaking, there were three main roadblocks at the time.
- The first was that mRNA was very unstable; it degraded too quickly.
- The second problem was the amount of output, e.g. the translated protein, was minuscule, too small to scale up for use in widespread clinical therapy.
- And the third problem was that mRNA therapy was proving to be immunogenic in human cells. In other words, it created an unwanted immune system response, a surprising result because immunogenic responses had not been seen in animal studies before – not even monkeys, which normally have a similar immune response as humans.
After a Boom in Scientific mRNA Research, Now Came the Bust.
Without a major breakthrough in mRNA research, Karikó saw the academic interest dry up. Researchers who had quickly jumped into mRNA research were now leaving the field in search of the “next big thing.”
This shift came at a bad time in Karikó’s career at Penn.
Elliot Barnathan left the Cardiology department (he now works at Johnson & Johnson), leaving her without a mentor and research colleague.
(Their paper on the overexpression of urokinase receptor cells in mammalian cells following the administration of IVT mRNA was published in Nature in 1999.)
Even worse than Barnathan’s departure was the funding issue for Karikó’s research.
Karikó had not been able to secure an all-important R01 grant to fund her research in mRNA-based gene therapy, and, as a result, the administrators at Penn demoted her from the professor tenure track in 1996.
Despite this, Karikó took this major setback in her stride, vowing to be more determined than ever to pursue her mRNA clinical therapy research goals.
She landed a research position as an adjunct professor in the Neurology department at Penn’s Perelman School of Medicine, a position she was to hold for 17 years. Here, she worked with neurologist David Langer, seeking to use a nitric oxide encoding RNA to treat a patient with a sub-arachnoid brain hemorrhage as well as experiment with delivering antioxidant enzymes to stroke patients. A side benefit of these experiments was they significantly improved the protein yields from the mRNA therapy, helping make progress on one of the three major mRNA issues mentioned above. (Karikó, et al., 1998 and Karikó, et al., 2001)
A Chance Encounter with Drew Weissman Leads to a Successful Collaboration
David Langer left the lab, leaving Karikó once again without a collaborator.
Subsequently, however, a very fortuitous and serendipitous meeting took place in the basement of the lab building where she met her future collaborator (and co-Nobel Prize winner) Drew Weissman at the copier machine, where she was making copies of the latest research papers.
As Karikó notes, had it been just a few years later (when research papers became widely available online), she might not have run into Weissman at all.
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As Karikó recalls, “It turned out that he (Weissman) wanted to make an HIV vaccine – and I was working with RNA – and so we thought that we can try to work together and see if the mRNA would be a good vaccine.”
They published a paper (Weissman, et al. 2000) that demonstrated they could deliver mRNA to the cells, creating significant amounts of antigen proteins. This also created a very strong – and unwanted – immune response that negated the benefits of the intended therapy.
“I was not happy that this RNA I am making was inflammatory!” Karikó explained. “So we decided to that we will figure out why it was happening… (but) how could the RNA which I am making be inflammatory, given that it is the same RNA as inside of our cells?”
Thus began their crucial experimental research that eventually led to a Nobel Prize and lucrative patents for the University of Pennsylvania.
Weissman and Karikó decided to extract “natural” RNA from living cells and put them into different types of immune cells to see if it created an inflammatory reaction or not.
By 2005, they discovered that some natural RNA created a very strong (e.g. unwanted) immune response while others did not. Curiously, the transfer RNA (tRNA) by themselves was not inflammatory.
This unexpected tRNA result led to an insight: because the tRNA has a lot of nucleoside modification, perhaps they could use nucleoside modification to suppress the immunogenicity of mRNA.
Then the question became, which type of nucleoside modification should they choose from?
As it turns out, there are 108 naturally occurring modified nucleosides found in human RNA, created when the body synthesizes different enzymes to alter the RNA.
They decided their next move was to replicate naturally occurring nucleoside modifications to the RNA in vitro (e.g. in test tubes) and then see if they created a strong immune response (or not) when injected into living cells. To do this, they used different commercially available triphosphates to modify the nucleosides.
Here, Karikó recalled a bad outcome of a clinical trial from the early 1990s that resulted in the deaths of 5 out of 15 volunteer patients who were given fluorinated uridine containing a Fialuridine nucleoside. (McKenzie, et al., 1995)
Thus, Karikó decided to avoid using any unnatural nucleoside triphosphates (such as Fialuridine) to avoid a similar fate.
Experiments injecting mRNA with different kinds of nucleoside modifications into isolated primary identity cells showed that while many were immunogenic, those made with modified uridine were not. Among these, pseudo-uridine also had another surprising (and very welcome) benefit: not only did it not create an immune response, it also translated 10 times more protein output compared to their previous yields. (Karikó, et al., 2005)
Solving the immunogenicity problem and increasing the protein yields by an order of magnitude were huge breakthroughs that went a long way towards solving the three main roadblocks of using mRNA in clinical therapy mentioned above.
The next set of experiments by Karikó and Weissman helped solve a contamination problem they had observed in their output. Over two years, they devised a high-performance liquid chromatography (HPLC) purification process to produce very high-quality RNA output. (Karikó, et al., 2011)
The next challenge was to see if they could use the purified pseudouridine-mRNA to perform protein translation in vivo, e.g. in a living animal; in this case, it was laboratory mice injected with mRNA to create erythropoietin (EPO), a hormone that protects kidney cells.
Here, Weissman and Karikó thought they had come up against one of the three roadblocks (see above) in using mRNA for clinical therapy, e.g. that it degrades very quickly. (At that time, they didn’t have access to lipid nanoparticles to protect the mRNA, having to rely on commercially available TransIT-mRNA instead.)
At first, they didn’t see the expected amount of EPO proteins in the mouse blood. But they redid the experiment with mRNA coded for erythropoietin (which is responsible for red blood cell differentiation), and within 30 minutes, they could measure the epoch present in the bloodstream.
Karikó noted that this meant the RNA translation into proteins happens very quickly; she estimated the speed of translation at 5 amino acids per second. (Karikó, et al., 2012)
The Green, Green Grass of (a new) Home Beckons: Karikó Joins the BioNTech Startup in Mainz, Germany
In 2012, the University of Pennsylvania asked Karikó to “retire.”
Essentially, she was fired.
What to do now?
To pursue her goal of making messenger RNA coding for therapeutic proteins, Karikó decided (along with her Penn colleague Hiromi Muramatsu) to take a position at the recently established pharma startup BioNTech in Mainz, Germany, co-founded that year by the Turkish husband and wife research team of Uğur Şahin and Özlem Türeci.
Despite the challenge of leaving her husband and daughter behind in the suburbs of Philadelphia, Karikó was excited about the new position because BioNTech already had an mRNA-based cancer vaccine in clinical trials.
Karikó recalls, “I thought that if I wanted to see modified RNA used as a therapeutic in my lifetime, I needed to go to Germany and try to do it there.”
Her first research contribution at BioNTech was a project to create bi-specific antibodies that could eliminate large tumors in mice. Here, mRNA was an elegant solution. Rather than having to re-inject the animal (or eventually a human) with antibodies again and again, the RNA generates antibodies internally, reducing the need for external injections or the use of an injection pump. (Stadler, et al., 2017)
Karikó also worked with Muramatsu to optimize the overall mRNA IVT process, such as improving the purification to increase the protein output and the mRNA capping process.
The appearance of the ZIKA virus in the late 2010s became an important proving ground for BioNTech research. Norbert Pardi was able to take the pseudo-uridine mRNA particles (encoded to translate ZIKA virus-specific proteins) and encapsulate them into a lipid nanoparticle droplet.
Just one injection proved very effective in creating an immune response that protected mice (and later monkeys) from the challenge of ZIKA virus exposure. (Pardi, et al., 2017)
This was a very big deal.
Karikó said it was regarded by many as the most important paper published in the 10 years – and it subsequently helped pave the way for the future Covid-19 vaccine.
Pardi and the team at BioNTech were also working with Pfizer on a nucleoside-modified RNA-based clinical therapy for influenza (in 2018) as well as a clinical therapy for HIV.
Meanwhile, researchers at Harvard had investigated using nucleoside-modified RNA-based therapy coded to create VEGF-A proteins to help patients with heart failure recover. (Zangi, et al., 2013) The first patient was injected during a clinical trial in 2018.
AstraZeneca and Moderna followed with their own mRNA research, seeking to heal necrotic wounds of diabetic patients and treat heart patients.
Clinical Development of the BNT162b2 Covid-19 mRNA Vaccine
Karikó reminds us that all of this research noted above was well underway before the Covid-19 virus was first identified in China.
Once the Chinese scientists published the genetic sequence of the Covid virus, Karikó and the team at BioNTech was very well prepared to create an effective mRNA-based vaccine.
All their previous research and lessons learned came into play:
- Encapsulating the fragile mRNA in a lipid nanoparticle helped protect it.
- Using pseudo-urine nucleoside mRNA prevented unwanted inflammation – as well as increased protein yields.
- Advances in production, including the high-performance liquid chromatography (HPLC) purification process, to deliver a pure product.
The vaccine went from the design stage to delivery in a year, a record time, thanks to the no-expenses-spared approach of conducting so many processes in parallel, including vaccine clinical testing, manufacturing, and supply chain management, as well as the all-important “cold stream” that kept the lipid nanoparticles at very low temperatures to prevent degradation.
The Future of mRNA as a Clinical Therapy Platform
While the Covid-19 vaccine was in development, Karikó continued her research. She was the co-author of a paper investigating using intratumor injection of mRNA for cancer treatment in mice. This therapy is a little different in that the mRNA is not coding for a cancer antigen; instead, it induces the creation of a mixture of cytokines that attack the tumor. (Hotz, et al., 2021) This research is now in clinical trials.
So how does Karikó view the future?
In her address to the AAAS in 2022, she said she believes that mRNA has created a new class of medicine.
She notes that there are numerous preclinical studies using mRNA, including:
- Vaccines against malaria, HSV, and ZIKA
- Tolerization therapies for autoimmune diseases, such as Multiple sclerosis
- Genome editing of genetic diseases, including sickle cell anemia, HIV
- Treatments for edema, heart fibrosis, and bone healing
Some laboratory research has already reached the clinical trials stage, including:
- Acute diseases, such as heart failure and wound healing
- Cancer vaccines
- Infectious disease therapies for HIV, vaccines for flu
- Genetic disease therapies using CAS-9 mRNA for genome editing
Career Advice from Katalin Karikó: Never Give Up!
In closing, you might be curious about how Karikó feels about the twists and turns in her career.
In her typical blunt style, she offers her assessment and advice for her fellow scientific researchers:
“In the academic setting, I was not successful. I was demoted. I never received a single RO1 grant. And in general, I was not popular with those who followed conventional science.
But I have a message.
It doesn’t matter. The circumstances, the skepticism around you. What matters is your conviction, conviction. How hard you work, pursue your passion. And yourself that you believe that you can achieve those goals.
As scientists, we work at the bench in the laboratory, performing experiments day after day. And we hope that one day, maybe in our lifetime, we can witness advancement.”
With that important advice, we offer our sincere congratulations to Katalin Karikó and her colleague Drew Weissman for winning the 2023 Nobel Prize in Medicine or Physiology.
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