Scientist Stories: Katalin Karikó, Developing mRNA for Therapy

Hello again. I'm Susan Amara, the president of AAAS and the chair of this year's annual meeting. I'd like to welcome you all to today's plenary session featuring Dr. Catalin Carrico. We're thrilled that Dr. Carrico can be with us to discuss her remarkable career and one of the most important scientific advances of our generation, one that is truly leading us to the other side of a global pandemic. Today's session is brought to us by the generous support of Johnson Johnson, a member of the AAAS Corporate Circle and a longtime supporter of the AAAS Annual Meeting. J&J's support of some of AAAS's flagship programs, including our Mass Media Fellowships, Awards, now in their 45th year, and tomorrow's Minority and Women in Science Award Ceremony have provided really critical funding to our programs that powerfully advance our mission. Johnson Johnson is represented by a longtime friend to AAAS, Seema Kumar, Global Head of the Office of Innovation, Global Health, and Scientific Engagement. Following Dr. Keriko's talk, Seema will lead us in a fireside chat. It is now my pleasure to hand things over to Seema.

Thank you. Thank you, Susan. And hello, everyone. It is such a great pleasure to be here at this AAAS meeting. I wish we could have all met, you know, in person because I always loved, enjoyed so much being in person at AAAS and doing all of the networking. Unfortunately, we have to do it virtually, but thanks to technology, we can. And so I want to, first of all, thank you for joining us virtually. I hope everybody's staying safe and well. Well, this plenary is going to be inspiring and energizing, to say the least. And it's also going to be a reminder that science is 1% inspiration and 99% perspiration. Perseverance is sometimes half the battle of science. At the end, it's always worth it, as you will hear from Dr. Kathleen Carrico. I'm so honored to be here speaking with and introducing Kathleen Carrico, Dr. Kathleen Carrico, who I met actually September of last year when we presented her, as well as her colleague, Dr. Drew Weissman, with the Johnson Johnson Dr. Polly Hansen Award for Biomedical Research. The two of them were honored for their groundbreaking work on mRNA research that enabled the development of several COVID-19 vaccines. And of course, it also holds tremendous promise for future applications in both vaccines as well as therapeutics. The work of Dr. Karikó began several decades ago, and despite her belief in the power of mRNA, her research was not fully supported or funded. And flash forward to 2021 with, you know, more than 4.79 billion people across the globe having received at least one dose of the COVID-19 mRNA vaccine, the world could not be more grateful for her perseverance and for her belief in continuing to stay the course on mRNA research. We'll hear a little bit more about her story during our fireside chat, but in the meantime, I will first turn it over to Dr. Carrico for her presentation.

Catlin, can I turn it over to you? Yes, thank you. Thank you. Thank you, Sima. So I am very delighted to be here. Actually, I am in Philadelphia, and I wish that all of the listeners and watchers of this program would be here. But today I will present, and if I can have my slides here on the screen. Then I can tell you about the development of mRNA for therapy. Do you see it? So I just put here, there, a quote: the path of scientific discovery is never straight line, It has twists and turns in every juncture. And path of scientific— and what I suggest to those who are at the beginning of their career, to stay on the track, and no matter how long and winding that road might be. So can you make it that I can see my slide? Because I can just see Sima at present.

Ah, let's see.

I don't know. I can, I can navigate from here. I have, so talking about, you know, the mRNA development for therapy, so the next slide shows that actually it started with the discovery of the messenger RNA, and this was in 1961. And it took 60 years that we had the first mRNA vaccine developed and approved by the FDA. And during this 60 years, you know, the major important milestones were 1978, when isolated mRNA was delivered into mammalian cell and it could be translated. In 1984, when first time messenger RNA could be synthesized in a tube. In 1990, when the messenger RNA was injected into, uh, in the mice, and then the mRNA translated to the protein. 2000 was when we discovered that, um, uh, the actually the mRNA was inflammatory. And 2005, we come up with the, you know, a solution for that. And 2017, when LNP-formulated modified RNA formulated in lipid nanoparticle was used as a vaccine. I mostly remember all of these, the first one not in '61, I was just 6 years old and I couldn't even read in Hungarian, not that English, so I was not reading Nature and other journals, but the rest of it I remember because I was following this field for that long. And even before discovering the messenger RNA, the next slide shows that scientists wondered about that, what could be that, how could it be that in the nuclei the chromosome is present and then actually the translation occurring somewhere in the cytoplasm at the ribosome and how the information is coming from the nuclei from the chromosome. To the ribosome. And so just making interesting experiments and of course realizing that the ribosome has some RNA. And so what they did actually using the bacteria, the next slide shows what they have done is that they isolated DNA from bacteria and then they also isolated RNA from the same bacteria. What was interesting is here showing that these different bacteria has different GC content. And when they looked at the RNA, the RNA had the same GC content. So the scientists, actually this was Spirin and colleagues in Russia, they realized that this RNA cannot be, you know, the messenger RNA because it seemed like it was all composed the same. Today we know that it was all ribosomal RNA. So the scientists had to look further. And the next slide shows that in two, they published in two papers. The next slide shows is that it was published in actually in Nature. And what was interesting, you can see that both of the paper mentioned that it is an unstable molecule because, you know, in that case, for example, in bacteria, it is a couple of minutes and the RNA degrades. So of course, scientists know that based on these discoveries established what we know today. The next slide shows it, that what is the information flow. So that, you know, the information is coming from DNA. On the next slide, please. And it transcribed to the RNA, and RNA is translated to the protein. Of course, the scientists could, you know, identified RNA, but they, they could not synthesize it. So what they did, they could isolate, and that's what they started to work with. So the next slide shows that what they have done. So they realized that reticulocytes, which is the precursor of, of the red blood cells, enriched for one type of RNA, and they could separate this RNA. It come separated, it was between the tRNA and the 18S RNA, and this fraction fraction. They collected, and actually this contains the beta globin, which is very enriched in the reticulocyte. And then they translated this in rabbit reticulocyte lysate, and they identified that it is mouse hemoglobin. They used, they used the radiolabeled amino acid to detect that. And in later studies in '71, and it was shown that when they— the same isolated RNA injected to the frog oocyte, it also translated to this protein. So these were the first RNA delivery experiments in the very early '60s. While scientists, you know, tried to develop assays that how to deliver, other scientists, the next slide shows, were working on to figure out what could be the structure of this RNA, this new messenger RNA, which was and in 1975 probably was the year of the CAP because they discovered independently here in New Jersey, in Japan, and in Australia that it has a 5' very interesting, they called in this paper, in the bizarre 5' terminus. And I put here the picture of, uh, Yannick Thomas, who is the organic chemist, and he synthesized the CAP structure and sent it to scientists to identify, so they have a reference material. And, uh, Janu Thomas became my PhD advisor. So when I started there in my PhD in '78, actually, I knew that the messenger RNA has a cap structure. Here I may mention that in the same year, Bernard Bornstedt at NIH also discovered the enzyme, identified that vaccinia virus has a specific protein enzyme which can cap the viral RNA. Scientists advanced further, and the next slide shows with the delivery, because they were excited to deliver the RNA. So, you know, many of you are surprised that the RNA was delivered, and which is something very new, but you can see here that 1978, they already have done that. In this state, at that time also, they used isolated rabbit globin RNA, and this was from a rabbit reticulocyte lysate, rabbit reticulocyte. And what they did, actually, they put it into the liposome. The liposome was actually invented in the same time that the RNA was discovered. It was 1961.. And Oleg Benkham was the father of the liposome. And here the scientists put around the RNA and this protected this liposome, which was phosphatidylserine ethanolamine-containing phospholipid liposome. And they put, and it helped the RNA to fuse to primary cells. Actually, they use here mouse spleen lymphocytes and they could detect with immunoprecipitation that the RNA was translated. So this was published in two papers, paper in Nature in 1978. So I consider that was the first RNA delivery experiment. We were reading about actually in this experiment and was excited. And the next slide shows now that my involvement is started here, because in 19— 70s, I started to work in the lipid laboratory first, and there we did generated liposomes, and we delivered at that time it was plasmid, we did not know how to, or we didn't try to isolate RNA, but for me I am mentioning this time because it was so important, this was the first experiment I was doing with mentors, and it influenced me later, I was keep thinking about this very first experiment, And so, I did a lot of lipid-related research, but when I finished my studies and I started my PhD, actually, I went to the RNA laboratories. And here, actually, not working with RNA, next slide shows, it was, I was working with a small 2'-5' linked oligoadenylate. And the next slide, you can see that.. And these molecules was, you know, I started in '78, and they were just discovered in 1977. Ian Carey in London discovered that this short RNA, 2'-5' RNA, is responsible for the interferon-induced antiviral effect. And we decided that we very much need antiviral compound. And, you know, even today we know that how important antiviral compound is.. So my role was to make this molecule enzymatically, chemically, and this oligonucleotide synthase was the enzyme which can, in our body, can polymerize from ATP. And I might mention here the oligonucleotide synthase is very important antiviral molecule. This is an enzyme in our body, and those who actually could synthesize less of this enzyme, they were more prone to get seriously sick from coronavirus, which turned out. So the function of this small molecule is that it activates RNA cell latent nuclease, and then it degrades the RNA, all of them, not just the viral RNA, but that was what was believed at that time. So my love for RNA started here in the '70s because I learned how to handle them, how to label them, and so it was an exciting time. I moved to the United States and I still worked on the same 2'-5' link molecules, but when here the important antiviral compound would have been needed, that, you know, the HIV was very serious in 1985, '86, and but we, this molecule, this 2'-5' linked molecule was not good for that, so when we, in the next slide shows that in this point, you know, here in '86, so we started to use double-stranded RNA, this was a mismatched double-stranded RNA, and we tried to induce the patient interferon system, Unfortunately, it did not have the HIV patient, because, you know, the interferon mechanism is compromised, and so we couldn't induce that. So, but by that time, it is about '88, when I am already working with the short RNA, with the long double-stranded RNA delivery problem and others, and Meanwhile, in the field, at the same time, you know, started to move on, and that was the, you know, the mRNA, because as we, I'm showing the next slide, in 1984, when mRNA could be synthesized in a tube. So it happened, as the next slide shows, in the laboratory in Harvard University, Douglas Mattonen and Paul Krieg performed this experiment and published in 1984. In 2004, interestingly. So, what was important is that they realized that using phage RNA polymerase is the answer to make RNA in vitro. Prior to that, you can see publication where bacterial RNA polymerase was used, but those, you know, problem was to promote the fidelity. Here, the phage polymerase required very short promoter, it has very high fidelity, and so that's what they used That first RNA what they synthesized and published, it was coding for human beta interferon, and they did the capping with the capping enzyme from Bernard Most, and then so the RNA had the characteristic 5' UTR, 3' UTR, poly-A tail, and the coding sequence, so when they synthesized, they injected, the next slide shows, into the throat or site,. And when they cultured these, they injected a couple of provocytes, and when they cultured these provocytes, those secreted functional interferon beta, and they were functional, they inhibited viral proliferation. So, this was a very important breakthrough, and not just because now that we could make RNA in a tube as we designed, But it was also because this was a time when molecular biology advanced and became the tools. For example, here the SPC RNA polymerase was commercially available in the same year when this was published. In addition, I might mention here that at that time, I was working at University of Pennsylvania, and I requested Paul Krieg for this plasmid, and he put in an envelope and he sent me. There were no MTA was signed at that time. So it was like the old days when everybody tried to help the others. The next important step in the next slide shows that instead of, you know, injecting individually a cell, that's of course is not feasible, but, you know, the lipofectin was introduced. And it was 1987 again when publication came out, then the product was commercially available. And other products the same way. The trizol, for example, Chomchinsky published it, how easy now to isolate RNA with it, and it was also available. And what was important that this lipofectin is a positively charged lipid, and the nucleic acid is negatively charged. It was mixed, protected, and then delivered to the mammalian cells. And the first time RNA was— next slide shows— there was Robert Malone and colleagues delivered with RNA to the mammalian cells. So compared to the '78 publication where I demonstrated that, you know, RNA was delivered in primary cells, here it was the RNA was not isolated, but rather it was in vitro transcribed, but otherwise it was a very similar experiment. So next step was in our field was 1990, as the next slide shows, And the following slide, because that was when, when John Wolfe published that he injected mice with luciferase protein RNA, and it was a naked RNA because the lipofectin was not really used. It now, lipofectin helped move forward the project in cultured cells, but in animals those were toxic, so most of the in vivo experiment was in naked naked RNA, just like here. And subsequently in the '90s, next slide, I listed a couple of major findings. In '92, when vasopressin mRNA was used in rats, and it was Floyd Bloom at Scripps could fix this special, uh, Rottweiler, Rottweiler border rats and treated them. Successfully. '93 and '94 was the two papers when first time messenger RNA was used as a vaccine for infectious disease, and that was for influenza. For Martinon and Maulien, they were in working in France, and then actually they used liposome similar what we were doing in Hungary, so, and, and that delivered messenger RNA. And subsequently, most of the studies were focused on cancer vaccine, and that was in the '05 was the first time, first paper came out, Currier, Conry, and Alabama, they used naked RNA for vaccination with a cancer antigen, and Eli Gilboa at Duke University did a lot of studies, and he used human dendritic cells, and he generated the vaccine ex vivo, so that he moved on and established a company, Merix. That was Merix, was in 1997, was the first RNA company, and they initiated clinical trial where they generated human dendritic from human PBLC dendritic cells and treated them ex vivo with tumor-specific messenger RNA, and those were injected back to the patient. And later this Merix became Argos, the company, but unfortunately their clinical trial were stopped due to futility. While all of these scientists were working on in vivo studies, I joined cardiology, and the next slide shows in University of Pennsylvania, and I also started to use messenger RNA together with Elliot Barnathan, who is a cardiologist and actually working in Johnson Johnson. And, but at that time we worked together at the University of Pennsylvania and we were interested in intima thickening. And that's what we generated, messenger RNA coding for urokinase receptor. And I am just showing this example. We did other studies, but the urokinase receptor to be functional needed intensively modified, and so glycosylated the GPI link so that— and mRNA was delivered to the cell, and then cell knew what to do with it, and then we could measure functional receptor on the cell surface. So at that point, we thought that, yes, the RNA will be good for something. But unfortunately, Elliot left, and so I moved over to neurosurgery. And the next slide shows that what we tried to do here, we did a nitric oxide encoding RNA, tried to treat for— goal was to treat a patient who had subarachnoid hemorrhage and get a major blood vessel constriction, and maybe local delivery of the nitric oxide synthase would help what we argue. So again, mRNA is not the drug. The mRNA codes for a protein, and usually that has the beneficial effect, could be therapeutic. But in that case, for example, it is an enzyme which codes for a product which has like the nitric oxide with the millisecond half-life. So that case, the nitric oxide is what is the drug. We also synthesized different kind of RNA because, you know, these are antioxidant antioxidant enzyme encoding RNA catalase glutathione peroxidase, superoxide dismutase, because it was thought at that point that maybe the stroke patient would benefit if, you know, antioxidant enzymes would be higher level present. So we did a lot of experiments, and we, meantime, we constantly improved the mRNA on a way that more protein was produced from it. So I was working on neurosurgery. And actually, I worked there for 17 years, so all of the way to 2013. But meanwhile, when David left and I met Drew Weissman, actually, and the next slide shows that I met Drew at really at the Xerox machine. And this was 1997 when we still copied papers because it was not available digitally. And if it would happen 2002, where I, all paper I downloaded digitally, so I wouldn't met him. But here he was, this new guy on the floor, and then we were started to talk. And then turn out that, you know, he wanted to make HIV vaccine. I was working with RNA, and we thought that we can try work together and try whether mRNA would be a good vaccine. And so generating the gag mRNA and delivering to human dendritic cells— Drew was working with the human dendritic cells. This was very new at that time. Ralph Steinmann discovered, and so he generated from PBMCs, and, and when he delivered the mRNA I generated for this GAC-specific mRNA. He was very happy because a lot of protein was produced from the RNA, and all of these activation markers of these dendritic cells was up, and the inflammatory molecules were secreted by these cells. And of course, me, who was working in neurosurgery and wanted to use RNA for therapeutic purposes, so coding for therapeutic protein, I was not happy that this RNA ion-making inflammatory. And so we drew, we decided that we will figure out why it is, because we were in, you know, even in 2000 when we published this paper, 2000, there were not known, only just we knew that double-stranded RNA induced interferon, but even that time it was not known how, because it was 2001 when the first time Toll-like receptor 3 was discovered and understand that double-stranded RNA activate Toll-like receptor factor 3. And this is how interferon is induced. So, we tried to figure out also that how could it be, you know, the RNA which I am making, and I thought that the RNA I am making is the same which is inside all of our cells, is inflammatory. And then we argue that maybe because the RNA is coming from outside to the immune cells, maybe it senses as injury-related or invader or whatnot. So we wonder that whether the RNA which is inside the cell, whether if we would take out and then now try to put into, you know, immune cells, whether it will be inflammatory. And this is what we did. And the next slide shows when we isolated them, generated a monocyte-derived dendritic cells and isolated different kind of RNA from the mammalian cells. And in purple, you can see that those are the in vitro transcribed RNA, which which I was always doing in a simple in vitro assay, and the isolated RNA in blue, that those were not as inflammatory. We measured TNF-alpha as a marker for it. And the E. coli RNA was also quite— total RNA was quite inflammatory. And so, we thought that maybe these RNA during the injury released from the cell, you know, they can instigate some inflammation. But what was more interesting for us that the tRNA, you can see that the tRNA was not inflammatory at all. And in the next slide, you can see that the tRNA, and we knew that, has a lot of modifications, nucleoside modifications. So this is the thing which gave us the idea that could it be that nucleoside modification could suppress the immunogenicity of the RNA. And this is, we try to answer that. And of course, how, how we could do that, how we could make our mRNA and made incorporate a modification, and which kind of modification we shouldn't, we shouldn't induce. And the next slide, just to scare you a little bit, I show here what kind of modification there in an mRNA. And there are more than 100 different ones, and these are incorporated after the RNA is made from the 4 basic nucleotides synthesized in our body, different enzymes, they change and modify, and those enzymes were not known, you, we just couldn't order to put somehow to our RNA and see that whether it will be less immunogenic, and so we didn't know what, how we should proceed, and came up with the idea that we might have to order nucleoside modified or triphosphate, the modified nucleoside, which is triphosphate, and try to incorporate to the RNA. So that was what we have done. And the next slide shows that what was commercially available, those 5, 10 different nucleoside triphosphate. And why we ordered this one only because we insisted that we are just working with modified nucleosides which is normally present in our body, and the reason for that because I remember from the early '90s, '93, when it was published that fluorinated uridine-containing, it is called thialuridine nucleoside, when they delivered to a human from a clinical trial, 5 patients out of the 15 volunteers died. And they did not understand for more than 10 years that why it happened, because this molecule was not toxic, not to the mice, not to the monkeys, not, not even monkeys. So it took, you know, in 2006 when finally it was realized that there is a special nucleoside transporter, which is only present in the human, and this middle of the coding sequence has a mitochondrial localization signal, and what happened is that all of these unnatural modified nucleosides were pumped into the mitochondria and the patient died from acidosis, so we don't want to do any kind of unnatural one, we want to use only these 10 different nucleoside triphosphate to make RNA with it, because it is present in our body, and human body would know how to handle. Of course, we didn't know that whether it can incorporate by the enzymes. So that's what we tried in the next slide shows, when we were successfully could make at least 5 of them to incorporate to the RNA. And the next exciting step was to find out whether these RNA now that containing these different modified nucleosides, whether they will be less immunogenic. Of course, you know, we were excited because we didn't know at that time, you know, whether we need all of the modification which was in the tRNA or one of them. We didn't know what we would need. And the next slide shows that we were very happy finding that some of these nucleoside-modified containing RNA were not immunogenic. These were now measured in isolated primary cells, these isolated primary dendritic cells, so it was not generated, so it was isolated from the blood. And then we found that those which had modified uridine, those were not immunogenic. And so we wondered that maybe uridine is somehow nature selected that detecting, and the immune cells detecting to as a foreign material, but when it is pseudouridine, 5-methyluridine, 2-thiouridine, or whatever was the uridine was modified, it was not immunogenic. Of course, we wanted to use, I wanted to use the RNA for coding for therapeutic protein, Drew wanted to use for vaccine, but both cases we needed that this RNA should be translated. So the next slide shows that luckily, we found that some of the nucleoside-modified uridine-containing RNA was translatable. What was surprising is that the pseudouridine-containing RNA translated so efficiently, 10 times more protein was produced than from the conventional, which shows in the blue bar there, but the pseudouridine translated very well in primary cells, and whereas the 2-thiouridine was not translated. So subsequently, now that we focused all of our study to the pseudouridine-containing RNA, but when we did some experiments, we noticed that we have some contamination present. So the next slide shows that when using the double-stranded RNA-specific antibody, we could detect in a dot blot assay that double-stranded RNA was present in all of our RNA transcripts. And so it took about 2 years and a lot of work to figure out how to get rid of the double-stranded RNA, which finally we could manage in 2011. And we established an HPLC purification procedure, and we could purify RNA. It was very important because in the light blue, the blue bars, you can see that how much translation was improved when the RNA was purified, modified. So finally, we could get a very high-quality RNA, which was nucleoside modified, which was purified, and we We moved on, and the next slide shows that we want to see that whether we can— this RNA can be translated in vivo. At that point, we used— in the next slide shows that we used Transit to deliver in vivo to the animals. In that time, we didn't have the lipid nanoparticles. We just had this Transit, which is commercially available.. And what you can see in the low panel is that when we use the pseudouridine-containing RNA and very small amount we injected to the animals, 0.1 microgram mRNA, for a couple of days we could measure erythropoietin actually in the, in the blood. When we did experiment and we just injected 0.1 microgram messenger RNA coding for erythropoietin, which is responsible for red blood cell differentiation. 30 minutes after injection, we already could measure the EPO present in the circulation. So the RNA is taken up by the cells instantly, translates instantly. The speed of translation is like 5 amino acids per second. So depending on the length of the protein, you can detect very, very quickly. You can see on the blue line there that when we use the conventional uridine-containing ePO mRNA, that not translated as efficiently, and these were all HPLC-purified mRNA molecules. On the right panel, you can see that when we injected this pseudouridine-containing RNA, the hematocrit of animals increased, and weekly injection, the very right panel shows that the weekly injection of erythropoietin-coding RNA, small amount, could maintain hematocrit at high level. So the RNA we injected, it was functional. I have to emphasize, of course, we didn't inject it in the kidney. We injected it intravenously. We also did some studies where intramuscularly or subcu injected, and it didn't matter where we injected, we will always could measure EPO circulating in the blood, and also the hematocrit increase. The very important panel is in the middle one to show that even in animals, the nucleoside-modified RNA was not immunogenic, whereas the uridine-containing RNA was. This was 2012, and in this time, you know, it seemed that I have to move to move on, and I moved to BioNTech to Germany, and next slide shows that although the new building was not there yet, we were on the campus, and we were a very small company, and I moved there because I wanted to make messenger RNA coding for therapeutic protein, and Uğur Şahin established this company, and he already in 2012 already messenger RNA entered in clinical trial for as a cancer vaccine, and I thought that if I want in my lifetime to see nucleoside-modified RNA to as used as a therapeutic, I have to go to Germany and try to do here. Here I'm just showing one of the example when I was there, and I'm still there actually, at the company that we started to make bispecific antibodies. And these are very short half-life this protein has. So the advantage of the mRNA in this case, not only that it is very quickly, cheaply the animal can translate the RNA and generate this bispecific antibody, but also that this short half-life, you know, that due to the short half-life, very frequently had to inject to here in the animal, but in human already the protein, the bispecific antibody is human, human use, and those patients need a pump put to the arm, and then those even the pump had to be filled up every second day with this protein. So it seemed that if they would have the RNA, they would not need a pump, and then they do not need such a frequent injection. So here we show that it is very effective, and it was the bispecific antibody recognized partially the Claudin-6 tumor antigen and CD3, so that it put close proximity the T cells and, and, and the tumor cells. And we have done a lot of study here. Actually, we moved there with the with my colleague Hiromi Muramatsu from Penn, and we did a lot of study, and at 2 years we were just doing the same thing we did at University of Pennsylvania, but now we had to do it in a scalable manner, and so that was, you know, one of our focus there, so that the purification, like the HPLC purification, couldn't be scaled up, so we had to come work with other type of purification. We also had to, you know, the capping of the messenger RNA, which is very critical to have a Cap1 here, also shown enzymatically we put there, but it was very tedious, so that we had to come up more improvements. So the next slide shows what we were doing at that time, you know, doing a lot of optimization, and the 3' UTR, 5' UTR and the coding sequence. So we did, and when we did all kind of optimization purification, we could, as the panel shows, we could have from the mRNA made more protein, longer time, higher level. On the next slide, I will just show you that what some codon optimization could result. So that we used pseudouridine in our experiments. So that when the inflammatory uridine are there, you know, with showing in blue here that, you know, the pseudouridine-containing RNA would not be immunogenic. But, and some used instead of using pseudouridine rather than eliminate uridine from the messenger RNA, and however, and that making the RNA GC-rich. But one problem with this one that you cannot 100% eliminate uridine from the RNA because because there are amino acids, and those here, that half of the amino acid needs uridine to code, and some, you know, the phenylalanine needs minimum of 2 uridine, so in every time if you are doing codon optimization with this way, your, the RNA immunogenicity depends on what is the composition of the protein you try to encode, and I also mentioned here 2 reason why I wouldn't code and optimize, actually, for infectious disease-related vaccines, because you might lose with this optimization out-of-frame cryptic epitope, which is very valuable, or you are generating a new epitope, which is useless. But So that's optimization, codon optimization had to be handled very gently. While we were working actually in, in Germany, my colleague here in the Norbert Pardi, the next slide shows he was proceeding with formulating modified RNA to the lipid nanoparticles, and he published this paper together with Drew Weissman, and I was also included in this publication where messenger RNA encoding the Zika virus-specific proteins were done, and it was one, this was one methyl pseudouridine-containing RNA just like in the COVID-19 vaccine. The lipid nanoparticle was very similar, made by Aquitas, were formulated, and these were tested out in mice, and it was also in monkeys, and it was very, very effective. And a single injection of this mRNA, this LNP mRNA, protected the mice. And very small dose, 50 micrograms, was also sufficient to protect the monkeys when it was challenged with the Zika virus. And I might mention that today the BBVA Award, which given out by Portugal, and for the best paper of the year for the looking at 10 years, you know, what was, what was those paper which had the most impact. And today this paper get the award for 2021 from the BBVA award, because the importance that that was the first paper when lipid nanoparticle-formulated nucleoside-modified mRNA was demonstrated that how powerful vaccine it could be. So from, uh, we went on with Norbert and published and performed more experiments. This was for Zika virus, it was also later with influenza and HIV, and we also demonstrated and had proved why nucleoside-modified RNA was critical. And as recently as this year, we also published why it is— was the lipid nanoparticle is critical. It is not just wrapping up the RNA, helping to protect, helping to deliver to the cell, entering to the endosome in the immune cells and helping to release it, but it is also containing the— it is an adjuvant. The ionizable lipid acts as an adjuvant because the mRNA, messenger RNA, is not immunogenic. And why it is important? Because the messenger RNA would induce, if it is conventional RNA, it would induce interferon. And if the interferon induced, as we demonstrated, follicular T helper your cells will be not present, and then result that there will be no good antibody response. Maybe you put cellular response, cellular immune response can be generated, but not the antibody response. So this was the importance of LNP, the nucleoside modified RNA. And as the next slide shows, and I don't have to tell you more about this because we were sitting in front of television and we knew when was, you know, the sequence became available for the SARS-CoV-2. We watched what happened when all of these companies started to— 150 companies at the same time started to make a vaccine, and how the phase 1, the phase 1, 2, and phase 3 trial ended. And so as a result of it, we know that within a year, we already we could have a vaccine which is very safe and very effective. And last year, August, was when it was fully approved, the BioNTech-Pfizer vaccine. And I might mention that it is a public domain that BioNTech started to work with Pfizer in 2018 to develop an influenza vaccine. So when people learn about the development of messenger RNA for vaccine, they thought that this, this is the first time, you know, that compound, this messenger RNA, is used. And as I told you already, were many, many studies were done, but many of you will not know, and the next slide shows, that actually the nucleoside-modified mRNA was already in clinical trial. The first patient actually was injected on February 5th, 2018, with the messenger RNA coding for VGFA. And this study started actually from Harvard University when Lior Zlange and Kenneth Chin, you know, injected VGFA mRNA to the failing heart of mice, and they demonstrated the beneficial effect. And AstraZeneca and Moderna proceeded with this finding, and the STUTE clinical trial was already underway, one of them using for wound healing for necrotic wound of the diabetic patient, and the other was the heart failure treatment which started prior to the COVID-19 vaccine. And the next slide shows that this was the most advanced, actually most advanced mRNA therapy, because it was already in a phase 2 trial. And this year, January, we learned that the patient who participated in this trial, and this was a elective coronary artery bypass surgery, where during the surgery they injected 30 times naked RNA coding for VGFA to the heart. And this on the slide on the marker on the right, you can see those labeling. And they injected the RNA, and in the link there, shown there, is you can Here the patient who are talking about their experience that how their life was improved and their heart was performed so much better, and so now it is in this clinical trial advances into phase 3, so there is a success already in, in other field of this nucleoside modified RNA, this was also one method of pseudouridine containing RNA. And another field is not— this was for the heart. Another is what the next slide shows for— that was for the liver. And in this case, if the next slide please, in this case, Cas9 mRNA treatment was used by the Intellia company to treat this patient who who had amyloidosis, transthyretin amyloidosis, and this is a very serious disease, and there were already the RNA, actually when they developed siRNA, that was the first what they targeted to treat this patient, and what happened is that they have a morphological functioning protein, and coded in the liver. And this protein, when it is made, it is aggregated, and then interrupting this production of this toxic protein would be very critical. And of course, on-island targeted messenger RNA, but here, the Intellia used the Cas9 RNA, and the next slide shows that they This mRNA, what they deliver, they contains the mRNA codes for the Cas9 protein. The next slide, please. And then guide RNA, when it delivers, you know, inside the cell, the Cas9 RNA translates to the protein and complexing with the guide RNA finds the critical target in the liver cells, the hepatocytes. And maybe the next slide shows that, how it interrupts and eventually this toxic protein is not produced. And the result of this trial, which was published last year, is shown that the patient in the blood, in the blood, the level of this very toxic protein was dropped, and so here already 3 patients were permanently treated and cured, so that's another big success of this nucleoside modified RNA use. I mentioned here one more example because the next slide shows when intratumoral injection of mRNA was used for cancer treatment, this is not the vaccine, because the messenger RNA is not coding for cancer antigen, but rather it was cytokines, a mixture of cytokines. We performed this experiment, and here is shown only in animal studies, on the mRNA coding for IL-12, interferon alpha, GM-CSF, and IL-15 sushi. And this messenger RNA, when injected to the cell surface, for close tumor like melanoma in an animal setting, then not only— so what happened is that the tumor cells of this close tumor, which is cold, became hot because these cytokines locally were secreted. Immune cells were rushed there, and then when they were in the circulation, then they could eliminate distantly located tumors here in the The picture shows on the right that it eliminated the tumors from the lung. This is just the animal studies I've shown. This is a Sanofi. It is advanced to human trials, and hopefully the patient will be cured by that. So we have to wait for the result. And so then finally, I want just to mentioned to you that in the last slide shows what is now is 2022 and what is beyond. So I mentioned all of these clinical trials which were successful. In addition to that, there are other trials are ongoing when messenger RNA codes for monoclonal antibodies. We already heard, you know, in the news, we can hear that for HIV, for flu, already clinical trial using nucleoside-modified RNA LNP is already initiated, and first patient already injected, and more genetic disease are targeted with using enzyme which can edit the genome. The clinical, preclinical trial ongoing, and those are, and let's just, I try to mention here, even 4 years ago, we already on the messenger RNA therapy meeting, we already heard about using messenger RNA to vaccinate against malaria. Herpes simplex, I showed the data for Zika virus. Last year, we demonstrated that using multiple sclerosis-specific autoantigens we could induce tolerization in two different multiple sclerosis model animals. And so this is also a new field. We published how VGFC can help formation of new lymphoid vessels and fight edema. And more papers, like just Last week was one about bone healing using the BMP-specific mRNA. So this is a platform, the messenger RNA therapy is, this is a platform, many, many diseases can be treated. There are, luckily more attention was paid. Now it is the advantages that doesn't matter it is a vaccine or any kind of RNA, you you can use the same settings. Only the order of the nucleotides which is incorporated based on the template is different, but otherwise the messenger is the same. Of course, the formulation is important and it is different because for, for a vaccine, when we are using messenger, when we are using the formulation lipid nanoparticle, those are containing the adjuvant and maybe You know, it is not useful for all. So for other formulation, for other treatment, we have to use other formulation. For example, for tolerization where we didn't want to activate the immune system. So a lot of effort was put on to make the formulation targeted to selective cell groups or organs, and this is the future to make it more specific, the therapy. But the other advantage of this mRNA therapy is that it is affordable because the human or the patient will make the drug translate, which is the protein-based therapies are very expensive because purification screening of the protein is very tedious to figure out what is, even if it is protein is the final drug for screening, it, the messenger RNA can accelerate tremendously the screening time, because, and put through, so many, many things can be done, and you will, and we will see the future that what it holds, but know that more attention on the field, more money is coming. So hopefully, a year from today, we can talk about more successful clinical treatment. And with that, the next slide just shows all of my colleagues. And I want to thank them all for helping me on this journey and, and those long, long winding road that I stayed.

And thank you very much for your Thank you. Thank you, Caroline. That was just such a thorough and extraordinary talk. You took us along on this whole journey from and throughout your own career and finally ending up at where we are today. So thank you for that. What I wanted to ask you actually is since we've covered your science in great detail, I wanted to ask you questions about, you know, the career, career in science and women in science and things of that nature. So let me start by asking you, first of all, you know, you, you left Hungary in 1985 and you moved to the United States and continued to pursue your research. What lessons would you share with others who want to immigrate to the US and continue to pursue a career in science? And how does your experience compare to those who are trying to come to the United States and work in science today?

'85 was a long time ago, and of course a lot of things changed. And so for me, when I think back that how important was for me, like being at the University of Pennsylvania, the number one importance for me was to listen to those lectures, that the best scientists came, and I was just amazed. And every week I had to limit myself to listen not more than two lectures because otherwise I have to work in the lab. And so today you can be in your home country and you can listen to all of these lectures and, and, and you can have access to all of these publications and others. So that's also a very important part. And, but as, you know, the process that you are coming to a country and then all of a sudden everything is new, you have to adopt so quickly, you learn these things. And what is science? Every time we have a new problem, now we have to purify, how we would make it. And then, you know, it is a good exercise to to learn that how quickly adopt and how quickly you can come up with solution for anything. First in life, because you had to adopt a new home, new environment, and then it is similarly in the laboratory, you know, that when you are doing different things and, you know, it is much— so what happened is a lot of people like to You know, it is very comfy corner and they don't want to leave and they don't want to. And then it, you know, if they leave, then, you know, that's immediately have a lot of experience. I personally never wanted to come here, but now I don't mind, obviously. You're back and forth always now. Yes, but So it is, I like the open-minded, you know, here in the United States, for example, that, but maybe, you know, it changed also in other corner of the world. But, you know, that everybody was so helpful, as I mentioned during the presentation, that people who I never met, you know, they just sent in an envelope, we received the plasmid cut out and soaked in, you know, the little bot mump because they were just want to help.

And so it was very uplifting. Yeah, you know, that speaks to an important topic that I know is close and dear to AAAS's heart and, you know, being discussed all around the world, and that is the importance of international collaboration in the scientific enterprise. What are your thoughts on that? Because, you know, especially during the pandemic, we saw a little bit of a rise in nationalistic sentiments, but really science flourishes when there's international collaborations and cross-country, cross-border science is very important, right?

Your thoughts on that would be so welcome. Yes, so this pandemic, and you know, I put science in the spotlight and then a realization that the collaboration is a must. And so that we can see all already even every level, not just the bench scientists who try to help each other, but we could see that even the large companies, you know, if the other company had a lipid expert and reached out, you know, to how to scale up the lipids. And so it was everybody's input. And if you see that our fellow Chinese scientists sequence, and then they distribute the data, and how it's helping everybody, you know, to work together. And for me also, moving in Germany, actually, that in my team is very international. BioNTech is very international, like 57 nations was working there. And women and men. I have more women work there. And so it is, um, and then those reaching out for more in the home country. I also try in the, in, now that I have more time, you know, to reach out back to Hungary.

And maybe it would be very effective. Well, yes, that is, that is great. And, you know, I think that this is what we've always seen and it continues to see the international collaboration. You brought up a really great topic that's very close to my heart, and that is the topic of women in science. So you are one of those women in science who has just, you know, made it, you know, to a place where people recognize you. Because a lot of times when you ask somebody to name a woman a woman scientist, a famous woman scientist, people may not always think of that.

What do you think we must continue to do to encourage more women and girls into careers in science? Yes, so I think that, you know, the children are curious and then, and boys and girls equally curious, and in school, you know, they are advancing and I don't know where the girls will fall out from this process, but one obviously, of course, when they, you know, give birth and, you know, they— it is hard for them, you know, to handle all of the job they need. And so for me, it was very helpful that, you know, we had affordable child care, quality and affordable. That's what, you know, one would be very important to have. And, and I don't know that, you know, I try to encourage also children and the girls and boys, it doesn't matter, you know, that to study, you know, science, because they have to realize what is this school is for, you know, that to realize that what do you like, what, what you would like to do in rest of your life, and that they have to find that. And that's the school for, you know. They do sports, do that and that, and they realize that what they like. And so that, of course, if they like this spotlight, spotlight, which I am, you know, probably they want to be an actress or some performing art. But, you know, they like to solve problems and having fun with this and coming up every time to figuring out something, you know. And having fun, then they should be a scientist. And so that's what I recommend them. And I don't know if they want to be rich. I don't know what they want. They should, you know, what they would do with the money. You know, it is more having fun is, I think, is more valuable. And having a goal to that your research at the end helps somebody is the, you know, ultimate reward.

Yeah, that is great. That, you know, that it's because at the end of the day, science is fulfilling when it reaches society and solves a problem. And you must be so thrilled to have, you know, something that you worked on almost your entire life to actually have had such a huge impact on the world. What do you think the future is for biomedical research?

Both here in the United States and across the world? So I am very optimistic because, you know, I can see who are around us that, you know, very bright young people. And what we need more because there are so many things to discover. And so I hope that— I know that I was not funded, but I can understand also that You know, it is, you know, I was, I don't blame anybody for that. I just blame myself. I did not have, not done enough preliminary data or not explain well enough and whatnot. So I hope that more money will be spent. And because we realize, you know, that here is a virus, you know, we cannot see it. You need certain assay to detect, and then it can stop our life on the way we knew it. And so that we, not just because of this, but that is very important to invest into science. And there are so many diseases could be cured if we would put more emphasis on it. And I know that the basic science is important, and, but the applied science, and, and, and I was a basic scientist, but I was always thinking what it would be good for, and good for, you know, so that the, who is performing the experiment, maybe that's the person who can think the application most likely.

That is, uh, that is excellent, because you're right. I mean, we have not yet, uh, scratched the surface of all of the healthcare challenges we need to solve. There are so many diseases that still need therapeutics and vaccines and cures. I have one last question because one of the things that we've all talked about is despite this great, wonderful thing about vaccines and the vaccines that are available for people, there is still hesitancy and there is still a little bit of hesitancy in believing the science. What do you think we as a society, especially as a community of scientists, can be doing to increase and enhance trust in science?

Yes, so I see my— I realize that we have not done a good job in the past because we did not educate the public. We didn't talk about what the molecular biologists is doing. What is the— what, what— where our science today, you know, on— maybe it was the papers or something, or articles about, but not a simple language. And we— and maybe those who are making, you know, films, they were not doing about science. So that, that we have to do that, and we have to explain some simple, simple way to to the people that what is the science, what is the vaccine, how it acts. And the one thing is also that to tell them that what we know, this we know, and that we don't know yet. And this is a process, this is, you know, and then the honesty, which, you know, we always as a scientist, we are always honest. And but, you know, maybe it didn't come through to the people. And then those people had more time who were just, you know, hopping from studio to studio and telling stupid things and, you know, anti-vaxxers, because they didn't have to go to the laboratory to work.

And so they spread things which was absolutely not true. Yeah, you, I mean, you bring up a very good point about, you know, in the old days of molecular biology, we probably could have done a better job of explaining what is molecular biology and how it works. That's a great point. Your hope for the future, what is your biggest hope for the future?

And do you think that mRNAs will turn into therapeutics and are you working on that? Yes, of course, I believe that the RNA will be help for a lot of treatment. Of course, it will be not good for everything, the RNA, matter how you wrap it up, it won't go through several layers of cells, you know, that, but it can have a lot of treatment of a lot of diseases. And I am very optimistic. I was always very optimistic and believe that when I can see a science and, you know, it really is not a belief because, you know, we are scientists and we know the data and we look at the data and then we can conclude things. But I am very optimistic, but we have to work also to, you know, inspire the new generation and explaining to them that what we are doing. And so being in the spotlight right now, that's what I try to do, you know, when I go out to to different countries to school children and to tell them about, you know, the excitement and what as a scientist we can experience. And I encourage them to find what they like to do because, you know, if they like to do something else, then that's what they should select.

That is just fantastic advice, and I'm so glad that you're using your time in the spotlight to to continue to inspire the next generation into careers in science. And I know that everybody watching today and the entire AAAS community and the entire Johnson Johnson community will be very happy because we are all about inspiring the next generation of science, as you know. And I also wanted to let the audience know that even though we are out of time, I hope that you enjoyed, you know, Catiline's just phenomenally thorough, very good presentation, and also the conversation we just now had. If you wanted to listen more to the conversation, then please go to the J&J YouTube channel where, um, Kathleen and I actually had a much longer conversation with each other in September when she won the Polly Hansen Award. And so for that information, please go to the the J&J YouTube channel. And for those of you who are here at AAAS, in case you were not able to watch this, the replays are going to be available.

And so thank you very much to the AAAS community.