Scientist Stories: Thomas Cech, The Magic of RNA from CRISPR to Vaccines

Good evening, everyone, and thank you for joining us tonight for The Magic of RNA: From CRISPR to Coronavirus Vaccines. We're excited to be joined by Nobel laureate and distinguished professor Tom Cech this evening. We're going to allow another few minutes for attendees to join us, and we'll be getting started momentarily. Some quick housekeeping items before we get started. All attendee lines will be muted throughout the presentation. Attendees may submit questions by clicking on the question and answer or Q&A button located at the bottom of the Zoom window. We may not have time to get to your question, but we'll certainly do our best to address the themes and ideas that are most common. And as a reminder, today's presentation will be recorded. This recording will be added to the CU Boulder Retired Faculty Association's website. And now, please welcome Dave Cassoy from the Retired Faculty Association.

Good evening, everyone. I'm delighted to have an opportunity to welcome you to the third of our distinguished faculty, uh, faculty series lectures, uh, organized by the Boulder campus of the Retired Faculty Association with the help of the Office of Faculty Affairs and support from our colleagues in the Strategic Relations and communications operation. Today's event is an example of how the association brings a community together to witness and take part in all that CU Boulder has to offer. And more specifically, with our distinguished faculty series, we hope to showcase some of our finest professors and their extraordinary research and scholarly work. I'd now like to introduce Provost Russell Moore to get us started this evening. Russ?

Thank you, David, and my thanks to the Boulder Faculty Retired Faculty Association for their sponsorship of this distinguished faculty series and this particular event with Tom Cech. My thanks too to my colleagues in Faculty Affairs and Strategic Relations and Communications for your hard work on this event as well. I want to invite folks to watch for future events sponsored by the Boulder Campus Retired Faculty Association, which is doing a great job of advancing the work of some of CU Boulder's leading faculty. And of course, when I say leading faculty, there's no one more readily— who more readily comes to mind than Professor Tom Cech. Tom joined the faculty here at CU Boulder in 1978. Became a Howard Hughes Medical Institute investigator in 1988, and then a distinguished professor of chemistry and biochemistry in 1990. In 1982, Dr. Cech and his research group announced that an RNA molecule from Tetrahymena, a single-celled pond organism, cut and rejoined chemical bonds in the complete absence of proteins, creating an understanding that RNA was not restricted to being a passive carrier of genetic information, but could have an active role in cellular metabolism. This discovery of self-splicing RNA provided the first exception to the long-held belief that biological reactions are always catalyzed by proteins. In addition, it has been heralded as providing a new plausible scenario for the origin of life, because RNA can be both an information-carrying molecule and a catalyst, perhaps the first self-producing system consisted of RNA alone. In January 2000, Dr. Cech moved to Maryland as president of the Howard Hughes Medical Institute, which is the nation's largest private biomedical research organization. In a day, in addition, HHMI has an $80 million a year grants program that supports science education at all levels K-12 through medical schools, and in international research. In April of 2009, Dr. Cech returned to full-time research and teaching at the University of Colorado Boulder, where he until recently was the director of the BioFrontiers Institute. Dr. Cech's work has been recognized by many national and international awards and prizes, including the Heineken Prize, of the Royal Netherlands Academy of Sciences in 1988, the Albert Lasker Basic Medical Research Award in 1988, the Nobel Prize in Chemistry in 1989, which made him CU Boulder's first of 5 Nobel laureates, and he also won the National Medal of Science in 1995. In 1987, Dr. Cech was elected to the U.S. National Academy of Sciences and also awarded a lifetime professorship by the American Cancer Society. Beyond his achievements and awards, Tom embodies all the best things about the life of science and the way we approach science at CU Boulder. He is devoted to interdisciplinary work, dedicated to teaching undergraduates, focused on mentoring graduate students and postdocs, including last year's Nobel Prize winner in chemistry, Jennifer Doudna, and centered in a life of inquiry, collaboration, and discovery. He does all of this with the generosity of spirit and work that is remarkable. He is, in my mind, not just a great scientist, but a great person who happens to be a scientist. It's my honor and pleasure to introduce our presenter this evening, Professor Tom Cech.

Thank you, Russ, for that very generous introduction. You've been a good colleague and a good friend since I've come back from Maryland back to Boulder 12 years ago. So I'm going to share my screen here and Can someone confirm that that is visible?

Yes.

Yes. Okay, good. So tonight I've decided to share— so first of all, thank you for joining us. And tonight I've decided to share some information about RNA that is mostly not our own work, but that I thought would be of more general interest to the public. And this is the CRISPR genome editing and messenger RNA vaccines. These are two of the most spectacular scientific achievements since putting a man on the moon.

And I think that the public deserves to share in this excitement because after all, it's your tax dollars that funded the RNA research that paved the way for these great technical achievements.

Now, before I start the talk, I just need to say that this is a picture of the Caruthers Biotechnology Building on the East Campus of CU Boulder, where we are privileged to work. And I'm also required to disclose my relationships with Merck and Company, where I'm on the board of directors, and Storm Therapeutics and ICON Therapeutics, where I'm on the scientific advisory So I think before we talk about these RNA technologies, we should ask, what is RNA?

Well, you all know about DNA, the famous double helix, and DNA, deoxyribonucleic acid, you can tell by the name, must be quite similar to ribonucleic acid. Now the DNA double helix, when it's copied into RNA, only one strand is copied.

The RNA is always single. This is something I pulled off the internet to try to amuse people, but since there's no laugh track on this talk, I can't tell if anyone is chuckling or not. The information content of the RNA is just the same as the DNA. The order of As, Cs, Gs, and Ts along the DNA is precisely copied into the RNA. Now the RNA can either be, can code for a protein, in which case it's called a messenger RNA because it serves as a message between the DNA and the protein synthetic machinery, or the RNA can be a non-coding RNA that performs some other function within cells. This is the first example of the CRISPR genome editing, and actually the RNA has a non-coding role. Now, this topic is of particular interest to many people in Boulder because of the very recent award of the Nobel Prize in Chemistry to Jennifer Doudna, who trained in my laboratory, at CU Boulder in the 1990s, and her colleague Emmanuelle Charpentier. There was a lot of celebration when her discovery of CRISPR genome editing was recognized at this level. Now, what does CRISPR do?

Well, it cleaves double-stranded DNA.

But it's not as simple as the red scissors.

This is where the RNA comes in. Because the CRISPR machinery carries around with it this non-coding guide RNA, and that searches in the DNA sequence for a match. When it finds a sequence that is complementary, to that of the RNA.

That means that G pairs with C and C pairs with G between the RNA and the DNA, for example.

Then it locks down and the protein component of the CRISPR machinery called Cas9 cuts both strands of the DNA so that now you have out of the entire human genome one place that has been cut.

Well, why is that of any interest or any use? Well, because at this point, cut DNA is something that biological organisms have learned is a deadly event, and so they have developed repair machineries that stitch it back together.

There are two basic kinds of these, and I'm sorry that they have such technical names, but this first one called non-homologous end joining just means that the two broken ends are glued back together. During that gluing back together, it's a little sloppy, and so very often a small insertion or deletion called an indel, shown in the bottom here in red, is introduced at the repair point. That can be itself a mutagenic event, can inactivate a gene. Now the other possibility is that if there is a sequence existing in the cell, or if the experimenter provides a sequence called a donor strand of DNA that has matches to the flanking sequences next to the CRISPR cut of the DNA, now you can use this homology-directed repair to introduce purple sequences of your choice into the human genome.

You can imagine if the original human gene, let's call it a gene in a blood cell that is producing the hemoglobin, the oxygen-carrying protein, If this individual had sickle cell anemia and had a mutation at a particular site, the CRISPR machinery could put in purple sequences, which would be the correct functional, the ones we want sequences at that position, and the sickle cell anemia would be alleviated.

What are some of these medical applications that many companies are now engaged in nonstop, research and development to try to make use of this CRISPR genome editing. Well, where might the first successes be? I think in addition to the sickle cell anemia that I mentioned, other genetic diseases that are extremely deadly, such as muscular dystrophy, progeria, these are really prime targets already for CRISPR genome editing. And also we can imagine that certain terminal diseases like cancer would be excellent candidates. Then what are some of the big technical challenges standing in the way of the implementation of CRISPR genome editing? Well, you've got to get this combination of the Cas9 protein and this guide RNA molecule, this non-coding RNA, into the relevant cells. That's pretty easy for us to do in my laboratory with cells growing on Petri dishes where you have really good access to the surface of the cell.

It's much more challenging to do in an entire human organism in the middle of the brain. How would you actually get it to the relevant neurons?

Then we also have the technical challenge of specificity. I have described the CRISPR genome editing as being entirely specific, but in reality, nothing is really perfectly specific. There is always some low level of off-target editing, and one has to try to minimize that and also be aware that that off-target editing could have unforeseen side effects. Finally, we must be concerned about the ethical implications of changing the human genome. These are perhaps not very great if we're changing a gene in a blood cell or in a liver cell because that wouldn't be passed on to the next generation. It would die off when the person finishes their lifespan. But germline editing, which would be inherited, is a whole nother matter. And there is great concern in the scientific community and in the general public that this germline editing could be used to make designer babies, not just determining, uh, the eye color or the hair color of your child, but perhaps their height and weight, a group of superhuman beings. That is something we see in science fiction movies. Now with CRISPR genome editing, it is probably technically possible and certainly something that we're not ready to embrace at this time. Now, much has been written about these medical applications of CRISPR, but I want to talk about something that's already extremely useful, extremely valuable, and that's the research applications of this technology. Because for the first time, we can, with surgical precision, edit particular sites of the human genome. We're doing this, have been doing this for half a dozen years in my own laboratory. Even undergraduates have mastered this technology. It is that straightforward, and robust. Of course, the undergraduates are really good too. Now, 3 sorts of research applications. We can use CRISPR to inactivate a gene to help understand its function. We can edit a single base pair in a gene to test what effect a mutation would have or to erase a mutation and see what kind of a good effect that might have. Finally, we can add sequences encoding a fluorescent tag, a little molecular light bulb, to follow a protein as it moves around and explores a living human cell. Since this third application is so visually exciting, I thought I would focus on that tonight. Now, the particular system that we're going to put the light bulb on is called telomerase. Some of you have heard of this as the immortality enzyme. This is a little molecular machine that extends the ends of our chromosomes, and it consists of an RNA strand which was discovered by Liz Blackburn and Carol Greider, who won the Nobel Prize for discovering telomerase itself. Then the RNA strand also needs a protein to work along with it. This is called telomerase reverse transcriptase, or TERT for short. That was discovered by Joachim Lingner, a Swiss postdoctoral fellow in my lab here at Boulder. Just like the CRISPR machinery, the telomerase machinery doesn't work unless it's got both the RNA and the protein component. One of the things that the RNA does is it determines the sequence that's laid down at the end of the DNA, again, by the principle of complementary base pairing, A pairing with T, C pairing with G. Now, if you want to understand how this telomerase finds the ends of our chromosomes. I'm sorry, I forgot what the next slide was.

Excuse me.

I wanted just to mention briefly that this telomerase has a lot of medical interest also, because if you have too little of it in your stem cells, then they age prematurely. This contributes to another variety of human diseases. But if you have too much of it, then this is one foot in the door for cancer because this is the immortality enzyme and tumors are immortal. Most human tumors reactivate the otherwise quiescent telomerase gene, actually the TERT gene, in 90% of human cancers. The way that it's reactivated or the most common way was discovered by my friend Levi Garraway when he was at Harvard Medical School. He discovered a mutation in the regulatory sequence of the TERT gene, the telomerase reverse transcriptase gene, in 2013, that when mutated attracts some regulatory proteins that pumps up the action of this gene and it is found in so many cancers that it is now the third most common mutation in all of cancer. Back to this idea of tracking the telomerase around the cell, Jan Schmidt, a postdoctoral fellow in the laboratory, and Art Zogg, my longtime research associate, decided to do this live cell imaging using CRISPR genome editing. The one component that needed to be edited was a protein called the shelterin protein that binds to the end of the chromosome. That allowed us to mark the chromosome ends, and then we also had to mark the telomerase protein with a different color. Using CRISPR genome editing, we could insert the sequences encoding these fluorescent tags right in the human genome so that everything was expressed in the normal way. But when it was expressed, it carried along with it this little light bulb. In the movie that I'm going to show you here, What you will see is shown on the right is that most of the telomerase is exploring the human cell nucleus by rapid diffusion. Each chromosome end called a telomere is probed thousands of times per DNA synthetic phase of the cell cycle. Then occasionally, the telomerase sits down on a chromosome end, for long enough to actually extend it. You will be, I think, amazed as I was to see how much motion there is within these live cells. This is not sped up. This is real-time movie, 12 seconds in the life of a cancer cell. You see the green dots, which are the telomerase, are moving around rapidly. The red dots, which are the chromosome ends, look like they're static. They move very slowly over a long period of time, but they are in this 13-second movie essentially stationary. Now I'm going to play this once more and focus on this little yellow dot. This is where a telomerase has found a chromosome end, so the green is overlapping with the red, making for yellow. You can see that they are holding onto each other for the entire length of this movie. We think that that is the event that actually is important for extension of the chromosome ends. Now, when you're doing this research, you can't just look at the pretty pictures. You have to use tracking software to track the trajectories of thousands of these telomerase particles through the nucleus. Here you can see that they are really exploring most of the nucleus. Sorry for the technical term here, but there's one area called the nucleolus that is actually not being explored. There are no chromosome ends in the nucleolus, so it doesn't waste its time looking in there.

Then by analyzing these data, we come up with a model for the dynamics of how human telomerase finds this needle-in-a-haystack spot, this very end of the chromosome, which is such a small fraction of the chromosome length. It probes the telomere many times during a cell cycle, But if it doesn't find the very end of the chromosome, it gets released again. Then much more rarely through both protein-protein interactions with the telomere protein and this base pairing interaction between the telomerase RNA and the DNA at the end of the chromosome, which I showed in that earlier painting. Now it's locked down long enough to actually do the extension. Using this technology, we can even examine the effects of various cancer drugs that have been developed against telomerase, such as this Giron drug, which binds to the RNA subunit, and we can see how that interferes with the recruitment of the telomerase to the chromosome end. That's one example then, of CRISPR genome editing as a research tool. And every day there are 100 different applications that are published in the scientific literature. It's literally being used by tens of thousands of laboratories around the world. So let's move on to the second topic, which is something that you see every day on the news and on your computer that vaccines against the coronavirus are obviously something that is really front and center right now.

So what is a vaccine?

It is something that primes our immune system. It sort of says, heads up, something dangerous is coming, right? You need to get rid of it.

In order to prime the immune system to recognize the coronavirus, you don't need to use the whole virus, although you could.

That was a traditional vaccine strategy. But all you really need is this so-called spike protein, which is painted by the artist colored red in this particular rendition. It's not really red.

That is the part of the coronavirus that is first seen by our immune system.

If we can prime the immune system with just that protein, that is enough to vaccinate one against the virus. The nice thing about using just the spike protein is that without the RNA heart of this virus, which is the infectious part, the spike protein is completely benign. It's not infectious by itself, so that's a good thing. What does your immune system do when it sees the spike protein?

Well, it activates two kinds of activities within the immune system.

The B cells, which are bone marrow cells, make antibodies which can bind to that spike protein and thereby prevent the— cover up the spike protein, prevent it from entering human cells, or the immune system can activate T cells, thymus cells that give a cellular response to attack and kill infected cells, which is also a good thing, not for that particular cell, but a good thing for the survival of the whole organism, namely us.

Now, before I go on with the mRNA, let me just remind you, if you don't, haven't seen this before, that vaccines are generally the safest of all of the approved drugs that you've heard of. Well, why do they require so much scrutiny? Why are the safety standards for vaccines so much higher than for other drugs? Because they're being given to large numbers of healthy people. If you have a 1% chance of getting a serious illness from the vaccine, that's completely intolerable if you're going to be giving it to a million people. We're going to eventually give it to children.

That's why these vaccines have to be tested especially thoroughly before the FDA will approve them.

Then that's not enough. They are monitored for many years, for their entire lifetime after they are marketed. If you look on the CDC website, you can see fantastic data that's been collected, for example, from the last 50 million kids who've received the chickenpox vaccines, there's been less than one serious event per million vaccinations.

And by the way, that number of serious events is much lower than what you would get if you had a million kids with the chickenpox.

That would have a much higher level of serious events.

So I urge you, if you have some neighbors who are hesitant about vaccines to take a look at this CDC website. Now, what are the ingredients that you need to mix together, not to make cake, but to make a coronavirus vaccine?

Well, there's several different recipes that can be used. Some of these use, like the Johnson Johnson vaccine, recombinant adenovirus, a cold virus encapsulating a DNA. Some of the Chinese viruses are weakened forms of the coronavirus itself.

But I'm going to talk about these two at the top because I am Dr. RNA and I love RNA, and I'm so excited that the first vaccines to be approved were the messenger RNA vaccines, wrapped up in a greasy little ball called a lipid nanoparticle. Now remember the first story, the CRISPR story, was the non-coding RNA.

Now we've got the messenger RNA, which is the coding RNA, and what do you suppose that messenger RNA needs to code for? It needs to code for the spike protein. So the way we normally read about information transfer in biology in a high school textbook or a college textbook is that the gene—

there's a gene for every protein. The spike protein would have its specific gene, double helical DNA that is made into messenger RNA, which then orchestrates the production of that particular protein.

But what scientists at Moderna and at BioNTech-Pfizer realized was that you could short-circuit this pathway. All you needed was the messenger RNA because it could make the protein.

You don't need to mess with the DNA at all.

Given that there were zero examples of mRNA vaccines ever having been successfully developed, and approved, what would be the chances that the first one would be successful?

I don't know, but I think there was some luck involved that we really lucked out that the first one was successful because this doesn't usually happen when you step up to the plate for the first time and hit a home run at the first ball that is pitched.

The reason that I'm excited to share this story with you is because it's your dollars, your taxpayer dollars that funded the fundamental research into how RNA works, independent of knowing about that there would even be a coronavirus or a pandemic, independent of any applications. That was the reason that these companies were able to do this so quickly and so successfully. So some of the technologies are as follows.

RNA sequencing, which was done just a year ago, was first revealed in January of 2020, revealed the sequence of the spike protein that was the target for the mRNA vaccine.

Virology was critical because it enabled us to understand Why the spike protein was the thing we needed to cap off to prevent the virus from entering cells. Nucleic acid chemistry was critical to make the RNA stable and safe. RNA biochemistry, how to make a ton of RNA in vitro. That's a Latin term.

It means in glass.

Now we use plastic test tubes.

But we still call it in vitro for old time's sake. These lipid nanoparticles that package the RNA so that it could get into cells. All of this was worked out by fundamental scientists who didn't know what the application would be, but we're just following their curiosity and knowing that RNA was going to be really important. Now, there turns out to be an enormous contribution from CU Boulder to these technologies. So Professor Marvin Caruthers in biochemistry invented chemical synthesis of small fragments of DNA. The technical term for these are oligonucleotides, and these are absolutely essential tools for sequencing the code of the coronavirus spike protein. So he did this, he developed this in the 1980s, and it has applications to absolutely every biomedical problem, including of course sequencing the coronavirus. Professor Aki Ullenbeck, also in the biochemistry department in the 1980s, chemistry and biochemistry, developed and really modified and tuned and really got this in vitro transcription of RNA turned into a robust technology. And this turned out to be the same method that Moderna and BioNTech-Pfizer used to synthesize the mRNA and are using right now to synthesize the mRNA for their vaccine. And again, Uelenbeck had no idea that this was going to be important for fighting a pandemic. He just wanted to make specific RNAs for research purposes, which many of our laboratories do all the time. Now, the other thing that these pharmaceutical companies should be credited for is the way that they overlapped the development of these vaccines. Normally, these different blue bars, the clinical trials, the manufacturing, the FDA approval would all be done. You'd finish blue bar number 1, and then you'd start blue bar number 2, and then you'd start number 3. The blue bars would be lined up in a straight row, but by overlapping them, they were able to achieve an approved vaccine in 1 year instead of the typical 5-8 years. That it would take. So it was a record, not just by a small amount, but by a huge factor. Now remember, we don't have problems, we only have challenges, and there are some challenges with these mRNA vaccines. They are not perfect tools for fighting the virus. First of all, because mRNA vaccines are a new modality, manufacturing and supplying them has been more challenging than it would have been with a traditional vaccine.

Uh, it—

a cold chain is required, which means you have to keep the vaccine, depending on which one it is, either frozen or at least very cold, um, the whole time it's being shipped and stored.

And this is particularly a problem for distributing it to rural areas.

The durability of the vaccine means how long after you're vaccinated are you still protected. Well, we'd all like to know the answer. Right now, because we fast-tracked it, all we know is that it's good for 6 months. Could it be good for 10 years?

Possibly.

Could it be good for a year at a time so that you would need a booster shot like an annual flu vaccine? That's possible, but we don't know yet, and we will know within another year how durable this response is. You've heard a lot about the variant viruses, the mutant viruses that are being spun off all the time by the coronavirus, and some of them are partially resistant. None of them so far has been completely resistant to the vaccine, but some of them, like the South African variant, are partially resistant, and that's not good news. But with an mRNA vaccine, any undergraduate in my laboratory, if they were given the sequence of the new variant, could design a vaccine against it in a week. So that's another very exciting component of this mRNA vaccine technology, that it's tunable to however the virus changes, you can tune the vaccine. And then another challenge is a lot of anti-vax sentiment, people who think that they don't need the vaccine and don't realize that their reluctance to get it is actually having a big effect on their friends and relatives and the whole community. And that anti-vax sentiment is strong even in the Boulder Valley. And so nonetheless, despite those challenges, I think it's great to have a light at the end of the tunnel. And I would like to thank both the people within my own research group, and the people who I talked about for the mRNA vaccines, uh, Aki Ollenbeck, Merck Brothers, the dedicated scientists at Moderna and BioNTech-Pfizer, and for J&J as well. Um, I mean, I love mRNA vaccines, but I think that one should get any vaccine one is offered. And we also thank the Howard Hughes Medical Institute and the National Institutes of Health for funding, and the Damon Runyon Cancer Foundation funded Ian Schmidt when he was here. He's now on the faculty of Michigan State University. And I thank you for your attention. And now I would like to introduce my postdoctoral fellow Linnea Jansson, or Linnea Jansson-Frisberg, if she could reveal herself at this time. I first met Linnea when I was giving a research seminar at the University of California at Santa Cruz, which is a wonderful center for RNA research, much as Boulder is. And I was able to get her interested in applying to come to my laboratory for her postdoctoral work. And you probably can't tell that she's actually in the area because she could be Zooming in from anywhere, but she in fact does work in the lab, and she will tell you a bit about that before she fields the questions. Linnea?

Thank you, Tom, for the introduction, and good evening, everyone. As Tom mentioned, my name is Linnea, and I'm a postdoctoral researcher in Tom's lab, and I'm just going to quickly mention what I do in the lab. So I study how RNA regulates epigenetics, and epigenetics refers to the DNA sequence independent changes in how genes are expressed. So all the cells in your body have the same genetic materials, but epigenetic marks determine which cells are turned on or off to produce a specific cell type, which is why a skin cell and a muscle cell, for example, even though they have the exact same DNA, look and behave very differently. Specifically, I look at how RNA regulates the enzymes responsible for adding methyl marks onto DNA. DNA methylation is an epigenetic mark that often serves as a signal for genes to turn off. This is important because abnormal DNA methylation, it's one of the earliest signs of cancer. I'm currently looking at how RNA regulates the activity of a protein called DNA methyltransferase 1, or DNMT1 for short, and I'm interested in how these protein-RNA interactions vary between both healthy and cancerous cells. So I will serve as your moderator for tonight's discussion. We will start with a couple of questions that were submitted during registration, And we will then move on to questions from the audience. And if you have a question that you think of— so Tom, if you want to pop back up on the screen.

I'm here.

So here we go. Could CRISPR be used to enhance or establish metabolic pathways in microbes so that a colony could efficiently consume greenhouse gases, for example, in smokestacks, to produce more sustainable and useful byproducts besides biomass?

Yes, quite remarkably, the CRISPR gene editing machinery is active in bacterial cells where it was originally isolated from, but also in crop plants, rice and corn, also in the yeast that is used to make bread and beer, also active in all mammalian cells in which it's been tested. This is not something you could have necessarily predicted, but it turned out, again, sometimes it's good to be lucky. It can be used. To re-engineer bacteria. And of course, then the other part of your question, which would take a whole hour seminar to cover, is what— which genes exactly would you like to modulate or add to bacteria to allow them to have these positive effects on the environment? I'm going to keep all my answers rather, rather brief so we can get to as many of your questions as possible.

I think that is a good idea. We have 50 questions so far. So I'm going to ask another of the pre— the questions that were asked during the registration, and then I'm going to move into the questions, the Q&As that have been asked right now, because some of them are overlapping with the early ones. So we have a person asking, does the fast-track production of the coronavirus vaccine create a bad precedent for the development of future vaccines?

Well, does the fast track create a bad precedent? Certainly, there was during the approval of it, a sort of a trade-off between, between being really careful and methodical about the, about the trials and the, the international need really for a vaccine as people were dying every day. And so just as in most decisions in life, there's— you need to balance the two things. I'm not sure though that it's going to turn out to be a bad precedent. I think that it may turn out to be a good precedent and reveal that Some of the extra excess time that we've been taking to test some of these medicines has not actually been adding much value but has been slowing things down. And now that the first mRNA vaccines have been shown to be safe and effective, I would imagine that when the next mRNA vaccine comes along, scientists are going to say, you know, we do need to do some safety and efficacy studies, but we have a track record of success. Maybe we can accelerate these processes all the time instead of just when there's a pandemic.

Great. So now I'm going to move on to questions from— that were submitted during your talk. The next one we have is, and I'm sure this has been something that a lot of people in the general population have been curious about. Anti-vaxxers are claiming that the COVID-19 vaccine changes our DNA. Please comment on this. Thank you.

Well, one of the beauties of an RNA vaccine is that the possibility of DNA getting into your chromosomes is gone because there's no DNA being introduced in the vaccine. The vaccines that have DNA in them, like the J&J vaccine, that there, there is DNA in those vaccines, the, the J&J and the AstraZeneca. And so there is a hypothetical possibility that that DNA could find its way into the human genome. I guess what it would be likely to do if it did that would be to produce some of this spike protein, which would then hopefully give one continued protection. But it wouldn't be something I would want to see happening, and that's why so much safety study had to be done before approval of those vaccines. We do not see that event occurring with the DNA vaccines, and I would argue that it's just not even a possibility with the mRNA vaccines.

Thank you. Um, so here's a question that is similar to a question that was asked during registration, so I'm going to attempt to combine the two. Um, so a viewer has a question: my wife has asthma and is easily allergic to things. Is the vaccine unsafe for her, and are there any precautions she should take? And I'm going to combine this with a person that also asked a similar question about autoimmune disorders and if there are specific precautions people with autoimmune disorders should take.

It's an important question, and when I'm giving a talk to 1,000 people, I'm certainly not going to give medical advice. And so, and also, I am not qualified to give medical advice, so please talk to your physician about this. I think an answer that you are likely to get from your physician is that if you've had— if you've tolerated other vaccinations pretty well, there's no reason to think you won't tolerate the coronavirus vaccine equally well. If you've had severe reactions to previous vaccinations, then that would be a reason for special caution.

Great. So a general question here just about RNA. We have an asker, or somebody is asking, what decides if RNA is coding versus non-coding?

Yes. First of all, the coding RNAs have to get exported from the cell nucleus to the cytoplasm where they're read out into a protein sequence. Special signals on the RNA that allow it to get exported from the nucleus to the cytoplasm is one answer. The other thing is that there are little codes in the RNA sequence itself that determine whether it's going to be coding. The first of this is the triplet, A-U-G, which means start making a protein right here. All of the mRNAs would have that AUG or occasionally a related sequence followed by codons for the individual amino acids. Lysine, tryptophan, phenylalanine, et cetera, going down the chain. And the non-coding RNAs don't have that code embedded in the sequence of the RNA. Good question.

What are your thoughts on using CRISPR to break down tumors?

Well, I think that CRISPR can be used to inactivate genes and it can be used to add information. There are genes called oncogenes which enable a tumor to divide rapidly. CRISPR inactivation of those oncogenes, perhaps even the TERT that I talked about would be a good target. That's just— what should I say? Logically, that would work. The logic is fine. How easy that would be to implement and how successful it would be requires a lot of development and a lot of clinical trials. On the other hand, there are tumor suppressor genes that help us survive tumors that are often inactivated in cancer. In those cases, reactivating the tumor suppressor gene would be something that CRISPR in principle could also achieve. I think lots of potential and much research needs to be done before we know how practical such approaches are.

Great. Another COVID question. So why are health experts saying that even those who already have had COVID should get vaccinated? Why would the vaccines be more efficient or more effective at giving people long-term immunity when the actual, actual viral particles themselves may not?

Yes. So the, the very perplexing and unusual thing about this particular virus is the range of severity of infection. Many people, probably 90% of people, certainly most of the young people who get infected have a very mild infection and they don't produce a lot of antibodies. What's been found is that the vaccine produces 10 times more antibodies against the coronavirus than one of these mild infections does. That's the reason that they are recommending vaccination for people who have already had the disease. I suppose if you had an extremely severe case, a near-death experience with a real high viral load, then you might be okay. But that's a very small percentage of the infected people who'd be in that category.

Great. Are the mutant viruses showing variants only on the spike protein for which the current vaccines target?

No. RNA viruses are sloppy replicators. They make mistakes all the time when they copy their RNA genome. They make mistakes throughout the 30,000 subunits called nucleotides of the RNA chain. It's just that most of those changes are not displayed on the exterior of the virus, only the ones on the spike protein. The spike protein is what the virus needs to get into the cell through something called an ACE receptor. That's why all of the focus is on the mutations within the spike protein.

Great. Will mRNA be the new standard for creating new vaccines, do you think?

I think there's a— I think this is really revolutionary. And so should— can we see a much better flu vaccine, better tuned to the flu that is originating in Asia and coming in our direction in the future based on mRNA. I think that this has opened a door and the possibilities, maybe even a cancer vaccine of some sort might come out of this. So I'm hopeful, but it's too early to declare victory. And we need more students and research fellows like Linnea and others working on RNA before we can really explore all of these myriad of possibilities that have now been opened up for us.

And so this is a bit of a bureaucratic question, but we have somebody asking, since these vaccines are currently emergency use authorization only. Do you have any thoughts on the timeframe for, for when the FDA will actually approve them?

Um, that's a good question. I have not read up on that particular, um, question. Um, I would imagine that they might want to see, um, a year or two of track record for safety and efficacy before they would give it regular approval rather than this emergency use authorization. But I don't have— that's just a hunch. I, I don't know an exact number.

And here we have a few, uh, people asking a similar question. So we have people— since you talked about both CRISPR and the mRNA vaccine, uh, people are asking if CRISPR was used at all in the development of the mRNA vaccine, or is there any link between the two?

There, there is no direct link, except that they both had this CU Boulder connection, which I pointed out, and they are both involved my favorite molecule, ribonucleic acid.

I agree. How long of a DNA sequence can be inserted by the Cas9 protein?

Um, yes, well, the, the, uh, they can be quite long, but, but, uh, the efficiency, uh, can be quite low if you try to insert a very long sequence. So you might have to try multiple times before you get success, which is fine for research purposes, but a problem for medical applications. Let me just say that this protein light bulb that we inserted to make the TERT protein fluorescent is not a very small molecule. It has— I can't remember exactly. Let's say it has 1,000 amino acids in it. And each amino acid requires 3 RNA nucleotides to code for it. So that would be 3,000 nucleotides that would have to be inserted into the DNA. And so we were able to insert 3,000. So it doesn't have to be a small change, like for sickle cell anemia, where you'd want to change just 3 of these units. You can change thousands.

Great. So this is an interesting question. So how does one reverse engineer mRNA from knowledge of protein like the spike protein?

Yes, this was figured out in the 1960s in a heroic effort by Marshall Nirenberg, who was at the NIH, and Gobind Khorana, who was at the University of Wisconsin and then at MIT. And they won the Nobel Prize for deciphering the genetic code. And so once you have this table of codons, like AUG means methionine, UUU means phenylalanine, This table, which was figured out in the 1960s and is in all of our textbooks, and you can find it on the internet. Now, once you have that, you can go from mRNA sequence to predicting very accurately what the protein sequence is, or you can go from protein sequence and figure out how you would make an mRNA to code for that protein. And that's the kind of thing that in our classes, in our— when we teach biochemistry or molecular biology, that'll be a test question, right? On the test, we'll say, if you want to make this protein, what kind of RNA do you want to make? What sequence? And the students figure it out.

Great. So it's 8, so I'm going to ask 2 more questions before we go to closing remarks. So this has been— this question asks something that has been a topic of discussion in the scientific community for a while, but with the rise of the popularity of CRISPR, what measures are in place ensuring that the creation of designer babies is prevented?

Well, first of all, the scientists have gotten together and themselves put a prohibition on germline editing. Now, of course, that doesn't— just because a group of influential scientists decides that this is something that shouldn't be allowed doesn't prevent a rogue scientist, and there was one in China who went ahead and made designer babies a couple of years ago. He was put in jail in China for having done this. But just like any other technology, good people can make the right decisions and the right prohibitions, and it doesn't really give you an ironclad guarantee that in some rogue country or with some rogue group of scientists that someone might try to break through those ethical barriers. But we're doing the best that we can.

Great. And so to end on a hopeful note here, I'm going to ask this question. Um, are there other diseases you feel might be a good target for future mRNA vaccines?

For future mRNA vaccines? Well, yes, I think that, um, particularly anything that changes rapidly, um, through— by mutation, um, that this will end up being a, a very good approach because the vaccine can be changed so rapidly to match the mutated virus. And so many RNA viruses, including the common cold and flu virus might be finally better attacked with vaccines through this technology. I think that the more pie-in-the-sky but exciting possibility would be some cancer vaccine using the information of oncogenes, the sequence of the oncogenes, and the fact that they are expressed by, by, by in tumor cells as a possible lead, but that's more speculative.

Great, thank you. And thank you everyone for listening. And now we're going to have some closing remarks from Dave Kazoy.

I'm very pleased to have an opportunity to thank Professor Tom Czech, Dr. Linnea Jansen Fritzberg, and all of the people who joined us tonight to get a little bit of education about all of the things that these experts have been talking about. We're really pleased to offer these kinds of seminars in the future. We'll have another one on April 21st. We're in the, uh, the fourth of our distinguished faculty series, and we're delighted to be able to offer that to a very wide public. Stay tuned. Thanks very much. Good night. No, I was trying to figure out how to do two things at one time.