[Dr. Church]: In order to understand how something works. And also in order to develop new technologies, you need to be able to, to write and edit. It's like reverse engineering, electronic circuit or some software. So I have no idea what this code does. Let's, let's change it in case of the biology. You'll, you'll, you'll take a piece out and now it no longer handles glucose.
You say, okay, that's part of the glucose monitoring system. And, and you can just get through that and you can get to more and more nuance changes for discovery's sake, but it's often entangled with not just discovering, but making useful, uh, synthetic biology, um, that there you'll have a challenge that you'll have out there, and that will drive the reading and writing technology forward. It will drive our creativity in terms of how these things can positively influence, uh, society and ecosystems. [Dr. Patrick]: Hey, everyone. Today I'm extraordinarily excited to bring to you, Dr.
George Church. It's really a special opportunity to talk to Dr. Church because he's one of those rare living historical figures, whose work is so vastly influential that it can change our perspective on the potential of an entire field. In my opinion, this gives his generally optimistic take on technology and the future a very special weighting. Through his work in the Human Genome Project, in particular, he has directly contributed to exponential shifts in understanding feasibility and capability in the field of biology. The Human Genome Project began as a $3 billion moonshot shortly after Dr. Church
first pioneered a method of direct DNA sequencing in 1984. The goal, to sequence a single reference genome for humans. Completing the initial aim of that project, Dr. Church and his collaborators and colleagues ultimately set us on a path to where we find ourselves today. Sequencing is now over 10 million fold cheaper, and most people can get their genome sequence inexpensively if they so desire. But where do we go from there is the question. Arguably one of the most important geneticists
of our time, Dr. Church helped initiate the Human Genome Project in 1984 and the Personal Genome Project in 2005. His lab was one of the first that showed CRISPR-Cas9 worked for precise gene editing in normal human cells. And he has been behind countless other scientific
innovations and disruptions, specifically in the world of precision genome sequencing. Dr. Church has described the key theme of his lab as technology development, radical transformative technologies. So, let's talk about those. George, it's hard to know where to start, large genome-writing history, universal donor cells, multiplex editing, and the ability to perform thousands of edits in a single cell, organoids. But maybe we can just start with the
Human Genome Project. What is the back story, and how did we get to present day, an era of writing? [Dr. Church]: Oh, thank you, Rhonda. Let's see. Let's start with...I think we didn't realize that we were on an exponential when we started sequencing. I got introduced to it through RNA sequencing. There wasn't DNA sequencing. And then Wally Gilbert was my mentor as a graduate student, and he and his team invented it in 1977. Independently. Fred Sanger published a paper that same year.
It took a little longer for the Sanger method to get implemented. But what happened was, very quickly, we got to a point where we were talking about doing a whole genome, mainly at the Department of Energy's bequest, in 1984. They asked a harder problem, which was, how do you estimate mutation rates to the consequence of energy? And we felt...you know, a handful or maybe 10 scientists in what would later be called genomics said, "Well, we can't do that, but what we might be able to do is get one genome, a reference genome." That consolation prize was big enough that Charles DeLisi at the Department of Energy just started writing checks. I mean, he didn't wait for an act of Congress or anything, just
had money for this kind of R&D because of health effects. And then it took about three years. I was transitioning from postdoc to professor, and my lab got one of the first two Genome Project grants. And then the NIH...it took about three years before the NIH got involved, but they got involved in a big way because they felt they were a more appropriate vehicle for anything health related than the Department of Energy. And they did it, kind of, in a teamwork with maybe 30% DOE and 70% NIH in the United States component, plus lots of international collaboration, really starting in 1990 with a 15-year goal. There was a lot of talk of cutting corners at the beginning. I didn't necessarily call it that, but there was a lot of people trying to do 1X coverage, meaning doing every base pair...reading it exactly once. And I didn't like most of these corner-cutting things, but I was
the most junior member of the project from the beginning. I didn't have a lot of sway. I also felt that we should put technology development upfront because that could reduce the price, and then we could do a lot more than one genome for a lot less than $3 billion. As soon as some of the senior members of the visionary team, like Jim Watson, who came in later, started representing...drumming up support in Congress, it became evident that we would have $3 billion. And then the motivation for bringing the price down disappeared for a few years...a decade. Then instead of...some of the corner cutting was to not worry too much about the repetitive sequences, which in the case of the fruit fly was about half the genome.
Yeah. You know, it doesn't matter. And at one point, they were going to just do the coding regions, which is 1%. It turns out, we still haven't identified the 1% coding regions that would not have been a shortcut. So a lot of these shortcuts were really ill-conceived. But fortunately, we did get a decent 92% of the genome and declared victory.
I want to make sure we've got that before we go on to writing genomes. That's a whole another topic. [Dr. Patrick]: Do we need more reference genomes? And what are your expectations of finding new tools elsewhere in the evolutionary tree? [Dr. Church]: Well, so, yes, we certainly need more genomes. It's not just the reference. It's the population variation that's important. The variation is at least as important as the reference, and it helps us make sure we've got a good reference. So you can call that the reference. It is growing recognition that we can represent the reference as a diversity.
We are finding tools in the genomes. One of the nuances that developed, the first kind of recommendations for maybe 1984, 1985, 1986 was the Human Genome as if there were one and as if there weren't any other genomes. And I kept advocating for genome comparisons because, when you compare two genomes, that's almost as good as an experiment, but it gives you a richer formulation for exploration. And we have...part of that genome comparison has resulted in new tool discovery, and so it's kind of a positive feedback loop. You sequence some genomes, you find some tools, use those to read and write genomes, find some more tools, and so on.
I don't know where that ends, but I do think that synthetic biology is probably ultimately unlimited, while the diversity on earth, even though it's vast, is more limited. Almost by definition, we can explore more than currently exists, at least initially in narrow corridors, where we're looking at, you know, specific tool-building ecosystem, restoration, and medical consequences. I think there's a rich field of...let's say you had one book, and that's the only book you had. You could read it and reread it and reread it, and you keep learning more and more. But as soon as you start writing books, now you've got millions of them. That's how I think of the synthetic biology or writing of genomes. [Dr. Patrick]: I've read a quote...kind of reminds me of the quote that I read
from you that stated, "I have speculated that essentially everything that we can currently manufacture today without biology, we will be able to manufacture with biology and with potential advantages. Biology is intrinsically atomically precise, and it's scalable to cover the whole planet essentially for free." That's pretty revolutionary, I mean... [Dr. Church]: Yeah. That's accurate reflection of how I felt then and how I feel today. Why is it reasonable?
They are atomically precise. Biology does not yet gracefully use the entire periodic table or all the chemical bonds that you might want to make out of that periodic pairs of elements. It comes pretty quick...pretty close. It uses a lot of inorganic bonds that might surprise some people. There are biological systems if you look widely enough. And now we're not talking
about necessarily, you know, your enzymatic tools, which might have been implied in the previous, but, you know, all the things that...all the chemistry and physics that biology uses. They can make things that are fiber optics like and sponges. You can make semiconductors, ferromagnetic materials that help like compass. There are
all kinds of dichroics and gratings that generate colors, you know, and the list goes on. Materials that are used, either naturally or where the enzymatic apparatus that is used actually can...if you give it a new set of elements, it will incorporate those. You could say misincorporate them. But the point is they're atomically precise in that you can
reproducibly make a molecule with thousands of atoms, and the next molecule over has exactly 1000 atoms and exactly the same configuration, you know, off by less than an atomic bond in length. This is not something that happens in Silicon Valley or other, you know, worldwide manufacturing of silicon-based circuits or any other inorganic circuits. It is so far unique to biology. Another thing that's unique to biology is ability to replicate, so you can make a copy of yourself. So to make a copy...you know, the idea that a cell phone can
make a copy of a cell phone is ludicrous so far, but there might be a use of hybrid system where we use biological inspiration...electronics inspiration to make hybrid devices that can replicate use of the full periodic table and do, you know, a few things that electronics is a little bit better at, better at telecommunications at certain wavelengths, very hazardous wavelengths like X-ray and gamma, as well as the other end of the spectrum, the radio. [Dr. Patrick]: Let's talk about how writing the human genome may help us better understand it. So Francis Collins described the working draft of the human genome as the first glimpse
of our own instruction book. But today, many scientists believe that to truly understand the instruction book, we also have to write it. Can you explain why that is? [Dr. Church]: Right. Well, I'm not sure I would say, have to, but it is certainly very advantageous. I should mention that we don't even have the full instruction book
of any human being yet. We declared victory in 2001 on a kind of a rough draft of 92%. Actually, it was considered the final draft of a rough draft in 2001. It was final draft in 2004, but it was still haploid, meaning it was just one genome, while all...essentially, all of us are diploid, inheritance from mother and father, except for our gametes. So, the sequence we have, the one human genome that we have is not of a gamete. It's of a strange haploid cell.
But that's not the big barrier to understanding. The big barriers, as you say, is...in order to understand how something works and also in order to develop new technologies, you need to be able to write and edit and alter. And you understand it because you'll say, "Gee, I have no idea..." It's like reverse engineering electronic circuit or some software. "So I have no idea what this code does. Let's change it." And then you say, "Oh, that changes the calendar. Okay. So then that code does calendar." Or in case the biology, you'll take a piece out, and now it no longer handles, you know, glucose. So you say, "Okay, that's part of the glucose monitoring system." And you
can just get through that, and you can get to more and more nuanced changes for discovery's sake, but it's often entangled with not just discovering, but making useful synthetic biology. You'll have a challenge that you'll have out there, and that will drive the reading and writing technology forward. It will drive our creativity in terms of how these things can positively influence society and ecosystems. [Dr. Patrick]: What's the goal of writing a large or a whole genome or an entire chromosome?
[Dr. Church]: So there are a few ideas that have come up, where something at a genomic scale is more desirable than a single gene. So a huge fraction of recombinant at the end, so the GMOs and synthetic biology, historically has been changing one or two genes. It doesn't make sense to synthesize whole genomes if you just want to change one or two genes. But more and more, we're seeing advantages of changing so many genes. You might as well rewrite the thing. As an example of that, we have a project to change the genetic code to make any cell resistant to all viruses. And we just published a paper where we think we did that. And
the way that it works is that the virus...all viruses, as far as we know, depend on the host genetic code, the translation ribosomal machinery. You can change the code without hurting the host. The host could be a cell. It could be an organism. So far we've only done it in one
industrial organism, E. coli. But anyway, if you change that enough, the virus can't mutate. There'd be too many changes that are required to get the virus to be back to its healthy state. And we think that this is completely general in that, essentially, every plant, microbe, and animal on earth, shares a very similar genetic code to one another, and in any case, have a genetic code that they share with the viruses. And if you take it offline, change it enough, like sometimes as few as two codons, let's say two codons, a code for serine, leucine, and arginine are our favorites ones because they have so many codons for each. They're triplets of ACG NT, so like AAA codes for lysine,
the amino acid lysine. There's 64 of those, and if you change one, you get a new genetic code. If you can change two, and now you get something that's multivirus resistant. So that's an example where you have to make so many changes, tens of thousands of changes genome-wide. And they're interspersed throughout the genome. You might as well just synthesize it, and that's what was done.
Another example is of the extinction. There are a number of changes you might have to make in order to bring back some physiology like cold resistance and all the traits that go along with cold resistance, may be scattered around enough that you're...you can think of it either as highly multiplex editing or as a complete rewrite. And even when you do a complete rewrite,
you're not changing every single base pair, all 3 billion times 2 bases. You're leaving them mostly intact. You've chemically synthesized it, but it's still useful to think of it as a lot of edits. The maximum number of edits we've done by editing, meaning having an enzyme that's targeted at a particular place, is 24,000. The maximum we've done by synthesis is almost the same amount, although we have synthetic projects which are now getting close to done at 60,000. But then we're going to take the editing up to a million pretty soon. They go back and forth. There's a technical leapfrogging that goes on between editing and writing of genomes.
[Dr. Patrick]: This, sort of, moonshot goal of changing genomes or writing large genomes in a way writing, editing them, where, you know, as you mentioned, you make let's say...you take a human cell in a Petri dish, make it resistant to viruses or, you know, make it capable of synthesizing essential micronutrients that we usually have to get from our diet. Like, even if it just sits in a Petri dish forever, and that's the only place it goes, to me, there's something very just fundamentally, you know, awe-inspiring about that. Is it, kind of, like, along the lines of your thinking with doing some of those things? [Dr. Church]: Yeah. I think the community, the synthetic biology community has responded in the same kind of awe-inspiring the initiation of this kind of project. I hesitate to call it a
moonshot because I actually think the moonshot was not as inspiring to me as the satellites, the GPS satellites, the weather satellites, and, you know, surveillance of land. And the same thing goes for other big projects. The Genome Project wasn't as impressive to me as reducing the cost project, $1000 Genome Project, sort of, the technology development. And the Manhattan Project was certainly not as attractive to me as say the projects for nuclear fusion, which could have...all of these things could have started much earlier on. They sound may be a little bit harder, but what they have in common is they're very much more consciously aimed at positive societal consequences. And I think it's a little easier to get everybody excited about these, sort of, things.
And I think being able to make industrial microorganisms, plants, and animals are important for ecosystems and agriculture and human stem cells. They won't stay in that Petri plate. They will make their way into cell therapies in humans. And if we're going to fix something that's broken that you can fix with blood cells, you might as well have those blood cells be resistant to all viruses as well if that is shown to be safe and effective by the FDA and similar organizations. [Dr. Patrick]: You, kind of, alluded to this earlier, but how do you think the Vertebrate Genomes Project will affect the field of genetics and biology? [Dr. Church]: So the Vertebrates Genome Project, I think, is just indicative of
our wish to sequence the whole biosphere. Vertebrates in particular are helpful because they often constitute keystone species in the wild. And I hope...I think there's a reason to believe that we will be restoring more and more of the non-urban environment to wilderness. Certainly, you can see about 1000 successful
rewilding projects, a local rewilding. So, the most famous one is probably restoring the wolves to Yellowstone after 70 years. They had a typical keystone effect. That ripple effect was anticipated and worked out, which was they changed the abundance of large herbivores, which then changed the abundance of the willows and other trees, which changed the beavers' behavior which changed the lakes, which resulted in aquaculture. So just introducing one vertebrate had all this ripple effect. That's one reason to do it, but there are many others. And if we are causing the extinction of many species, we are also causing the hybridization, which is the creation of new species. It's not clear that we're making extinction faster or more significant than hybrid than new species. I think our
gut feeling is that we are, but it's not proven yet. But in any case, we need to do that survey to see in detail what we're doing, and in some cases, we need to freeze away as many organisms as possible. But we shouldn't be confused that freezing it away or putting it in a database doesn't mean that it's going to be easy or even possible to restore. We need to do everything. We need to document, freeze, and protect what is already there by shrinking our agricultural use possibly by, you know, 10 or 100 fold. I think that's totally
feasible to do with synthetic biology and other new tools that we have. [Dr. Patrick]: Can you talk about the advantages of perhaps computer-aided design of genomes, the, sort of, aspirational software, heart and soul of the Genome Project, right? So in particular, I'm curious about advances in AI like those coming out of DeepMind such as the AlphaFold, and if they have special relevance for this, sort of, complex work.
[Dr. Church]: Right. So the genome consists of, 1% of it codes for proteins, and an AlphaFold is focused mostly on the proteins. There's some software for folding RNA and folding even the genome itself. That can either be predictive or it can be measured. So there's a lot of software that's used for looking through microscopes and
determining the structure and try and correlate that structure, again, by synthetic biology. You say, "Let's change the shape, not just the sequence, and see how that...see what function that affects." And that trial and error can go very quickly or even exponentially. Once you get going, you see the patterns and you start testing more and more sophisticated hypotheses.
But AlphaFold is not the only way to do it. So there are other machine learning-based methods, in fact, machine learning plot coupled with multiplex libraries, which can be in the millions or billions of synthetic molecules that act as...there are subtle variations or sometimes not so subtle various ones together. If you do machines and everything plus mega libraries, you're focusing on functionality rather than on structure. AlphaFold predicts a 3D structure. And to illustrate this, you have...let's
say you take a serine protease. It's called a serine protease because there's a very key serine right at the active site. And that serine has an oxygen that's part of the mechanism. If you change that oxygen, that hydroxyl to a hydrogen, it now becomes an alanine, and it's completely functionless. But the three-dimensional structure is completely preserved. It is
atomically precise throughout the structure, but it's a dead enzyme. So what's more interesting, I think, for most practical applications is studying what functional consequences are of substituting. And that applies not just to proteins, which AlphaFold predicts, but also the RNAs and DNAs. You want to know what the landscape of functionality is. And that can be done partly by phylogenetic evolutionary trees, where you line up. We now have tens of thousands
of examples of almost every major macromolecule in the cell, proteins, RNAs, and DNAs. And then using that, or you can...if you feel that's not enough that evolution hasn't provided you with enough diversity for your machine learning, you can generate your own data set. So, when they were learning chess, and Go, they would have the computer play these games against itself to generate more data. Big data is good in the case of machine learning. And in our case, we use these mega libraries, these millions and billions, even trillions, that act as a kind of a wetware computer. It can do all this computing, and you can read it out in terms of the sequencing
that you were talking about earlier and barcodes. So you can barcode all these molecules and combinations of molecules. And so you can think of these synthetic biology libraries as an honorary computation device, what you use together with the machine learning, which is typically done on a classic von Neumann machine, meaning an ordinary kind of computer that most of us would recognize. [Dr. Patrick]: In your opinion, how has the idea of biology as a software,
reading, writing, programming, and debugging, sort of, held up over time? [Dr. Church]: Well, metaphors are imperfect. I think the advantages outweigh the disadvantages of using these metaphors. I'm a programmer since, you know, the mid-60s as a pre-teen, and I've been programming both computers and biology. And I find that the metaphor really works for me personally. Where it breaks down a little bit is when you say that your goals should be set by the goals of the metaphor, in other words, that in the early days of synthetic biology, there were multiple camps. And one of them was the camp where we're going to have "AND" gates and "OR" gates, and "if-then-elses," and all the Boolean logic that might characterize a certain category of computation electronics. And I felt and I still feel that there's a lot to be...a lot of interesting biology
that occurs with analog circuits. And we've kind of lost track (or some of us) have lost track of that key component of electronics, but it is there. But anyway, the analogs, there's the evolution, where you, - typically, when you make a cell phone, as far as I know, you make a very small number of prototypes that are very similar to one another, and you test them out. But in biology, like I've said a couple times now, you can make billions and trillions, and you can do accelerated evolution, while with most, you know, bridge building and building trains and jets and cell phones, you really don't have that luxury of making trillions of them and seeing which one works best. [Dr. Patrick]: How fast is the field of synthetic biology advancing? Are you excited about where the field's going? Do you have any concerns or fears? [Dr. Church]: Yeah. I would say both excitement
and concerns. And I think that applies to all technologies. I think that we need more radical and just... You know, it could be positively disruptive, but you have to think of all the potential negatives. So, it is happening exponentially. How fast? It's doubles at least once a year. Sometimes we'll get a factor of 10 per year as measured by both reading
and writing DNA. Most of the 10, 20, 30 million fold has occurred within the last decade or two. So it's potentially faster than Moore's Law for electronics. And it has, kind of, this atomic limit that's very comfortable programming precise atomic positions using biology. Now, those sorts of tools we've got. We're getting more and more. Now, on the negative side, we need to be...we have good government agencies that we should be very supportive of, both intellectually and financially like the FDA, the EPA, the USDA, and their foreign equivalents.
These are not sufficient, though, because they're things like equitable distribution of technology. We want everybody on the planet to have...at least have a chance to, not only theoretically have access to because the price is right, but also have the education or the dialogue that allows them to evaluate whether they...to know that it exists and to evaluate, what, they want to use it or not and whether it's good for them. So, it's not sufficient to just, like, lob over a free piece of software like, you know, GPS software, and they don't know what the satellites are doing and so forth. Now, modern uses of GPS like Google Maps is fairly accessible, but there's almost no technology that's completely equally accessible. You know, clean water, roads, you know, cell phones are getting accessible in remote parts of the world. The only thing that is truly
accessible equally that I can think of offhand is a biotechnology, and that is smallpox. It's completely extinct, and so you don't have to constantly be bringing out a new drug or a new vaccine that maybe not everybody can afford, every government can give out for free. But smallpox extinction is something we can give out for free generation after generation. So, I'm looking for more and more of those. You know, bringing down the price of reading and writing DNA by 10 million fold is just a start.
We should look for that in almost everything we do with synthetic biology. [Dr. Patrick]: Do we need the NIH to embrace the Human Genome Write Project like they did the read? Or is that, sort of, already happening? [Dr. Church]: I think it would be lovely if they did. I think we need to
pursue multiple routes, philanthropy, industry, government, multiple...again, having DOE and NIH in the game was helpful, but there's a number of others that are interested in the Genome Project Write, NSF, ARPA, DARPA, and IARPA. And these have supported it in various forms, have supported synthetic biology, Genome Project-write, has, yeah, been...it's the heir of all those wonderful funding sources. As long as it has a vision that includes something that is net positive for society, there will be a way and hopefully multiple different ways for different flavors of it. One of the early flagship challenges is this resistance to all viruses in multiple organisms. I think that's something that can be clearly articulated, and
it has, I think, a much higher positives and negatives. And most of the negatives, I think, we can mitigate by thinking of all the possible downsides and how to protect against them. [Dr. Patrick]: Let's take a dive into some of the gene editing tools and whatnot a little bit. Over the last 10 years since Jennifer Doudna and colleagues first developed CRISPR gene editing, there's been a lot of excitement about it. Your lab was one of the first to show that gene editing using CRISPR-Cas9 could be done in normal human cells. So acknowledging the undoubtedly, like,
revolutionary impact of CRISPR, do you think it's possible it's been overhyped from the standpoint of the public at large not having a more comprehensive or appropriate understanding of where it, sort of, fits within the existing toolsets of synthetic biology? [Dr. Church]: Yeah. I hesitate to use the word hype because it implies somebody is being hyperbolic. It was kind of a team effort of...it's wonderful that we're bringing it, any part of reading and writing genomes in synthetic biology to people's attention or science, for that matter. This is one of the more exciting things in science right now is getting people... It's not just about CRISPR. First of all, you can't really edit if you can't read. So I think the big revolution here is being
able to read the genomes. You read them at the beginning to find the tools. You read them again to decide what your goals of editing are. And then you read it a few times to make sure your editing is going well. Then you read it again to see that the edit that you made has the physiological consequences, which increasingly we're using DNA reading as a way of or RNA reading to see how the physiology is going, the so-called epigenomics for physiology. So, that reading is important. Another thing that's important is there was some pretty good editing methods that are still in use to predate CRISPR, notably homologous recombination, which Smithies and Capecchi got the Nobel prize for decades before Jennifer and Emmanuelle.
I'm a big fan of Jennifer and Emmanuelle, by the way. We've started a few companies together, Jennifer and I. But there's homologous recombination, which is very powerful. It's precise and over large distances, while CRISPR tends to be imprecise and/or small in scope. Another one that dates back two decades before CRISPR is SSAPs or lambda red as it's sometimes called is a way of getting precise editing. And that's what we actually used
to...around 2009 to make libraries of billions of edited cells in a day, a single person. That shows some of the power. And the other evidence of its power was that that was...the first completely recoded genome was done mostly a combination of SSAPs and recombinases, which is also very precise. CRISPR was basically a hatchet, and I sometimes call it genome vandalism.
So, I think we need to embrace all of these methods and a few more that are coming now, deaminases that can be done with and without CRISPR and more sophisticated SSAPs and integrases, transposonases. So, it's a rich... I think it's okay if the public just latches onto one aspect of it, but it'd be nice...it is nice whenever a more nuanced and visionary form where it illustrates the importance of reading and other more precise and larger scale editing and writing where you write...synthesize something from scratch, and usually pop it in by some...it
could be popped in by CRISPR but more commonly is popped in using recombinases or integrases. [Dr. Patrick]: What about some of the existing capabilities of, you know, gene editing therapy, you know, things that have been done, you know, in transgenic models for, you know, a decade at least or more, you know, so deleting versus addition, you know, of a missing gene? [Dr. Church]: Right. Yeah. So, you can think of CRISPR as a subset of editing. Editing is a subset of genome engineering, and genome engineering is not a subset of, but it's kind of a Venn diagram overlapping set with therapies and GMOs and so forth. So, most gene therapies that have been approved are adding genes, and this is done typically without CRISPR. And, you know, when you have a genetic disease, you're missing a gene, so you don't really want to edit necessarily. You want to add it back in. As you grow older, a lot of your gene
products...your gene expression is dropping down. One way to deal with that would boost it back up, and we've explored these sorts of things. The use of gene therapy, putting in a missing gene, and, in fact, editing for that matter for rare genetic diseases is by its nature expensive. It's millions of dollars per person over a lifetime, partly because the R&D costs and the palliative care and all sorts of health care for someone who has a very severe disease that might have died young years ago, but thanks to the Orphan Drug Act and others, they can now lead closer to normal life but at millions of dollars. It's great that we'll keep developing these gene therapies and better ways of delivery. Oh,
I forgot to mention, delivery is another thing that's sometimes missed when people just shout CRISPR. You have to get it to the right place, the right dose, the right time, maybe to turn off when it's done its job. So keep it off target...keep it off target, so minimum. So anyway, the delivery. An alternative to this expensive solution is a much lower cost one, which is genetic counseling, where you basically tell people before they get married, before the preconception or sometimes post-conception, that they're at risk. They themselves are carriers. They are healthy. They will be healthy. But if they marry someone that has the same carrier status, they put their children at risk. So there's the two methods.
I think a lot of the Western world tends to go towards the interventionism, you know, reactive medicine where we'll spend millions of dollars, you know, by not pursuing preventative medicine. But the preventative medicine, in this case, is, you know, low hundreds of dollars just to know yourself to know how to keep your children healthy by making preconception choices. [Dr. Patrick]: Yeah. I will probably circle back to a little bit more of that in a minute, but since we're talking about...you mentioned a few other types of, you know, gene editing, the deaminases, and you've talked about this multiplex editing.
What does it mean to be able to go, you know, to performing 26,000 edits or you said, I mean, a million...potentially a million edits in human cells, you know, versus the previous record of something like 62? I mean, what applications does this most impact? Is it, you know, the large genome creation or tissue engineering or germline? [Dr. Church]: Right. Our previous record of 62 or 42, depending on how you count it, was in pigs, and it was for tissue engineering. It was germline. So germline is kind of off the table for humans, in part because there is no clearly articulated medical need, and the time for discovering safety and efficacy is over a lifetime which is, you know, unaffordable and ill-advised. So anyway. But germline certainly work, gets into humans via pigs. This has been...the idea of transplanting
organs from animals to humans goes back at least to the 1960s, where a chimpanzee kidney survived for nine months in a school teacher who went back to teach and, you know, was normal for nine months. That was the exception then and it would be the exception now except for the synthetic biology that we do on the germline of pigs, which now made it into many pre-clinical primate transplant trials, pig-to-primate, and a few pig to human trials that are going on. Primate survival looks like around 600 days so far, and they're still...a
couple of them are still alive at 500, 600 days. We're going to keep improving these. That's in the order of 40 to 60 edits per genome in the germline. The multivirus resistance requires more than that. Some things that are done for diversity and ecosystem maintenance may involve even more. They're a type of tape recorder, something that's called a flight recorder, so it's analogous to planes, that record a lot of data. But typically, you don't read it, so a lot of writing, not much reading,
unless the plane goes down. Then you'll look at selective regions for debugging what went wrong. That same thing could be put into the bodies of plants, animals, and even humans because it's a very compact reporting device of the physiological states of every cell in the body. We've shown this works, sort of, in the scale of 60 to 24,000. That's probably...our first effort at making a million edits will be in the form of these molecular flight recorders. So those are a few examples, but the number
will grow as soon as we get more than a handful of people working on these visionary projects. But we'll see a blossoming of all sorts of creative uses of making multiplex editing. I think non-multiplex editing will become the exception. [Dr. Patrick]: So as you mentioned your lab, you know, gene-edited pigs, and you enhance them by
making them resistant to some retroviruses. Do you think, you know, as a more visionary, kind of, question, that you could use, you know, more precise gene editing, the deaminases or CRISPR, whatever, to eliminate viral spillover events from livestock to humans? So, I mean, there's a lot of viruses that originate from livestock when we're raising animals in captivity. [Dr. Church]: Yes, this is important. The viruses that we got rid of were endogenous retroviruses,
meaning they're built into the pig genome of every pig on the planet. And they have been shown to infect human cells and to replicate and go into other human cells. So this is particularly bad scenario in immune-compromised patients. And the FDA recognized this decades ago and really was, I think, pleased to see progress being made on eliminating them from the germline of the pigs. But in addition to viruses that are built into the germline of animals and humans, there are viruses coming in from outside. And we just published the first example. This is with Luhan Yang's team. She was a graduate student and a postdoctoral fellow in my lab and
co-founded eGenesis and Qihan for making cell therapies and organ therapies. But anyway. As a side project, we published a paper on getting rid of African swine fever virus by making CRISPR to attack the viral DNA. What CRISPR originally evolved to do is to take out bacterial viruses. We think this is the first case of using CRISPR in a practical sense for eliminating mammalian viruses from the environment. It's using CRISPR against mammalian viruses. But zoonotic diseases is bigger than that. If we could make a huge fraction of plants, animals, and humans resistant to those viruses because of their genetic code, that actually anticipates viruses we haven't even seen yet. It should handle all natural viruses, so like, you know, Marburg, Ebola, HIV, CRISPR. These would not have been surprises. They would
have been surprises to scientists but not to this cancer resistance...sorry, virus-resistant cells. [Dr. Patrick]: So it sounds like CRISPR seems to be uniquely positioned for that, you know, type of use. [Dr. Church]: Well, not necessarily... so I like CRISPR. I love CRISPR. I personally benefited from
it, but it is...I like to balance it. There are other nucleases that some people claim are more specific, less off target. There are deaminases that don't involve CRISPR. So I wouldn't say...term unique is too strong. We have a lot of tools in the toolbox. A lot of it has to do with delivery and testing too. Testing is a big deal, which is somewhat swept under the rug
when we're just...it's just like, "All we have to do is design, you know, CRISPR to take care of everything." But there's a lot of reading and, you know, synthesis which isn't CRISPR. And then the delivery and testing. So it's integrated whole. It doesn't require CRISPR. [Dr. Patrick]: So another technology would be base editing, which, you know,
doesn't involve double-stranded breaks and DNA. And I know there's a phase 1B trial with the PCSK9 target. They're targeting at gene target for the liver as a potential treatment for the hypercholesterolemia familial form. You know, I just read about this recently and pretty excited. You know, I know people that are taking the anti-PCSK9 antibodies, which are very expensive, and you have to get them every two to four weeks. It'll be interesting to see, you know, if the base editing could be a one-and-done treatment, do you think? [Dr. Church]: That is one of the advantages of gene therapy in general, whether it's editing or adding genes.
Yeah, I think that a lot of our diseases are diseases of wealth. I mean, we used to have much more active vegan diets, you know, low in overall carbohydrates mainly because it was low in calories altogether. And so diabetes and some of the cardiovascular diseases didn't affect us. Also, we didn't live as long in general. So it was less of an issue. But PCSK9 is...it looks like it's shaping up to be a terrific example of something that basically all humans can be thought of as having the same disease, and therefore it's a large market could be low cost. Aging is another or a variety of age-related diseases that might have a common core
where we are programmed to die at a certain age. The mice diet at two years old, bowhead whales at 200, humans somewhere in between. And so that's probably negotiable. Now, PCSK9 is not solving aging in general. It's a very specific thing that may be common to most humans. It was de-risked because there were a few humans that were walking around that were basically double null for both copies of their PCSK9 from mom and dad. That kind of showed us that it was going to be safe and effective, although there's still quite a bit of long-term studies that have to be shown to make sure it doesn't cause early onset neurodegeneration in the particular way that we're implementing it, which is not germline, which is how it...the
people that previously had PCSK9 nulls for germline via natural mutations. [Dr. Patrick]: Since you mentioned aging, it sounds like...and I think you think aging is fundamentally a program. It's a really interesting idea, one that's probably...it's got many implications, especially when we're thinking about whether or not we can mitigate aging or potentially cure it. So could you talk
about your perspectives on that, what you think it might mean for the future of human aging? [Dr. Church]: Well, so what we're mostly aiming for is serious diseases of aging. They may have relatively little in common in terms of what organ is affected, you know, what system. There may be 9 or 10 different pathways that can be affected, the so-called hallmarks of aging. So it's a great diversity. There is a school of thought that they have a small core set of systems biology, systems medicine that if you get at that core, you can change the clock. You can make it shorter
as in mice or longer as in bowhead whale. And then you can rejuvenate. There is rejuvenation that occurs whenever you go through gametogenesis and fertilization, sort of, normal reproduction. You reset the age clock, and you also reset it when you do something unnatural, which is cloning where you take the nucleus from an old animal and put it into rejuvenating environment of an egg.
And there's also a rejuvenation process that occurs unnaturally when you use transcription factors. These are DNA binding proteins that regulate the expression of genes. Four of them so-called Yamanaka factors or OSKM as an abbreviation. These will very convincingly take a very old cell and turn it into a very young cell, maybe like, say, a skin cell from 80 year old. It will take on many of the characteristic...most of the significant characteristics of an embryonic
cell in that it can produce almost all the tissues of the body, probably all of them, except for the extra-embryonic. And the parts aren't part of the body that contribute to the early embryogenesis. So those are a few, and there are many others that shown that the blood...what's in the blood of older and younger animals can influence one another. The older blood makes the younger ones old, and the young blood makes the older animals younger by a variety of biomarkers and disease-related things. I fall into the...there's two schools, at least two schools of thought here. There's a damage
school where you have to go in there and kind of micromanage a surgery to fix the damage as your surgeon might fix a damaged broken arm. Then there's the epigenetics school where it says that if you convince the cell that it's young, it will fix itself to a large extent. There will be some exceptions. And we've seen that over and over these, you know, fertilization, cloning, and OSKM factors are three. Again, the bloodborne factors are four examples. And we need to reset all of the mechanisms, all nine hallmarks of aging in probably all of the tissue types of the body, at least the stem cells for each of the body parts, to have a shot at...we're aiming for youthfulness, lack of age-related diseases, so you should be youthful at an age where you normally would be unhealthy, even if you're not dying of any particular disease. That's what we're aiming for. It will be approved by the FDA
for specific indications for specific diseases of aging. But then if it really is getting at the core of aging, will it be immediately applicable to almost all of the diseases of aging? And aging just affects everything, almost every morbidity, mortality, even like accidental death. Infectious diseases like COVID has a very...and its cognitive consequences have very steep increases at around 60 years old. [Dr. Patrick]: So I recall, like, one of your former publications. I forgot what year. I think it was a PNAS one where you did gene therapy and added three
transcription factors to rodents, to mice, and there was some reversal of aging or biomarkers. And it was a TGF beta receptor and FGF21 and... [Dr. Church]: Alpha-klotho. Yeah.
[Dr. Patrick]: Klotho. Yeah. [Dr. Church]: Those were not transcription factors. Those were soluble factors. [Dr. Patrick]: That's right. Okay. [Dr. Church]: But we also did a separate experiment where we took three transcription
factors, OS and K, of OSKM, separate experiments, but delivered in similar ways, adeno-associated virus. Then we did some other experiments with follistatin and telomerase, so it affects the ends of the chromosomes, the telomeres. Follistatin is mostly muscle. But each of these has, you know, reproducible impact on hallmarks of aging, on biomarkers of aging, and diseases of aging. And it affects multiple diseases with about seven different categories of diseases that we've done now in mice. A subset of those have been tested in dogs now, aiming for a veterinary product.
The three that you mentioned, I think, have slight advantages, the fibroblast FGF21 and TGF beta. I should mention, that is an...the other two are natural alpha-klotho and fibro FGF21. But the TGF beta receptor is normally membrane-bound. We made a soluble form of it. So all three of them tend to be soluble, and they effectively act like the young blood in rejuvenating these mice and dogs, and hopefully, soon there'll be in human clinical trials. And that has the advantage that we don't yet have a good way of delivering to every cell in the body or every stem cell in the body. Remember I said delivery was very important and it's so important. We need to fix it. But anyway, in the meantime, we can deliver the genes to a subset of cells in various parts of the body, and then those subset will deliver the proteins, those three proteins you mentioned, more broadly. And so you can, in principle, affect the whole body by that
combination of two kind of tiers of delivery. That's the idea behind that. And the dogs is a particularly good conduit to humans because they're large mammals like humans. They live often in a human environment, eat humans...like sometimes eat human food. They have similar kind of emotions and bonding and eye contact and all the rest. And the owners can really sense
their states so they can get at more subtle, positive and negative consequences earlier. So anyways. And it's a product now that people care deeply about their pets. So, I'm very excited about, you know, Rejuvenate Bio, and Noah Davidson was a postdoc in my lab, and he started Rejuvenate Bio. It seems to be shaping up to be a good product line. [Dr. Patrick]: Yeah. It'll be exciting to follow these results. You kind of answered
one of my questions, which was, you know, a lot of the rodent research, particularly with aging, not a lot of it translates, you know, to humans. And, you know, one thing, in particular, I think, that is important to consider with human aging is that, you know, humans are exposed to disease and viruses. We're not in this, like, sterile lab environment, and we have these periods of real, like, illness and muscle disuse, and it's just very different than a rodent. But there's advantages to studying...to using rodents. What do you think? Why should we use rodents to study aging? [Dr. Church]: So, as a prelude to the experiment that you mentioned where we use three soluble
factors in dogs, we did 45 different gene therapies singly, one at a time, in rodents, mice to make sure...to find the subset of three that we wanted to test in rodents in combinations...various combinations. And then once we had settled on the 3 factors out of 45, then we moved into dogs, and then we'll next move into humans. So, you shouldn't blindly expect the rodent model to work. They're advantageous because they only live two years, so it's easy to see a longevity effect. We're not always
looking for longevity. We're usually looking for aging...reversal of age-related diseases because that's what the FDA wants as well. But we do occasionally measure longevity in the case of the follistatin and telomerase treatments. Those did show a pretty significant...very significant longevity effect on the rodents. So, even primate trials can be deceptive. There's a lot of differences in the way that
they're treated. In fact, in certain ways, dogs have, I think, a more similar environment, maybe, you know, more to their liking, more natural for them since they've been our companion for tens of thousands of years. But even dogs are not an ideal of larger... You know, pigs are very close to humans and their organs. That's why they're being used as transplants, but they're also imperfect. So an alternative to all of the animal models is human organoids, and those are getting increasingly accurate. Also, we can basically skip a lot of the developmental biology and go straight to a particular organ. We can't go via normal human development because there's a ban on letting human embryos develop past 14 days in a dish. But it's considered ethical to make an isolated heart
or even heart plus lungs, plus muscle, plus liver, plus neurons, but not a whole brain. As this is rapidly developing, we're exploring collectively with diverse set of voices, you know, how to do this in a way that's humane to the animals or develop all...you know, completely animal-independent strategies for both testing therapies, but also being the therapies. The organoids are increasingly moving their way into clinical trial. So for example, we showed restoration of a demyelinating disease in rodents by putting human organoids, brain organoids that contain...that remyelinate and are protected against the demyelinated mechanisms. So they're
supercells and that they are not just replacing the cells that were damaged because they just get damaged themselves, but they are resistant to the damage. And I think that is repeated over and over again in both cell therapies and organ therapies we're developing, is that the goal is not just to deal with the organ shortage or it's to have something that's enhanced is immunologically superior that is less rejected, resistance to pathogens, resistance to cancerous senescence, cryopreservation. All of these things have been demonstrated in animals, and now we want to either get them into the humans via cell or organ transplants. [Dr. Patrick]: If I remember correctly, you enhanced the brain organoid to...I think you edited it from APOE4, which if you're homozygous, you have like a 20-fold increased risk for Alzheimer's to APOE3, so making it more resilient against Alzheimer's, I guess.
[Dr. Church]: Correct. So that's somewhat...depending on how you look at the composition of various genes, now that particular case E4 is not the predominant allele. And so you might call it an enhancement. PCSK9 is very rare in the population, and so if you make everybody or a large fraction of population PCSK9 negative, that could be called an enhancement relative to the average. But it's not an enhancement relative to the minority. In the case of APOE3 or even APOE2, which is...that is rare than the E3 plus E4, and that would be an enhancement. But E3 over E4 is probably about closer to average.
But this whole definition of or this whole obsession of an enhancement seems odd because a huge fraction of our popular technologies are enhancements, you know, a smartphone makes us smarter in a certain way. It can also make us dumber. But the point is it has the capability of helping us navigate, help getting access to the world's facts and factoids. Cars, jets, so forth enhance our ability to locomote. So I think increasingly, we're going to recognize that the biotechnologies we're producing are not just reactive medicine where we're putting out fires. They're preventative medicine where we're...by enhancement, we're protecting ourselves. Like vaccines is a beautiful example of enhancement that protects us. We're far healthier than our ancestors were because of vaccines.
[Dr. Patrick]: I kind of would like to just move into...a little bit to the germline editing. We've kind of talked...alluded to it a little bit here and there. But you've said and previously that you felt like an obligation to be balanced. But you've also, of course, said it's important to focus on outcomes and not to rationalize addictions to future. And you're even involved in calling for a temporary moratorium on germline editing? [Dr. Church]: No. [Dr. Patrick]: No.
[Dr. Church]: I was opposed to the obsession with moratorium because we already have a moratorium on all new drugs. We don't allow anybody to use new drugs that haven't been through the FDA testing. So, yeah. So it sounds subtle, but I was concerned that we would be developing
germline where there's no need. But there's also no need for a moratorium because we have very good regulatory mechanisms for preventing that, sort of, thing from happening at a market scale. Now, a moratorium would not do anything more at the market scale and also would not do anything more at the individual scale. Both the FDA...and, in fact, most laws do not work on individuals that want to break the laws that are willing to accept the consequences or think they're above the consequences. And that's what happened in the case of germline. Someone either misinterpreted
willfully the guidelines or didn't think it was a law. In fact, he didn't get convicted of germline manipulation. This is He Juankul from China. He got convicted of, you know, not following the rules for getting the consent of the funding agencies and the patients and so forth. He actually did a pretty good job of getting the consent by some criteria. He spent an hour of videotaped counseling to make sure they understood what they were getting into.
But anyway, as far as I know, he was not convicted of germline therapy, but something more nuanced. And he's out now. The three years is up and he's out. And as far as we know, the children are healthy, which is more than you can say for the most revolutiona
2022-08-27