CRISPR-Cas3 May Save Us from Antibiotic Resistance. Podcast with Paul Garofolo

If you are alive today, you’ve benefited greatly from humankind’s ability to deftly handle infectious agents via antibiotic medications. In fact, perhaps the single greatest achievement of modern medicine remains our ability to thwart (many) deadly microorganisms.

But our high use of antibiotics has put great pressure on these pathogenic bacteria to mutate for their own survival. As a result, deadly strains of bacteria have become more virulent and more resistant to our medications, creating so-called “superbugs.” For instance, an increasing percentage of tuberculosis cases worldwide are attributed to bacterial forms that are resistant to multiple drugs and require more complex treatments with an array of different medications. Eventually, former wonder drugs, like penicillin, can be rendered ineffective.

This is a very concerning problem that is not likely to go away on its own. We need new ways to control bacterial infections. And we need them fast. This brings us to my guest today.

In this episode of humanOS Radio, I speak with Paul Garofolo. Paul is the CEO of Locus Biosciences, a biotech company that is developing a novel class of antimicrobials that take advantage of the CRISPR-Cas3 system. If you are unfamiliar with how this works, I’ll briefly explain here.

CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) is a family of DNA sequences found in bacteria that play roles in bacterial defenses. Basically, bacteria capture pieces of DNA from attacking bacteriophages (bacterial viruses), and these snippets are integrated into CRISPR arrays. These arrays enable bacteria to recognize viral invaders in the future – a bit like our how own adaptive immune systems remember previous infectious exposures to protect us from future exposures. Bacteria generate matching RNA snippets from these arrays that can target viral DNA and then use CRISPR associated (“Cas”) enzymes to cut the DNA and disable the virus.

There is a variety of new and emerging biomedical applications that use this system, perhaps most notably (and controversially) for human genome editing. But Locus Biosciences is going in a different direction. Instead, they are designing RNA segments that direct the enzyme Cas3 to chop up the bacterial DNA.

This could eventually form the basis of powerful antimicrobial therapeutics. First, it would offer a viable alternative to conventional antibiotics, and would presumably be less subject to the known mechanisms of antibiotic resistance. Second, this method could be targeted to specific pathogenic bacteria, thus leaving your friendly bugs alone. This seems like a win-win that could be both lifesaving and health promoting (depending on the context).

Locus Biosciences just raised $19M in series A funding for a human trial, from Artis Ventures, Tencent, Abstract Ventures, and North Carolina Biotechnology Center, so we are a little way off from this being made available to the public. But it’s truly exciting work, and if it pans out, it might be just in time. Listen below to learn more!

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Paul Garofolo - 00:00: I think it's fair to say within the next 10 to 15 years that's the new realm of drug products that are going to be coming out for humanity. It's pretty exciting stuff.

Kendall Kendrick - 00:19: HumanOS. Learn. Master. Achieve.

Dan Pardi - 00:33: Paul Garofolo, CEO of Locus Biosciences, welcome to HumanOS radio.

Paul Garofolo - 00:38: Hi Dan, how are you? It's great to be here today. Thank you for the invite.

Dan Pardi - 00:41: You are working on some of the most interesting stuff happening in science at the moment. Today, we're going to have a conversation around CRISPR and your company. You've recently raised around from ARTIS Ventures, and Tencent, and there was a few others in that series.

Paul Garofolo - 00:56: Abstract Ventures in the North Carolina Biotechnology Center.

Dan Pardi - 01:00: Right. Tell us a little more about CRISPR and the Cas3 system that you're working on.

Paul Garofolo - 01:05: I guess the best place to start is, what is CRISPR? Because I think more people come and they think about CRISPR today as a molecular pair of scissors that can be used to do gene editing in human cells. But actually what the origins of CRISPR are is a bacteria cell's immune system.

Dan Pardi - 01:25: Okay.

Paul Garofolo - 01:25: Fifty plus percent of the bacterium in the world actually have an indigenous CRISPR system inside of them that defends the cell. Unlike a human body or a larger organism, bacteria cells have to survive by themselves, and they come equipped with all the machinery that's needed to ensure their survival. What Locus is all about is our scientific founders, and scientific team we've surrounded them with, has essential learned how to hijack that immune system and bacteria, and turn it on itself.

02:06: CRISPR-Cas3 is a novel platform and existing system that's inside of most of the bacterial targets we're going after, that can be tricked to kill itself. We're working on a large group of drug products that are hopefully going to replace broad spectrum antibiotics, to be able to address the global threat of multidrug resistance.

Dan Pardi - 02:32: Wow.

Paul Garofolo - 02:32: We're pretty excited about the company and pretty excited about the technology. I think that's what attracted really visionary investors like Stuart Peterson and the ARTIS team, as well as David Wallerstein and the crew from Tencent. Couldn't be more pleased on that round and the partners that we now have on the team.

Dan Pardi - 02:50: Let's go a little bit deeper into CRISPR. So, CRISPR's an acronym: Clustered Regularly Interspaced Short Palindromic Repeats. This is inside of bacteria as a way to basically provide protection against what?

Paul Garofolo - 03:02: Well, a bacteria cell's gotta do all kinds of things by itself that normally a larger organism would work and consort with the immune system and other biological tools to do things like defend itself from invading viruses, or figure out how to potentially keep records of things that have attacked it in the future. It can defend itself against those if they show back up again. What CRISPR basically is, that whole notion of the short repeat. It takes a snippet, or a memory, of every single invading facier plasmid that comes into that bacteria cell to attempt to do something to it, normally kill it. It remembers it, it takes a small snippet of it's DNA, it adds it to a long list of other invaders that it's been able to ward off over time, and it essentially builds a memory or a vaccination card, if you will, that helps it to protect itself moving forward.

04:10: CRISPR is the system that's evolved over millions of years in bacteria to help ensure that bacteria cells survive and evolve. Pretty crazy, sophisticated, natural system, to be honest. I think a lot of science is now honing in on that CRISPR set of functionality, and really exploring a lot of ways of not just leveraging it inside of bacteria, but really porting that set of tools over to the human genome and trying to figure out how to do some pretty exciting things inside human cells as well.

Dan Pardi - 04:50: When was CRISPR discovered?

Paul Garofolo - 04:52: It think most people would generally agree that CRISPR was discovered inside of a set of scientific labs at DuPont. It was discovered in 2005-2006. It was in Dr. Barrangou's lab at DuPont that essentially the discovery was made, that these repeats that they were seeing inside of bacteria genome's was actually self-defense mechanism. It published a paper, probably 2000, don't quote me on the years, but I think it's 2007 that some of the original publications came out on CRISPR. Of course, what DuPont and others were really trying to do with the initial set of discoveries for a long time, was to figure out how to harness that immune system to make food better. To ward off invading viruses that would attack bacteria to help bacteria defend itself with even greater strength than the existing CRISPR systems.

06:03: DuPont used it in things like yogurt and cheese, to be able to do things like elongate the food spoilage process. And worked with the technology for what was almost a decade before it hopped out into a larger scientific purpose beyond, I would say, its bacterial origins.

Dan Pardi - 06:25: So we see that bacteria will add sequences, based off of the virus that it's exposed to. And how does it use that system to defend itself against that virus going forward?

Paul Garofolo - 06:35: What CRISPR has the ability to do, it to recruit different enzymes to be able to come and do different jobs. They've been able to find that in some bacterial species a particular set of Cas's exist, and in some bacterial species, multiple Cas's exist. Probably the most well known Cas is Cas9. The way that Cas9 works in the CRISPR addressing capability, it uses essentially a location that is defined by a pam to be able to identify a base pair location on a genome. Then it recruits the Cas9 enzyme, which that tool's function is to essentially behave like a pair of scissors. It makes a clean cut across both strands of the DNA at the exact location that you identify.

07:31: That particular Cas is really used to repair a cell. It can go in and actually cut a DNA at the location and open up an area for some repair to that cell to happen.

07:47: There are many different enzymes that not only have been discovered, but are likely yet to be discovered, that are part of this CRISPR system. The one that we've been able to leverage and harness is the one that's most prevalent in nature, which is Cas3. The way that a Cas3 works, is it uses that same addressing ability to go in and use and use a pam to identify a base pair location. Instead of making a double strand cut, it recruits what behaves like a PAC-MAN. It makes a small nick on one strand of the DNA, recruits that PAC-MAN, and it unzips, unwinds, and permanently destroys one strand of DNA around the entire genome of a bacteria cell. Effectively rendering the cell dead.

08:42: We use the Cas3 mechanism of action to spearhead a brand new portion science around a new type of programmed cell death. Data about it.

Dan Pardi - 08:53: So the Cas is the enzyme that finds the right location in the genome, and does the double stranded DNA cut. In this case the Cas3 acts like a PAC-MAN to then chip away the base pairs, and then in this case, it will actually kill the cells.

Paul Garofolo - 09:06: Yeah, that enzyme is really the tool. It might be a blunt edge pair of scissors, like a Cpf1, that sorta cuts off-setting cuts. It could be a Cas9 which does a precise cut. It could be a Cas3, which uses a PAC-MAN. At this point, there's probably at least, I would say, five to seven fairly well defined Cas's that folks know what they are and what they do. I'm not sure anybody actually knows how many more Cas's are out there, and what potentially that the tool could be used for.

09:42: It is pretty exciting, some of the activities that are happening in academia to figure out what all these different enzymes are that can be recruited, and what they can potentially do, not just in bacteria, but actually what they might be able to do in cells that they get ported over to. You know, human cells are probably the most exciting thing that germa can add.

Dan Pardi - 10:04: What are some of the more common applications with the Cas system? What's a range of utilities?

Paul Garofolo - 10:10: There are certainly efforts under way, not just to make modifications to genes in the human genome. But there's certainly a tremendous amount of work in the agriculture and food safety arenas to use that some functionality to enhance certain traits in crops, to fend against bacterial infections in food, and to potentially even use different CRISPR systems to enhance some of the yields in pretty much any bio-reacting process that might be out there industrially.

10:54: I think the favorite tool of most is certainly the Cas9. I think that provides a great tool to be able to edit a target. But there's certainly other Cas's out there to be able to do other types of functions. I guess the easiest way to think about it is, CRISPR is seemingly a toolbox. You might have a scalpel or a pair of scissors in there, but you probably also have a hammer and a screwdriver, and maybe a Phillip's head and other types of tools that may very well one day be able to do all kinds of fascinating things. Human cells, plant cells, the soil, and undoubtedly anywhere that bacteria exists, is a key target for the science to move.

Dan Pardi - 11:41: You also have application for modifying genes in living organisms. Is that correct?

Paul Garofolo - 11:46: In theory, absolutely. I think there's a tremendous number of companies that are actually working on that. I think technically, the way ... and again, there's probably a lot more advanced PhD level discussion that can be had on this. But, the interesting that about the was Cas9 works is it makes a single cut across a strand of DNA. If you're trying to edit a genome, and let's say, take a gene or take a string of genes out, you need to make two cuts. You need to make sure that that little snippet that you've essentially tried to remove doesn't repair itself. So, the challenge on the technical side for human genome editing is, I mean, it's fantastic. I think the promise of it is absolutely amazing. The thing to keep in mind is that the Cas9 as a tool makes this very precise cut. What you do after the cut to actually drive a repair or a deletion, repression, activation, those are the things that I think are really exciting.

13:04: I would say that what a lot of teams are looking to do is to actually modify sections of the genome, so that you can essentially do something like, let's say you're going after HIV. What you would be looking to do is to sort of make two cuts that are in the right locations to basically remove a string of genes that have embedded the HIV into the genome of the human cell.

13:34: When you look at things like cystic fibrosis or Duchenne's muscular dystrophy, the progress that these teams are making is phenomenal. Super exciting inventions are coming out that would allow you to look at those mutated cells and be able to potentially ... and this is a little bit out of my league in terms of what the company is focused on ... but to be able to go in and catch it early enough that you would be able to modify those cells so that the body can essentially repair itself. I mean, that's just amazing stuff. I think it will take a little longer to get those things approved. It will take time to get that through the FDA. But I think it's fair to say within the next 10 to 15 years. That's the new realm of drug products that are going to be coming out for humanity. It's pretty exciting stuff.

Dan Pardi - 14:29: I was reading about something very interesting this weekend. MIT researchers developed nanoparticles that deliver CRISPR genome editing system to modify genes in mice. What they were looking at is mutations in a gene called PCSK9 that regulates cholesterol levels. So this is a gene that is associated with a rare disorder called dominant familial hypercholesterolemia. Right now the FDA is looking at some antibody drugs that can inhibit that gene. But they have to take those antibodies for the rest of the patient's life. With this new nanoparticle delivery of CRISPR, they were able to permanently edit the gene following a single treatment. Instead of having to take these antibodies every single day, or whatever the measurement is, but forever, you could just in one swoop, edit it. And this thing that causes you to have too high levels of cholesterol, it's done. That's an example of the power of the system and why it's so interesting and cool.

Paul Garofolo - 15:18: Yeah, I agree with you wholeheartedly. The opportunity to be able to potentially be sitting on cures instead of treatments is really exciting. I think that delivery of Cas9 into target cells and the repeated delivery that it will take, amount of exposures to the drug product to be able to permanently change the human genome, and what the body is actually reproducing. That's going to take, more than likely, a single shot and be done. But will be, with time. I think, if you did a treatment for six months to a year and that was able to drive your care, I think you'd be really, really happy with that outcome. Rather than being on some kind of lifelong treatment to be able to address your disease state. No question about it.

Dan Pardi - 16:13: I also heard of a Chinese group that was knocking out the myostatin gene in beagles. Myostatin is a gene that inhibits muscle growth. So you can look at these Belgian blue bulls that have been genetically bred to have this enormous amount of muscle. With a myostatin gene missing, muscles just keep growing. And they look like they are about to win Mr. Olympia, they are so buff. They were able to successfully modify it. I did now hear of a human trying to modify myostatin in himself. What are your thoughts around the ethics involved in CRISPR technologies, and how is that going to play out?

Paul Garofolo - 16:46: I think, maybe in my perspective, escape by being in the field, but my sense of what technology has done for humanity through the course of time is that it helped to drive the process of evolution. To say that various technological advancements over the millennia have not had direct impact on the evolution of the human body is probably putting your head in the sand and hoping that time will pass you by. I look forward to the day when we're able to actually do things along the lines of edit the human body in early stages of life, before the onset of genetic disease takes effect. From the perspective of being able to save lives and/or meaningfully change lives, all of those pieces, I'm 100% bought into.

17:50: I'm sure they represent some challenges that need to be overcome. Delivery is probably one of the largest challenges, to make sure that we deliver correctly to the right targeted cells, and that that delivery is effective consistently. That's what I would say a lion's share of companies are focused on, and we're no different. Delivery is certainly something that needs to be commanded, and we need to make sure that that's right.

18:21: You get into this next level question with modifying the human genome, which is an ethical question about should the human body be enhanced in order to change the future of humanity. And that subject requires more discussion. I think that most people would agree that wholesale editing of the human body to, let's pick one that is likely highly controversial. Let's say you wanted to make super soldiers. And you wanted to create a new group of human beings that were essentially genetically programmed to fight. That's many decades away, to know which genes you would need to suppress, and which genes you would need to activate, in order to finely tune that type of outcome. But I'm not sure I would be supporting of that type of experimentation on actual human beings. I think the thing to keep in mind, though, is that not surprisingly, there are many regions of the world that are not going to agree with that.

Dan Pardi - 19:36: Yeah.

Paul Garofolo - 19:38: You're going to have to get into human genome editing competition, and there is an argument to made that you'll want to be in the front of that, and not in the back.

Dan Pardi - 19:50: What I fear more is access. If you have the right amount of resources, you can make your children smarter or stronger, taller. You could accelerate the caverns between the haves and the have-nots that way. And that's something that needs to be constantly part of the conversation.

Paul Garofolo - 20:06: I would agree with you wholeheartedly. Undoubtedly it will be for the few and not the many at the initial steps. How is that governed, if governed? Maybe that thing that I think is something to make sure you remember is these are not U.S. based decisions. You might look to the FDA to put their thumb down on being able to control the discussion and the debate. But it's a global technology, and frankly, just about every middle school and high school science student these days has access to CRISPR kits for their biology classes. This technology's available in every nook and cranny of the planet. There are going to be a lot more people than just civilized cultures that are working on some of these things, and you know I think honestly that's gonna have to be part of the discussion as well.

21:05: I think the other thing, though, that balances some of that discussion is the practical reality of the difficulty involved in doing some of this. Knowing exactly what genes to manipulate, that's still needs tremendous amounts of work, although certainly there in certain fields of study. The ability to deliver it to that location and get the exact outcome that you're looking at, that is very difficult science, and it'll take a good amount of time to get there. Which should, hopefully, provide the right amount of time to be able to have those moral and ethical discussion. I think they need to happen on a global scale, so that we get alignment on an international level to be able to determine which way do you take some of these powerful technologies.

22:00: The CRISPR train has certainly left the station. Science all around the world is now picking up that technology and running with it. It's not stoppable. The next sets of scientific breakthroughs in biological realm are very likely to come from CRISPR technologies.

Dan Pardi - 22:19: These are some very interesting and more controversial subjects. But you're not focusing on editing the human genome; you're focusing on modifying antibiotic resistant bacteria that we have now part of our world as well. Give us an example of one condition that is scary in terms of the degree to which it is resistant to antibiotics, and how the CRISPR-Cas3 system platform that you're working on could help to protect us.

Paul Garofolo - 22:47: There's probably a couple examples I could maybe give you to set the scale of it. Let me start with maybe an easier one that folks would be more familiar with. You know Hugh Hefner, I presume. You know that he recently passed away. He actually passed away from an antibiotic resistant strain of e coli that he contracted while he was in the hospital for other ailments. It was that multidrug resistant strain that e coli that unfortunately was the final straw for Hugh. The reality is, there's a number of bacterial strains, pathogens, that are out there that are now developing pretty massive resistance to standard antibiotics. Not just the standard ones, but the ones that are the more potent, higher tox profile drugs that are called "last line of defense" drugs.

23:49: Another really common, very familiar pathogen that's a pretty massive threat is MRSA, or multidrug resistant strains of staph. These are the things that really have unfortunate breakout opportunities. Staph: there's actually quite a few drugs that are being worked on for MRSA and BRSA infections. Maybe I'll start with some of the easier ones, but C. Diff, or Clostridium difficile, which is a pretty violent pathogen that you can get in your gut, if it's treated with a front line, you're in pretty good shape. But for the 25% of patients that, actually, that front line or standard of care has no effect on, you get into trouble pretty quickly. Of that group of 25% that moves into secondary, you end up facing a pretty long battle to try to get rid of that pathogenic threat. The end of that line is death. A fair percentage of those that get recurrent C. diff actually do pass away.

24:55: Acinetobacter, pseudomonas, even klebsiella, shigella, all of these bacteria that the CDC would list as essentially very high profile risks and/or threats. Those pathogens initially have very low MDR rates, maybe in the 5% to 7%. Over the course of 10 to 15 years, or at least the last 10 to 15 years, many of those are starting to creep up into the 20's. Some of them even getting further up, into the 30's.

Dan Pardi - 25:31: What's an MDR rate?

Paul Garofolo - 25:32: Multidrug Resistant rate. So of the number of infections that might be tracked, that percent which do not react to multiple antibiotics are considered MDR. So if you tried two or three different antibiotics and you can't get rid of your bacteria or your infection, you've got a problem. That would be considered an MDR strain of that bacteria that you're infected with.

25:58: You can get infections in pretty much any site of the body. Your back, the lungs, your mouth, your skin, your throat, sinus cavities, I mean, all the common places where you would get bacterial infections and common bacterial infections, that are now starting to be multidrug resistant threats. These are big risks to humanity. I think the one that maybe hits home for most is if a common staph strain can't be fought off by antibiotics, you can't have operating procedures anymore. You can't use a scalpel and cut open the human body and try to fix something else that's wrong, because you [inaudible 00:26:40] to get a staph infection from the operation, right in the hospital.

26:44: That's the one that has, I think, most government entities ... that reality. That might not be staph, that might be pseudomonas or that might be, unfortunately, something called CRE, or Carbapenem-resistant Enterobacteriaceae. CRE is the scientific name for something that most chronically know as what's called a hospital super bug. When CRE strains break out, they tend to be 50+% resistant to even last line of defense antibiotics like colistin. You're in serious, serious trouble if you're up against a CRE infection.

27:23: I think with time, expect to see these MDR rates rise. That has most folks in the medical community concerned that novel discoveries to combat these pathogenic threats are not keeping pace with the advent of these infections.

27:44: I'll tell you another one that's really scary, Dan, is gonorrhea. Gonorrhea's gotten out to be about 70% or 80% resistant at this stage. That's been a pretty exponential hike. Years ago, maybe 20 - 30 years ago, you could get a shot of penicillin and knock that out routinely, anywhere in the world. Now, if you get a gonorrhea infection, those first line treatments that you come up against are only about 20%, 30% effective. That's over the course of maybe like a 10 to 15 year period. Those are pretty scary numbers.

Dan Pardi - 28:25: Very. These multidrug resistant bacterias, they're getting more virulent, more resistant. And the solution is probably not creating more antibiotics, but probably something that is outside of the box. That's what you guys are doing. Instead of trying to focus on using CRISPR to modify the genes within animals, you're looking at modifying the genes within bacteria to kill them.

Paul Garofolo - 28:44: These multidrug resistant strains, all those essentially can have isolates that are taken. Think of getting a cotton swab in your mouth to be able to essentially identify that bacteria. Well, once you have all of those isolates back, you can actually sequence them and you can look for conserved genes. Genes that are common amongst a number of different strains. What our technology has the ability to do is to take those conserved genes and actually build RNA guides that, when they're injected into the target cells, if they find those conserved genes present, they'll recruit a catch 3 PAC-MAN and essentially trigger program cell death or suicide in that cell.

29:33: What our technology essentially does is for those infections that get past the first line of defense antibiotics. When you get a secondary or tertiary infection, and they've maybe tried one or two antibiotics, and they don't work, you can use our drug product to go in and very specifically remove that pathogenic threat from the human body. We can essentially program it to go after those MDR strains.

30:04: There are companies that are working on viral solutions with CRISPR systems. In fact, Stuart and his team at ARTIS have invested in that Cas9 play excision, to actually go after just that, although they're attacking HIV as a first indication. So, super exciting stuff.

30:22: We do exactly how you describe that. We would do that to go after, let's say, staph, or clostridium difficile, or C. diff, or pseudomonas, which is advanced pneumonic, let's say pneumonia in the lungs. Those types of infections in the human body, you may take a pill one day to be able to go into remove that pathogen from your stomach. You may inhale it from a standard inhaler, like you might take if you have any type of, let's say, asthma. You might spray it on if you got the infection on your skin or if maybe you had an operating incision site that you were working on. We hope to one day soon also have an IV drip so that you could have an IV bag that you could go straight into the blood stream and circulate throughout the body.

31:17: What we like to do is pair what's called that route of administration with the site of infection. If you have it in your gut or your lungs or your mouth, the trick is to get the drug product to the infected cells, so that you can inject your CRISPR commands and recruit the enzymes inside the targets, or put the enzymes into the target and activate the tools to do the killing.

Dan Pardi - 31:42: Will these ever be like a vaccine? If there are certain bacterial infections that many harbor, could you give a CRISPR pill to then address multiple potential bacterial infections that are in the body?

Paul Garofolo - 31:56: You would logically think that you could give something that's considered a prophylactic. We actually have some animal tests coming up to see whether a prophylactic approach would work more effectively, the same effectiveness, and/or less effectively as one of the treatment regimes. We hope to, by probably the middle of 2018, have a really strong sense of whether or not you could do that. In theory, because CRISPR is essentially a vaccination card, if you could somehow embed the right vent into that vaccination card, you could protect that potential cell moving forward. Unfortunately, that's the cell we're trying to get rid of.

Dan Pardi - 32:45: Right.

Paul Garofolo - 32:48: For our technology, however, there's a possibility that you could do something like that. But maybe I'll step back real quick, and do at least an intro to biology from maybe a basic level, since that's certainly in my forte, the basic description of biology.

Dan Pardi - 33:08: Please.

Paul Garofolo - 33:08: You have two types of cells that are inside the human body. You have human cells, and you have bacteria cells. That's essentially what's sitting inside of the human body. Each of those cells types has a natural predator. For human cells, you have viruses. For bacteria cells, you have something that's called a bacteriophage, which is essentially a bacterial virus. But the two of them have evolved together to try to keep balance. If you get Hepatitis C or HIV or one of those viruses, the really nasty ones, what they're trying to do is, they're trying to inject themselves and their commands into human cells, and then wreak havoc, which they do.

33:49: On the bacteria side, you have these bacteriophages, or some people like to call them phages. But you have a bacteriophage which is a virus, and it does the same thing. It attacks a specific type of bacteria and it injects, usually, copies of itself with commands to replicate inside that host. So you have this natural growth of the bacteria and this natural growth of the bacteriophage that sort-of try to out compete each other and bring bacterial infection back into balance.

34:24: What we're doing is we're actually hijacking those phage and putting our CRISPR RNA guides and actual Cas3 enzyme into those bacteriophage, sending those into the human body, using that as our delivery mechanism, and then injecting the CRISPR systems into the target cells. Your ability to stick to either the bacteria side or the human cell side is pretty strong. I think most scientists, if not all, would agree that phage inject their payloads to bacteria and cannot inject them into human cells. And I think the reverse is true. I think that our virus is unable to inject its payload into a bacteria cell. There's some exceptions to this rule with intracellular pathogens, but for the most part, that law of nature stands.

35:21: This sorta gets into where we think is Cas3's benefit to medicine. The fact that our CRISPR systems cannot and do not infect human cells inherently takes us away from that ethical and moral discussion. So we can't with Cas3 modify the human genome. We can only affect bacteria cells that are in the body. We don't have to get into any of that moral or ethical debate to figure out whether our science should be used.

Dan Pardi - 35:56: Yeah.

Paul Garofolo - 35:56: Moreover, because phage only inject into bacteria cell and we can't edit, the human genome, the chances of our PAC-MAN getting into a human cell and wreaking havoc on the human body is zero, because we can't deliver it to the cell. Even if you could, there's other reasons why it wouldn't work. That inherently means that the only thing you're going to remove from the human body is bacteria.

36:23: We didn't get into on-target and off-target pieces yet, but I'm sure that's coming. The nice thing about us is that our competitor is essentially a broad spectrum, carpet bombing, antibiotic that indiscriminately kills good and bad bacteria throughout your body. Our technology has the ability to go in and precisely identify and remove bacterial targets without any risk at all to going into the human cell.

36:57: So it's a safe place for us to begin to use CRISPR technologies, because it's maybe an easier technical challenge than what Cas9 teams are faced with in addressing human gene editing.

Dan Pardi - 37:10: So the Cas3 system would be able to edit mitochondria within cells?

Paul Garofolo - 37:16: We do have some discovery efforts that are underway to try to figure out whether it's possible, and there have been others that have tried to figure it out. But the protein structure that needs to be embedded into the eukaryotic target, or the human cell target, is [inaudible 00:37:33] so you have questions of how to deliver it, and how to get it to express itself inside of a human genome, or a human cell. You have the challenge of getting the mechanism to actually recruit the enzyme and do the right job.

37:49: One of the most wonderful advancements on the human cell side was when Jennifer and Emmanuel made the single guide Cas9, because it created this fairly small, single protein structure that more easily could be injected into a human cell to do its job. On the Cas3 side, Cas3 goes along with a protein chain called Cascade, which actually can be anywhere from four to six proteins that you would need to string together. The delivery of that into a human cell may not be impossible, but it's virtually impossible. I think that there's some benefits there.

Dan Pardi - 38:32: I see those benefits, and I'm also hopeful that you'll be able to solve that, because removing mutant DNA from mitochondria has been shown to reverse or slow aging, as in a work from Caltech by Bruce Hay. That ability to remove mutant DNA from mitochondria as we age could keep our mitochondria that do exist younger, there's less clutter that builds up within the mitochondria, and they've been able to genetically manipulate the mitochondria, and they were able to preserve youth in animals that were aging. So this sort of an application combining CRISPR and having the right targets could be youth preserving within our lifetime, which blows my mind.

39:11: Also, the real comparator here is the carpet bomb that kills all the bacteria. And we know we have symbiotic bacteria that not only are neutral in their effects in the human body, but are also symbiotic. For example, the bacteria in the microbiome with synthesize vitamins, they will extract calories and produce short-chain fatty acids, and they do all sorts of things that affect our hormones. Have you thought of any application that would directly affect the microbiota specifically?

Paul Garofolo - 39:39: The C. diff drug product that we have is one that we think is an ear-play microbiome approach. In reality, C. diff is a little different that some of the other pathogenic threats that are out there, because C. diff as a bacterial species is not necessarily developing resistance to an antibiotic. The issue with C. diff is, at least it's commonly believed by academia and science at large, that when you use vancomycin to treat C. diff, you indiscriminately kill the good bacteria that are in the lower intestines, that actually keep the existing C. diff cells in check. When you're doing this sorta indiscriminate carpet bombing of your gut, you're taking out the very bacteria that plays a very healthy balancing role inside of your body.

40:37: One of the reasons why we lose C. diff as an indication, even though the space can be considered to be a bit crowded, is that we are the only mechanism of action that reaches into the human body and selectively removes just the bacteria that we're after, leaving the rest of the microbiome untouched. We're pretty excited to be able to prove out that Cas3 mechanism of action in the C. Diff indication, because it will prove exactly that. That the capability of this CRISPR system is to go into the body, in any location, and be able to pull out bacteria that you've programmed it to remove.

Dan Pardi - 41:16: That's incredibly exciting. The simplification of our intestinal colony of bacteria is one of the reasons why pathogenic bacteria like C. diff can get more of a foot hold and grow. Carpet bombing them, it's like a short term solution that sets the stage for a worsening of that situation in rapid order.

Paul Garofolo - 41:34: Exactly. And there's other ailments too that ... the really exciting part about Locus as a company isn't necessarily just the pathogenic threats that we're working on right now, but the medium range opportunities to be there as science begins to fully understand what good bacteria and bad bacteria are inside the body, and what they're doing. You've seen and probably, with the show that you run, Dan, you've probably heard of various bacterial theories of things that are related to central nervous system disorders, or diabetes and obesity, or things like colorectal cancer and long term exposure to the bacteria that sits in nitrates that sit inside of processed meats.

42:25: I think we firmly believe that all of those things are real. We firmly believe that there are good agents and bad agents that the body is exposed to environmentally, and that the manipulation of those good and bad agents is something that could change the future of science.

42:44: The one bad part, or maybe the second very bad part, of the discovery of antibiotics, is the lack of academic research that occurred over the last 100 years in bacteria. When you don't have to worry about a bacterial infection because you have 26 different antibiotics that can be used to attack it, you don't study it. People don't fully appreciate or understand what the long term exposure, or even short term exposure to certain bacteria, does to the body for things like dementia or cancer in the gut.

43:23: I think that most people would probably logically agree that there's certain foods out there that are probably causing some inflammation in your gut, and that that long term exposure to inflammation is probably doing some bad things. What if we could take those bacteria out of your gut? What could we do, and what's the link between and bacteria and the human body and the brain? There's got to be some link. What is that link, and what happens when science catches up and is able to say, "hey this group of bacteria is causing this issue"? Well, now we'll have a technology platform that can go in and take that bacteria out of the body.

44:02: That's the really exciting part about where we are. We're sort of right on the cusp of the wave, Dan, and if we're able to get the FDA through working with pathogenic threats to improve the Cas3 mechanism of action, hopefully by the time that approval comes through, science is beginning to catch up with other opportunities that we might be able to approach.

Dan Pardi - 44:22: Over the course of this conversations, I'm having an increasing appreciation of what will become a necessity, which is the coevolution of CRISPR technologies with an understanding of the omics, or microbiome. If you understand what bacteria are producing, the good effects, vitamin synthesis, and suppression of inflammation and short-chain fatty acid production, versus the ones that are more pathogenic, we're gonna have some sort of interesting relationship where we have technology that can help us understand what's going on inside our body. We have precision medicine, and we end up putting ourselves in a better situation to thrive.

Paul Garofolo - 44:58: To go down that road, if you could put yourself out 50 years from today, do chemicals sit at the cornerstone of medicine, or do proteins? Does the DNA editing and capabilities that are beginning to be born today eradicate the use of chemicals in medicine? Is that possible to do to chemicals in medicine what green technologies are going to do to oil? It's possible, Dan. It's possible that precision medicines that are nanomachines that are able to reach into the human body and change cells at a DNA level, that certainly 100 years, that is how medicine's going to work.

Dan Pardi - 45:44: Yeah.

Paul Garofolo - 45:45: We'll probably look back at this era in time and think about, "why did we put those chemicals into our body when we really didn't understand what they were doing?" The future holds a lot more precision than what we currently have to deal with today. In fairness, I think it won't just be humans that that's a true statement for. As we start to look at the application of CRISPR outside of human medicine, I think that's certainly an area where we'll really start to see some very disruptive change in a number of industries where biotechnology sits at the center of them.

Dan Pardi - 46:21: You are at the forefront of that movement, Paul, with Locus Bio. You just raised $19 million, as we talked about earlier in your series A. What are you looking to do with those funds in the coming months to years?

Paul Garofolo - 46:34: The primary use of those funds is to advance Cas3 into human trials. We have two, possibly three, opportunities in front of us where we believe that we'll be able to prove the Cas3 mechanism of action can reach into the human body and selectively remove a pathogenic threat at will. The $19 million is earmarked to be able to complete that needed pre-clinical studies, to gain FDA approval, and to be able to execute that human trial. That's the efficacy human trial to be able to prove the Cas3 mechanism of action. Pretty exciting times ahead.

Dan Pardi - 47:16: Paul, thank you so much for joining us, your explanation of CRISPR, it's potential applications, the futurist perspective of where this all might be in 50 to 100 years from now. It is fun to talk about how technology might completely change our world's stretches of time and the distance, but what I'm hearing is that in pretty short order, we might be benefiting from at least early versions of CRISPR therapies. Thank you for your work, and thank you for your time today. We really appreciate you coming on to the show.

Paul Garofolo - 47:44: Dan, thank you for having us. We appreciate sharing the story.

Kendall Kendrick - 47:50: Thanks for listening, and come visit us soon at humanOS.me