The Translational Mixer
The Translational Mixer
Episode 3: Kiran Musunuru, gene and base editors hit the clinic and a Bloody Mary mix
UPenn's Kiran Musunuru, a human geneticist and practicing cardiologist who has pioneered the translation of gene- and base-editing approaches, talks to JC and Andy about the latest clinical results and modalities discussed at the 2024 Keystone symposium on Precision Genome Engineering.
4:07 Impacting patients
6:44 In vivo editing in different liver diseases
11:37 The FDA stance on programmable therapy
19:41 Base-editors march into the clinic
25:54 Multiplexing with base editors
28:57 Reaching broader patient populations
33:32 Investigator-initiated trials
39:27 Prime and epigenetic editing
44:34 Excitement around Bridge RNAs
47:15 Kiran’s mocktail
Bloody Mary 3 Ways
4oz (120 ml) tomato juice
1/2oz (15 ml) fresh lemon juice
1/4oz (7 ml) Worcestershire sauce
1/2 barspoon (3 ml) prepared horseradish, or to taste
2 dashes Tabasco, or to taste
Celery stick, for garnish
Salt and freshly ground pepper
Your choice of pickled vegetables, skewered on a cocktail pick, for garnish
DIRECTIONS: Add the tomato juice, lemon juice, Worcestershire, horseradish, and Tabasco to a shaker tin with ice and gently shake for 5 seconds. Strain into a chilled double rocks glass over a large ice cube. Garnish with the celery stick, salt, pepper, and pickled vegetables and serve.
For alcoholophiles, add 2oz (60 ml) vodka to the tomato juice, lemon, Worcestershire, horseradish and Tabasco. Enjoy!
Refs:
Gilmore et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N Engl J Med 385, 493-502 (2021) DOI: 10.1056/NEJMoa2107454
Chiesa et al . Base-edited CAR7 T cells for relapsed T-cell acute lymphoblastic leukemia. N Engl J Med 389, 899-910 (2023) DOI: 10.1056/NEJMoa2300709
Longhurst et al. CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema N Engl J Med 390, 432-441 (2024) DOI: 10.1056/NEJMoa2309149
Durrant et al. Bridge RNAs direct modular and programmable recombination of target and donor DNA. https://www.biorxiv.org/content/10.1101/2024.01.24.577089v1
Keystone Meeting on Precision Genome Engineering
Somatic Cell Genome Editing Consortium
The Mixer music “Pour Me Another” courtesy of Smooth Moves!
Andy Marshall: Hello everybody, welcome to The Mixer. I'm your host Andy Marshall, here again with my buddy Juan Carlos Lopez.
Juan Carlos Lopez: Hello Andy, good to be here.
Andy: So JC, who's our guest today?
JC: Andy, today we have a very exciting episode of The Mixer. We have Kiran Musunuru, who is a professor of medicine at the Perlman School of Medicine at the University of Pennsylvania. As you know, Kiran has been a pioneer in the study of genome editing, and he has been instrumental in bringing base editing towards clinical application. He recently co -organized a Keystone meeting on precision genome engineering, and he's going to share with us some of the insights that transpired at the meeting.
Andy: That sounds great. We should probably also mention he's a scientific co -founder and advisor to Verve Therapeutics which is a biotech that's pioneering some of these base editing therapies. So let's get going.
JC: Let's go... (music) —
Andy: Welcome, Kiran. It's great to have you here on The Mixer. So we wanted to talk to you because you recently co-organized a keystone, which was called Precision Genome Engineering. So it would be interesting for our audience to hear a little bit about the meeting itself, like what was its genesis, whether it's part of a series, and some of the aims of you and the other co-organizers had for the meeting, and then some of the key themes perhaps that emerged.
Kiran Musunsuru: Absolutely. Well, first let me thank you, Andy and Juan Carlos for inviting me to be on The Mixer and let me get started.
So the Keystone Symposium on Precision Genome Engineering, it's one of a series as you suggested. It's been going on for quite a few years now, so it's an annual event. Not all Keystone Symposia are annual, but this is one of the more popular ones, one of the very well -attended ones, and it has become an annual fixture. And the other distinctive feature of this particular one is that often Keystone Meetings are paired up with other keystone meetings, joint sessions. The idea being to, if there are overlap or some commonality between two themes, have them occur the same place, same time, and then you can have some joint keynote sessions, but you'll also have the separate sessions. And so it does foster some intermixing of the communities. And so this year, it was actually paired with basically mRNA delivery, or RNA delivery, or I should say nucleic acid delivery, so more broad than just RNA, mRNA. And that actually, if anything ended up being a little too close in the sense that I found myself really wishing, wow, I'm really enjoying what I'm seeing here in the precision genome engineering room. But, at the same time in the other room, just right next door, these really cool talks on delivery that I really like to see just because genome editing and delivery are really so intertwined.
So I felt like, wow, it's kind of too bad that these two meetings were together in the sense that, you know, in a way, I would have loved to have gone to either conference at different times and to have it all in one place. For me, at least being the organizer of one, I was basically stuck with one. And so missed out on a lot of what I'm sure were very interesting talks. I know they were interesting because other people who were not so, didn't have the fidelity I had to have to genome engineering, you know, got to go back and forth between the rooms. And so I got to hear, you know, sort of second hand what was going on in the other rooms. Like, wow, I wish I could have been in there, but sadly I could not.
4:07 Impacting patients
So to answer your question, you know, like some of the some of the exciting stuff that one, at least on my side of the resort, so to speak, and precision genome engineering. What I think this meeting emphasized in a very powerful way is just how much progress is happening on all fronts. So I can't say there is one single astonishing new discovery that just kind of blew everything up, right? That just upended the field. That was not this kind of meeting. There wasn't like an announcement. in the sense that, wow, we have like a whole new editing modality along the lines of say a base editing or prime editing.
What we saw was a lot of elaboration and a lot of progress and the editing modalities that already exist, both on the technological side, but perhaps more importantly on the clinical side, clinical translation, actually starting to see some of these technologies actually make it to the clinic, actually have an impact on the patients who have been enrolled in the clinical trials. And of course, we're all aware of exa-cel (Vertex’s exagamglogene autotemcel; Casgevy), the sickle cell and beta thalassemia treatment, getting approval from the FDA not that long ago.
And then even for those technologies that are even newer and haven't even had the chance because they haven't been around that long to make it all the way down the clinical development path, you can see they're going to get there. You can see that progress is being made in pre -clinical models and mouse models and non -human -human primate models. And then it's, you know, it's only a matter of time before they get into the clinic and human patients start to receive doses of these, you know, various types of gene -editing technologies. And it's going to greatly open up the horizon in terms of diseases that can be treated with editing.
Andy: Maybe we could take one step at a time If we're thinking about therapeutic modalities in terms of gene editing here. And so obviously, you know, there's, there's CRISPR classic; Cas9. And then since 2016, we've had base-editing technologies that have come online and we're going to get into that, I think a little more in terms of some of those are now already moving into the clinic. And we have prime editing, which has a lot of flexibility in terms of the types of changes that you can introduce, opening up this opportunity to actually do knock-ins in a manner that's perhaps less chaotic than Cas9. It's really powerful technology…but we've also started hearing about serine integrases, people are talking about transposases, even people have been talking about gene writing and retrotransposons. So could you fill us in a little bit about what you've seen there and what you think is exciting?
6:44 In vivo editing in different liver diseases
Kiran: Absolutely. I can go through those one by one if that works for you. There's a lot to say on each of those ones that you mentioned. So let's start with CRISPR 1 .0 as a lot of people think of it, standard nuclease editing. I think what was really exciting to see on the clinical side. there was a nice presentation by Intellia Therapeutics showing two different drug products. One intended for a transthyretin (TTR) amyloidosis, the second intended for an entirely different disease, hereditary angioedema. So two very different diseases, their clinical presentations, their pathophysiology, everything about them is different. They have nothing to do with each other, except for the fact that in each case, if you inactivate a gene in the liver, you could have a therapeutic benefit.
And so what they showed very powerfully, and we already knew about transthyretin amyloidosis, the original paper came out in the summer of 2021. So you know you're already talking, you know, two and a half years ago, showed great impact in terms of reducing transthyretin, the toxic protein levels in the blood and the first patients who were dosed. And they're, you know, all the way into phase 2 at this point; they did dozens and dozens, maybe, you know, at least 70, maybe even up to 100, patients by this point. So that's been going well. So that's not news in and of itself. What's newer is the fact that they're now starting to discuss data from the second indication from hereditary angioedema.
And so what was nice to see in the talk was the juxtaposition of the two. And that's very powerful because what it really emphasizes is the programmability of the technology, what they were able to do is take their one drug for transthyretin amyloidosis and take that essentially the same exact drug and change just the guide RNA. Everything else is the same. The Cas9 nuclease is the same. The lipid excipients of the lipid nanoparticles are the same. Everything is the same, except that guide RNA. And now all of a sudden you have a different drug product that works for hereditary angioedema.
And so you have a series of patients who are treated with this second drug, patients with hereditary angioedema, and what was really exciting to see, and it's not absolute new data that was unveiled at Keystone, it's been out for a few months, and the New England Journal of Medicine paper came out actually right after the Keystone meeting, but showing the cohort of patients, the first cohort of patients who got this drug, had an amazing clinical response.
Before that they were having pretty frequent angioedema attacks, which it's not necessarily fatal, or you can along those lines, but it is very detrimental to quality of life. And in almost all the patients, no more attacks in the few months after they had gotten treated. And so I would say this is actually the first demonstration of an in vivo gene editing therapy actually demonstrably improving the quality of life of patients. Transthyretin amyloidosis, that was a great success, but that was all based on the biomarker, seeing toxic protein levels in the blood go down. But it's harder to measure clinical outcomes. That takes a lot longer, that takes years. You have to do randomized controlled trials to really be sure what's going on. Whereas with hereditary angioedema, you really could see the impact in the sense that these patients are just having much better lives.
And this sort of parallels what we saw on the ex -vivo side with ex-acel, with the sickle cell / beta thalassemia drug, where those patients were having sickle-cell crises, they were having, you know, needing transfusions, having a lot of pain episodes that required treatment hospitalizations and so forth. And most of those patients have not had any such events since they got treated. So now you have what is clearly an in vivo success, really improving patients’ lives to go along with the ex vivo success. And to me, that's awesome, right? You know, like, we're now really ripping and roaring, right? Out of the gates because, you know, it's ex vivo and in vivo. It's like, now you have the full spectrum of disease.
Andy: That's a really key advance, yeah? To show clinical outcomes with an in vivo editing therapy.
Kiran: Absolutely, absolutely. That's a real advance to show that they're in vivo. clinical improvements. Like this is not just a drug that's changing a biomarker or this or that. It is actually impacting patients' lives. Like there's no ambiguity about it.
But then the other message that I think in a way is, to me at least, more exciting and has broader implications, as I said, is the programmability. Because now you've done it for disease one. It seems to be working.
You've done it for disease two. It's definitely working. And you know, it doesn't take much imagination to say, Hey, let's just tweak that guide RNA and target a third gene and activated gene and then you can treat some other disease. And so we'll see that happening.
We'll see that happening with Intellia. I'm sure we'll see with other companies that are largely focused on nucleases. You know, CRISPR Therapeutics, Editas, Metagenomi, which just went public, they're all sort of in this nuclease category. Now, the limitation there is that there are only so many genes to inactivate, particularly in an organ like the liver. There are only a handful of diseases, right? The vast majority of genetic diseases are even acquired diseases, so that strategy is not going to work. And so then you have to go to the next -generation CRISPR technologies, right?
11:37 The FDA stance on programmable therapy
JC: This issue of programmability, you alluded to. I agree with you that it's super interesting and a real advance. Now, the question is, from the point of view of clinical development, has regulatory science caught up with this programmability? Because you could say the way, the way it sounds is that you could essentially swap the guide RNA and have a new drug, but the regulatory science will still require you to do all the safety and toxicity studies, everything that you need to do before you can go into the clinic, right? And in the gene-therapy field, as you know, the NIH has this initiative called PAVE in which they are trying to precisely avoid this. Let's keep everything the same and let's just put a different payload in the virus. And that way we can save ourselves a lot of preclinical development when projects get to the clinic. So are the regulatory agencies thinking about it in the same way?
Kiran: Yeah, you hit the nail right on the head, with with that question. It's very much on my mind. So by way of disclosure, as an academic investigator at the University of Pennsylvania, I'm the recipient of funding from the NIH from their Sematic Cell Genome Editing Consortium, which is intended to fund investigators like myself, largely on the academic side, to take in vivo genome editing therapies—this is restricted to in vivo— to the clinic, through IND enabling studies to the clinic. And so one of the programs on which I'm working, and it does tie to the key... Keystone because a couple of my students presented on this work. So it's technically, you know, included in the Keystone update, if you will. But something we've been working on is the disease (PKU) phenylketonuria, a very well known genetic disease, patients who manifest disease. It's picked up on newborn screening. It's actually the reason newborn screening was instituted in the 1960s. It was for this disease and the recognition that you start to incur neurological damage from day one if you don't actually try to manage the disease. The disease is caused by very high phenylalanine levels in the blood because the enzyme that breaks it down is defective and if it builds up high enough it's neurotoxic.
And you can somewhat control it, not totally control, but somewhat control if you have a strict low protein diet. There are a few medications they don't necessarily work that well or they're challenging to take or they're expensive like daily enzyme or co-factor injections. So there's still a great amount of unmet need in this disease. And so something we've been working on is developing base -editing therapies to directly correct disease -causing mutations. Now the challenge with a disease like PKU and most genetic disorders is that there are a lot of different mutations are more properly speaking a lot of different variants genetic variants in the responsible gene That can cause a disease, right? It's not one size fits all in contrast to sickle cell, where there's one variant that's responsible for all cases of disease. So you can have one size fits all solution there. And so what we've been doing is asking the question, okay, we can make different therapies for different variants. They have similarities. And this gets to your question, Juan Carlos. In some cases, you're using the same exact editor, whether it's a base editor as we're doing, or whether it's a prime editor or a nuclease editor or XYZ, and you're only changing the guide RNA and so you have these two drugs say for two different variants and two different parts of the gene but it's the same disease and the editing works in the same way it's just the guide RNA is different the variant that's being fixed is different but the fundamental mechanism and all the characteristics of the drug are otherwise the same. And so one advantage of my being on the academic side is I don't have to have the same sensitivities about my interactions with the FDA that companies do, right? For a company, it's proprietary, it's a trade secret, like you'll never get them to fess up to exactly what went on in a discussion with the FDA. As an academic, I couldn't care less about that. I'm actually happy to share with the world exactly what I learned from the FDA.
And so I actually had a discussion meeting with the FDA a couple of weeks ago, not that long ago. And one of the questions I posed was exactly this question. We have these two drugs for PKU that are very similar. Essentially, you know, like just changing the guide RNA, changing, tweaking the messenger RNA a little bit, because it's not exactly the same base editor, it's a little bit different, but it's largely the same, you know, at the mRNA level 99 .5% identical. And we asked the FDA this question. Can we streamline the regular process? Can we include these two drugs in the same IND application to allow us to start clinical trial? Or are these going to be treated like two different drugs and we have to do the full suite of IND -enabling studies and whether mouse studies and monkey studies and so forth for these two drugs. And we could tell that the FDA is still very much thinking about this, right? They don't have definitive answers for any of this. And we're probably one of the first probably not the first, but probably one of the first , to approach them with this kind of question, even though it's kind of in the air. And the response they gave back to us was that if the editor is not the same, so even if it's slightly different, that it's going to be treated as two different drugs, they cannot be part of the same IND. However, there may be situations where if it's only the guide RNA that's different, it can be included in the same IND.
Now, that's very high level blanket advice, you know, the devil's in the details, and it's hard to know, like, you know, what are the circumstances where it would be allowable? What are the exceptions? I think we're going to have to figure that out. And the FDA is going to have to figure that out as we go forward. But there is some openness to the concept of programmability. It's just not totally clear what shape that's going to take, what sort of evidence the FDA is going to need from us, as the investigators, and, you know, from companies as well. to convince them that it makes sense to take advantage of this programmability because we understand from what studies have been done that the drugs, yeah, it truly is the same drug. It's biodistribution is same. It's toxicology is the same. Just changing the guide RNA doesn't change any of the fundamental characteristics. It's going to be just as safe , regardless of which guide RNA you use to go forward with it. So that's kind of where we stand right now.
Andy: I guess Kiran, a corollary to that is the biology also needs to match between the case that you're using and then extending it out. Because, for instance, if you're going after a different cell type, there's all kinds of different variables here that have to be taken into account. So it's not only the modality itself, it's the biology pathomechanism behind the disease, how we understand that, that , all of these things are going to come into that calculation yeah?
Kiran: They will, right? So, you know, one interesting question is, you know, we presented the sort of the simple case of, well, they're both for PKU, it's the same disease, it's the same organ we're targeting the liver, right? You're right, that simplifies things. If we had gone to them and asked, well, we have two drugs that are exactly the same, guide RNA is different. but it's two different diseases. You know, one is PKU and one is another metabolic disease galactosemia. Is that permissible to include in the same IND? And so we're going to be approaching the FDA over the coming years with exactly that same question, because our ambitions go far beyond PKU, we want to tackle a whole variety of diseases.
As I understand it and Intellia was not in a position to ask these questions of the FDA with its programs, because one was so far ahead of the other. And they basically did the transthyretin amyloidosis program and it had already gone to the clinic and was having success by the time their second one was ready. But it's the same sort of situation. You have the same drug, you're just changing the guide RNA, same target organ delivery, but it is two different diseases. So what it might look like to have them in the same IND, to have them part of say an umbrella clinical trial. Who knows, right? It's the Wild West , but it's going to be very fun to try to figure this out. A fun challenge. Let me put it that way. It won't be simple, but I think it'll be a very interesting space to watch.
19:41 Base-editors march into the clinic
JC: Yeah how about we go back to the other modalities.
Kiran: Well, I already sort of got into base editing by virtue of my example of PKU, but other things that are going on. And in the same way that it did for nucleases, let's talk about the clinical success that we're starting to see with base editing.
So I can talk about two and they were both presented at the Keystone, one by Waseem Kasim from University College London. So he gave a nice talk on the work he's been doing ex vivo therapy. So now you're talking about outside the body taking cells, in this case T cells outside the body, and then treating them in this case with base editors to to effectively create cancer immunotherapy.
And he was able to get this to work very well. And if you've been at all paying attention to the field, you've heard of Alyssa, the young girl who had refractory leukemia, was basically had gone through all her treatment options. There was nothing left. And, and Waseem was able, and his team , was able to craft this new therapy using base editors, which at a time was only three or four years old, . using that to engineer these cells, knock out different genes, to engineer this therapy, this immunotherapy, CAR -T, and then administer it to Alissa, and it worked and she's in a remission, and she is healthy to all appearances.
An incredible success, especially coming not that long after the success with nucleases ex vivo deployed for sickle cell and beta thalassemia. And so, you know, it's great to hear him give updates on that work and his ambitions going forward.
The other talk and by way of disclosure is talk I gave by virtue of my involvement with a company called Verve Therapeutics. So my disclosure is I'm a co -founder of Verve Therapeutics and continued to be a scientific advisor to the company.
I don't speak for the company and I did not speak for the company. I have to be very clear about that. I was not representing the company at the Keystone, but I did summarize some of their publicly presented data. And the real excitement there is the first in vivo base editing therapy, administered to patients with familial hypercholesterolemia, genetic condition that causes them to very high blood cholesterol levels. So high they have very bad heart disease. So these are really, really sick patients. And 10 patients have been enrolled in the phase 1 clinical trial with a base -editing therapy delivered by lipid nanoparticles in the same way as Intellia’s therapies. So using messenger RNA, encoding a base setter, using a guide RNA, in this case, targeting the gene PCSK9, which is a cholesterol regulating gene, with the purpose of inactivating it. So it's kind of like a nuclease in that the goal is to turn it off. The difference is that here you're only changing one base, one letter out of the three billion letters in the genome, but you're doing an exact exactly the right place to turn off the PCSK9 gene. So the effect is the same, you're turning off a gene, but it's in a cleaner, more precise way. And this was administered to the first 10 patients at varying doses. It was a dose-escalation study, single ascending dose, where you start with a very, very, low dose, almost a homeopathic dose just to make sure it's safe. Do that in a few patients, go up to the next dose, do a few patients, and so forth.
And what was evident, just... from the first 10 patients , is that there is a dose -dependent effect. The higher the dose, the more reduction of LDL cholesterol levels, the so -called bad cholesterol levels in the blood. And the patient so far who has received the highest dose had a 55 % reduction of LDL cholesterol levels, fell by more than half as a result of getting this therapy. And what's remarkable, not surprising at this point, because Intellia has shown data like this as well, but what's remarkable is that in the data that's been presented publicly so far by Verve therapeutics, that reduction in LDL cholesterol has been rock steady stable for more than six months so far. Like it comes down and then it does not budge. It is down 55% and it just stays there. So we're at six months as the latest data release. My expectation, my prediction is it's gonna be there for the lifetime of that individual. So you can see the potential here. Wow, okay, so base editing, it's now been deployed in human beings. It is working. And so that's great as a cardiologist, 'cause I am a cardiologist, th`at makes me very happy, 'cause now we have this powerful new tool to tackle heart disease, which is the leading cause of death worldwide.
But again, there's a bigger picture — just like with Intellia and transthyretin amyloidosis and hereditary angiodema there's a bigger picture — which is that now that we know base -editing works in human beings, and base editing has the unique advantage over nuclease editing in that it can make precise changes, we can do much more than just turn off genes. With Alyssa,
with CAR-T, it was about turning off genes. In the hypercholesterolemia trial from Verve, with PCSK9 it was about turning off the gene, but... very, very soon, what we're gonna see is base editing being used to correct disease -causing variants in the body.
And that will open up treatments for a whole variety of rare genetic diseases. Where the key is not to turn off a gene, 'cause that's not gonna do anything for you. But it's actually fix, correct, repair, whatever words you wanna use the gene, restore the function of a faulty enzyme or some protein that's in there that has an important function that's somehow driving the disease, and then effectively be able to, we don't use the word cure lightly, but potentially cure diseases and potentially do it at a very young age. So we've already talked about PKU, that's one such disease where, you know, as I, as I mentioned, we've been able to show at least in mouse models that we can correct two different PKU variants in different parts of the gene with base editing. Correct that variant. And what we find is when we do this with lip and nanoparticles, with messenger RNA, encoding a base editor with guide RNA, within 48 hours of treatment, the disease has entirely resolved those phenylalanine levels that started super, super high, are now down to normal . 48 hours, that's all it took, entirely corrected the disease, stunning. And just imagine doing that in a human being. And so hopefully my academic group with the funding we have from the NIH Somatic Cell Genome Editing Consortium in a few years will complete the IND enabling studies and get permission from the FTA to actually take this into human beings.
25:54 Multiplexing with base editors
Andy: Kiran, this is a really fascinating point that you're making and you know really speaks to the transformational aspect of base editing. But the other thing that maybe we should kind of of refer back to Wassim Kazim's work is the point about the safety, the increased safety of base editing. So obviously with traditional Cas9 you're creating these double -strand breaks in the DNA and if you're creating multiple edits then you kind of get this problem of unwanted recombination in the in the chromesome that you really want to avoid. So can you just speak to that other interesting advantage that comes along with base editors?
Kiran: Yeah, so the potential for multiplexing I think is what you're really getting at. The idea that you can mix several guide RNAs with an editor, that editor will now go to several places in the genome and make the desired change. If it's a nuclease that means double -strand breaks. And so you're going to get multiple double-strand breaks throughout the genome. And any time you have that scenario where there are multiple breaks at the same time, there's the potential for what I think of as genomic mischief, pieces of chromosomes coming back together in the wrong ways, which may be okay, right? It all depends on context. So if you are in a highly differentiated cell type like neurons, like hepatocytes, the whole genome is there, it's just not strung together in exactly the right way, but you know, it's probably not going to have a big functional effect. That's, you know, not a given. If you happen to hit an oncogene, if you happen to hit a tumor suppressor gene, maybe there are negative consequences, but you have to get pretty unlucky for that to happen. If you're in a stem cell, like say a hematopoietic stem cell, it's kind of a different scenario, because if you have a reshuffle genome and that cell has to give rise to it. a whole niche of cells, like, say, the entire hematopoietic system through many, many cell divisions, even with the first division of that stem cell that has a normal genome, you're not going to get normal genomes segregating into the descendant cells, right? You're going to get imbalances,you're going to get aneuploidy, and so forth. And so there's the potential for carcinogenesis arising from that, right?
So context is important. I hate to, you know, like paint with tube, brought a brush, and say, "Nuclease pad !" base editing good," because it's not nearly as simple as that. But you're absolutely right. Like base editing and prime editing, and you know, those are the two precision editing technologies. They have the advantage that you're not, you know, at least not directly making double strand breaks, you can multiplex and get single letter changes in say several different places in the genome. And what's happening in one place doesn't affect the other. Or the way I like to think of it is, you know, what happens in in Vegas stays in Vegas, you know, like what happens on chromosome one stays on chromosome one, what happens on chromosome six stays on chromosome six, right? They don't interact with each other in any way when you're using something like base editing or prime editing.
And so we've seen that principle applied very nicely by Wassim in the treatments that he's making for patients like Alyssa.
28:57. Reaching broader patient populations
Andy: So one other point that I’d really like you to elaborate on for our audience is I think with the PCSK9 story, there's this argument that's being made that this will kind of lay down a marker for base editing being useful for broadly available conditions rather than these kind of very rare conditions. And, And this brings in the practicalities of manufacture, distribution, pricing, all of these issues that tend to, if you're thinking about ex vivo gene edited cell therapy, it's really difficult to think of that as something that will be broadly available to many, many people. Can you talk a little bit about the elements of the base -editing platform for PCSK9 that you think are going to allow it to be more broad?
Kiran: Yeah. So, I mean, it really comes down to a few things, right? And you can really tie it to the components, right? There's the basic delivery vehicle. And by and large, we're talking about lipid nanoparticles. There are other ones in play, right? There's the more traditional and adeno-ssociated vector (AAV ) delivery approach, and that's fine. For some organs, you don't have a choice. That's the only way to deliver there. If you're talking about the liver, lipid nanoparticles really seem to be the best option, at least for now. There are virus -like particles of various types that are being tested. We actually saw some presentations of that on more on the delivery side of this Keystone Symposium.
I didn't necessarily get to see all those talks, but as I said, but a lot of of energy there and using virus-like particles and attaching different doodads, so to speak, antibody fragments or chemical modifications to get them, you know, lipid nanoparticles or virus-like particles to go to different places in the body. So that's one very important element is like, where are you directing your therapy?
But once you've gotten it there, that's a big assumption that you can, but let's assume you can get it to where you want to go. Then the editing machine actually becomes repurposable or programmable, as I said before, right? So you have your messenger RNA that encodes the base editor. And the base editor has come in different flavors, but it's pretty straightforward to just test a bunch of different variations and then figure out which one is best matched to the variant you're trying to correct or the gene you're trying to inactivate.
And then you have the guide RNA, right? So you have your lipid nanoparticle or your virus-like particle or RNA. Then you have the base editor and you have the guide RNA so it simplifies down to a pretty you know reasonably handled mix of things there aren't that many variables you have to worry about in contrast to prime editing which we can talk about in a moment which is more complicated but at the same time more versatile. But what's really remarkable to me and you know again this this really came out of Keystone there was a whole session a workshop where there you know half a talks from different groups that are all all using base editing to go after different diseases.
We already talked about CAR -T for leukemia, we talked about hypercholesterolemia, we talked about PKU, but we saw spinal muscular atrophy in one of the talks, we saw thoracic aortic disease in another talk, we saw a rare cardiovascular disorder called pseudoxanthoma elasticum in another talk and so on and so forth. So you can see, wow, the base editing technology, at least at the pre -clinical stage, now the tools are there, and there's a clear path to the clinic. It's been now proven out in human beings, seeing a lot of energy from a lot of groups around the world saying, okay, well, let's take the disease where we think we can make the most impact. Let's take these tools. It's not too hard to figure out how to get base editing to do what you want it to do on, you know, within its technology. technological constraints. And you can quickly, rapidly demonstrate its efficacy in mouse models. And then you can kind of take it from there. And so I think to your fundamental question, which is about cost and access, I think, a lot of the accessibility is going to be tied to streamlining. Can you actually streamline the process of discovery?
Can you make it very, very straightforward to say ‘Okay, I have a variant in in gene X, and I need to target an organ X, and are there enough, you know, well -validated pieces lying around that I can just kind of, you know, like, switch out whatever I need, take from this toolbox, take from this toolbox, and within ideally a matter of weeks, and if not weeks, at least a few months, hopefully, be able to come up with an optimal solution for that variant, that disease in that target organ, make your drug, and then be able to give to the patient?’
33:32 Investigator-initiated trials
Kiran: Now, of course, that brings in the second piece, what Juan Carlos referred to earlier, which is the regulatory piece. You need to work with the FDA and come up with a streamlining, so you don't have to take that new drug and spend, you know, three years and 10 million dollars to prove that one flavor of a base-editing drug, but instead be able to rely on precedence and say, well, it's already been done, you know, half a dozen time across all these different diseases, we understand the drug itself, we understand it, its safety profile, we can be satisfied that it's safe to go forward and then get an expedited regulatory process.
And so I think if you cut out a lot of the work involved in discovery, and you cut out a lot of the work that's entailed in regulatory approval, that's what we have to do to be able to get this dwn to an affordable timescale and affordable price and be able to greatly increase accessibility to a large number of patients. And I'm not just talking about a one -size -fits -all drug like sickle cell, which is where most people go when they have these conversations. They say, "Oh, wow, we have exa -cell. It's miraculous. It's going to change everything," except it's $2 .1 million. And how are we going to afford that? The real accessibility issue from the price? And that's a one -size -fits -all thing, right? It's the same drug you're giving over and over again to potentially hundreds of thousands of people if you could actually scale.scale. It's a more complicated problem if you're talking about more bespoke personalized treatments. But again, I think the same principle applies. If you can streamline the discovery process, streamline the regulatory process enough, I think we will get to the point where it becomes almost like a CAR -T, right? Like a CAR -T therapy is a personalized therapy. You're taking someone's cells, you're processing them, and it takes some time, it takes some money, but you can make the drug and deploy.
Now that's been deployed to hundreds of thousands of people. around the world. We're starting to see with personalized cancer vaccines. I think it's only a matter of time before we get gene editing drugs of various types, particularly base editing and prime editing to that point.
JC: I think it'll be quite interesting to see what the interplay ultimately is going to be between the traditional development pathway of randomized controlled trials, etc. and the investigator-initiated trials, because the way way that you're making it sound is the programmability of the editors is such that one can be very quick at introducing changes to the sequence, put it into the patient. And if you have an investigator-initiated trial, you can start treating patients that have specific mutations that you're capable of treating. Of course, that is not going to result in a regulatable product, but you'll be curing people. And at some point, there needs to be some change in the mindset in terms of how we currently view investigator-initiated trials, and instead of how we should be seeing them as true trials that can inform clinical development of products that can be then regulated by the FDA and paid by payers, et cetera, et cetera. To me, that interface, I get the feeling that these advances are going to make it necessary to have this conversation about how we turn these investigative shared trials into more valuable pieces of the development pathway.
Kiran: Yeah, I think that's exactly right. I mean, you can envision a couple of different ways to try to go after it. You can envision as more like these are n -of -one therapies and the clinical trials with your single patient in front of you. And so it's almost like single -patient INDs.
The other way to look at it is you want to bundle all these single patients together in a larger sort of almost like a master protocol, like a more platform approach, and then be able to have the flexibility, right? So I mean, I think there are a couple of different avenues to pursue with regulatory agencies like the FDA. And I, you know, as I said, we're going to see some very interesting things happening in that space in the coming. We all see it coming.
And the FDA, they're very well aware that there's anything that lends itself to a platform approach. It's gene editing. And so they know the score and they have signaled their openness to work with investigators like myself, certainly, to figure this out.
Andy: One thing I have heard, a counter -argument that I've heard hearing is that if you're in a kind of therapeutic program, very often there's some engineering that has to be done with the editing system that you're using. This is the kind of difference between the kind of translational world and the world of research. In the world of research, you kind of, oh yeah, I'm going to do this. It's not necessarily optimized for my particular system. But obviously, in the therapeutics context, the issue of I want my agent to give me the best possible outcome I can possibly get. So how do you deal with that?
Kiran: No, it's a great question because there is a tension between optimizing for every single scenario versus finding an adequate solution or set of solutions that broadly work across the patients you want to treat, right? Because it gets back into the questions of affordability and access. If you're really insistent on, "I need to find the very best solution for every single patient who comes before me," that's a lot more challenging than finding, you know, like a satisfactory solution. It may not be the best solution, but it will do the job. It will greatly improve that patient's quality of life.
But it might not be the very best solution. best, most efficient, absolutely squeakily clean, safest herapy you can make. Invariably, there is a trade -off there, right? There's, you know, externalities that come into play.
39:27. Prime and epigenetic editing
Andy: I'm kind of aware of time —I could talk about this the whole day I find this so fascinating. But maybe in thinking about rounding things up. moving on to this world of knockins and insertions. Do you want to talk a little bit about what excites you there?
Kiran: Yeah, keeping my eye on the clock as well. So we talked about nucleases, we talked about base editing, and we've already exhausted the hour. But I do want to hit upon prime editing and its related technologies. And I also want to hit upon epigenome editing, because that was the other area where I think there was a lot of excitement and a lot of interesting stuff that came out.
So let me hit upon epigenome editing first quickly. So this is yet another form of editing. Does not make double-strand breaks. It doesn't even nick the DNA, but it's modifying the context of the DNA. It's not changing the DNA sequence, but it's modifying, you know, making chemical modifications to the DNA bases. You're affecting the chromatin. More than that, you're actually focusing on the methylation status. And so what was really neat to see at this Keystone meeting was several talks, a couple from companies, Chroma Medicine and Tune Therapeutics, and one from the academic side, showing a lot of progress with epigenome editing. The two companies have been using CRISPR -based epigenome editing, and they showed off some great data, some non -human primate data targeting the PCSK9 gene as it happens, and showing that in non- human primates, you can use lipid nanoparticles, you can deliver this CRISPR -based epigenome editor, you use a guide RNA or guide RNAs to target the PCSK9 gene promoter and put lots of methylation groups there, silence the gene and seeing almost 100% silencing at the messenger RNA level because the methylation turns off gene expression, and seeing in mice and in monkeys persistent effects out to months or even as much as a year, which suggests that if you're trying to inactivate a gene, well we know we can do that with nucleases, we know that we can do that with base editors, both proven out in human beings, and I don't think it'll be long before we see that starting to be done with epigenome editors.
The flip side is that epigenome editors aren't going to be able to do much for you besides turn a gene down or potentially do the opposite, turn a gene up. So it really has to be tailored to your particular therapeutic application.
The third talk that was not from a company, it was from an academic group, showed a clever way to not have to use CRISPR for epigenome editing, but going old school, actually turning the clock back maybe a couple of decades, using zinc fingers and showing that with epigenome editing, it might actually be zinc fingers that may prove to be more useful because they're smaller, they're easier to pack into viral vector. You can actually pack several of them into viral vectors. So you can tune several different genes at once. They actually seem to be better tolerated in cells. And so it's kind of funny to think, wow, we've come all this way and yet we're going back to zinc fingers because that's actually might be what's gonna work best for at least epigenome editing. So that's epigenome editing. It was nice to see the progress on various fronts there.
And then prime editing, the most noteworthy talk was from Prime Medicine, where I got to say I was just so impressed because it was Andrew Anzalone, who's basically the guy who invented Prime Editing in David Liu's lab, and then went on to be a co -founder of Prime Medicine, and he was representing the company at the Keystone. And he gave an update on the preclinical progress of Prime Medicine's various programs. And I swear, he must have gone through a dozen of them, and they all look fantastic. Anyone, any presenter doesn't matter if they're company speaker , doesn't matter if it's me as an academic , we always like put our data in the best possible light sure but still it's very impressive to see wow this prime-editing technology they're just redeploying it in different variations across all these different disease states and really like having impressive results whether it's ex vivo whether it's in vivo whether using it to correct specific nucleotide variants in the same way or similar ways base editing can, whether it's putting in small insertions or deletions, whether it's tackling trinucleotide repeat expansions, there were a couple of examples of that like Huntington disease, Friedrichs ataxia, or as you alluded to doing more sophisticated things like doing gene insertion, like being able to do a multi -step prime editing or prime-like editing enabled process to actually be able to catalyze the insertion of large fragments or even full genes into the genome.
And so it was actually a very impressive demonstration of all these different flavors of prime editing, all the different variations in one talk across a dozen different diseases. Now, we'll see how quickly any of those get to the clinic.
Prime Medicine has guided their first ex vivo application, they're hoping to take it to the clinic later this year. We'll see. But it's clearly headed in that direction. And even if it's not Prime Medicine, there'll be other companies that are sort of in the space and using prime like technologies to do similar sorts of things. I mean, this is gonna be an area to watch for sure because of the versatility of these technologies.
44:02 Excitement around Bridge RNAs
Kiran: The other thing I would add to this is the potential for metagenomic mining to find just really cool things that we just didn't even imagine were there. And one very impressive example of this came from Patrick Hsu from the ARC Institute. He presented something new that he had mined out of the genome, which he's calling bridge RNAs. It's a totally different mechanism from RNAi, from CRISPR. It's RNA -enabled, but it uses an entirely different set of proteins, an entirely different mechanism. And so, these bridge RNAs basically come out of these mobile genetic elements, and it would take me too long to try to explain everything in gory detail, but he's released it as a preprint that was timed to his talk at the meeting, so, you know, it was a little bit buzz -worthy because of that. But basically, taking advantage of these mobile genetic elements that naturally exist and figuring out how they work, how they jump around, and discovering that there's this complex bridging RNA that basically is almost like bispecific. One portion of this bridge RNA specifies the target site in the genome, and the other part of this bridge RNA specifies the donor DNA. And so with this one complicated RNA bridging RNA molecule, it specifies both the donor and the acceptor or the target site in the genome. genome. And what he was able to show is that, at least in bacteria, it's all in bacteria for now, right?
So we'll see if it eventually makes the jump to mammalian cells, which would be very exciting. But it could show you can take one piece of DNA and stick it basically anywhere you want into the bacterial genome. You can actually engineer it so that the donor is one part of the genome and the acceptor site is another part of the genome. So you can catalyze inversions or do complicated, funky stuff like that. that. And I'm watching this, I'm thinking, "Wow, I mean, there are some potential therapeutic applications." And when this actually gets the mammalian cells, because now, you know, there are lots of diseases that are caused by chromosomal rearrangements, like hemophilia, for example, like something like, you know, half the cases are due to one particular inversion on the X chromosome. It's very stereotyped because of homology between two different portions on the chromosome. So, yeah, that's it. if you had a way to engineer just exactly the reverse flip You could treat a lot of diseases that otherwise wouldn't necessarily be so easy to treat, right?
So I think it just goes to show that there's a lot still waiting to be discovered. And so, you know, we're thinking about nucleus and base and prime and epigenome. But you know, I could foresee in 10 years like a dozen of different new things that we aren't even anticipating that's going to come out of metagenomic mining.
And, you know, some of them will be restricted to bacteria, they're not going to make the jump to the million cells, but some of them will make it to the million cells. And, you know, in their own time, I think we're going to have therapeutic potential.
41:13. Kiran’s mocktail
JC: Well, this has been very interesting here. And we, it's exactly what we were hoping to hear about. It's where probably one of the most exciting areas in, in biology. As I told you, in my email, the podcast is called The Mixer because we mix our interest in science with our interest in cocktails, but we know that you don't drink, so that's fine.
Andy: But maybe Kiran has a mocktail? Is it just plain old H2O or is it some other concoction, Kiran?
Kiran: Bloody Mary Mix, perhaps?
Andy: There we go. Fantastic. Fantastic to hear from you, Kiran. It's been a while since we last saw another face -to -face. Hopefully, maybe I'll be down at UPenn one of these days. There's so much going on down there. It's an exciting time for sure. Thank you so much for your time here and your insights. We really appreciate it.
Kiran: Thank you so much, Andy. Thank you so much, Juan Carlos. It's been a real pleasure.
Andy: How about that JC? So much going on in this field at the moment. And its really interesting to see how far into the clinic the field has come since we first met Kiran, what was it, over a decade ago at the Brigham?
JC: Yeah, very striking to see the progress. It's been really inspiring and I'm certain that the best is yet to come to be honest. I'm sure there will be a lot of excitement in this area for years to come.
Andy: And what about the cocktail choice? Are you inspired by the choice of cocktail?
JC: Yeah, I must confess that I'm not a fan of the Bloody Mary. I really am not. No, no, no. I've never made it, and I don't think I ever will. However, there's a rich bibliography about the Bloody Mary, and we're going to share with our listener a particular video that I find very informative. It's from a website called cocktailchemistrylab .com run out of San Francisco by a guy called Nick Fisher. He shows you three different ways of making the Bloody Mary, the basic, the expert, so the best way to make the Bloody Mary is by clicking on the link in the description below. What he calls the chemist level. And if you can pull that one off, my hat's off to you because it's a beautiful drink to make. So we'll share that with our listener.
Andy. Great, so everybody enjoy the bloody mary and thanks so much listening. Until next time. Cheers JC.
