A Find-and-Replace Approach to Fixing the Mutation Underlying Sickle Cell Disease

October 22, 2021

Gene editing is an emerging therapeutic area that promises to correct the underlying genetic causes of diseases. Graphite Bio is readying to enroll its first patient in a phase 1/2 clinical study of its experimental gene editor GPH101 to correct the mutation in the beta-globin gene that drives sickle cell disease. Though the condition can manifest itself differently from patient to patient, it can cause painful episodes due to the clumping of sickle-shaped blood cells that obstruct blood flow in small blood vessels, as well as other acute complications including stroke and infections that can contribute to early mortality in these patients. We spoke to Josh Lehrer, CEO of Graphite Bio, about the company’s experimental sickle cell gene editing therapy, how it works, and what makes it a next-generation gene editor.


Daniel Levine: Josh. Thanks for joining us.

Josh Lehrer: Very happy to be here. Thanks for having me, Danny.

Daniel Levine: We’re going to talk about sickle cell disease, Graphite Bio, and the company’s elf efforts to develop gene editing therapy to treat the condition. Well, let’s start with sickle cell disease. For listeners not familiar with the condition, what is it?

Josh Lehrer: Sickle cell disease is actually one of the more common genetic diseases and what’s interesting about how it developed as a genetic disease is that it was actually selected for. It’s a mutation with a single misspelling in one of the most important proteins in the body, in one of the proteins that codes that composes hemoglobin to deliver oxygen to tissues in red blood cells. So, there’s a single change, a single mutation in hemoglobin and in the carrier status in people who have one copy of this beta globin gene with this mutation and one normal regular copy, those individuals are actually resistant to malaria. So this mutation has occurred several times through human evolution. It’s been selected for because of the competition between humans and the malaria parasites, and carriers have an advantage. For that reason, most individuals who have sickle cell disease, when, for example, two parents who are carriers have kids. Sickle cell disease is when an individual has two copies of the mutation and that occurs mostly in individuals of African descent, because that’s where the carrier status has been selected for. When two copies are inherited, what happens is that the hemoglobin protein, which normally is soluble in the red blood cell and efficiently finds oxygen in the lungs and delivers it to tissues—that protein now has this misspelling, this abnormal amino acid, which is a sticky hydrophobic amino acid and the sickle hemoglobin clumps together and damages red blood cells. It pokes holes in the membrane and results in red blood cells that have this very characteristic sickle shape under a microscope. These cells can then sludge in the blood vessels, can decrease oxygen delivery to the tissues, and can cause many severe symptoms in individuals who have both copies of the mutation. Another interesting point of background here as it was actually described as the very first molecular disease by Linus Pauling beause it’s the first disease where we actually understood the reason at the molecular level, this single change, single mutation in the protein that causes this stickiness and how that then leads to the disease and the manifestations that occur in patients.

Daniel Levine: Well, how does the disease manifest itself in progress?

Josh Lehrer: So, the disease has actually changed. This used to be primarily a pediatric disease, with death in infancy, and children untreated die in general of infection because the blood doesn’t flow well through the spleen, you lose spleen function and then kids are at risk for overwhelming bacterial infections. In the U.S. and in Europe, and where there’s access to care, including in areas that are increasingly able to treat this in Africa with penicillin and with preventive care, kids can be brought back, brought through this higher risk period and live onto adulthood. But the disease doesn’t stop, unfortunately, getting through this sort of higher risk pediatric setting, and that’s because there’s really ongoing damage to the red blood cells. There are these two features of the disease that are called hemolysis, which means destruction of red blood cells that results in not enough blood cells in the blood vessels to deliver oxygen and the consequences of anemia, and then something called vaso occlusion, where these cells are damaged. They don’t flow through the vessels well, they stick and they sludge, and they cause backups in the small blood vessels that lead to really all the tissues in the body. And that results in really two types of symptoms. One is when there’s acute pileup of these abnormal red blood cells that causes an acute pain crisis or a vaso occlusive episode, you can think of that almost like a heart attack, but it can occur in all kinds of different tissues. It can occur in bones and it causes really excruciating pain, and it can also result in organ damage. And then separate from those acute events, there’s really an ongoing, inadequate blood delivery to tissues due to not enough blood cells and damaged blood cells that causes really multiple organ dysfunction. Most devastating consequences often are in the brain, including stroke, and it infarcts in the brain. And then resulting, even in children who survive that, early high risk period, in general, early mortality. So in the U.S. and in Europe, the average life span for patients hasn’t really improved in the past several decades and is in the range of 40 to 50 years. Now, what’s the prognosis for patients with the condition today? So, with this severe form of sickle cell disease with really the best available care, and good access to care, the life expectancy is in that range of 40 to 50 years. Unfortunately, along the way, there’s still a significant morbidity despite really the best available therapy, nothing that really prevents these kinds of acute pain episodes that are so disruptive to patients’ lives and require frequent prolonged hospital stays. And then, chronic organ damage, kidney failure, increasingly heart failure, and lung damage that led to early death and severe morbidities. The other really important morbidity in patients, even who survive longer, is cognitive dysfunction, which is fairly widespread due to either overt strokes or chronic inadequate oxygen delivery to the brain.

Daniel Levine: You’ve been a practicing cardiologist previously. You were the chief medical officer at Global Blood Therapeutics where you led the development of Oxbryta. I suspect you’ve had a chance to understand this disease, not only at a medical level, but at a patient level, what’s it like to live with this condition?

Josh Lehrer: It’s a real challenge and the exposure that I’ve had to patients in training, in a position, and then at GBT, and the ER, really is what’s motivated me to work in this area. Just one story or a couple of stories: One is that during my training I spent time in the lab working on how to figure out the structure of different kinds of membrane proteins. I worked down the hall from Max Perutz, who won the Nobel prize for solving the crystal structure of hemoglobin that explained how this one change that makes hemoglobin sticky can cause the disease. That was earlier in my career and I always felt that was such an amazing and elegant scientific story. And then also in other ways, so tragic and appalling in that the discovery was made in the 1950s and the way when I made my way to the wards and my medical training and saw patients, the way that we were treating patients really had barely fundamentally changed since those times—I mean, just offering essentially pain control and blood transfusions. It was, you know, a group of patients who had been neglected despite the fact that we knew more about this disease than almost any other disease. So, that was one of the reasons we worked with a sense of urgency at a Global Blood Therapeutics. We worked with patients and understood that this is a, it’s a medical problem, but there’s a lot of systematic bias and racial discrimination that patients face on a daily basis that compounds the cut of the medical aspects of the disease. So, we learn from patients that you’re not only having the acute pain crisis. You think of a patient, you know, a kid in school or in college, you might miss a month or two months of class and have a lot of trouble catching up. But in addition to not having good options for treatment, even just getting pain control that we know that African American patients are about 50 percent as likely to get prescribed adequate, analgesic or pain control for the exact same diagnosis. We would hear heartbreaking stories of patients with a huge pain attack coming on. Their first thought was not getting to the hospital as quickly as possible. It was going into their closet, choosing their best clothes to put on and putting on makeup so that when they arrived in the emergency room, they would be taken seriously and not be viewed with suspicion or viewed as someone there just drug seeking, but viewed as someone who actually had a medical problem in need of attention. So, it’s a disease that’s challenging on many levels. And, thinking about just the medical options developed for patients, as I mentioned, there have been advances recently, which are great new options for physicians, including Oxbryta, which I worked on. But there really is nothing that can stop this disease in its tracks—from the acute pain episodes, the mortality. The only way we know of that can cure the disease is a transplant procedure where stem cells from a matched sibling donor, often with that carrier status, I was talking about earlier that was normal, where were stem cells that are either normal or single trait, which is the carrier status, are used to replace the sickle stem cells in a patient.

Daniel Levine: You mentioned that this was the first disease to be understood at a molecular level. There’s a lot of therapeutic development going on today, substantial investment in new treatments and potential cures, but this was also a condition that was long neglected for a variety of reasons. What’s changed to account for all the activity we’re seeing today?

Josh Lehrer: Well, I think a couple of things. One is that there’s finally a recognition on the part of the pharmaceutical industry. Also, the FDA has played a big role in this—that there’s both an unmet need and that we could make progress from it in the drug development ecosystem, working with regulators to treat sickle cell in the same way that some other serious diseases have been treated to really spur development. So, like cystic fibrosis, I mean, using regulatory flexibility, thinking about new endpoints, that’s something that we worked really hard at a Global Blood Therapeutics. Sickle cell disease was one of the very first FDA meetings that was part of the patient focused drug development effort. I think that is partly a success in FDA and regulators working with industry to keep that investment going that had been really lacking and present in other serious diseases. The other is the advent of gene therapy, of genetic therapies, and genetic approaches to have the potential to cure diseases like sickle cell disease to take the potential to extend the same kind of benefit that can result from a bone marrow transplant from a sibling, but to start to do that with a patient’s own cells. Sickle cell is, as I mentioned, one of the most common monogenic diseases. So it really is at the top of the list when you’re thinking about what you would do with these emerging gene therapy tools.

Daniel Levine: Graphite Bio is developing both gene replacement therapies and gene editing therapies to address a range of rare diseases. How do you determine whether a gene editing therapy or a gene replacement therapy makes sense as a therapeutic strategy? Is one better than another in the case of a monogenic disease?

Josh Lehrer: Yeah, so the Graphite Bio platform and what we’re doing, and what’s a little bit what’s different than other approaches. So CRISPR and the CRISPR Cas9 nuclease are, of course, a revolutionary scientific development. And you can think of CRISPR as really a very precise pair of scissors for cutting target genes, and it’s been deployed in that way for knocking out or disrupting, genes and trying to improve genetic diseases in that way. What we’re doing goes one step further. We have an approach that really builds from the initial way CRISPR has been used to precisely cut a target gene, but then to give the cell a template for when it repairs that break and the target gene, and during that repair process, we can do what you’ve mentioned, where the template can provide the instructions to actually correct a misspelling in a gene or can contain an entire replacement gene. So, we really have been looking for areas we can make a unique clinical impact in a disease by instead of knocking something out, changing something back to normal. In sickle cell disease, because every patient with severe sickle cell disease has the exact same misspelling, the same single letter-change from an A base to a T base in the DNA. We can actually make that change back to normal by correcting the gene and leaving the normal gene, intact, essentially changing it from sickle hemoglobin to normal adult hemoglobin. So, we use a gene correction strategy in that case, and it’s a higher bar. It’s more difficult to directly correct the sickle globin gene and try to reach a cure that way then indirect approaches that are, for example, trying to take the brakes off the fetal form of hemoglobin and try to reinduce that expression. But it’s always been viewed as really the gold standard for cure because we know that carriers of sickle cell disease are normal. They don’t have disease. So by using gene correction, we actually now for the first time have the potential ability to essentially change someone at the DNA level from having sickle cell disease to having something like sickle cell trait or even better.

Daniel Levine: Your lead therapeutic candidate is GPH101. You call this a next generation gene editing therapy for sickle cell disease. What makes it next generation?

Josh Lehrer: So, what makes it next generation is really this combination. I was just speaking about using CRISPR first to cut, but then delivering a template to the cell to actually have a precision repair. So the results, I mean, you can really think of it instead of cutting or knocking out a gene, think of the word processor analogy. It’s more like find and replace. We’re using the precision of CRISPR, using something called an RNA guide, to find the target gene. So scan the whole genome and remarkably find just that one area of the sickle globin gene that has that mutation and then use template DNA to actually replace that misspelling and correct it and change it back to normal. So, what’s next generation is the fact that we’re using CRISPR for gene correction. That we can actually change a mutated gene to the normal gene and restore normal biology. The first-generation approaches, the ones that are in the clinic and are emerging, very encouraging data, make a lot of sense with those kinds of tools. With a pair of scissors, you can’t actually correct the mutation changes to normal. You can try to target other areas of the genome and essentially knock them out or get rid of them, and through that approach, try to indirectly make the situation better. So that’s really the idea behind fetal hemoglobin induction. Our approach has been really with the idea in mind that if we can change things to normal, that would really be the best approach and the best outcome for patients.

Daniel Levine: Walk me through the gene editing therapy, how it works, and what’s the process for treating a patient.

Josh Lehrer: Yeah. So, the process is similar to other gene therapy approaches using stem cells that have been advanced in diseases like sickle cell disease or related diseases like beta thalassemia, where the first step is we mobilize, the stem cells from the bone marrow of patients and then remove them from the patient. Then once we have those stem cells—these long-term stem cells are remarkable because they can essentially survive for the life of a patient and self-renew and they can give rise to all the cells in the blood immune system, including red blood cells, which cause a disease in the case of sickle cell disease. Then we deliver the scissors part, CRISPR, to these cells. We create this precise incision in the sickle globin gene.  Immediately after that, we deliver a DNA template with the correct instructions. We deliver that with a viral vector, that template is there just as instructions. It doesn’t actually integrate into the cells’ DNA, and then the cells own machinery correct the sickle mutation. Then we prepare these stem cells. We expand them and then deliver them back into the patient. Before delivering them back into the patient to engraft back into the bone marrow with the goal that these corrected cells will produce normal red blood cells for the rest of the life of that patient, we need to eliminate the disease stem cells in the bone marrow. So that’s called bone marrow conditioning. So, that’s sort of the whole process. After the patient has conditioning and receives these edited corrected stem cells back into the bone marrow, there’s a potential to then repopulate the bone marrow with completely normal stem cells and cure the disease in that manner.

Daniel Levine: And I know it’s early days, you’re just preparing to begin a clinical trial, but what is known about the safety and efficacy of this approach, and is there a concern about off-target effects at all?

Josh Lehrer: Yeah, obviously this is an area we’re gaining experience in the work that has been done by others, including the first-generation approaches, like the CRISPR/Vertex experience has been, you know, very encouraging and has shown durability and really shows us that you can use CRISPR to edit stem cells, have them engraft and persist in a patient and have what looks like some significant benefits. So that we think is actually significantly de-risking for really the whole field. And in addition, for our approach with safety in mind, we did design our platform to use an engineered version of Cas9. So it’s not the standard CRISPR that was first discovered. We’re actually using an engineered version that is more selective and has, in our hands, 30-fold less off target effects, so very low off target rates. We then carefully characterize where this off-target cutting is happening. We do careful preclinical studies and animal studies to make sure that these human cells have the function where they can survive and engraft in animals and don’t lead to problems in these animal studies. So, as we treat our first patients, we obviously will be monitoring and getting clinical data, but we have a lot of confidence in the robustness of this approach and in the potential for this to be curative from what we’ve seen in human cells, from patients, and also in animal models of sickle cell disease.

Daniel Levine: And what does the initial clinical trial look like? How big a study will it be and what will you be looking at?

Josh Lehrer: Yeah, what’s really amazing about these kinds of therapeutic approaches is that every single patient is almost like a clinical trial. We’re aiming for cure. We’re trying to really achieve sickle cell traits. So we’re going to learn a tremendous amount from every single patient. It doesn’t take a lot of patients to understand whether we can get to curative potential in this disease. So our initial trial is designed as 15 patients, we’re beginning in adults with severe sickle cell disease and then after the initial adult experience planning on expanding into adolescents and the younger patients. Really the ultimate goal in sickle cell disease is to be able to treat and cure children before they start having this chronic irreversible organ damage and strokes, to really prevent the progression of the disease early on. We’ll be working towards that goal in this trial. And the kinds of things we’ll be measuring will be the acute pain events that I mentioned earlier. Those are relatively easy to count. Not every patient has those events, but we can enroll patients that will have pain events and show that those pain events are not happening, that those resolved. Then it’s relatively easy to draw blood and really show that the red blood cells are functioning, that we can measure hemoglobin levels or red blood cell counts that are coming into the normal range. And we can show that the normal hemoglobin compared to sickle hemoglobin are at the levels that you would see in a patient with sickle trait. Then there are additional things we can measure over time that really kind of further give us competence in terms of the function of those red blood cells or showing that we can deliver oxygen normally to different organs and different tissues over time.

Daniel Levine: Sickle cell disease is a global problem. It’s got a higher prevalence in parts of the world where an ex vivo approach may not be feasible. How do you think about delivering and affordability as you design your therapies?

Josh Lehrer: I mean, it’s a critical point. This is a relatively common disease for a rare genetic disease, in developed countries. There are millions of patients with sickle cell disease in countries in Africa and in India. The way we think about bringing these kinds of curative options more broadly is by really making progress on our manufacturing approach, moving towards a closed system. As we scale up in terms of manufacturing and supply, those costs will come down. The other really key advancement that we’re coming increasingly focused on is what I mentioned on the conditioning and the accessibility of these kinds of curative treatments to patients outside of the U.S. and Europe will be greatly improved if we can do that preparation of the patient and the bone marrow conditioning in a less invasive way. Right now the standard approach is really to us, chemotherapeutic bone or conditioning myeloablation like would be used in any other transplant procedure. And that means a stay in the hospital. That means advanced, relatively intensive nursing and hospital care, and accounts for a significant percentage of the overall cost of the procedure. And our vision is really a day where we can prepare the patient in a simple outpatient, non-invasive way, basically giving an antibody that can remove those diseases, stem cells, and then give back edited cells that can cure a patient in a way that could bring this to more patients in developing countries as well.

Daniel Levine: Are you looking at in vivo approaches at all?

Josh Lehrer: We’re certainly looking closely at that field of monitoring that field, some of the advancements that are really exciting around an in vivo liver targeting of gene editing approaches, including the recent Intellia data, for diseases like sickle cell disease. It’s very early days. So it’s very different types of biological hurdles, a lot of biology really to solve and sort out. And if you’re thinking about editing stem cells and finding a stem cell in the bone marrow, it’s like a needle in a haystack. It’s one in a million or fewer the cells in the bone marrow compared to 80 percent of the cells in the liver, and you’re targeting those. So, it’s early days. We’ll be very interested if we can find the tools that can lead to full gene correction, we are going to be able to use our targeted approach. So I think it’s really an emerging area.

Daniel Levine: Graphite raised more than $270 million in an IPO in June that was on top of $150 million venture financing earlier in the year. How are you using that money and how far will it take you?

Josh Lehrer: We’re fortunate to be well-capitalized. That cash runway should take us through 2024. Obviously, job number one is advancing our program in sickle cell disease really rapidly for patients for all of the reasons we went over earlier. There’s a real pressing need for these kinds of therapies. This gives us the resources to advance our sickle cell program, not just proof of concept, but really into late stage trials and preparing to bring that to patients as a commercial product, a curative option. And then what this also lets us do is really, in parallel, start to advance pipeline programs, which use the same approach that takes full gene correction or gene replacement or precise gene integration, and then can offer potentially one-time cures for patients with other diseases who don’t have curative options. So, it’s really an ability to advance the sickle cell program and then the broader platform and pipeline behind that.

Daniel Levine: Josh Lehrer, CEO of Graphite Bio. Josh, thanks so much for your time today.

Josh Lehrer: Thanks very much. It was great to be here.

This transcript has been edited for clarity and readability.


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