From the evolution and structure of human hemoglobin to gene therapy, Dr. Glassberg discusses past and present clinical management approaches and future directions for improved outcomes.
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Welcome to grand rounds. It's my pleasure to introduce Jeffrey Glasberg. He is a tenured professor of emergency medicine medicine and hematology and medical oncology at the Icann School of Medicine at Mount Sinai and the director of the Mount Sinai Center for Sickle cell Disease. Doctor Glassberg is the first emergency physician to run a sickle cell program where he practices in a unique way, seeing patients in the emergency department in the hospital and in the sickle cell clinic, he has grown the Sinai Sickle cell program into one of the largest and most successful in the world with nearly 1500 patients and more than 25 million in NIH. Funding Mount Sinai's program is also one of the largest providers of access to cutting edge treatments for sickle cell disease, including monoclonal antibodies and gene therapy cures to mitigate the critical shortage of expert sickle cell physicians. Doctor Glassberg created the Mount Sinai Sickle Cell Fellowship, one of only two dedicated Sickle Cell fellowships in the United States. Doctor Glassberg research spans the translational spectrum including pulmonary complications of sickle cell disease, novel biomarkers for vaso ACLS crises, novel gene therapy targets and agents for the prevention and treatment of vaso occlusion. Please join me in welcoming Doctor Glassberg to grand rounds and ok, thank you very much for having me. I will stay close to the microphone. Um This lecture goes through three of my favorite projects that are going on right now and I framed it around a sort of past, present and future in sickle cell disease and I want to finish early. So I'm going to, there's my consulting and disclosures. I'm going to buzz through the background about sickle cell disease and assume that we don't need to cover um board review type of stuff. So this is about sickle cell disease and hemoglobin and the gene mutation that people have uh fetal hemoglobin. I'm skipping all of it. Uh This is about what sickle cell disease is and how it was the first genetic disease discovered as a, a single mutation with a single incorrect letter and how that gene gets translated into RN A, um transcribed into RN A and then translated into a protein, et cetera, et cetera. We have these different genotypes. Um Even though everybody has the same family of mutations, we see a lot of diversity and how sick people are. Um And that has to do with these other factors uh beyond sickle cell disease. But the bottom line is we see manifestations in every organ system because sickle cell disease is a disease of the blood and the blood feeds the whole body right now, we think the life expectancy is around 50. Uh It's a little hard to tell because some of the best studies were done before. Um Hydroxy was widely in use for young people. But 50 is, is a kind of useful estimate to benchmark where we think the life expectancy is. Ok. So, um I'm gonna go through sort of how things were in the past. Um and how we're trying to rectify that with one of our research projects. So let's talk about hydroxy hydroxyurea is uh a drug that is approved for the use. Uh and treatment of people with sickle cell disease. Basically, it has three mechanisms of actions. It raises your fetal hemoglobin levels, it reduces your leukocyte count, all, all all forms of leukocytes and it also has a nitric oxide on the side. So it's a nitric oxide donor which helps with vasodilation. Um In this New England Journal of Medicine article, in 1995 it was shown to reduce the frequency of pain crises. Three years after that doing my journal article, the drug was FDA approved. Um and 10 years after that, um it was still basically being used uh as a drug only for people who met the criteria in that New England journal article, which was people who had a lot of pain crises. So even though dozens and dozens of articles had been published, showing that this is a drug that actually improves organ function mitigates the complications of the disease. Doctors were still using it basically for people who had a lot of pain, which didn't make much sense. And I was, uh at this point, a resident, uh in training and it never made that much sense to me because it's a drug that treats the pathophysiology of the disease. Not everybody with sickle cell disease has frequent pain crises. Uh Some extremely sick individuals have very few pain crises. So it didn't make sense to me that we weren't using it for everyone. Um By 2008, there were consensus conferences reviewing all this literature and still the community didn't really come down on the side of we need to use this drug for a broader population. Um At this point now, this is, we're, we're talking about data from 2017 20 years after the FDA approval and we're still um going back and forth about who we should be using this drug. But by this point in my clinic, when patients ask me if they should be taking hydroxyurea, and they would say things like, well, I'm not sure I need it. And, you know, there's stuff on the side effects and I would say to them, this is actually a pretty simple decision to make. If you wanna live longer, you should take the medicine. And if you don't, you shouldn't any questions about, well, you know, I, I don't have a lot pain crisis or I'm not sure I want to take a pill or what if I have a side effect, take the medicine. If you wanna live longer. If you have a side effect or an issue, then you can re evaluate. But we know that this drug will make you live longer. So if that's something you'd like, then you should take this medicine. So this issue of having taken, I mean, the drug was really discovered in 1977 I believe. Uh but having taken 40 to 50 years to figure out that a medicine uh makes people live longer to me was, was really not ideal. This is Bernard Herrick, uh uh or sorry, James Herrick, uh the hematologist who discovered sickle cell disease in, in 1909. Um And so, you know, we were, uh I felt like sort of innovation was, was really stagnant and sickle cell disease. Uh We were taking too long to figure out which drugs worked. There was a lot of research and this is actually my own research where we were pulling drugs off the shelf and seeing, well, maybe this will help. And so this was our trial of, of inhaled steroids, which um we did a small pilot study and we show that people who took inhaled steroids who do not have asthma um did better. Their biomarkers improved. So we're very excited about this. And uh that led to an ro one, we did a a trial, 80 patients who were randomized, they took inhaled steroids for a whole year. And in the end, um it basically looks like inhaled steroids are worse. So, um I wasn't super hopeful anyway, you know, the, the idea that some asthma medicine that we had pulled off the shelf is going to transform the landscape of sickle cell treatment. Um was really, uh I, I knew that was never gonna be uh the truth. But um you know, this was just another one of many uh efforts to find new treatments that didn't work. Um But then at this time, we were having a lot of uh pharmaceutical companies that were developing agents for sickle cell disease. And uh by 2018 we had three more uh FDA approved, they work by all different mechanisms. Some are anti cycling, some are um uh anti adhesive, some are antioxidant. Um So we had all these new medicines. Uh One of them is Ryan Lizama. Um That is a Pectin blocker, you take it once a month. Um And you can see here on the right, the Kaplan Meyer or sort of uh hazard ratio curves where people who get um the high dose medicine have a longer time to first pain crisis. Basically, individuals who took this medicine, about half the number of pain crises with sickle cell disease. So this was an exciting drug that got FDA approved, I believe in 2017 or 2018, there's voxel to which also came to mark, this is a small molecule. It's an anti cycling agent, you take it and basically kind of gets in there with your hemoglobin inside the cell and prevents it from sickling by raising the oxygen affinity. And also just sort of getting in the way of the hemoglobin sticking together inside the cell. And this is a, you know, sort of cartoon uh uh depiction of, of what the molecule does. And then you have happy biconcave red blood cells. Um patients who take this medicine since they're not sickling, they're not Healy and they have higher hemoglobins. And you can see these waterfall plots that patients who took the highest dose had uh the largest hemoglobin response. We've had uh pretty good responses with this drug as well. Um We have a lot of patients on both of them. Uh Sometimes you can have a patient go from 7 to 11 or 12 hemoglobin taking this drug. And also very important people did not have more pain, they had slightly fewer vas occlusive crises on voxel to. So now we have this new problem where we have all these drugs. When a drug gets FDA approved, it does not get compared to other newly approved drugs. Um It gets compared to the standard of care. So, hydroxyurea was in some of these trials, but there was no comparison of the drugs together. And furthermore, there was absolutely no usage of these drugs in combination. So, while they're all FDA approved for sickle cell disease, there is no precedent for how to use them together. Um, other than some basic like drug drug interaction, do we have ac YP 450 issue or something like that? Um, so we have a new problem and I certainly didn't want things to go on for 40 years. Well, we're trying the way we did with hydroxy, um, taking that long to figure out that the drug because it treats sickle cell disease makes people live longer. So looking at um the inclusion criteria from some of these trials, I basically saw that the problem happening over again, right? The, the the clinical trial that led to FDA approved approval of Crisan Lizama required that you had to have a certain number of pain crises. And what happens when you do that with your New England Journal trial? Well, when the drug gets FDA approved, even though it's approved for Sickle Cell disease, all the insurance companies basically have their pharmacists pull the new England Journal study and write a coverage policy that's based on the inclusion criteria from that article. So you're already headed towards a situation where only people who have a lot of pain, which is not everybody with sickle cell disease is going to be able to access the drug. So that is what led me to uh write this grant, which was called real answers. And it's based on leveraging a large registry um uh consortium that we were in that has these eight sites, uh 2400 patients in the registry. And we've got all these very nice validated data elements and Pr Os and, and labs and, and all kinds of stuff. So I said, well, maybe we can sort of modify this this multi center registry to something that actually can figure out how to use drugs together and, and which drugs are better. So I, you know, we, we made sure we're collecting the outcomes from these clinical trials like hospitalizations, et cetera. But also I wanted to look at the outcomes that matter to us as clinician. And you know, what is it that drives survival and sickle cells. One of them is, you know, organ injury. So we think about blood as an organ, blood injury, we call that hemolysis. So we're gonna look at hemolysis and certainly hemolysis has been correlated very, very strongly with survival and sickle cell disease. So we have ways of uh dealing with the fact that markers of hemolysis have a lot of col linearity and we basically distill them into one score. Uh Pro BNP is another biomarker we follow in sickle cell disease. It it is strongly correlated with mortality regardless of whether or not you have a lot of sickle cell pain. And then another really nice uh biomarker we use in sickle cell disease marker of kidney injury is how much protein you're spilling in your urine. And one of the really nice things about these my biomarkers like hemolysis, uh protein spilling and pro BNP is they're disease sensitive, they're not cumulative, right. So, creatinine is, is a little different than protein spilling because creatinine has, has somewhat of a cumulative nature to it that over time your creatinine will go up, can go up and down uh with acute issues as well. But in general, until you're at end stage, your uh micro albumin to creatinine ratio is a very disease sensitive biomarker. If you put someone on a drug and they're spilling protein at 200 it might go down to 100. And so these are biomarkers that we felt we could use to assess the effect of these new drugs on organ injury. And we, we like that they're sort of distal biomarkers like the select an inhibitor. We actually see protein spilling get better, but it's not like a drug that's affecting the kidney. What you're seeing is the disease is, is getting better and the uh organ stress is improving. So we're not gonna conduct a randomized controlled trial for every possible combination of these drugs. In fact, if you go back to your Combinator x, which my kids are learning in school, um you've got four drugs. Um There are 15 potential ways you could combine those four drugs. And then if you wanted to compare each of those head to head in a in a randomized controlled trial, combo, one versus combo two, combo, one versus combo three. you'd be, you'd be doing clinical trials for about 150 years and this is a rare disease. So, it's just, it's a nonstarter. This is not gonna happen. And yes, RCTS are better. The evidence is high quality. But, um, it's just a, it's a fantasy if you think that you can do randomized controlled trials to figure out which, which of these drug combos are the best. So what do you do in that situation where you can use something called target trial emulation. And so what, what we're doing here is we're taking this registry framework of these eight centers, thousands of patients. And we're collecting the same data that you would collect. If these patients were in a clinical trial, you would get regular labs, your adverse events would be adjudicated, every hospitalization would be recorded. And then what medications you are on all your concomitant medications, especially the disease modifying sickle cell medicines would be followed with a very high level of scrutiny. You're not just doing chart reviews to say, oh, this person is prescribed hydroxyurea and Azithromycin from 10 years ago. And things like that, you're actually checking for the, for the disease modifying drugs. You're checking, like did this person pick up their medicine or did they get this dose of uh, Crisan Liz? And then you use basically sort of back end data techniques, uh like propensity score matching and things to create what we call cloning or pseudo randomization to figure out. So here are just the the biomarkers that um are the drugs and what we do know about the drugs. And basically hydroxy is the only one we know anything about. Um And this is the sort of predicted consort diagram of how these thousands of patients will probably flow through the study. And so we're collecting all this data and the, the additional thing that we decided to do is uh DNA. So we're going to collect uh DNA from patients once during the study to do whole exome sequencing. Uh And we'll look at all these uh markers, we'll look at all the stuff from that that they would look at in the clinical trials like how many pain crises. But we're also looking at organ injury, which I think is very important and just some preliminary looks at our own data when we, when we dove really deep into our own data and, and checked, you know, for patients who are truly taking medicine A versus medicine B and we have a, a period of time, we can analyze where we're 100% sure the patient actually got the drug and we have biomarkers before and biomarkers. A after we actually see some trends here uh that Crisan Lisa looks like a better drug. Um in terms of PRO BNP, if you look at the Delta for patients who started voxel to versus Kris and Lizama. It's a small group of patients about 2020 in each group or 25 in each group. You're seeing a delta for Riz and you're not seeing a delta for pro BNP, same for uh urine albumin to creatinine ratio. We see a pretty traumatic drop um with, uh with people who start cry em up and then vor for hemolysis. This one really surprised me because this drug inhibits hemolysis. Um But what we see is that it seems to really be driven by super responders here that, you know, we have some patients who will take the drug and shoot from 7 to to 12, but there's a lot of non response in velt to and, you know, we are seeing that clinically. Um We're also gonna do some secondary analysis of breaking out the different like sub phenotypes of sickle cell disease, high hemolyzing more vas occlusive phenotypes, et cetera. And this is just more of that vel to data with the super response. So, you know, we have um some people who just basically have these huge responses. So we'll look at that genetically as well. Um And we plan to do some, some deep dives on those patients looking at, you know, single cell rnaz and, and the transcriptome, et cetera. So that study everyone's ir be approved. Uh We should be probably launching in the next few weeks. And so, um call me in five years and I will let you know which, uh, which drugs are the best. But for the present we are, you know, we have this problem of, we have drugs. We don't really have a good way to measure sickle cell disease. We have tons of labs that we kind of follow. We watch them go up, we watch them go down. It's not ideal. And so, um, another project that we are uh actively in is um trying to measure sickle cell disease severity in response to treatment through the eye because the eye is a transparent window to a disease sensitive vascular bed called the retina. So what we had discovered is that if you, you know when you go to your retina doctor, you probably get OCT A done um which is just a picture of the blood vessels in the back of your eye. But we realize that if we take multiple pictures uh from an oct a machine and then layer them on top of each other. With the image processing algorithm, we can identify blood vessels that have disappeared and then reappeared and quantify how often that happens and those episodes where a blood vessel is there. And then two minutes later, it's not, that's, that's a vaso inclusive episode right there, there's a vaso inclusive event and we actually have some microscope data where we can show it happening. Um So what we created was a sort of a metric of, of um how often this happens and it just comes out as a number basically the percent of the retina where we're seeing these types of events. And you can say this is a, this is a patient before and after hydroxy oops and um those flickering uh blood blood vessels, you, you see less of them coming in and out on the posttreatment. So this metric, you know, we had a few different ones, but the one we're using is the one on the left, we call it intermittent flow index, intermittent perfusion index. And you can see that people without sickle cell diseases, they don't have this, you know, the it's the people with sickle cell disease are having these events. And so, um and then when we sort of looked at this intermittent flow index across the different types of sickle cell disease, it really seems to correlate very nicely with your disease severity. Um So this is hydroxyurea. Yeah. And you can see the computer basically highlights these episodes red is where a vessel disappeared, blue is where a vessel that wasn't there suddenly appeared. Uh And then we quantify that and you can see the patient before Hydrea, 2% of their retina had this. And after um 0.74 well, what about when you cure people with gene therapy? What do you get? Well? So this patient um had an uh intermittent flow index of 2.83. Um He uh and that was after uh an exchange transfusion. So probably more than 50% of his blood was hemoglobin a at that time. Um He was in the Crisper trial um after a second exchange transfusion is uh intermittent flow index went down to 0.73 after the second exchange you probably 80 85% normal blood. Um And we think of exchange transfusion as I wouldn't say it's secure. But, you know, right after an exchange, uh most of your blood is you have, you have less sickle blood than a patient with sickle cell trait. So it is as close as we can get with a treatment to making somebody not have sickle cell disease. And then he got uh your CRISPR therapeutics, gene editing and his post uh gene therapy, uh fetal hemoglobin is around 50%. And then um you can see his intermittent flow index stayed right where, where it was after uh two exchange transfusions. And it's been like that since. And um this patient does not have pain crises anymore. And all his, his organ function has completely normalized. He has some residual um bone injury from, from uh uh arthritis. He, he accumulated. Um we also to, to understand what is actually happening when these vessels are disappearing. We have something called an adaptive optics microscope where we can actually take microscopic videos of the cells and you, we are seeing different types of things. And so this is a video of a sick old red cell. This is a little bit of a clump. Uh Actually, you can see a few cells in there um including blood flow. And so we've identified um uh a few different mechanisms for the way that blood gets stuck in blood vessels. But um we have confirmed that it's, it's red cells that are basically not moving in, in the, in the vasculature. Uh Interestingly, it's, it's usually not white cells which uh some of our mouse mouse models, there's a seems like a bigger role for leukocytes. So, so that is that project. How am I doing on time? So, this is all great. But uh sickle cell disease is a genetic disease that is very amenable to potentially being cured with gene therapy. And so that is, yeah, I think we'd all what we would like the most would be to put ourselves out of business and um have this just be a disease that you, you may be born with, but you'll be cured and, and that's that and it's really not much of a thing. So there are, there are a few different ways that gene therapy is being used to cure people with sickle cell disease. Uh go over a little bit with the um fetal hemoglobin approaches. Uh That's the CRISPR therapeutics is using that one. But basically, uh uh when you're a fetus, you're making fetal hemoglobin, which is uh it's transcribed to, to from proteins, uh genes that are at different spots on the genome than adult hemoglobin. Those that fetal hemoglobin does not have a mutation. Your mutation is in your adult hemoglobin uh genes. And so if you can get your body to just decide that it would make fetal hemoglobin. Instead of adult hemoglobin, you could theoretically be cured. And the more the better we know that there are certain uh genetic abnormalities where people make 100% fetal hemoglobin. And it is a completely benign condition. There's nothing that fetal hemoglobin can't do. It doesn't even cause problems during pregnancy. Um So the more the better and you can see that basically, there's all these transcription factors that kind of get together in different ways to regulate that switch from fetal hemoglobin to adult hemoglobin. BC L 11 A is a big one. So how do we do this? Well, um the two big companies on the market are coming to FDA approval are CRISPR therapeutics and Bluebird Bio and Bluebird Bio uses a Lent virus. And so basically what they do is um they are inserting a trans gene. So the, the lent virus carries an entire gene for a normal hemoglobin and it will invade the cell and drop that gene somewhere. It's actually relatively random where it goes, there's no specified place. But by and large, uh when that is done that you have, it's completely benign and then you uh start transcribing this normal hemoglobin and it's actually a tagged hemoglobin. So they know how much of it you're making. It's, it's called 787 Q. It's not just normal adult hemoglobin. It's a, it's a little bit sim more similar to fetal. Uh it has a higher oxygen affinity, which is a good thing. Um, because that prevents sickling. Um, CRISPR therapeutics is using, um, is using CRISPR, they're editing a spot uh on BC 11 A called plus 58 and basically BC 11 A is a gene that turns off fetal hemoglobin. So if you turn off BC L 11 A, you effectively turn on fetal hemoglobin production. How is this actually done? Well, you bring the patient into the hospital to an apheresis unit, you put a large line in um you give them a stem cell mobilizing agent, not G CS F because that makes people with sickle cell very sick. Uh Plexor is what we use and you harvest as many hematopoietic stem cells as you can CD 34 positive stem cells. Those are taken to a facility to a manufacturing facility where um either the CRISPR special sauce is added or the Bluebird Bio special sauce is added and those cells are edited and then they are expanded using proprietary cell expansion protocols until you have basically a product that has around half a billion stem cells that are going to make blood, that is not sickle cell blood, it's still the patient's blood. But it has been edited, then you myelo, the patient, you bring them to a bone marrow transplant unit. Um Right now, all these protocols are using Busulfan which is very toxic MYOB blade of agent. Um There's radiation as well. Um And it's actually very important that you do a, a pretty complete myelo you are not. Um even though the patient doesn't have cancer. Um There's two things. First of all, you, you wanna kill as many cells that are going to make sickle blood as possible. And also the partial my ablation trials where they gave lower doses of bulan, you basically had injured cells with DNA damage that survived. And so there was more issues with um potential malignancies and such. So the my ablation is complete, uh which means that the patient will be very sick, very vulnerable. They will be in a uh bone marrow transplant unit for a month, month and a half. Um But you receive this myo ablation and then we basically hang a bag uh of this product which has come back from this manufacturing facility. Um Those cells will go into your body uh because you've opened the bone marrow itch by, by giving you Suan, the patient will slowly start to make their own blood but blood that does not have sickle cell disease. Um These are the results from the Bluebird Bio trial. Uh CRISPR look pretty much exactly the same. Basically, once you've had the therapy, you stop having complications of sickle cell disease. Um There is some like lead in period. Some patients with chronic pain may, may continue to have certain issues, but by and large, you stop having complications. I've had a couple of people who've had Bluebird Bio. Their hemoglobins are uh above 13.5. You get about 8 mg of the 787 Q hemoglobin from the treatment, um CRISPR therapeutics, the fetal hemoglobin levels are not uh quite as high as Bluebird Bio. So if you, if you check uh a fractionation of Bluebird Bio patient, it's gonna be 75% 787 Q. This gene edited hemoglobin uh CRISPR therapeutics patients. They make about 50% 40 to 50% fetal hemoglobin, but they're both cures. So you, you're above this kind of threshold where it's basically a curative treatment problem is that it is not a cure for everybody. Um First of all, 90% of patients or, or more probably would not opt for this because there's a risk that it can kill you that uh you know, risk is probably around 1 to 2% just from the abuse. It's not the gene therapy, it's the Bulan. Uh and, and the uh how sick you'll be after the myelo A and then there's a secondary cancer risk with the Bulan. Most patients don't want this. Most patients are not fully ready to risk their life to be cured. And uh most patients want to have Children. So um uh that's another major barrier barrier. So what's the real uh uh you know, holy grail for, for sickle subsidies in vivo gene therapy, which would be a product that you inject into the patient and it goes and edits the cells that make blood in the patient rather than having them taken out. This is a lot tougher. Um because when you're doing it outside the body in a dish, you have the advantage of. First of all, you have 100% cell selectivity, you don't have to have some kind of targeted lent virus or anything like that because you've selected the cells before you administer the therapy to them. Um And number two, you have the ability to sort of check what you did, right? If you edit these, these cells in the dish, then you expand them, you can sequence them to check that I did. I insert that lent virus into any tumor suppressor genes or anything like that. And if something goes wrong, well, you just don't have to give it back to the patient. Anything you do in vivo. If it's something that you regret, it's in the wild and you ha you are, you know, you're done. So um we very much, you know, need need improvements. So our research, we are looking for a gene, this is Jim Beaker and, and me uh looking for a gene that has some selectivity uh in intrinsic to it. So, one of the problems with BC 11 A, which is what CRISPR therapeutics uses, that gene is expressed in leukocytes. That gene is expressed in the brain, that gene is expressed everywhere. And so, um you would have uh it's proto-oncogene, you would have concerns about editing a gene like that in the body without having a product that could really home just to the cells that you want. Um In fact, you don't even want to get to stem cells, you wanna try to get to cells that are making, you know, red blood cells, which um is a tremendous challenge. Whereas KOF one is a gene that has similar functions. Uh It's a regulator of erythropoiesis, but it is expressed uh exclusively in red cells. So if you were to edit KLF one, you wouldn't have uh in cells that were not going to be red cells, you would not have such great concerns. And part of the reason we know this is that there are human beings who have mutations to KLF one where they are insufficient in it. And these are benign mutations, but these people make high levels of fetal hemoglobin. So we tried to find an edit that uh where can we edit KLF one that would cause it to mimic what these populations in, in Malta and um Asia have where it's a benign condition, but they make a ton of fetal hemoglobin. So the first place we went after was called intron one. And the reason we uh targeted this site is because there's a very highly conserved region. If, if you follow that red a down across these, all these different species, everyone except for the dogs shared this um base. And uh so I thought, well, maybe this is important, let's see what happens when we cut it out. And so um one of the things about CRISPR is, is uh just because you have the sequence, you have the guide um very often you still just don't get editing. And so we had to tweak the system quite a bit. And we wound up with basically using two different guides to come uh different uh from both sides to try to chop out this one base. And uh uh using that approach, we were finally able to get very good deletions of that area greater than 99%. And when we looked in uh cell lines, we were seeing reductions in KLF expressions. So we were thinking, OK, well KLF is, is, is a gene that if we turn it partially off, it'll be like these people who are hap low insufficient and they'll make more fetal hemoglobin. Um It worked actually in se in leukemia cell lines, these like immortalized cell lines. But when we went to human CD, 34 hematopoietic stem cells, we made the edit and it basically did not change the behavior of the cell. Just continue to make, you know, maybe 20% more fetal hemoglobin, but definitely not something that would be curative. So that was uh three years uh and a disappointing result, but then we went after a different spot called um ehs one, which is another regulator region of the KLF gene. Um where if you were to take out the whole eehs one, you'd have like a 100 fold drop in expression and that is actually not what you want. So, one of the big challenges with KLF one is if you're completely insufficient in it, that is uh you're gonna have a cell that basically doesn't produce blood. So you need a way to sort of turn it down. So what we started doing is saying, all right, well, ess one is a very potent enhancer, maybe if we just try to take out a small piece. So we designed a bunch of CRISPR guides to try to take out small pieces of, of EHS one and we did find one where our RN A expression was where we want, you know, the 3040 uh percent drop in in uh KLF expression. So OK. Now let's check this one to see if are we seeing um increases in fetal hemoglobin? So you can see over here uh control cells are cells that are just edited with scramble CRISPR. Um Our positive control is the BC 11 A edit that is in vertex Therapeutics and that raises fetal hemoglobin about four times our EHS one edit alone raises fetal hemoglobin sevenfold. And then when we actually combine it with, with the original um intron one edit that we've been trying for several years, it raises fetal hemoglobin about uh 20 fold. So this was very exciting. We're super happy. We now have a target that you can edit. And the beauty of this target is that um if you were to send in some kind of gene editing vector and you wind up editing the liver or the brain or you know, off target, basically everything except you definitely don't want to go into the germ line. Um This could work and it would, it would be much more likely than BC 11 A to be safe. So how do you do that? Well, uh lipid nanoparticles is the approach that we're pursuing at this point. Um you got viruses, viruses is one way to do it and nanoparticles is, is the other predominant um approach that you can take. Uh viruses have the benefit of being they have specificity. So you can sort of send them to home to certain thing. The downside is that you can have immune response to viruses. Um Generally these are treatments you can only get once with a virus and then you have formed an immune response and, and that's it. So with the nanoparticles, you can just give them over and over again. They have a variety of formulations that are non immunogenic. So it's um something that can be given the challenge that we have um come across is that, well, there's a few things. Um, first of all lipid nanoparticles, we know they're fantastic at delivering messenger RN A. That's, that's basically your COVID vaccine is A, is a lipid nanoparticle with a messenger RN A. They're not as good at delivering gene editing messenger RN A because a lot more needs to happen. Right. You're sending in an RN A that's gonna be translated into a CRISPR protein. That protein has to hook up with a guide RN A that has been sitting in that cell. Um That's about a 24 hour process. And then that uh combined guide RN A and CRISPR protein, which is called an R and P then has to go into the nucleus and edit. Um So in situations where you're seeing, you know, 80% expression of just RN A, you may see 10 or 15% expression uh with your gene editing. Um the, the, the other issue is that these LNPS are not self specific and lipid nanoparticles, when you inject them into patients, they tend to wind up 98% in fenestrated organs, meaning liver and spleen. So if you have a, a disease that is uh you know, like hemophilia or um transthyretin amyloidosis, where, where, what you need to edit is in the liver, that's great. But where, when we, we need to edit the bone marrow. Um So what we are uh looking at is two approaches. One is to take a nanoparticle that's, you know, just a garden variety of nanoparticle, but inject it directly into the bone. And for me, as an emergency physician, I'm very comfortable with this. The hematologists who do bone marrow biopsies are also probably quite comfortable with this. Um We put intraosseous lines in the, in the er, all the time when we uh cannot get access. And so um we are exploring that and the advantage of that is that these lipid nanoparticles are really today's technology, we can make them, we can manufacture them, we can turn them into a drug. Um And so if the, if the, the key is just injected directly into the bone, then you really are very close to a drug that could be put in a box sent wherever, including Africa uh and just administered to people by Andros injection. The alternative is homing nanoparticles, which are uh it's a little bit more of a future proposition. Uh We have them today, you can, you can acquire them and do your research and get your nature paper, no problem, but in terms of manufacturing them like uh and, and being able to GMP the product that's, that's not quite there yet. So we're hoping that we can get this working with a non antibody conjugated nanoparticle so that we have uh less of a, a hurdle on our way to hopefully having a gene therapy. So that is that and I'll be happy to take any questions. It is 25. It was really nice. Thank you a little bit later. Um Right. So hot. You know what it's like? So. Mhm Yeah. So I, I've done a lot of adaptive design stuff. We all love bas and adaptive trials. Um The, the difference here is that this is not actually a trial at all. It is actually, it's a extremely fancy cohort study. It's just an observational study. Um Well, I if you were gonna do an adaptive design, that would mean you are assigning patients to treatments. We are not, we are just letting doctors prescribe uh and you know, whatever regimens the patients are on. And one of the cool things is that we find that patients wind up on multiple different regimens sometimes because of indication bias, but sometimes because of like access and insurance, cut this patient off and we'll get these natural sort of uh crossover events because patients are being uh denied care by their insurers and things like that, which is a horrible thing. But we hope to take advantage of that. Uh But yeah, so because we're not, we're not assigning anything. We're just using data on the back end. I'm definitely going to try some Bayesian statistics because what we found is Bayesian statistics is, is very powerful for small rare diseases. You know, things where you have sparse cells in your mega regression and things are just not conversion, you can still get estimates of like what does this mean when you're using Bayesian statistics? So if the field seems kind of right for evaluating combinations about driving rational and that by the marketer type. Yeah, strategies um are, are these drugs you, how, how do clinicians select which drugs to use is what, what's the cost of it? Yeah. Um Well, I'll do the cost first. The vel to is 100 and $25,000 a year. And, um, Prisma up is $80,000 a year. And hydroxy is, I don't know, it's 25 cents a month or something like that. Um, and, uh, and Dari, which is this protein powder is $30,000 a year. So they are used in combination by people like me and the other people at these centers whenever we can, the way we get people on combination therapies is you get somebody approved for one and then you get the insurance company, uh, the, the drug company to give you free medication for a year on the other. And, um, Novartis and pfizer are both doing that for their drugs. I think they're, they both think they have the best drug. And so they're hoping that patient a will go on, uh Risan Lizama when they're already on vel tour and when their year is up, they'll decide I'll have my insurer because the insurers will pay for one drug in addition to hydroxy, we'll have a lot of combo data there. But the events where you have somebody on three or four are usually based on free medication or somehow patient, just like coasts through. I like, can never understand why it happens, but sometimes we prescribe a 3rd $100,000 medicine and they just approve it. So it's, we have enough of those events that we do think we'll be able to look at these combinations. And I think if there's a signal there, then we would maybe we would pursue some kind of trial where it's actually an interventional trial where assigning patients to things and then, you know, basic in adaptive designs would be I think that type of design is, is crucial for rare disease research. Yeah, I I definitely through the gray talk, obviously a lot of the risk of the bone marrow um treatment which sounds very effective is, you know, your bone marrow, it seems very, you know, not in is there any potential work out there? Yeah. So I think the intermediate thing between, you know, these in vivo gene therapy where you're just injecting. Um there are antibodies right that can ablate um CD 34 cells without all this like toxicity of bolt fan. And there are uh there's a few companies that are uh basically their myelo a regimen is an antibody. So those will be probably over the next few years. And I do think that those will replace Bluebird Bio and Vertex. I, I'm not sure if Bluebird Bio and Vertex are doing an antibody based trial. Um, but they should because certainly once those are available, everyone is gonna want to be, you know, up and running with a, a gene therapy that uses an antibiotic because it's less toxic. Ok. I sit early. Yes. Yes. So, first of all, how did I get a good result on the first trial? And um and then basically share the opposite in the second. So the first trial, we did not have an enrollment criteria that you were on a stable dose of hydroxy. This was earlier in my career when like basically every week I was seeing half new patients, half follow ups. And I think a lot of patients in the original trial were getting titrated up on hydroxy and it was not an enrollment criteria that you have to be stable for us. So I think that um some of the benefit of inhaled steroids in the first trial was due to confounding with hydroxy titrations. We know that steroids is uh in systemic steroids and sickle cell disease. The minute you stop it precipitates crisis, it raises your leukocyte count. Um Our hope was that if you decrease inflammation in the lungs, that is the organ that reverses sickling and, and, and oxygenates the blood and that, you know, with better oxygenation, you would have lower um complications and that the dose would be low enough that you wouldn't have these other issues with, um, steroids. What I think you're seeing here is the real harm signal is with acute chest syndrome, which is essentially people showing up with infiltrates on their chest x rays. So, uh probably it turned out that whatever beneficial effect we had from sick from inhaled steroids was trivial if, if not nothing and then people were getting more pneumonia. And so that's where you see this acute chest syndrome. Signal question you chat. Um you mentioned cost of those drugs. So the the question is, could you address the role of racism in the time to hydroxyurea approval? And you? Yeah. Um so I I think that what you're dealing with is, is the fact that um if there's not a pharmaceutical company that stands to make several billion dollars on the approval of a drug, the ability of the sickle cell community to advance that forward is, is vastly inferior to the pharmaceutical industry. There was no money in it for hydroxy. This is an off, you know, patent drug. Um And it was really left up to the community of researchers which historically has way less funding, things are getting better now. But um if you, we are always compared to cystic fibrosis, right? Cystic fibrosis has the CF foundation is a multibillion dollar organization. They fund centers, they fund research, they have their own patents. Our foundations had no money and, and so uh especially foundation funding and federal funding were underfunded. And our ability to move a drug like hydroxy forward was, you know, trivial in comparison to what CF and the CF Foundation could do for its patients. And that is certainly a form of structural racism, right? It's a or a health disparity. Um But with these newer drugs, we have found that, you know, pharmaceutical companies have realized that um with a biotech start up, you can have a business model where you will make a tremendous amount of money, right? Glyco memetics was Cry Cry Aliza before it got bought out by Novartis. Um And so, you know, we're seeing that in lots of rare diseases that there are ways for biotech start ups to make money. And so, um that has been um like a, an improvement in the situation. Let's say that your time.
