Dr. Matthew Heeney - Sickle Cell Disease

Sickle Cell Program and the director of the Blood Disorder Center at Dana-Farber Boston Children's Hospital. So, he's here to talk to us about um sickle cell and novel therapies and um uh it's a pleasure to have you. Thank you. Thanks, Faro. Oh. Hold, hold your applause till later. It's a pleasure to be back. I seem to make it every five-year journey back to your grand rounds, but it's, uh, it's fun to be able to tell you, hopefully about some new updates. I have far too many slides, so I'm gonna skip over a lot of them, some of which you may have seen before and get to sort of the perioperative management if there is a little bit extra there. There's not a lot new, I gotta say. And then there's a lot of new, uh, path physiologically-based therapies that hopefully we'll get to. As a result of those new path physiologic-based therapies, I'm involved in a lot of clinical trial work. And so here's, uh, some of the disclosures. So we'll review the path physiology of sickle cell disease, we'll review hopefully the preoperative management of sickle cell disease and discuss some of these new path physiologic-based therapies. So as you all know, hemoglobin is a hetero tetramer. It's made up of 4 protein subunits. There are 2 alpha-like globins and 2 beta-like globins shown here in purple and yellow, and each one of those globins is bound to a hemoiety, the iron-containing ring, which of course binds oxygen, which is the whole point of hemoglobin. It can also bind carbon dioxide, irreversibly carbon monoxide, and also nitric oxide. And the synthesis of this protein globins and the hemoides must be very balanced. Um, otherwise, any imbalance leads to a disease state. And so if you have an imbalance of alpha or beta globin, these are the thalassemia syndromes you've heard of. The imbalances in the creation of heme, these are the porphyrias, uh, or yoblastic anemias, all of which are, are potentially deleterious. And so these alpha and beta globin genes come from the alpha-like locus on chromosome 16 and the beta-like locus on chromosome 11. And what I show you here is just uh from 5 to 3, there are a number of gene duplication events over our species' evolution. And these are expressed at different times during our life. And so zeta, an alpha-like globin, binds with epsilon, a beta-like globin to make one of the embryonic, uh, hemoglobins found in the first trimester called Gauer one, and alpha and epsilon or zeta and gamma make these other really clinically not that useful, uh, hemoglobins to us. But the dominant intrauterine hemoglobin is fetal hemoglobin, or alpha 2 gamma 2 here in the green squares. Shortly before birth, Gamma is silenced, which we'll talk a little bit more about later, and beta gets turned on, such that now alpha 2 beta 2, or hemoglobin A1 or adult hemoglobin is the dominant hemoglobin through the rest of our life. And so another way of looking at this is, uh, in this graph. So the origin here is conception. This vertical line would be a term birth, and you can see zeta, the alpha-like globin, disappears into the early first trimester and is replaced by alpha globin for the rest of our existence. In utero, the dominant beta-like globin, as I mentioned, is gamma globin, and these together make fetal hemoglobin. And just before a term birth, gamma gets silenced, beta gets turned on, and the so-called globin switch occurs, such that you make hemoglobin A1 almost entirely by the time you're 1 year of age. So from a hematologic or hemoglobin point of view, you're an adult by 1 year of age. And this globin switch has been, uh, hundreds of millions of dollars of NIH funding into trying to understand what causes this globin switch, and we'll get back to that hopefully toward the end of the talk. So what is sickle cell disease? This is a beta globin, uh, disorder. And so, as you would imagine here, diseases of beta globin have no, uh, manifestations in utero, um, whereas alpha globin disease like alpha thalassemia can be a disease, uh, of the of the fetus. And so sickle cell doesn't become a problem really until halfway through the first year of life as beta globin becomes the dominant beta-like globin. And so it's a single amino acid substitution. A glutamic acid is replaced by a valine. As many of you know, avaline is a hydrophobic amino acid. And so when the uh hemoglobin molecule goes to the periphery and delivers its oxygen, there's a confirmational change that happens as it releases those oxygens. And what happens is that that valing, that hydrophobic valing gets exposed on the surface, surface of the hemoglobin molecule. And you remember from soap bubble experiments that hydrophobic things like to go towards other hydrophobic things in aqueous solution, and that's all the red cell is, is a bag of salty water. And so as a result, in the deoxygenated state, these hemoglobin molecules tend to bind together in a non-covalent polymerization process. And so in this cartoon, uh, my hemoglobins here are now represented by circles. And if I decrease the pH, um, increase the acidity, uh, increase the hemoglobin concentration, decrease the temperature, I push this reaction to the right, such that these begin to bind together until you You have this little nitus uh of uh polymerized hemoglobin and then very quickly, it becomes this very geometrically precise 14 stranded helical fiber. And this polymerization process in the deoxygenated state is the basis for all the subsequent downstream path physiology. And I tell the kids in clinic, this is just like your blood is Legos. And so this, uh, cartoon also from uh Frank Bunn shows your hetero tetramer 2, alpha 2 beta 2, where you take away oxygen, you expose that valen here represented by an indent and outcropping, and they snap together much like Legos in a very molecularly precise way. And so inside the, the blood cell, these hemoglobins, um, form this, uh, an electron microscopy here. You can see this nice sort of twisted strand. And the results from a cellular level is that the biconcave disc eventually takes on the sickle formation as all of these polymers line up. And here's an EM of a sickled erythrocyte. The one concept that's important is that of delay time. So on the bottom of this picture here shows a biconcave disc, uh, erythrocyte that's been cut in half, and you can see the little dots in there representing the hemoglobins. And as this red cell traverses the capillary into the postcapillary venule, the longer it takes for that to occur, the more time there is in the deoxygenated state for these hemoglobins to polymerize. And if that is a, that traversing of that capillary is so slow, you can ultimately end up with a sickle cell. And this is why in uh vascular beds which have incredibly high flow like the coronary artery, there's almost never a sickle vasoocclusion because the red cells out long before it's had a chance to polymerize. So this molecular polymerization leads to a distortion of cell shape into the so-called sickle shape. Eventually, as a red cell goes in and out of your left ventricle 6 times over the next minute, it's gonna change shape back and forth, back and forth until eventually those polymers damage the membrane of the red cell. And this damage leads to an abnormal permeability, such that water leaves the cell, and as a result, the hemoglobin concentration goes up as the cell gets more and more dehydrated. And eventually it can't take it anymore and it becomes irreversibly sickled at about 7 days of age as opposed to the normal 120 days of age or a red cell lifespan, and it hemolyzes prematurely. And so you end up with the two sort of pathonomonic findings in sickle cell disease, which are hemolytic anemia and tissue infarction related to those sickled cells blocking blood flow and leading to downstream, downstream decreased oxygen delivery and nutrient delivery shown here in these cartoons. So what's pretty clear is there are many other path physiologies that contribute to that vasoocclusive event, not just the red blood cell and its change of shape. The hemolysis here, shown like my red cells throwing up here, results in two problems. One is that free hemoglobin is pours into the plasma, and another intra erythrocyte enzyme called arginase is also released. And the, the combined effect of this is to decrease the availability of nitric oxide in the, in the vasculature. And you know, nitric oxide, formerly called endothelial-derived relaxation factor, is important to keep, uh, uh, blood vessels, particular arterials, um, relaxed. And so the result of having a low nitric oxide state by arginase breaking down the substrate for nitric oxide synthase and by directly eliminating nitric oxide is to have vasoconstriction. So now you've got sickle cells moving through a smaller tube. This is a setup for disaster. So we have this cellular pathophysiology beyond the red cell. We end up with vascular, um, endothelial dysfunction and vasoconstriction. The sickle red cell binds to the vascular endothelium more readily. It has more intracellular adhesion molecules. Because this is a hemolytic anemia, the reticulocyte count is elevated, reticulocytes are bigger than your average red blood cell, and so just physically, they, uh, block more. White cells have a variety, as you know, of intracellular adhesion molecules. Their job basically is to be a little Velcro ball looking for, uh, things to bind onto. Platelets and sickle cell disease are activated and they become hyperactivated during a vasoaccclusive event, which we'll talk a little bit more about in terms of therapies. And if you ever did a D-dimer on a patient with sickle cell disease, it's always positive, um, and that shows that there's also some thrombin degeneration. Add to that this nitric oxide low state, and increased transit time, which means you're more likely to hit the delay time, more oxygen extraction, more sickling, and you get this sort of vicious circle. This graph is just to show you that the probability of severe disease goes up as you have an increasing white count. So the white cells are clearly very important in the vasoocclusive event. And if you add to the high white count, severe anemia, which we now know as a high hemolytic rate and probably more nitric oxide consumption, your risk of severe disease goes up even further. And so we'll come back to that a little bit about how the white cell is involved. Actually, I'll come back to it now. And so here are the, uh, sort of classic idea of a, a white cell rolling along the vascular endothelium. Uh, and there are many, many, uh, intracellular adhesion molecules which mediate this, particularly the rolling by P selectin. Then as it becomes adherent, E selectin is important along with VCAMs and ICAs and other, uh, well-known intracellular adhesion molecules. And so, it's this combination of events, uh, and the contribution of the white cells which make them particularly damaging in sickle cell. And so I'm hopeful this is gonna work. I didn't have a Back time to look at that. Well, let's see, I'm gonna, I'm just gonna show you the real video cause it's here. Oh, recommended settings. So this is a video of a um a cremaster muscle of a mouse, which has been pulled over a light microscope. You can imagine that's delicate surgery for you. So this has been inflamed to induce what would be a sickle crisis. But in a sickle trait mouse, you can see there's excellent blood flow. The white cells are rolling along, some are actually adherent. But that's the normal state. Now, if you take a mouse that's been engineered to have sickle cell disease in this next frame, there's a freeze where it shows the white cells shown here with the triangles are now adherent to the vascular endothelium. And you can appreciate here, although it's a smaller vessel, although there's not a complete stopping of blood flow, no thrombosis, you can appreciate here there's a significant slowing of the red cells compared to the previous image. And this is decreased, just that decrease in flow there will result in a significant decreased oxygen delivery downstream. So, sickling leads to vasoocclusion, vasocclusion leads to regional hypoxia, acidosis. The red cell, as it gets older, it gets dehydrated. Those three things together make it more likely to sickle. And this leads to then the shortened red cell lifespan, the hemolysis, the release of free free hemoglobin into the bloodstream, consumption of nitric oxide, and the consequent endothelial activation. Inflammatory mediators with a reperfusion injury, um, all of which contribute even more to a vasocclusive event. And this is the vicious cycle that we try to break, uh, during a sickle crisis. And so the epidemiology of sickle cell disease, as you know, this is an autosomal recessive disorder. Both parents need to be at least carriers. This is the most common single gene disorder in African Americans, with about 1 in 10 African Americans being a heterozygous carrier for the sickle mutation and about 1 in 400 being homozygously affected. We, it's not a reportable disease, so we don't have an exact number, but we believe it's between 1000 and 120,000 Americans are affected by this, and this makes it an orphan disease in the United States. It's one of the biggest orphans, if you will, but it's, uh, certainly why it's of interest to new therapeutics. We now believe there are about 2200 newborns born annually, but, uh, there are many patients who move to the US or to industrialized nations when they have a, a diagnosis. But it's important to remember in cosmopolitan cities such as Boston that other ethnicities are also affected, those from the Indian subcontinent, those from the Arabian Peninsula, and those of Hispanic origin, whether that be from the Mediterranean, South, Central America, uh, or even the Caribbean. And why is that? Uh, it's because we know that the global distribution of hemoglobin disorders roughly mirrors the, um, distribution of malaria, endemic malaria prior to the 20th century. And Haldane and others put forward this hypothesis that in these regions of the world, that heterozygous forms or carrier forms of sickle cell, thalassemia, and other red cell disorders confer some sort of fitness, that patients or people who have this carrier state are much less likely to die from, particularly falciparum malaria. And therefore, even though 1 in 4 of their offspring will have a disease that will certainly shorten their lifespan, the advantage of the carrier state, uh, results in the selective advantage that allows these these uh abnormal hemoglobin diseases to continue on. And so just here to show you in sub-Saharan Africa, this is where uh malaria is endemic and yellow, and here's the allelic frequency of the sickle mutation. You can see there's almost a perfect overlay, sort of indirect evidence of that hypothesis. So at this point, I've really only been talking about SS disease. That's where you inherit the S mutation, the glutamic acid availing substitution one from each parent. But there are many other ways you can get to a severe sickling disorder. You can inherit an S from one parent and a blank or a beta zero thalassemia mutation from the other. So all you can make is sickle. So it's essentially the same as SS disease. SC, our next most common disease, is also a position 6 mutation, except it's a lysine instead of a valline that's not hydrophobic. The hemoglobin C does not participate in the polymerization. Um, but it does, uh, dehydrate the cell further. And there's a variety of other esoteric combinations, the SO, SD, SE, uh, and as we, our melting pot, um, uh, is bringing together all sorts of new, um, combinations, we don't really understand the, uh, um, the natural history of some of these combinations. So let's talk briefly about some of the manifestations of the disease and complications that arise. In my mind, I think of these that are acute and those which are chronic. And you can almost break this down as these are the ones that happen most commonly in pediatrics. And these are the consequence of the uh chronic vasoocclusion that happens, and it can affect almost any organ system in the body. In fact, any organ system. I can describe a complication wherever the blood flows, except I suppose the lens of the eye. So I break down those acute events as to those which are related to the vasoocclusive blockage of blood flow and those which are non-vasocclusive, really more related to hemolysis. And we'll talk about a few of these ones in red here, the more common ones, pain, crises, acute chest syndrome, stroke, and then bacteremia of sepsis. So pain, this is what we think of mostly for sickle cell disease. It can happen very early on is that um gammaglobin decreases and fetal hemoglobin goes away and the sickle mutation becomes the dominant beta-like, uh, expression, uh, around 6 months of age, that's when we begin to see problems. Pain can be very, uh, unpredictable, although there are definitely triggers such as dehydration or, um, hypoxia. So, Uh, and cold exposure. So this is right around the time of year when we find some poor undergrad who's gone off to college and she's wearing a miniskirt, going on a pub crawl, getting dehydrated from the, uh, from the alcohol, and then this triggers a crisis. Pain is the most common cause of ED visits and admissions to the hospital. It's our focus and clinic of the home pain management plans. And what's very clear is how you manage or how effectively you manage pain very early on in life really does affect the family and the patient's ability to cope with pain. And this is your touchy-feely statement here that uh the management of pain requires a rational, non-pharmacologic, pharmacologic, and psychosocial intervention. And so, on our service, we're pretty good at the pharmacologic. We have a good psychosocial team. Um, we wish we had a psychologist, but also other non-pharmacologic approaches are extremely beneficial. I skip over that. So often when we have sickle-related pain, it's not just the pain that you typically see in the long bones, the arms and legs, the sternum, the ribs, or the vertebrae. The flat bones of the skull and the pelvis tend to be spared. But you can have uh pain in the chest, but you have to make sure it's not an, uh, signaling something else, such as acute chest syndrome, which we'll talk about next. Fever, uh, and, uh, associated with, uh, long bone pain, we often worry about infection, and these patients, I'll show you, are, uh, at risk for serious bacterial infections. And so we often have to worry about osteomyelitis. Perhaps more of interest to you guys is abdominal pain. All these patients can certainly get anything like, uh, appendicitis, just like any other, uh, child. Um, but we do worry about two certain complications. One is splenic sequestration, so pain in the right upper quadrant in particular with a large and tender spleen is something we worry about. Or a patient who has abdominal pain with ichthys, then we worry obviously about uh cholelithiasis or choleiddocholithiasis depending on the location of the stone. I don't need to tell you guys about pain assessments, hopefully. I think Jco has uh tattooed these somewhere in your body. I like the fruit scale myself. It's from South America, but I'm Canadian, so I've never seen most of these fruit. I think if you come in with a, I guess that's a watermelon, we want you to go down to like a papaya by the time we've started treating you. But, uh, nonetheless, it doesn't matter what you use, you just have to use it often. Uh, this is the, what, 6th vital sign, 7th vital sign, one of those ones. So what's our basic treatment, and this is what's uh most uh disheartening for someone who's been working on this disease for their career, is we treat with hydration. We give them a little over maintenance to try and break down that vasoocclusive event, and we give them a little local heat to allow some vasodilatation, and that is the end of our path of physiologically-based therapy. There is nothing after that, after 110 years. And so after that, all we do is give you medicine so you don't care anymore. And so we give you opioids, non-steroidal antibiotics, we give you everything else, and then we try and treat all the side effects until you get better by yourself. It's uh quite humbling. OK. And so I don't really need to tell you, I don't know why Aleve has shown up there. But, uh, in the analgesic ladder, you climb up this ladder with uh incomplete pain relief. So you start with non-opioids such as Tylenol, then you move on to NSAIDDs, uh, and COX-2 inhibitors, and then on to finally opioids and then parenterally, uh, as, uh, you get worse. And then you try to climb down this peeling off one at a time. Just because you go up a step, as I tell the patients, you don't stop the step below. So because you had an NSAID, don't stop the non-opioid, etc. So acute chest syndrome is just that, it's a syndromic diagnosis. This is a chest X-ray from a patient here. You can see over the course of one day has whited out one lung. I'm not a radiologist, but even I can see that. And so this is a syndromic diagnosis. As I mentioned, you need a new pulmonary infiltrate, a fever, and any respiratory symptoms. So that would be pneumonia in any other person, but is acute chest syndrome and sickle cell disease. And why do we care about that distinction? This is the second most common cause of hospitalization. In fact, patients who are in the hospital, immobilized, not taking deep breaths, they can get a little bit of adalectasis, and that adalectasis leads to a VQ mismatch, and that's when you get intrapulmonary sickling, and that can really blossom, uh, into acute chest syndrome. It's also the most leading cause of death in sickle cell disease in pediatrics and adults, so we have to treat this with great care. And this has important consequences for the postoperative state as well. And if you have comorbid asthma, this is a, this is an additive or even synergistic uh mortality risk. This is a study for, uh, from the acute chest syndrome uh group now from, gosh, uh, almost 20 years ago now. Uh, this must have been in the, uh, on the eve of the creation of the IRB, um, because they brought 670 consecutive cases of acute chest syndrome. And you can see that a variety of infectious agents were thought to be inciting chlamydia, mycoplasma, legionelle, a lot of atypical bacteria. Fat embolism, probably from a, a, a vasocclusive event and bone infarction, was responsible for a small percentage. But you can see almost 60% had either all studies negative or the studies were incomplete. And the feeling is this was in situ vasoocclusion leading to the acute chest syndrome. And so how do we treat acute chest syndrome? Well, if they're hypoxic, we give them oxygen. Um, if, even if they don't have a history of asthma or bronchospasm, we give them a bronchodilator trial to see if that improves their hypoxia. Often it will. We give them a judicious IV fluid. We wanna replete their, uh, correct their um deficit, but we don't wanna result in complicating matters by having Pleural effusions, control their chest pain so they can have good diaphragmatic excursion to prevent further analectasis. We treat them empirically with broad-spectrum antibiotics and the macrolide because of those potential inciting, um, atypical bacteria, and then we'll often transfuse them very early. Luckily, in the last 15 years, we've probably only intubated about 1212 times and probably half of those in one patient. Um, but, uh, aggressive ventilatory support is really needed and often daily bronchoscopy to remove what is really an acellular protinaceous goo out of the airway which accumulates and, uh, causes, uh, air trapping and problems with, uh, ventilation. So this Kappler-Meyer curve is just to show you that if you're asthmatic and have sickle, you have a significant increased mortality risk. And so we treat patients who have comorbid asthma very aggressively with inhaled steroids. Um, but this is also something to think about in the, for the anesthesiologists, uh, preoperatively. Stroke, I won't spend a lot of time on. I'm certainly not a neuroradiologist, but if you squint, you can appreciate there's fewer blood vessels up here. And if you look at the middle cerebral artery here on the right versus on the left, it's, it's really been obliterated. And this is, uh, in this case, Moyamoa disease, um, afflicting this child with sickle cell disease. And these are the various genotypes of sickle cell. Again, these Kaplan-Meyer curves showing every time it goes down, somebody's reached the endpoint here, which is stroke. And by the time they hopefully leave my care around 20, historically, those with SS disease, about 10% of those children will have had a stroke. So it's not affecting everyone, but it's certainly one of the more feared complications. And so how do we treat this? We need to exchange, transfuse them, basically take out their sickle blood, give them regular blood back. We don't really care what's happening in their head too much. If they have symptoms of a stroke, we assume it's a stroke and worry about imaging later. Um, we give them IV fluid but don't wanna have cerebral edema. And then we give them a simple transfusion while we're waiting to get 6 or 8 units of blood from denum often cause we have to use extended cross types. But ultimately, we need to get to an exchange transfusion. And so often we'll be asking you guys to help us put in a central line very quickly so that we can move to a rapid erythrocytoppheresis. How do we assess those who could be in that 10%? Um, we do transcranial Doppler ultrasounds where you inciate the major intracranial arteries, the circular willis, the, uh, middle cerebral artery, the anterior cerebral artery, the PCA also, and the internal carotid. And it turns out that this is the best way of integrating all the various risk factors, um, that you can, uh, assess and, and have a pretty good idea of who's gonna have a stroke over the next few years. And so if you look at the velocity, those with the highest velocity have the highest risk. And so if you're over this threshold of 200, you can see here that about 60 or 40% of patients over the next 3 years will have had a stroke. And so, SOP study took those patients who are over 200, started them on monthly transfusions to suppress or eliminate their sickle hemoglobin. And you can see here that these curves separated. And now, the standard of care is to screen all children annually with a TCD. Those with a high velocity get to put on chronic transfusions for life. And this is a pretty good way of, uh, eliminating, um, the, the chance of them having a stroke. And in fact, this was published and the NHLBI put out an alert about this, that all children with sickle cell disease SS should be, uh, screened annually. And this is what happened to the stroke rate in California, but this is true across the country and across the developed world that use TCD. Um, that you can reduce the rate of stroke and sickle cell disease to virtually zero. And this was so effective that the overall rate of stroke in children went down just because of the effect this had on the sickle population. So sepsis, so children with sickle cell disease, their spleen, uh, becomes, uh, um, uh, non-functional by about 1.5 years of age. This is a technicium liver spleen scan where you take the patient's red cells, heat them to damage them, label them with technicium, infuse them back into the patient, and they'll go to places, uh, and light up places which have phagocytic function. And so here's a normal patient. You can see the Um, reticular endothelial function of the liver, but there's, uh, some phagocytic function here in a normal child, but this is a 2 year old who has completely absent uptake of their spleen. So they're functionally asplenic at a very early age. And so one of the earliest studies in sickle cell disease, the PROP study, uh, showed that if you take patients, identify them early with sickle cell disease, start them on penicillin prophylaxis, that you can decrease the incidence of invasive pneumococcal disease, uh, significantly. And so this kappa marker has been flipped over. But every time the, the curve goes up, somebody got invasive pneumococcal disease, and you can see that those who are on penicillin got virtually none, and they had to stop the study early as it was unethical to continue. And this resulted in the recommendation from the federal government that prophylactic oral penicillin should be started by 4 months of age, and also that all children in the United States should be screened at birth for sickle cell disease. Uh, relevant to us, New Hampshire was the last holdout, uh, until 2007. Live Free or Die, I guess they chose the die part. Um, but nonetheless, um, they finally snapped too. And so, nowadays, really, penicillin is not probably as important as vaccinations. As you know, in urban areas like Boston, there's probably like 80% penicillin resistance among strep pneumo. And so instead, uh, pneumo, you, many of you lived through the area of pneumococcal sepsis, um, and many of you saw its recrudescence after the serotype spread around PCV 7 and now PCV 13. I suspect soon in the next few years, we'll see that there's gonna be a new PCV 18 or 21 or something of that nature. Menactra, the conjugated meningococcal vaccine, has made a huge difference. It's now also um eligible down to age 2. And then more recently, with the outbreak of serotype spread in meningococcus to the, the B serotype, there's now Bexsero and Trumenba, but these cannot be, uh, are not been shown yet to be effective under age 10. I assume over the next few years, we'll be starting giving this primary series of this Men B vaccine early also. Not to forget, Pneumovax, it covers obviously 23 serotypes, PPV 23, and there are some serotypes which are found in PCV 13, which are not in PPV 23, and of course, vice versa. And so it's important to make sure patients are fully vaccinated with these. And since then, I didn't show the Kappelnmeyer curve, but really, it's unlikely to die of a, of a, uh, of a bacterial infection and sickle cell disease, which really is the leading killer still in underdeveloped nations. Splenic sequestration, I hope I don't need to tell you guys where the spleen is. It's here in the left upper quadrant. Um, but it can become massive very quickly, and you know, it's often important to start palpating down in the right lower quadrant when you're looking for the spleen edge, for sure. And the clinical research diagnosis of acute splenic sequestration, uh, it really has its highest incidence in toddlerhood. Um, but you obviously have splenomegaly, and with that, splenomegaly becomes hypersplenism as the spleen gets big, it gets dumb, and it starts eating off the all the cells, uh, in the body before their, uh, their best before date. So your hemoglobin drops about 2 g per deciliter from its baseline. So in sickle cell, that might be 8 and so dropping to 6 or below. The retic count compensates and goes up from the baseline. The platelet count often ends up with a relative thrombocytopenia, and these patients have a high white cell at baseline, and so they may come into the normal range, but below their baseline. The spleen can get so big, you can have uh uh uh impingement on the diaphragm, leading to dyspnea, impingement on the spleen, leading down to the stomach, pardon me, leading to early satiety or even vomiting. And you can hide your entire blood volume in your spleen if you try. And so there's always a, a sort of resident case of a hypovolemic shock, but nobody can find the bullet hole or whatever it is and it's that's because it's just not circulating, but it's still in the body stuck in the spleen. How do we manage this? Well, we fluid resuscitate them first with crystalloid, but really the goal is to give them red cells, uh, judiciously small red cell transfusion as that will induce the spleen to auto-transfuse all that blood back into the body, and we don't want them to, uh, go above a hemoglobin of 10 or 11 afterwards. So, if they're at 4, we might shoot for 6 and hope that the spleen empties out to 10. It turns out that you observe them, this doesn't help. If you try to transfuse them until the mythical age of 5 when the risk of pneumococcal disease goes down, that doesn't help either. There's still a high recurrence rate. And that's when we often turn to you. Partial splenectomy really plays no role in this hemoglobinopathy and so ultimately, a total splenectomy is usually uh what we need to, to do in this case. Well, I'm not gonna tell you much about choleotiasis. There's an ultrasound. You can see all the stones. A hemolytic anemia produces lots of extra bilirubin. These patients are at very high risk. And one of the studies I was involved with early on, if you have comorbid, uh, sickle cell disease and Gilbert syndrome, which is quite common, you're the 7/7 genotype where you can't conjugate your bilirubin very effectively. Almost 90% of those children will have their, um, gallbladder removed by the time they leave our care. Avascular necrosis. Uh, there's no orthopods in the room, but this is a problem too. Uh, the way we treat this now is, uh, by doing decompression, um, by putting, um, the drill in here, drilling multiple pathways to try and decrease the pressure in the femoral head, and then at the simultaneously taking bone marrow from the iliac crest, centrifuging it. Into a low volume and uh inserting that back into the femoral head and that actually can help. And I'm, again, not a radiologist here, but preoperatively, you can see here in the left hip is very ratty and broken down. This child underwent the procedure from our group here and you can see there's a much smoother and healed uh as um femoral head in this case. And so this can actually uh be quite effective. All right. And then quickly, we'll talk about perioperative care. Unfortunately, since I've spoken to you last, there haven't been a lot of major uh uh improvements or, or changes to what you should be doing in perioperative care. I think I've shown you that sickle cell patients more than other children will be likely to meet you at some point because they're likely to get a cholecystectomy, a splenectomy, or potentially a joint replacement. Um, also, very typical to have, uh, Enlarged lymphoid tissue and, and need tonsillectomy or adenoidectomy because that leads to obstructive sleep apnea, hypoxia at night can induce sickle crisis or other complications. So, it turns out that the OR is an extraordinarily bad place to be if you have sickle cell disease. In the 50s to 70s, it turned out that uh people who went to the OR, there's a 10% chance of not coming back and about a 50% chance of coming back with a sickle related complication. In the cooperative study of sickle cell disease, which was really during the 80s, was a natural history study of both children and adults, it turned out in that cohort, 7% of all the deaths were perioperative. It turned out that there was a 1.1% mortality rate as you went to the OR. Luckily, children are hard to kill, but that still doesn't mean we shouldn't, uh, try to prevent that. Um, so. Let's keep trying. And if we think about the things that we know pathophysiologically and or emotionally with the patient that can lead or trigger a vasoocclusive event, a lot of these are in the operating room. The patients are stressed before they come. Um, they're exhausted because they didn't sleep the night before. They get stripped naked and put in a cold room cause surgeons aren't allowed to sweat, is my understanding during an operation. Um, And uh you can uh hypoxia, again, uh, bagging, or ventilator is never as good as your diaphragm, and uh fever postoperatively, infections, etc. All of these things are known to trigger vasoocclusive pain. And particularly what we worry about is acute chest syndrome, uh, as, uh, as I've shown you, the leading killer, but postoperatively a big problem. And so this, uh, study group now from, gosh, uh, 25 years ago now, um, looked at developing standard perioperative regimen, comparing the complication rates of two transfusion regimens preoperatively and looking at the risks of those and trying to identify predictors of complications. And so they took nearly 700 patients getting similar, uh, procedures and similar populations. They gave them all preoperative hydration, and this is why we bring people into the hospital the night before to give them overnight hydration. I'm sure at some point, the insurance companies will ask us to be doing that at home. We give, make sure that, uh, the, uh, anesthesiologists are aware of the case, that they do appropriate intraoperative monitoring, and then postoperatively that we make sure that they have good diaphragmatic excursion, good pain control if it's an abdominal procedure to allow that to prevent adalectasis even further. And so they took two groups, as mentioned. One group got an aggressive transfusion, uh, protocol, and one got a conservative protocol. Aggressive meant that they either got an exchange transfusion or multiple, uh, every two week transfusions with the goal of reducing their sickle percent from 95 or 100% down to 30%. Whoops. And the other was a single simple transfusion the night before the operation. Most of them got a single simple transfusion. They didn't reduce their sickle hemoglobin nearly as far, but they got their hemoglobin up to around the same 10.5, 11. And that, the, you can see the obvious thing here is that those who got the aggressive protocol got many more units of blood, many more blood donor exposures than those who took the conservative regimen. And so they put these head to head. And it turns out you don't really need to read this whole thing. But it turns out that there was really no significant difference related to complications postoperatively related to sickle cell disease. Um, and so now, we tend to use a conservative regimen unless it's a particularly high-risk, um, operation or particularly high-risk patient. However, what was seen is there was a significant increase in transfusion-related complications than those in the aggressive group, as you might expect. There were, um, almost, uh, twice as many patients who got a new aloe antibody. And this is the fact that, uh, at least in the United States, most blood donors are Caucasian and most sickle blood recipients are non-Cucasian, and there are subtle small, uh, minor blood group antigens, which we try to eliminate with uh extended cross-match, but these patients can end up with new allice sensitization. You can also see there's a 4 times risk of delayed hemolytic transfusion reaction, which can be quite scary in sickle cell disease. Not only do you hemolyze the transfused cells, but you can hemolyze. Um, the bystander, uh, endogenous cells. And of course, in an exchange transfusion, often you need a central line, and this doesn't even take into account the thrombosis that we so commonly see with central lines. So the conclusion here was a conservative transfusion regimen, a single, simple transfusion was as effective as an aggressive regimen in preventing the majority of post-operative complications, uh, and, uh, had less, um, transfusion-related complications. Also looking at other predictors of, uh, of risk. So those who had postoperative acute chest syndrome, if they had a history of pulmonary disease, so I've already shown you that asthma, comorbid asthma is a bad problem. And then the surgical risk category, which I must admit I'm not an expert, the higher that is, the worse off you're gonna be too. Turns out that if your, uh, age didn't really make as much of a difference as we thought, uh, but if you're a person who's admitted a lot because of sickle-related complications, you're more likely to have postoperative complications, which sort of makes sense. I'm gonna skip through that trial. And so currently, the National Heart, Lung and Blood Institute recommends that surgery and anesthesia be aware that the patient has sickle cell disease. That's important. Um, the patients with SS and S beta zerothal, the so-called severe forms, which have a baseline hemoglobin around 7 or 8, should be transfused by simple transfusion up to 10 g per deciliter. SC patients who are already at 10 or 11, you can't give them a simple transfusion. You can't get the hemoglobin too high or you run into viscosity problems. Those patients might need an exchange transfusion. And you need to use uh extended cross-match blood to try and prevent ice sensitization. And so regardless of genotype preoperatively, they need a good assessment of their hydration, their hemoglobin profusion, oxygen saturation, and transfusion. Intraoperatively, all the regular, um, monitoring, but the patients need to be kept warm. Um, they need to be well hydrated, uh, through the procedure. Um, and if there's a lot of blood loss, that needs to be replaced. And then postoperatively, paying a lot of attention again to hydration and saturation, oxygenation and using, uh, appropriate, uh, analgesia to allow deep, uh, inspiration. All right. So in the last 1516 minutes, I hope to look, talk a little bit about some of these new pathophysiologically-based therapeutic advances. But first, we'll talk about what's currently existing. So as I mentioned to you, fetal hemoglobin, there's this honeymoon period in the 1st 6 months of life where patients almost get no complications because of the amount of fetal hemoglobin is there to prevent, uh, the polymerization process from happening. Fetal hemoglobin has a few other features. One is it has a high oxygen affinity, of course, to draw oxygen off the placenta in order to feed the growing fetus. And fetal hemoglobin doesn't participate in that polymerization reaction. I tell kids it's like that Lego that your older brother, uh, took, and then he took a lighter to it and melted it a bit, so it doesn't fit on the other Legos as well. Um, and so in that way, it, it inhibits the polymerization process. And so here in this picture cartoon, your fetal hemoglobins are green. They can either interfere or block off, uh, this process, so you don't get as much polymerization and you end up having less cells shape change and less complications downstream. And this is also seen epidemiologically. If you have 8.6% fetal hemoglobin, in other words, your gamma globin to beta globin switch is leaky, and you make a little bit of fetal blood into adult life, you have about a 20-year survival advantage over those who don't. And so, just a little bit of fetal hemoglobin expression can uh have a huge outcome on the, uh, on the, uh, uh, on the disease. Historically, interestingly, back in the 80s, uh, Aura Platt, who's now the head of our clinical laboratories, David Nathan, who's, it never seems will ever retire, uh, Stu Orkin, George Dover, other giants from, from this institution noticed that adults who are being treated with hydroxyrhea, um as a really rather primitive treatment for chronic myeloous leukemia or other myeloproliferative disorders, I'm sure in the days before power charts, somebody accidentally ticked a box that said hemoglobin electrophoresis instead of hemoglobin. And they noticed some of these patients started making fetal blood. And they thought, uh, these guys have bigger problems. But Doctor Nathan in particular thought this could be a good treatment potentially for beta globin disorders. And so this is sort of a seminal paper from the 80s showing that when they take just 5 days of uh hydroxya, they could increase the fetal hemoglobin significantly here from 8 to 12 or from uh 5 to 7. And so as I mentioned, this is a very old drug. It's sort of synthesized two centuries ago. It was used in the middle of the last century as a very primitive, uh, antineoplastic. It's really not used much anymore. How it works, we have absolutely no idea. It was just really a fortuitous observation that fetal hemoglobin goes up. This decreases, uh, sickle polymerization, therefore, decreases hemolysis, increases the red cell lifespan, so your total hemoglobin goes up. And because we know that it was treating leukemias cause it made the white blood cell count go down. Um, this is also considered a side effect. We, it's dose limiting. But as I've shown you, I think a couple of times now, the white cells are bad. And so, in fact, by lowering the white blood cell count, we actually think this is a benefit, not necessarily a side effect, as long as you don't go too far. And so there's a variety of studies. Um, the IRB was formed, and so we started doing studies in adults first. And so this is a multi-center study of hydroxyrhea in the 90s that showed you could increase fetal hemoglobin, decrease hemolysis, improve anemia, um, and decrease your white count. And you had a decrease in painful crises by almost a half, a decrease in acute chest syndrome, the leading cause of death again by about 5, and the number and frequency of transfusions went down by 50%. And if you took the drug for a long period of time, up to almost 20 years, you had a 40% reduction in mortality. The once a day oral drug, off patent, cheap as dirt, and has this incredible benefit. And uh still, it's hard to get patients sometimes to take it. These studies were repeated essentially in, in younger age groups, most recently in the baby hug study, starting at 9 months of age with all of the same uh benefits have been noticed. And so, boy, this slides screwed up. But uh, who should get hydroxyia now on the eve of 2019? There's really no age restriction. It probably shouldn't just be the most severe genotypes, but we should consider it in all genotypes. We really don't need people to have frequent pain or acute chest syndrome anymore, which used to be earlier indications for it. And now we're getting parental requests. Hey, Johnny hasn't been in clinic in a while, what's he taking? And so we often will even see that. But this from 2014, uh, uh, evidence-based expert panel from the NHLBI, uh, declared that in infants nine months of age or older, um, regardless of clinical severity should be offered hydroxya. So that's an offered hydroxyurea, but it's severe a recommendation as the federal government can make. So the only other therapy we have really that's uh currently widely available are transfusions. As I mentioned, this can protect patients from stroke. It can also protect patients who've had recurrent acute chest syndrome from uh lung injury. But the problem with transfusions is that each unit of blood comes 250 mg or so of iron, and our species doesn't have a way of getting rid of iron. We lose a little hair and skin and menstruate a little, but we don't lose enough, not 250 mL or 500 to 750 mL a month. The other risk is I've also uh shown you is that, uh, patients with sickle cell disease are at high risk of allo sensitization, and then you have a, a, a narrower and narrower donor pool. And when you really need a transfusion, it's hard to get one. And then every year, there's a new infectious risk. The recent, the most recent is Zika virus, right? So anyone who's getting 3 units of blood a month, uh, is really interested, uh, what's going on in the blood supply. And in New England, actually, the biggest problem for us in these hyposplenic patients is Babesia. Somebody went down to the Cape for the weekend, had a really nice time, came back and gave blood on Monday, didn't notice the tick bite. They have Babezia in their bloodstream, and if you're hyposplenic or asplenic and you get babezia in your transfusion, it's, uh, can be almost lethal. And actually can be lethal in many cases. Here's the problem. This is ferritin. I see it goes almost straight up in patients who are treated for, uh, uh, stroke prevention. And so we have to get that iron out some way, and the only way to do that is pharmacologically. Deserral is the tried and true. It's been around for 40 years or more now. This is a very effective drug, um, but not very efficacious. Did I get that wrong? It works in clinical trial, but who wants to give them 10 to 12 hours a day subcutaneous infusion 5 nights a week? Nobody. Um, so it works. If you use it, your pee turns to looks like rust. It's really, uh, gives you a lot of positive feedback that you're getting rid of that iron, um, but nobody wants to do it. Diferrarone was the next drug. Uh, it's got a long history, uh, um, uh, related to it. Um, some very interesting lay books written about how the approval of diferrorone and how this hospital was involved in some of that. But it's a twice a day drug and requires, uh, biweekly or bi-monthly, pardon me, blood counts, which is onerous to the families. And difiroerro is the current really, um, The main drug that we use, it comes in two formulations. Exjade is this dispersable tablet, uh, or Jade new, which is a swallowable film-coated capsule, and most recently in the last year, they've come up with a sprinkles formulation, which is helpful for us. And in this case, you actually poop out the iron. Uh, let me tell you, it was not a very fun clinical trial to collect everyone's poop, uh, to see how much iron was in it. And so, as I look at the, the novel therapies now, I just wanna give you a little bit of a background how, why things are changing. Um, and so this landscape in the US and drug approval, there are two acts. Um, I'm Canadian, so all this is new to me. I, all I knew was that thing on TV where a bill becomes a, uh, you know, well. But anyway, the Pediatric Research Equity Act and the Best Pharmaceuticals for Children Act both had the same goal of increasing the availability of clinical trials in childhood to have her have adequate testing and safety for new therapies in, in, uh, children. And at around the same time, the Rare Disease Act and the Orphan Drug Act, we're trying to promote uh research into diseases for orphan diseases, which, as I mentioned, in the US that's uh less than 250,000 Americans are affected. And so, as I mentioned, sickle cell disease has about 120,000. So, it's really one of the biggest orphans because it's a big disease. It's a fair market in the United States, but then if you look globally, this is one of the most common genetic disorders of our species. So, in emerging markets such as India, Um, there's potentially a market, so companies are very interested in this. So I joke that I was the ugly kid at the dance, and now I'm like the male Swedish underwear model that all of these companies want to talk to all of a sudden, uh, thanks to this. Because if you have a drug which is currently already licensed for some other indication and you run a trial on sickle cell disease, it does not have to work. All it has to do, you have to complete the trial. That's all you have to do. And then they get 3 quarters of patent protection. And that'll become important to you. So I show you a couple of drugs which are making between 8 and $10 billion a quarter, um, that they are, uh, a million dollars a quarter that they want these trials to be, to be done. So, we can look at this a number of different ways. You can manipulate the oxygen binding of the hemoglobin. So if you hold on oxygen a little more tightly, maybe it won't, uh, cause uh polymerization. Maybe we can, uh, disable how the white cells, uh, interact with the endothelium and with each other. Maybe we can decrease the activity of platelets contribution to that vasoocclusive event. Maybe we can tamp down the inflammatory response or tamp down the antioxidant or oxidative stress that happens with the sickle cell disease. I'm not gonna really have time to talk about the endothelial dysfunction, um, a failed clinical trial, and hopefully, we'll get to talk about gene therapy, which is important for this institution. So you're probably all familiar with the oxygen dissociation curve, the sigmoidal shape due to the cooperativity of binding a 4 oxygen. Atoms. And so hemoglobin A is here in the middle. As you move the oxygen dissociation curve to the left, the P50, which is the partial pressure of oxygen, at which 50% of that hemoglobin is saturated, uh, becomes lower. And as you move to the right, it becomes lower affinity, that number goes higher. So, hemoglobin A is 26.5. Fetal hemoglobin, as I mentioned, is a higher affinity. It's more in the 20 range, and sickle hemoglobin actually has a low affinity. It gives up its oxygen, uh, too easily. And, uh, and then you'll end up sickling. And so you can imagine this would be a potential way of, um, ameliorating the disease. Turns out a company named Global Blood Therapeutics, amongst others, have developed small molecules to stabilize the, uh, hemoglobin in the oxygen-bound formation, the 10th state. And there's a dose-dependent increase in oxygen binding. It's highly selective for alpha globin. It's quite amazing. Uh, they have this, uh, small molecule which binds almost entirely to alpha globin and to no other proteins. Uh, it's quite amazing, at least so far in their clinical trials. And then a mouse model prevents, uh, it allows the mice to live through a hypoxic challenge. So here's the oxygen association curve, and you can see there's a dose dependent shifting of the curve to the left. So it does what it's supposed to do. And then in an early small trial of children, uh, of, uh, sorry, adults with sickle cell disease, it decreased hemolysis. And so there was a lower bilirubin. You can see the patients that were in purple who got the drug. The LDH also went down, although a little less excitingly. But overall, the total hemoglobin, after just, uh, I think this was only a 4-week trial, the hemoglobin went up by about 1 g and those patients were treated with 700 mg. So this spawned two trials, um, the HOPE trial and the HOPEI trial, uh, in kids. The HOPE trial is just finished and was, uh, uh, some of the results have been, have been shown at the Ash, which was just 2 weeks ago. So I'll show you this hot off the press. So in this, uh, study, there were actually 3 dose ranges. I'm only showing you the high dose in the placebo here. And this is a waterfall plot showing every single patient what happened from baseline to their hemoglobin. And you can see that 65% of patients went up by at least 1 g. Some went down, interestingly, but some went up a lot, and that may not be a great thing to have you go up that high. But you can see in those who got placebo, it was about equal up and down. When they looked, however, at the vasoocclusive pain rates, the number of vasoocclusives or the incidence of vasocclusive events from 3.4 to 2.77, it's a reduction, but it was not statistically significant. However, within three days of presenting this data, they had a common stock public offering I saw last night of $150 million. So they're hoping obviously that to get some uh accelerated approval from the FDA on the basis of this. We look now at the white cells, so here my cartoon shows you a leukocyte coming in. You can see the P selectin is thought to be start, uh, the early initiation uh that leads to rolling. That E selectin binds it more firmly, uh, and that it initiates sort of the diapeddesis and extravasation of that white cell, and that L selectin may be involved in intra, um, leukocyte interactions. And so if we could interfere with some of the selectin, uh, activity, maybe we could, uh, decrease the, the contribution of the white cell to this. And so there are two different approaches. A company called Glycometics, which is now in partnership with Pfizer, has a panselectin inhibitor, uh, affects all of those. Um, and this is used in the acute setting. So somebody comes in with acute pain, you give them this drug to try and decrease the vasoocclusive event. And another company, Celexis, which gave us the most expensive drug known to man, um, Solaris, uh, turned to sickle cell disease next, and they have created a humanized monoclonal antibody which affects only P selectin. And they have a preventative strategy to try and give this every month to try and prevent sickle vasocclusion. And so the initial Rivapansil small molecule pan selectin inhibitor trial was 76 patients at 22 sites, including children and adults with mostly sickle cell anemia, that's SS or beta 0. And just to show you the results of that, that this is the probability of vasocclusion occlusion resolution, and they decreased the time of a vasocclusive event from 63 hours to 41 hours. That was not statistically significant, but bordering on clinically significant, I would say. But more interesting if you look at this curve, those who got the drug in red used almost no opioid compared to those who in the 1st 3 days used, uh, veterinary amounts of opioids. So that was quite exciting and that's led now to the phase 3 trial called reset, which is open at this hospital. We're now about 320 patients into the 350 and hopefully, this will, uh, this will, uh, complete enrollment in 2019. The Celexis antibodies is this humanized antibody again towards T selectin only. The sustained trial, um. Uh, it was about 174 patients, uh, and, uh, young adults and adults with all genotypes, and this completed about 2 years ago now. They give the drug, as I say, there's a loading dose, uh, every 2 weeks for 2 doses and then every month, and the study completed in 2016. Just before the results were shown, Novartis bought the company for $665 million suggesting there was a positive result. Um, and then a few weeks later in the New England Journal, it did come out. So patients who got placebo are having three vasocclusive crises per year. Those with the high dose were getting about half. Um, so that was statistically significant. And the way that they often show this is the time to 1 and 2nd vasoocclusive crisis. So high dose here in orange. It took longer for patients to have their first vasoocclusive crisis with the high dose than with green who had placebo. Another thing to look at is platelet inhibition. So, as I mentioned, the cell, uh, hemolyzes, it releases arginase, it releases hemoglobin, it also releases ADP. ADP is a very potent platelet, uh, antagonist. It's actually what we use in the laboratory to, uh, for platelet aggregation studies. And this cartoon here from Alan Michaelson and our platelet research group shows ADP in the dense granules of platelets gets released in an autocrine fashion. It activates the ADP receptor, the P2Y12, and there are a number of drugs here. Prasagrel and Teagrelor are the ones that I'll mention to you, um, which are obviously already, uh, on the market. Takereor makes $8 billion a quarter. And so they're very keen to have a clinical trial. Um, I think in some smoky boardroom, they don't really care if it works, but the people on the ground really hope that it does make a difference. So the Dove trial, I'll mention briefly in the Hester trial, just one slide. I was the lead investigator for this multinational trial. It was quite exciting, uh, children only to meet those pediatric requirements. It was in 14 countries, uh, all around the world, which was quite exciting. Um, some novel features of this study was it really was the first study to look at both the developed and underdeveloped world or low resource countries with high disease burden, and one of the first to use an electronic patient reported outcome, uh, even where we had to give, uh, solar panels to families. So they had cellular service, but they couldn't get power. It was quite amazing. And so cutting to the chase here, um, the, uh, the patients had 2.7, uh, crises per year and it reduced to 2.3. And so there was not a statistically significant reduction. There was a reduction, but not enough. And so here's the Kael Meyer curve. You can see they're beginning to diverge. Although the numbers of patients go down, we wish that we'd lasted on for more than 9 months. But I think what we really wish, uh, in retrospect, is that we underdose the patients. With this drug for people who have stents, they get a 100% platelet inhibition. We ended up getting, the target was 30 to 60% inhibition. So the more to the left on this curve, the more inhibition you get. And so you can see the patients went from here to here, from the dark bars to the white bars. But there was a huge overlap. Patients were not even close to being inhibited when they were on the fully titrated dose. And so we clearly underdosed these patients. And so the Tyagrior study, um, well, so, so Eli Lilly said, great, thanks very much. $10 billion for us cause they only make $3 billion a quarter. And so they shut down the whole program. Um, and then Tyagoror, the AstraZeneca came along and said, hmm, that looks like a good idea. So we're now doing a basically a similar study, but the FDA of course, has asked us here to increase the, uh, here's the green line that's where the inhibition we had with the Dove trial. And looking back, they clearly underdosed it cause if one brain bleed would have shut the study down, they would have gotten no patent protection. I'm a little cynical if you can't tell. Um, and this red is where they have patients who have percutaneous stents, and so we're trying to get it a little higher here, hoping that we're gonna see some benefit. And in the last few minutes I'm gonna skip over fish oil. Sorry, Mark. It works a little bit. There's a phase 3 trial coming out, um, cause I wanna show some of the other things here. There's a lot going on, I tell you. Glutamine was FDA approved, but I just love this. The trial ended in 2014. Scott Gottlieb was chosen by our current president to be the commissioner of the FDA on May 11th. On May 25th, uh, uh, Ndari was approved. Before any paper had been published. Um, Scott Gottlieb was on the board of the company, um, and he hadn't divested himself as a financial interest. I watch a lot of MSNBC, but nonetheless, that seems a little bit odd. Um, all right, curative treatments. So allogeneic stem cell transplantation, as you're familiar with, you take, uh, stem cells from an HLA matched donor, preferably a sibling. You don't manipulate them in any way. You either get them from a sibling or from some other member of the family, and you put them back in and hopefully Those cells will grow up to make normal red blood cells. And so, this is a European study um from Elaine Gluckman. It shows 1000 transplants, uh, HA identical sibling transplants, and you can see uh about a 98% survival in patients who get uh um. Uh, cord blood and, uh, almost as good for those who get bone marrow transplants, a little less for peripheral, uh, stem cell source. But, uh, this is a, a valuable, um, treat, a curative treatment for patients who have an HLA matched sibling. The problem is with each sibling, you have about a 1/6 chance of that sibling not having sickle and being an HLA patch. So the vast majority of patients don't have this as an option. So what regulates this gamma globin to beta globin switch that happens? Could we use this therapeutically to push people back to make fetal blood? The mothers don't want them back in the womb, but if we could just, uh, um, get them, uh, to make more fetal blood, that would be good. And so, Stu Orkin, Vijay Sankaran, Dan Bauer, and others, um, from Genome-wide Association studies discovered that a transcription factor called B cellNA is responsible for about 15%, which is a huge amount of the natural variability of fetal hemoglobin levels in normal populations. And it's part of the nerd complex, which I joke I was in in high school. Um, and this is involved in the transcription regulation of the whole beta globin locus. And it turns out that if you knock out or knock down B cell 1A, um, here's a mouse with, uh, normal red blood cells with a nice round shape with a central pallor. Here's a mouse with sickle cell disease with the blue cells being reticulocytes and lots of sickle cells. If you knock it out conditionally, you go back to normal. And so this seems to be a good therapeutic target. And so now from a gene therapeutic approach, we can attack a number of different ways. So this is autologous, don't need a donor. Fantastic. No graft versus host disease, fantastic. The leading cause of complications in the allogeneic transplant. And we can either uh choose a viral integration approach using a lentiviral vector that integrates into the genome, or we can use a genome editing approach such as CRISPR Cas 9, zinc nuclease, uh, um, zinc finger nucleases. And we can either have a gene addition approach where we simply take a normal beta globin and put that in an additional one and add that, like it shows in the picture here, a beta A to the sickle mutations, or we could add a gamma globin to try and produce more uh fetal hemoglobin. At the other end, we can use these um editing tools to cause a base repair and just fix the sickle mutation. We're not quite there yet, that's sort of Star Trek, but we're getting closer. And another approach where you can either use the editing approach or with a viral integration approach is to target BCL-11A to either knock it down or to eliminate or delete an intronic enhancer in BC-11A, which abrogates its expression. And so, uh, Bluebird has, uh, created a lentiglobin. This is a therapeutic beta globin derivative. It has a single, um, amino acid substitution, which has anti-cycling properties and also is a good biomarker to see the transgene expression. And this was from a year ago now, uh, this, this, uh, paper of one patient. And you can see that the hemoglobin A that the patient was transfused with went down and it was replaced by the backline here of the, of the lentigglobin. And the total hemoglobin went from 7 or 8 in this patient to almost 12. So that was very exciting. And this was just from 3 days ago now. This is their most recent 7 patients, and you can see the 33 month, 3 month, 3 month follow-up. These patients still have transfused blood around here in blue, but the transgene in pink, as you get longer, uh, has an increasing proportion, about half of the, of, of the hemoglobin being produced, and the total hemoglobin here looked normal at 13.7%. So this is pretty exciting. We, however, had a slightly different approach, uh, building on the BCNA discovery from here is to try and knock down BC-11A. And so, here is our lentivirral vector, which has as its, um, cargo, a hairpin RNA which is targeting BC 11A. And it has a beta globin promoter so that it's uh expressed only in erythroid cells. And this short hairpin RNA has been disguised or pushed into a microRNA. Um, which allows it to be expressed appropriately in the right places. And so now, Doctor Williams' lab has, uh, uh, created this vector which we call schmear. I don't really know my favorite word, but short hairpin, uh, microRNA. Um, it really it's a physiologic gamma globin expression, and that's what I show here. So here is an untransduced control, the amount of fetal hemoglobin and the amount of, um, gamma globin produced. And you can see in patients, it's now like 90%, uh, from in vitro assays taken bone marrow from patients, you can almost completely, uh, reverse them to make gamma globin. And so we've now, uh, consented 4 patients, treated 1 patient who's now 6 months out. He engrafted, uh, in 22 days, which is quite quickly. He had, uh, complications related to myoablation. We still have to give these patients my ablation conditioning, but really no other medicinal, um, uh, uh, adverse events. You can see here the viral copy number, the number of copies of the transgen, and the peripheral blood per white cell, and so it's almost 1. So it's almost every cell has a copy of the transgene. And you can see that in peripheral blood samples in a month and 1 month and a half, there's no BCL-11A expression. So we really active completely suppress the BC 1A expression. And so in this one patient in months post-gen therapy, this is the transfused blood that went away as you'd expect over the 1st 3 months when we stopped transfusions. And the percent of fetal blood went up to about 30% and then it sort of held there. That was a bit disappointing. We're sort of hoping to see the 80 to 90% that we saw in vitro, but probably more importantly is the number of cells in the periphery, red cells which have any fetal hemoglobin in them at all, is close to 70 or 80%. And so that actually probably prevents them from sickling. All you need is 10 picograms of fetal hemoglobin in the cell to prevent that from happening, from, uh, polymerization. And so that's it. So that's kind of exciting. We have 3 more patients over the next 3 months who are gonna get treated um with uh this uh schmear. Um, but this is the CME question for you, uh, which of these is not like the others? So, as I mentioned, I'm Canadian, so, uh, we got invited, uh, because I have no idea why, uh, Celine Dion wanted to, but she gave us $2 million for this gene therapy program. And so the four of us who've, uh, certainly not been to Las Vegas that often got to go hear her sing. Uh, and meet her, um, and there's certainly more than one diva in this picture, but it's, uh, all right. Thank you. I'm sorry I went a little bit over, but, uh, very exciting times. Any questions? Well, Matt, the time is late, but I, I can't let you go without thanking you for bringing this great, uh, summary together. I think the introductory part on giving everybody a reminder of what he, what sickle cell anemia is all about was very helpful, and, uh, certainly the, um, advances have been made are really quite stellar, particularly the last one. So, so thank you for bringing this to us and if there Uh, questions we'll have people come up and ask you at the end. So thanks a lot, Matt. I Hi. Thanks. Is the pattern of the hemoglobin switch any differently propositions that either counter at the timing of the proportion.