Segments 1. W elcome, Disc losures Patr ick Sosnay Page 2 Program Goals 2. Yesterday- Patr ick Sosnay Page 5 Overview of Mutat ion Classes 3. Today- Stuart Elborn Page 7

Ivacaf tor and Beyond Smal l Molecule Therapy for Cyst ic Fibros is

4. Today- Meghan Ramsay Page 16 Management of Pat ient Expectat ions 5. Tomorrow- Joseph Pi lewsk i Page 19 Transforming CF Care One Mechanism at a T ime 6. Q&A Al l Faculty Page 30

Segment 1 DR. PATRICK SOSNAY: Good evening, everybody, and welcome. My name is Patrick Sosnay, I’m from Johns Hopkins University in Baltimore. I work in the adult pulmonary clinic and do genetics research with Dr. Garry Cutting, I help organize a website, a project called CFTR-2 where we have a database of CF mutations, and I’m pleased to welcome you tonight to our Ahead of the Curve celebration of 25 years of CFTR. This meeting is always an exciting event because we get to discuss and celebrate really the hard work that we’ve done over the past year, and this year we have an especially great celebration, that 25 years ago CFTR, the gene that causes cystic fibrosis, was recognized. We have some great presentations for you today that celebrates the past, what we’ve learned, where we are now and the exciting opportunities for therapies that directly address the mutation and talk about some of the future therapies, the further bit that we have to go to really continue to tackle this disease. A bit about our accreditation, you can get CME credit for physicians and nurses. Some specifics of the CME credit. So our learning objectives tonight:

• Explain how new CFTR modification advances point to changes that have to be made in clinical practice.

• Describe the utility of the

genotype/phenotype correlations beyond diagnosis in achieving more effective patient treatment.

• Integrate the patient into an individualized therapy regimen to improve outcomes.

A HIPAA disclosure statement. Our disclosures, Dr. Mike Boyle was on the planning committee for this, his disclosures are listed here. And we have to acknowledge really the Johns Hopkins Medicine and Johns Hopkins Nursing CME Departments who help you all get CME credits and help put together some of the educational portion of this talk tonight, and DKBmed who really from start to finish runs a great operation. So we have them to thank for putting this all together. Finally, the money that supports this event was paid for by Vertex, but all the educational content was provided by us. Really us at Johns Hopkins and Dr. Elborn and Dr. Pilewski as well, so Vertex put up the money but they’re not at all involved in the message here, that’s all directed by us. So stories have beginnings, stories have middles, and stories have end, and really the CFTR beginning is going to be displayed in this video here. So this is a video talking about the initial steps on the discovery of the gene. (Video being played) SUZANNE PATTEE: I remember

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my parents telling me that the doctors told them that I had a 50 percent chance to be five years old. KATIE REISINGER: My parents just weren’t given much hope for me to live a long and healthy life. MARGARET LEIGH: At that time most of the patients became very sick during childhood and a large number died during childhood. SHEREE BERKMANS: We lost children at eight, nine, ten years old. BERYL ROSENSTEIN: We had little in the way of hope, we had little to offer patients, there was tremendous frustration that more progress had not been made. SCOTT DONALDSON: Other than airway clearance and pancreatic enzymes, we didn’t have many tools. PAMELA DAVIS: We were pretty much still fighting a rear guard action against cystic fibrosis. We were dealing with the symptoms systematically one by one, each one as they arose, we were behaving aggressively because we believed that the gene would be discovered. FRANCIS COLLINS: I was a junior faculty member at the University of Michigan, got there in 1984, had developed a method to try to travel across chromosomes by jumping instead of walking, which at the time was an important advance potentially because it might enable us to get to the gene for cystic fibrosis from markers that were somewhere

nearby but were actually quite a distance away. MITCH DRUMM: A group in Toronto had just found that there were DNA markers that were associating with CF and so Francis got on the phone with Lap-Chee Tsui. LAP-CHEE TSUI: And Francis came in because he had the technology of chromosome jumping. So I said Francis, you’re jumping, but you’re jumping from a place of quite far away from the gene and so perhaps we can collaborate because we knew exactly where we were and then we have very good psychophysical map of the region, the gene, so we can collaborate. COLLINS: Jack Riordan’s lab joined the effort, given his expertise in other aspects that were going to be important like being able to look at sweat glands. TSUI: Jack was very important in the discovery of the cystic fibrosis gene because he had made a number of (indiscernible) libraries. JACK RIORDAN: We had sweat gland samples from patients in the CF Clinic at Sick Kids as well as people without CF and we cultured those cells. TSUI: The first piece of gene that we identified or we isolated was from his library on the so called sweat gland CDNA library. GEORGE RETSCH-BOGART: It really was this extraordinary collaboration between Francis

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Collins, Lap-Chee Tsui, and Jack Riordan, that pulled together really the most innovative and state of the art teams. COLLINS: And finally, after many fits and starts where many of us began to despair about whether this was a solvable problem, in the summer of 1989 the data emerged to say we found it, this is it. PATTEE: It was amazing the day they announced the discovery of the CF gene. DANNY BESSETTE: Being on the cover of Science magazine and knowing that they were possibly close to finding a cure for CF or at least understanding where CF comes from -- LEIGH: The researchers were excited, the families were excited, the caregivers were excited. BERKMANS: Especially, you know, working with patients day to day on the floor so we were excited for what was to come and what that meant. REISINGER: We were at home and the phone rang, my mom picked up the phone and another CF mom called to tell her that they found the gene and she just started crying immediately because she knew it meant there was hope. ROBERT BEALL: So it was really an exciting, exciting time for our community, for our scientists, but most of all, for the parents and patients. It gave them a new sense of hope.

TSUI: The first time I saw the cover of the magazine presented to me in a small celebration at a hospital, I mean I had tears in my eyes. BATSHEVA KEREM: On the day of the publication of the science papers there was a big ceremony in the Hospital for Sick Children, families of CF patients and CF patients, themselves, they came to the hall, and this was really, really touching. When we came into the hall, they were all standing and clapping hands. TSUI: And I felt a little bit of an accomplishment because I finally did something for the patients, for the families. DONALDSON: This was a huge breakthrough, not just for CF, but for genetics and medicine in general. So it was a big deal and I think everybody had a clear appreciation for how special and important it was. PAUL QUINTON: The discovery of the gene for cystic fibrosis was an exhilarating time. DAVIS: And we believed, somewhat naively at the time that from the gene would automatically cascade tremendous understanding of the pathobiology of the disease and then a cure. COLLINS: Shortly after we found the gene I think all of us had the expectation that this was a disease where gene therapy would be particularly appropriate. JEFFREY WINE: Nobody really fully anticipated the tremendous

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difficulties that lay down that path. MICHAEL BOYLE: Even once people began to realize, hey, this is going to be more challenging, the fact that we knew CFTR, we knew the cause, still said hey, this is the direction we need to go after, it just means it’s going to take more than we originally thought. TSUI: Because of the variation of the disease presentation among different patients, even patients with the same mutations, there must be other genetic factors influencing the presentation or the symptoms of the cystic fibrosis. So it was let’s look for the so-called modifier genes. COLLINS: It was several more years before the other approach, namely trying to develop a drug, a small molecule, really got fully pushed and I give huge credit to the Cystic Fibrosis Foundation and to Bob Beall for deciding to make a bet on that at a time when I think a lot of people thought the chance of success was going to be very low. But here we are. BEALL: The Cystic Fibrosis Foundation had to change how it did business. We used to support academic institutions, mainly with grants, but then we recognized if we really were going to translate that wonderful knowledge that we had learned from the academics and the test tubes to new therapies, we had to bring in a new partner and that was industry. PHILIP FARRELL: Well discovery of the cystic fibrosis

gene opened the door to not only a better understanding of the disease, but the application of the detection of the CF gene mutations for newborn screening. DAVIS: And we’re able to identify patients with CF or the vast majority of patients with CF very early in the course of their lives and it gives us an opportunity to intervene. FARRELL: This year over 10 million babies will be screened for cystic fibrosis. DRUMM: Evidence keeps accumulating that the sooner we get in and slow down the progression of the disease, the better off these kids will be. RETSCH-BOGART: Now we have patients who we’re seeing right at the beginning of life through newborn screening, we now have the promise of therapies that will be game changing in the sense of preserving lung function and improving nutrition in ways that we previously couldn’t do, so it is a completely different landscape. (End of Video) Segment 2 SOSNAY: So starting with what they learned when they discovered the gene that causes cystic fibrosis, it laid out a map that really we were going to use, that we continue to use today, and hopefully I can talk to you a little bit about going from that discovery of the gene to establishing the framework of

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understanding how different mutations may affect that gene, affect the protein product of that gene, and how we’re going to use that to address therapies that are going to fix the mutations. So our learning objectives:

• Describe how CFTR mutations cam be classified (by type, by class, by therapies).

• Discuss the variety of CFTR mutations.

Other things that happened in 1989, let me put a timestamp on this, 1989 was the year of Tiananmen Square, the Tiananmen Square demonstrations in Beijing. 1989 was the year that the Berlin Wall fell and the beginning of the reunification of Germany. In 1989 unfortunately the Exxon Valdez spilled oil off the coast of Alaska. And in this momentous year we also heard from our distinguish speakers in the video, 1989 was the year that the CFTR gene was recognized as the cause of CF. So you’ve seen these schematics in many different talks in this presentation and previous presentations and it’s really become an important guide for us because it helps us group the many different mutations in this CFTR gene into patterns that might be more amenable to group together and think about therapies in the same way. So class I mutations, these are mutations in which the RNA produces a premature termination codon, in other words, the RNA stops short. The RNA is the template that our cells use to

assemble the CFTR protein. And that template doesn’t go all the way to the end of where the protein should be. So the cell recognizes that it’s abnormal, and no protein is made. Class II mutations, the most common mutation, F508del, it’s a problem where the protein is made but it is assembled or folds correctly. The cell recognizes that it folds in correctly and gets recycled by cellular machinery so you can’t find much of protein in the cell, you can’t find really any protein up at the cell surface where it might act to conduct chloride. Class III mutations fold properly, assemble properly and do get trafficked and put in the apical surface, the top surface of the cell, but those particular mutations can’t be turned on, so they don’t conduct chloride. Class III mutations include the G551D mutation, the success story that ivacaftor is able to open up that particular mutation, conduct chloride, with really some dramatic clinical benefits that you’ve been, you guys have heard the clinical successes from that. Class IV mutations represent mutations in which the chloride channel is at the surface and it does conduct a little bit of chloride at baseline, but it doesn’t conduct enough chloride. So it’s kind of like a class III mutation except there’s a little bit more chloride conductance and you actually can turn those mutations on. And example of this is the common R117H mutation.

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And finally Class V mutations are mutations in which the protein functions effectively, conducts chloride effectively, but there’s just not enough of it at the cell surface. And often this is due to a splicing defect or the way that different pieces of RNA are put together to assemble the protein. There’s a class VI mutation that’s been talked about that are fairly uncommon where the problem is that the cell, the CFTR at the cell surface gets recycled more rapidly so it gets inserted into the cell surface but then gets quickly taken down from the cell surface. And the end result, similar to class IV, is that you’ve got less functional CFTR up at the surface of the cell. And there are 2,000 different mutations in the CFTR gene that are described, and it’s difficult to know with all of those mutations which class they will fit in. In certain cases we can make assumptions. If a particular mutation causes a premature termination or a premature stop, we can group that into class I. But for many of the missense mutations, where you have an amino acid substitution, we can’t tell if those are class II, III, IV, or even in certain cases class V mutations. And class is not always a simple assignment, there’s some mutations that may fit in more than one class. To make this more complicated, the naming systems change, and this is as we increase the use of genetics in medicine, the geneticists have changed rules with this as far as where we’re starting the nucleotide number.

And you can see that there are legacy names that you probably are most familiar with but a mutation could also be named by its nucleotide position or by its protein position. And this makes it particularly difficult to interpret new genetic testing reports. A simpler way to think about mutations and a simpler way to think about the classes of mutations, and actually Donna Peeler presented to me, one of the nurses in the pediatric CF clinic, is to think about it very, very simply as is there CFTR, is there CFTR protein at all, that’s the first category that needs to be met. And finally, if there is CFTR protein at all, does that CFTR protein work. So thinking about it a little bit more simply into is the protein there in the right place at the top surface of the cell, the apical surface of the cell, and does the protein conduct chloride. So I think the simpler framework is appropriate when we start thinking about some of our therapeutics, especially some of the therapeutics that are closest to being delivered. Segment 3 DR. PATRICK SOSNAY: For our next video we’re going to hear about the state of CFTR as of today and some of the exciting therapies that are now available.

(Begin video)

DONALDSON: Fast forward 20 years into the future, now we have multiple therapies that we can use to take care of patients.

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We know they work, we know the size of the impact they have in patients, and I think we’re at the beginning of having therapies that are truly breakthrough therapies. DRUMM: I can’t tell you how thrilled I am about the state we are in CF right now. ROSENSTEIN: The mood among not just the patients but among the staff treating patients with CF has turned around completely because we now have real hope. DONALDSON: You can see huge changes in patients’ lung function, their survival, their nutritional status, so it’s very clear that at a given age patients are doing so much better than they were 20 years ago. FARRELL: We no longer have to worry about children suffering, suffocating, being severely malnourished. STEPHENSON: For nutrition care it’s a whole new ballgame. Today it’s preventive nutrition, and when I first started it was rescue nutrition. ROSENSTEIN: One can immediately see the nutritional benefits, fewer hospitalizations earlier in life, parents who are part of the team now and come into the CF program not with frustration but with much more of a sense of teamwork and optimism. FARRELL: Fortunately, this is a new day for children and adults with cystic fibrosis. DRUMM: Clearly for patients with

gating mutations, we have a drug that’s available for them today, ivacaftor, and that is a total game changer. BOYLE: We finally have something that could potentially address and make a difference in our patients with the most common mutations. GUGGINO: Having worked in the laboratory on CFTR and its ion channel properties, to see a treatment come along based on activating this channel, it’s truly remarkable for me to see this. TSUI: I was very happy that finally a drug was identified for cystic fibrosis based on the gene discovery. WINE: Without the gene there could be no potentiators, correctors, CFTR-directed therapeutics. The development of all of those therapeutics depended on having cell culture assays made possible by having the gene product, being able to express it, and understanding how it worked. DAVIS: It was a complicated process and it really required I think the collaboration and cooperation of academic labs and industrial-scale screening and medicinal chemistry. It was a remarkable tour de force. CHMIEL: Ivacaftor only affects 4 percent of the CF population, but the reality is it shows that we can do this. BEALL: Our promise to our community is, we have to treat 100 percent of our patients with

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disease-modifying therapies. BOYLE: And again it sort of shows proof of this whole principle that as we improve chloride transport we see an improvement in outcomes. REISINGER: And seeing my fellow CF patients benefitting from this drug is amazing to me, and I’m so happy for them and hopeful for a day that it’s available for everyone with CF, not just delta-F508 mutation, but anyone who has cystic fibrosis. PATTEE: I feel very thankful to be alive here at 51. It was very hard to see that age when we were kids. BESSETTE: We’re in a new, whole new era. This used to be a pediatric disease and pediatric disease only. REISINGER: My pediatric pulmonologist was so important to my family. We had such a great relationship, and I remember not wanting to transition to the adult clinic at all. I was reluctant to leave. BERKMANS: When we talk to kids about transitioning now they’re like, no, I don’t want to go, I’ve been with you for 20 years, and we say, no, this is a good thing, this is a celebration, this is not a bad thing to be able to go to the adult CF clinic, because like I said, we didn’t have one when I first came to work here. DAVIS: None of what we’ve seen in CF would be possible without the enthusiastic participation of the patients in clinical trials. And

most of them do it with little expectation of benefit for themselves. REISINGER: Being involved in clinical trials is very important to me and I think for the whole CF community. PATTEE: It’s just part of my way of giving back to the community and giving hope back and give hope to others with CF. DAVIS: Some of them do it and they say to you right out of the gate, it’s for the kids. It’s for the next generation of CF patients. I’m here taking this chance, taking this risk for the kids. RETSCH-BOGART: Preserving lung function, maximizing nutrition have become even more important so that when a really, really effective therapy becomes available patients will, you know, benefit the most. REISINGER: As an adult I’ve been diligent about not only sticking to my treatment regimen, but exercise and making sure that I can stay as healthy as I can for as long as I can, and that’s what inspires me to sit with my vest on for hours every day or going running or do all the different things that we have to do. LEIGH: So we’ve greatly extended the lifespan of cystic fibrosis patients. We’ve greatly improved the quality of life of cystic fibrosis patients. And we have changed their expectations and their dreams. DAVIS: All but one of my patients is over 50, and I had the pleasure

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of hearing from an emergency room doctor when one of my patients developed acute belly pain and went to his nearest emergency room and we had a call from the emergency room doctor who informed me he could not possibly have cystic fibrosis because he was 62. And, you know, in some ways that’s a triumph.

(End video)

DR. SOSNAY: Now I have the privilege of introducing our first speaker, Professor Stuart Elborn from Northern Ireland. He’s here from Queens University and he’s going to talk about the present in CFTR. DR. STUART ELBORN: Thank you very much Patrick. Thank you to the team at Hopkins for inviting me to come and talk at this great symposium. Watching those videos has reminded me that my first fellowship involving the care of people with cystic fibrosis started in 1989. I was at the North American CF meeting when the first discussion around the discovery of the gene occurred in Washington in that year. I could remember the real sense of excitement that went around that whole conference and the sense of optimism that we could make a difference. The next time I felt that feeling was when the results from the first ivacaftor study were shared. It was a very dark, cold February day, and it was about eight o’clock in the evening because Bonnie Ramsey was the other person on the call with some of the team from Vertex, and when

we saw the data from that first ivacaftor study, truly the hair stood up on the back of my neck. It was a real sense of, well, the identification of the gene has now led to something that really makes a difference for people with CF. And for the next two or three talks, you’re going to hear some of the exciting data that we now have on treatment of people with cystic fibrosis based on understanding of their genotype and directing the right therapy to the right patient. So I’m going to try to:

• Describe how modulating and potentiating CFTR improves clinically important outcomes in CF.

• Describe indication for potentiator and combination therapy in CF.

I’m going to talk about the first two classes of CFTR mutations in the context of personalized, stratified or precision medicine for cystic fibrosis, and I’ll come back to this concept in a couple of slides to elucidate what we mean by it, because it’s a big drive across a number of diseases now, and I think it’s important that we understand this concept. But before doing this, I want to get into this groove of history because it is so important that we acknowledge where we come from as we try to figure out how we apply stratified medicine with these new therapies to people with CF. This graph has grown in my slide decks over a number of years. I want to make a couple of points from it. The first ne is that we owe

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a huge debt to the physicians and caregivers who observed what was happening in CF, did early observational studies, made empirical decisions about treatment, applied those decisions, and then tried to figure out how they might be working. The introduction of the sweat test, airway clearance, and then some of the therapies that began to develop around antibiotics and mucociliary clearance have made a huge impact and have been big part of why the prognosis for cystic fibrosis has improved over the last number of decades. The drugs that came out of these observational studies initially, and then in the last couple of decades through well-performed randomized, controlled trials with adequate powering for determining if we were making a difference, have given us a great armamentarium of therapies to treat infection and to improve mucociliary clearance. At the top you can see the last little bit says stratified or precision medicine. And we are moving into a new era where we’re not treating the downstream effects of the pathophysiology of CF in terms of infection and mucociliary clearance, but we’re getting right to the heart of the disease by being able to use drugs that now modulate the mutant CFTR that our patients have in their lung cells and improve the function right at that basic defect level. This is to remind you of the pathophysiology of CF, because I think that’s important as we go through the next number of talks.

In the healthy lung the CFTR channel shown in cartoon works properly, it keeps the airways hydrated so cilia work properly. As you go across the normal you can see that the cilia and the EM picture in the middle are nice and straight and they’re functioning well. That keeps our airways clear from all of the bacteria in the particulate matter that we breathe in with every breath, so that we don’t get infection and inflammation in our lungs. The lower panel illustrates the situation in cystic fibrosis, where we have dehydration, it’s a desert in the lower airways in terms of the hydration that’s required to keep mucociliary clearance working properly. The particulate matter and the bacteria that people with CF breathe into their lungs, right from the time they take their first breath, is not cleared properly. And that infection sets up an inflammatory response in the lung which for a lot of complicated reasons gets frustrated and is persistent. The immune cells, particularly the neutrophils that come to deal with infection, start to damage the lungs in people with CF and result in inflamed airways, as you can see on the far side of the lower panel, and blocked, mucus-plugged airways, which drive this injury in the lungs over many years. In thinking about the treatments we’ll now talk about, I wanted to come back to the concept of stratified or precision medicine. This slide illustrates how we have traditionally approached diagnosing disease in people and then how we’ve thought about

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treatment: people present with symptoms, we make a differential diagnosis, we do some tests to confirm that diagnosis, and then we empirically prescribe a treatment. For example, if someone has high cholesterol, we do a cholesterol test and then we put them on a statin. However that means that we end up treating a lot of people with not too much effect, because trials have shown us that there were responders and nonresponders in these sorts of general diseases that have been treated empirically. What we’re trying to do in medicine across a range of diseases and has been pioneered by the cancer field, is then when we make a diagnosis, to do some more tests that not tell us what it is, but tell us whether patients are going to respond to the therapies that we have available. That’s a diagnostic test that’s a bit different because it will drive therapy. And then that diagnostic test allows you to target therapy, which is now the key as it is for treatment for breast cancer, lung cancer, and a number of other conditions. In cystic fibrosis, the understanding of the gene and the understanding of the function of CFTR and the sorting out of the various classes that Patrick described to you have allowed us to develop a precision or stratified approach to therapy. The panel that’s come up gives you the sort of principles around what is required for a stratified or precision treatment. You need a biological mechanism that

differentiates different groups or stratas, you need multiple treatments that are appropriate to one but not another, and you need a clinical biomarker. In cystic fibrosis we’re now in that position: we have a fairly good understanding, although it’s still not perfect, of CFTR function; we have a clinical biomarker which is the patient’s genotype; and we now have a range of therapies, either licensed or coming through clinical trials that will allow us to select the right treatment for the right mutation in our patient groups. I want to pause for a second, though, and say this is really exciting, but we must remember that the therapies you saw in the first slide, the treatments for infection and mucociliary clearance still are important, because we haven’t completely sorted this out, and the mutations of the CFTR gene only account for some of the disease phenotype in cystic fibrosis. So it’s important that the conventional treatments that we have available are still used in our patients. Over the next 10 minutes I’m going to talk about class I and class II mutations. This is some data from the European cystic fibrosis registry published by Kris De Boeck and colleagues, which gives you some data on the prevalence of class I mutations and class II mutations. Class I are in the top panels, class II on the bottom panels, and the plots are the percentage of patients by country. There’s some quite interesting data in this which we won’t have time to dwell on. But,

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for example, in Israel, which is in panel A, the first bar, you can see that class I, which are stop codon mutations, are very common. But in Europe and similarly in North America, class I mutations account for around 10 percent of the mutations and the chromosomes of people with cystic fibrosis. And of those class I mutations, panel B gives you an indication of those that are nonsense or stop codon mutations. The lower two panels show you the prevalence of class II mutations. Panel C is all of class II mutations, and panel D is F508del mutations. And as you can see, these account for the vast majority of CF mutations in people with cystic fibrosis in Europe. Indeed, panel D indicates that the vast majority of these are F508del heterozygotes and homozygotes, and it’s about 50/50. So people with F508del account for around 80 percent of the patients, with half of those being homozygote and half heterozygote. Class I mutations are shown on this cartoon, and again, as Patrick has already described to you, in class I mutations, the process of translation in the ribosome to make the protein from RNA is disrupted because the mutation stops the process of making the protein. These are mostly caused by nonsense mutations with the X, which indicates the stop codon, but can also be caused by frame shift mutations. Generally patients with this class of mutations have classical or severe phenotypes.

And to show you in a cartoon of how this works, this is the process of translation shown in a cartoon with the stop sign being the premature stop codon. In the situation where this occurs, the protein is not made properly. You may make a small, defective bit of protein that never gets very far and this is brought back into the cell, or no protein may be made at all because the RNA and the ribosome become unstable. To treat patients with this particular class of mutations, an observation with gentamicin by Batsheva Kerem and Michael Wilschanski and Eitan Kerem — you saw Batsheva on the video —indicated that it might be possible to read through that stop codon. And subsequent to that, a company called PTC developed a drug related to gentamicin called ataluren. Ataluren attaches to the ribosomal complex making the protein. It gets to the stop codon and there it allows the ribosome to read through, and the notion is that you would then get full length protein which would be normal CFTR and get up to the cell membrane. This looked like quite an exciting approach to treatment in some early phase 2 trials that were nasal potential difference, and similar trends were seen in lung function, improved on a couple of different doses of ataluren. This graph shows the nasal PD where a downward deflection is an improvement, suggesting the drug had a positive effect on CFTR function. This drug has now been tested in a phase 3 clinical trial. It was

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published a couple of months ago in Lancet Respiratory Medicine and somewhat frustratingly, the phase 2 data was not confirmed in the phase 3 study. This is the lung function results from patients treated with ataluren on the green line compared to patients treated with placebo. There was a trend toward a benefit in the patients treated with ataluren, but the difference was not significant. This was looked at in a subgroup of patients who weren’t using inhaled aminoglycosides, primarily tobramycin, and there was some encouragement. So we know that aminoglycosides have some effect in themselves on read-through in the laboratory, and when patients who weren’t receiving aminoglycosides were looked at, there was a significant but still a small improvement in lung function. There are a number of potential reasons why this drug hasn’t worked, and there is some controversy around whether this drug does result in read-through. So there is much work still to be done in class I mutations, but it is encouraging that a further clinical trial with ataluren aimed at patients who are not taking inhaled aminoglycosides is about to start. I’m going to move on now briefly to talk about class II mutations. Patrick has outlined the biology of class II mutations where the CFTR protein is made, but it is very defective and most of it is degraded within the cell. However, a small amount does get up to the cell membrane and

that’s estimated to be around 2 to 3 percent. This is illustrated on this slide. This is the normal situation where normal CFTR gets up to the cell membrane and works, and in class II mutations, particularly F508del, it doesn’t get up to the cell membrane in any high number, but is degraded within the cells, thus being called the protease. In F508 there are a few CFTR channels up in the cell membrane, but even those that get there don’t work properly; they are blocked channels, even those few that make it. Here is some work from Fred Van Goor and his team at Vertex. He was able to demonstrate that the combination of a drug called lumacaftor in cells that were expressing two copies of F508del in combination with ivacaftor on the far right bar resulted in some increase in chloride transport in this bronchial epithelial cell model, suggesting that this combination therapy of lumacaftor and ivacaftor could get more protein up to the cell membrane, and then ivacaftor could open the pores and get chloride conductance. And this has been through a number of early phase trials, and you will have seen the phase 3 trials I hope at Michael Boyle’s plenary session and also presented today by Claire Wainwright. I’m going to give you the highlights from these two trials, called TRAFFIC and TRANSPORT that recruited over 1,000 patients and were almost identical in design, comparing two different doses of the combination

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of ivacaftor and lumacaftor against placebo. This is the, two separate trials with the lung function results showing significant improvements in FEV1 on both doses that were trialed in both studies. It is very reassuring that there’s been a consistent response to the combination of lumacaftor and ivacaftor and that both of the doses seem to work pretty similarly and both were significantly better than placebo and were sustained at 24 weeks. We now have data beyond that suggesting that this effect continues up to 48 weeks. There was also a significant improvement in pulmonary exacerbations. This is pooled data from the two studies, and in this slide you can see that the time to next pulmonary exacerbation was reduced in patients who were taking combination therapy. We do have some debates within the CF community about what exactly is a pulmonary exacerbation, and this was broken down in a couple of ways to try and harden this up. And the events requiring hospitalization or events requiring IV antibiotics were also significantly reduced. This is very interesting data, because the improvement in FEV1 is less than what’s seen with ivacaftor, but this is still quite a strong signal for reduction in pulmonary exacerbations. I think we now understand clearly that pulmonary exacerbations are a bad thing for our patients, so if we can prevent pulmonary exacerbations, we can reduce the

decline in FEV1 and also probably have a significant effect on survival. So this is a very important result from this clinical trial. There were some trends for improvement in body mass index and a modest but nonsignificant improvement in quality of life scores. In summary, the key endpoints from this trial showed a 3 percent improvement in lung function, a reduced time to next exacerbation, fewer hospitalizations, and some improvement in body mass index and quality of life. There is, however, some concern about the stability of this combination therapy on delta-F508 mutant CFTR. This has been a big discussion in the scientific and clinical trial sessions within this conference. But suffice to say that there are still some important aspects of CFTR function and stability that have to be understood so that we can further optimize treatment for homozygous patients with F508del. And it may be that a second corrector is needed to stabilize this molecule to get the best out of it in chloride ion transport, and hopefully further improvements in the clinical outcomes. It is so exciting over the past number of years to have been able to be involved in care and clinical trials in CF when we have a realization that CFTR is a druggable target. We now have treatments that will change the

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function of this protein. Ataluren has a limited impact at present, but I think there is still some optimism that drugs can be developed for class I mutations. Finally, the lumacaftor/ivacaftor data indicate that this is a very promising combination. We still have a way to go to get the best combination or combinations of drugs to continue to make a significant impact on this class of mutations in cystic fibrosis. Thank you very much. Segment 4 DR. PATRICK SOSNAY: Next I get to introduce my coworker, Meghan Ramsay, who is a nurse practitioner in the adult clinic. She started as a nurse practitioner when I was a fellow, so much of what I learn about taking care of patients with CF patients is from Meghan. She’s going to talk a little bit about the context of these exciting new therapies in respect to everything else with the burden of treatment that our CF patients have. MEGHAN RAMSAY: Thank you all. I hope everyone’s enjoying their evening. I’m going to talk with you about managing patient expectations. So to get started, we’ll go through the learning objective:

• Define better ways to manage patient expectations about new therapies.

I’ll give a little bit of insight, and I probably won’t have the full

answer, but I will work as hard as I can. This slide was used by Dr. Mike Boyle at a plenary session a few years ago, and bits of it have always been pulled out for talks, but this is why it’s not an easy question, there’s no one answer. Because when we look at the genotype and genetics, CFTR is, yes, part of the equation for the variation in lung function, but we still know and have heard, that even if we know the genotype, we still can’t predict heir lung function. Part of their variation in lung function is from CFTR, but it’s also modifier genes. The other whole 50 percent of their variation in lung function is environment, and we all know adherence is a big issue. A lot of research is going on about that right now. We also know that environmental exposures play a big portion, and their treatment and their access to medications and to care also plays in. There is also a whole other random element that’s we’re not even sure we have the full answer to in the variation to what’s causing the variation in the lung function. When I was putting this together I was thinking, when ivacaftor first came out, everyone heard the headline, the nurse coordinators and probably other people in the facility, and I got multiple emails, multiple calls. Everyone wanted to know when they could get started, what this meant for them, or they wanted to know what genotype they were. Many people didn’t know their genotype, I feel patients have become much more

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educated since ivacaftor has come out. We’ve also been able to find more genotyping in our patients, so we’ve had a lot more discussion about it. We knew some of the information that had come out from the research on ivacaftor. Obviously, everyone was extremely excited, and we were able to relay the research finding to our patients, but what were the patients hearing? What they’d been hearing now has been more buzzwords about new treatments coming out there. I did a quick Google search and found some of these words that we’re going to be up against about what our patients are hearing and somewhat expecting. One of them was that it’s a dream come true. “As a parent this is such a dream come true,” Camille said, she’s the mother, “Dylan has started growing and gaining weight and he’s missing fewer schooldays, and overall he’s feeling much better.” And then another title of an article was “it’s a new wonder drug that can heal lungs of CF sufferers.” So what brings us to this miracle or hope idea that I’ve written about here? I asked a couple of our patients who have gone on ivacaftor, and I asked them for one word they could tell me about being on the medication. Well, I didn’t get one word, I got probably two pages worth of information from both of them. I’ll go through their stories and we’ll talk about this miracle or hope. One patient who started on the medication had been preparing

herself for years ahead of the drug so that her lungs could be the healthiest they could. She nailed down her treatments, she started exercising aggressively, she improved lung function on her own before she went on this treatment. And a lot of this is because she had a sister who had cystic fibrosis and she saw her dwindle and wait three years for a lung transplant, and then she eventually did get the lung transplant and is still alive. So she had this in the back of her mind that some day, cystic fibrosis was going to cause this progression for her. So she wanted to get her lungs in the best state she could, she got as healthy as she could, she got on the medication and she says that everywhere in the community that she sees about cystic fibrosis on the blogs, on the Internet, is that the drug is a miracle. She says it has given her hope. She says she has been able to have hope. She has a family. She has recently become a new parent, and she feels that she has a hope to control her destiny with the hope that she is so grateful for for the CF community with the drug. She said she still works hard every day, she still exercises and does all of her treatments. Another patient was part of the clinical trials, and she decided about six months into it that she didn’t feel “CF sick” anymore. One of her online friends who had CF — it was not a person-to-person friend and I do trust this patient — had told her that she had lost her “CF card,” because she was on a therapy that was correcting the underlying defect.

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Our patient said, “I didn’t quite feel like I lost my CF card, but I finally feel like for the first time I’m not CF sick.” She says it’s still a trial every day because she still does her treatments, she is also married and has a child, as well, and she also said that this has given her hope. It helps her with her treatments and it’s given her hope for the future with her family. So what are we to do with hearing our patients who go online and hear about a medication? They hear these case stories from other patients, they hear that it’s a miracle and we know that only a little bit over 4 percent of the patients right now are on this therapy, and obviously on the horizon there will be more. Well, unfortunately we won’t be able to put a lot of people on the drug right now, so we’ve got to keep up with the treatments and do what we’ve been doing. A lot of that involves adherence, which is very difficult. We all know that’s what we struggle with every day with our patients, letting them know that we’ve got to keep their lungs healthy. This drug’s only going to help their lungs if they can get on a medication that’s going to help with the underlying defect; it’s going to help their lungs for the future. So we’ve got to keep them as healthy as we can, a lot of this is communication and the explaining what their genotype is and what we know about their genotype and possibilities. We let them know about the Cystic Fibrosis Foundation pipeline and where the research is.

I feel keeping them informed has been very helpful. I think it also decreases a lot of anxiety when you’re talking with your patients and communicating with them about what you know about the drug and what that means to them. This also means they’re coming together as your team and trying to figure out how you are going to disseminate the information to your patients. When a new article or headline comes out, suddenly you get a bombardment of emails asking “is this for me” or “what more do you know,” thinking I have a whole lot more information than what’s out there, you can proactively send them information or have a communication plan for your patients, or tell them what you are going to do when a new drug does come out. And I think that will help allay a lot of the patients’ fears and help us as providers tackle this. A lot of it is communicating with the patients and families and educating them on what genotyping is and what that means to them. We can also let them know we don’t always necessarily know the answer and we’re not hiding it from them, but we’re still working on it. In summary, I don’t have that miracle answer. There are some things that we can’t change when we look at the CFTR. Some patients are going to be eligible for certain medications and some aren’t, but we know that there are some environmental factors we can change. We might be able to change some

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of their exposures and improve their adherence, and this is something to be aggressive about, to talk with them about adherence to their treatments, because it does go a lot longer way than they may think it does. And again, communicating with the patients, getting them familiarized with the CFF.org, the pipeline, getting them familiarized with research and where we’re at and what their genotype means to them. Hopefully in the future of our patients and in the future of this presentation we’ll have a lot more hope when we leave here tonight. Thank you.

Segment 5 DR. PATRICK SOSNAY: Our next video talks about tomorrow and the future, and what are the next steps to hopefully make cystic fibrosis as much as possible a disease of the past.

(Begin video)

BOYLE: It’s great to look back 25 years at the discovery of the gene. I guess the real question is, what about looking forward? FARRELL: I’ve never been more optimistic or hopeful in my entire career. CHMIEL: We are at the precipice, and I think the advances that we’re going to see in the next 5 to 10 years are going to be exponential. WINE: The rate at which the

discovery process is moving forward here, and the fact that it’s now clearly a combination of industry and academic research labs, is unlike anything I’ve ever seen before in the CF field. DONALDSON: Now we know what can be achieved, and that tells us where we need to go. RETSCH-BOGART: I see the discovery of the gene, understanding the protein, understanding how the cell processes the protein, and the variations in the protein from all the different CF mutations that we now are aware of, these are all tightly linked, and continued success in developing therapies is going to be that tight understanding between protein structure, cell biology, and drug therapy. BOYLE: The results from the trial that were announced this summer basically demonstrated clearly that we can do the same thing in patients with F508del. The results showed an improvement in FEV1 and showed an improvement in how frequently patients were sick. Now, it’s not to the levels we saw with ivacaftor and patients with G551D, but we’re not surprised about that because we knew this was going to be a harder nut to crack. The reason we’re so excited, though, is obviously this is a much bigger group of patients that we have a chance to impact. KEREM: Probably ivacaftor could help other mutations from class III and class IV, and maybe can be even combined with a corrector

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for delta-F508 or an agent that will promote read-through for nonsense mutations. DRUMM: About 2,000 different mutations in this one gene can cause disease, and we know very clearly that one size won’t fit all. So a compound that works for one individual or one mutation may not work for another, but the fact that all these other drugs are in the pipeline means that we’re probably going to keep hitting more and more and more of these patients, getting us to the point where we’re going to be talking about semantics to say we have a cure versus we have a treatment for it. And we’re making really big strides, and I think that’s incredibly exciting. BOYLE: We know we’re not done yet. We know we need to get stronger agents, we know we want to increase to patients who have just one F508del mutation, the most common group, but we’ve been waiting for this for a long time. It couldn’t be more exciting to treat the underlying cause and see a difference in how they’re doing, it’s exactly what we were hoping for. TSUI: I’m quite hopeful because now the high throughput screening and the drug libraries are all available. It just requires a magic moment to identify this particular drug. BEALL: Obviously, now there’s hope that lumacaftor and ivacaftor will be approved by the Food and Drug Administration for homozygotes, we’re working on heterozygotes, and we’ve got a pipeline for the nonsense

mutations. RETSCH-BOGART: To me the excitement comes from the width and depth of the pipeline in terms of the kinds of agents that we are now looking at, novel approaches to managing lung infection, because that will probably still be an issue for patients even if they have a great protector potentiator drug that improves their CFTR function. LEIGH: I have worked with patients with cystic fibrosis for a long time and I, as much as they, I want to see the day when there’s a cure for cystic fibrosis. BESSETTE: Hopefully the next generation of patients with CF won’t even have to acknowledge they have CF because we’ll have found something to take care of it. VON BERG: My hopes for the future are that we have the magic pills to not help the 4 percent of people living with CF, but that we can help everyone living with CF. PATTEE: That might be what keeps me going, that hope and passion for the future, that we’re going to be have better treatments and be able to help more people with CF live longer. RIORDAN: The hope is to be able to restore enough normal CFTR function in any individual who has a mutation. REISINGER: It’s given so much hope for the CF community, and that inspires me to stay healthy, knowing that there are new drugs on the horizon.

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LEIGH: This is going to take money, it’s going to take research, but it’s possible. DRUMM: It’s a paradox in that we see how far we’ve come and yet how slow the process has been. It couldn’t move fast enough for these kids who were affected, so even though we’re making great successes it’s still not enough. BOYLE: It is important as caregivers that we send the message that this is an ongoing process that’s going to require steps, and that we’ve taken the first couple of steps, but it’s not going to be one big step. BEALL: But you know, I think at some point we’re going to look back on the small molecules and say, that was then; now what are we looking at. Now we have therapies that treat the actual gene defect and put in messenger RNA that might be mutation-agnostic, and we’re looking at gene editing and stem cells. So we are building a pipeline now, not for the next two to three years, but we’re going to bring in the technologies that will provide a robust pipeline for the next 10 or 15 years, so we can think not just of a daily therapy where you take these pills, but hopefully of a lifetime therapy and a lifetime cure. COLLINS: But the sense now is that we finally, after 25 years, built upon that fundamental knowledge of what the glitch in that gene is, what it’s protein product is, what it’s supposed to do, how to encourage it to do it, even in somebody with the

disease. And now we see at long last a sense that this disease may find its way into the history books. That’s the dream. DR. PATRICK SOSNAY: A round of applause, those videos were fantastic. DKBmed, Peter, you did a great job with those. Next we’d like to bring up Dr. Joseph Pilewski from the University of Pittsburgh who is going to talk a little bit more about some of the future therapies. DR. JOSEPH PILEWSKI: Thanks very much, Patrick and Peter for the opportunity to have this. It’s quite humbling to speak at the end of all these esteemed scientists. My task in the next 20 minutes is to very quickly give you a glimpse of what’s going on currently and where we might end up with new therapies. I have a few disclosures that are listed here and the title of this talk is “Transforming Care, One Mechanism at a Time.” I have these as learning objectives:

• Explain how new CFTR modification advances point to changes that have to be made in clinical practice.

• Describe the utility of genotype/phenotype correlations beyond diagnosis in achieving more effective patient treatment.

• Demonstrate how to integrate the patient into an individualized therapy regimen to improve outcomes.

In the next 15 to 20 minutes is we’ll briefly review pathogenesis, because we’ve talked about that

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briefly with Stuart, what causes mucus obstruction and susceptibility to infection in CF. We’ll get at personalized medicine, about the future of small molecule potentiators and correctors, where are we now and what is the status of the cell biology, the understanding of the protein processing that is crucial to developing the right combination of correctors for patients. Then we’ll talk about other approaches to correct CFTR; that is, gene therapy and gene editing, just so they’re not forgotten. This is a very quick review of the pathophysiology of cystic fibrosis. We know we have an abnormal gene that causes and abnormal protein, and then we have this part of the sequence that we presume based on in vitro studies and now more recently some in vivo studies, some patient studies like the GOAL study, we find links between ion transport and airway surface liquid depletion and delayed mucociliary clearance. This is going to be one lesson we’re going to take out of the GOAL study. Once we get to this point, we reach this common pathway of a vicious cycle where you have delayed clearance, you have mucus obstruction, and infection, inflammation; and then you get lung damage, lung scarring; and that over years to decades eventuates in end-stage lung disease. At the beginning of this it’s critical to understand the genetic defect,

and I want to remind everybody of this reference source, the CFTR2.org. This is a critical thing that Patrick and Garry Cutting put together. It’s a wonderful resource for us as providers and for patients as well. So with that as the pathophysiology, what do we understand about this problem? Observation started back in the 1990s, when people took primary lung cells that were filled with mucus as shown in this schematic and cultured the cells from these affected CF lungs, and then they cultured them in these inserts that allowed you to visualize what happens on the surface of those cells. And using a fluorescence dye, these very elegant studies, published in 1999, showed that when you put a fluorescent fluid on the top of one of these cell layers, you establish the fluid layer shown here and it’s maintained in a normal cell. So over that 24 hour period the fluid is retained, it doesn’t evaporate because there’s an ion flux process in the apical membrane that regulates and maintains this liquid layer. In CF that liquid layer deranged: it is added at time zero and by 12 hours, it’s diminished this much and by 24 hours it’s virtually gone. So the regulatory mechanisms are defective, and that’s because we lose an ion channel, the CFTR channel, that secretes chloride and bicarbonate and regulates sodium absorption. With that as the understanding, we have to go back to the genetic level and ask, how does this happen? This is where we get some glimpses of residual function mutations based

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on classes. When we think about therapeutic approaches, as has already been alluded to, we have to understand this well. Stuart beautifully talked about stop mutations and the potential for translational read-through as a therapy. We know a lot about delta-F508 CFTR biogenesis and synthesis that one has to do to get to the cell surface, and we’ll talk about that some more, particularly how correctors are being developed to address that. Then we’ll talk about gating regulation mutations, a lot of which have some residual function. These are mutations that traffic to the cell surface, where the protein is either abnormal in function or abnormal in degree of ion conductance. Those are the potentiator targets for CF drug development. We’ve already mentioned ivacaftor. This drug was approved in 2012, and we know that it does a lot of beneficial things for patients. We know that it restores about 35 percent of normal CFTR function and it improves lung function as shown here. This is the placebo versus the treatment group in a year-long study. There’s about a 10 percent improvement in FEV1 absolute in these large studies. In addition, there were improvements in FEV1 and a marked improvement in exacerbation frequency. This was the paradigm. This shows that it’s possible. With approval of ivacaftor the CF community said let’s use this opportunity to study more about the degree of CFTR correction

that it’s going to take to have clinical benefit. This slide shows a graph of CFTR activity versus sweat chloride. These are two markers of CFTR function, and it shows a correlation between them. We’re all familiar with these patients, who have high, high sweat chloride These are patients with typical CF; they have virtually no CFTR activity and very severe disease. At the other end of the spectrum we have patients who have normal CFTR.They have normal sweat chloride and 100 percent of CFTR activity. Carriers lie somewhere in the middle here. And then as you move down the spectrum, we have patients who have residual function mutations, where they may have sweat chlorides in this indeterminate range, they may have 20 to 30 percent CFTR activity, and they have milder disease. We know that from a variety of studies that many of those patients with residual function will live into their 40s, 50s, and early 60s. If you superimpose on that the results of the ivacaftor trials and their dose response effects here, this is what happened as the dose was increased with ivacaftor: you went from 25, 75, 150 mg, you moved down in sweat chloride and you moved up in CFTR activity. That moved us towards normal. We can think of that as a minimum layer or level that we know we have to achieve to get clinical benefit in patients. One way to think about this is to think about patients who have at least 30 percent of CFTR activity as those who are likely to benefit

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from drug therapy and move closer toward residual function and closer to normal. In contrast, in this gray zone, between 20 and 30 percent of CFTR activity, we’re not sure. Many of these patients treated with ivacaftor benefitted from that level of correction. Where we are now is drugs that are probably right here, right at that 20 to 30 percent of corrected CFTR activity, and the benefit of those will take some time to sort out. One of the things the GOAL study did for us was let us to take patients before and after initiation of ivacaftor and look at their mucociliary clearance to say what happened to the clearance of mucus from their lungs once they were put on drug. This left hand-panel, which we’ll show in a minute, shows the clearance of a radio-labeled tracer out of the lung, so patients breathe in for five minutes a radio-labeled tracer that isn’t absorbed into the bloodstream, and then with a gamma counter we can visualize that in a time lapse movie over 20 to 30 minutes. This left-hand panel will show the result before a patient’s started on ivacaftor. You see that most of the radio label is retained in the lung. Without any coughing the lung retains a lot of that radio label; very little of it ends up down in the stomach. In contrast, if you look at the panel to the right, so what you’ll see is that this tracer moves efficiently up into the trachea and down into the stomach, so at the end of approximately 30 minutes, most of the tracer is moved out of

the lung. This is a critical flushing mechanism that the normal lung does. We learned from ivacaftor that flushing restored at least some degree of ion transport in patients with the G551D mutation. This gives us some physiologic clues to where we are with these potentiators and correctors. This shows the summary data, looking at what happened to mucociliary clearance. This shows clearance in patients before and one month after initiation of ivacaftor. The baseline clearance was here and the clearance after drug was up here. The other summary shown here shows a significant increase in the amount of clearance. The other thing we know about the CF lung is, we make the supposition that abnormal ion transport leads to infection. We know that from a variety of in vitro models. The GOAL study gave us a chance to investigate whether correcting ion transport would help reduce or minimize the amount of infection. And in some work that Sonya Heltshe and others are going to show at a workshop tomorrow, they found that the odds of pseudomonas positivity in the year after ivacaftor initiation was reduced by 35 percent, with a significant odds ratio reduction after they adjusted for other variables. So this is evidence linking an ion transport restoration to a reduction in infection, and it gives us hope that by intervening early in patients with potentiators and eventually correctors, if we restore ion transport we’ll help prevent those downstream events that are so critical to the

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pathogenesis of CF lung disease. An important advance in the last year in clinical trials has been the application of ivacaftor to other mutations. We know from a variety of other very elegant in vitro studies that when you take different CFTR mutations and you express them in cells in the laboratory, you can determine whether they make the CFTR protein, essentially saying do they have any residual function; and if they have residual function, will ivacaftor augment that function to restore it closer to normal? This is some data from Fred Van Goor looking at a variety of different mutations. The left-hand panel shows that these mutations process the mature protein. The right-hand panel in black shows their ion transport before ivacaftor and in the open bars after ivacaftor. There’s a significant number of mutations here that have some residual function, and that function is augmented with the addition of ivacaftor in cells in the lab. One of the most common of these is this R117H mutation. I’m going to review with you some of the recent data that was presented at this meeting regarding the effects of ivacaftor in patients. A cohort of patients with R117H mutations was randomized 1:1 to receive either ivacaftor or placebo. They received the ivacaftor for 24 weeks followed by a three-week washout, and then they all had the opportunity to go into an open enrollment phase. The promise was revealed very nicely in this study, and we

showed a significant improvement in FEV1 in patients who were treated with ivacaftor. Here is the placebo group. During the 24-week period, there is virtually no change in absolute percent predicted FEV1. In contrast, the patients on ivacaftor enjoyed on average about a 5 percent improvement. Notably, after the washout period, there is an open label randomization or open label extension, and again, there’s about a 5 percent improvement in FEV1 during that gray period to the right side of the slide in the open label extension. This is very compelling data that the R117H mutation responds to ivacaftor, and patients with that mutation may respond well to therapy. The company is now seeking FDA approval, with a hearing later this month to try to obtain approval for using ivacaftor in patients with R117H mutation. The other population I mentioned are patients who have residual function. This shows the in vitro data looking at a variety of mutations and their responsiveness to ivacaftor. These are responsive mutations. Then there’s a number of other mutations shown in this panel where the protein is not synthesized, so these are nonfunctional. However, some of them, even with very little expression at the cell surface, will respond to ivacaftor. This was generated from these in vitro results to show that this is a panel of mutations where we think there is residual function. We

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think that by giving an exposure to ivacaftor they may derive clinical benefit. Jerry Nick did this study at Denver. It’s a somewhat complicated study design, but essentially what he wanted to do is to look at a variety of panels during very short intervals of drug therapy where he randomized placebo and ivacaftor in these cycles and had washout periods in between. At the end, all the patients received open-label ivacaftor. This period gave him a chance to look at a real-world application: if you put a group of patients who had residual function mutations on ivacaftor, what would happen to them? That was the study design, and it allowed him to determine a response at the end based on standard assessments and then go back and correlate that to potential short-term measures and markers. I’m looking forward to hearing his presentation tomorrow and what he’s learned from that, but he was kind enough to share with me some of the summary data that occurred in individuals in this study. This shows a somewhat dramatic example of a woman who had a 15 percent absolute increase in FEV1 during the ivacaftor phase that washed out, did not return with placebo, and then returned again with ivacaftor, and then again during the open label she had improvement in lung function. If you look at the aggregate data shown here, there was a statistically significant improvement in absolute FEV1 both during the two-week cycles

shown here, and during the longer-term, open-label extension shown in the right hand panel. This is evidence to suggest that ivacaftor will have applications beyond G551D and other gating mutations, to R117H, and to this other cohort of patients who have some residual CFTR function. Now we’re going to turn back to delta-F508 and summarize some areas where progress has been made to identify correctors for these compounds. Stuart nicely went through the data with lumacaftor/ivacaftor and the background of this mutation. These patients come to us with their tattoo on their forearm and they say what are you going to do for me, doc, and we say, we’re working on it; enroll in a clinical trial to help us figure this out. The challenge here is to get the channels out of endoplasmic reticulum. So shown schematically this is the movement of the CFTR protein from the endoplasmic reticulum to the golgi to the cell surface, and delta-F gets targeted to this proteosome. There’s been, a number of labs have very aggressively gone after this question of how we can do better with this. And so we know that ivacaftor monotherapy was not effective, we know that lumacaftor as well as another corrector called 661 that I’ll talk about in a minute, are both correctors that increase delta-F508 CFTR activity in vitro when combined with ivacaftor. So let’s talk about these studies of VX-661 in the phase 2 trial that we completed the analysis and

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presented today. If you compare 661 to lumacaftor, 661 has a couple of favorable properties. One, it has a longer half-life, and two, it doesn’t have the same drug-drug interactions that you have with lumacaftor. Lumacaftor is the CYP3A4 inducer, whereas 661 does not have those properties, which makes it an appealing candidate for long-term drug therapy. This was the study design, it was a dose escalation where patients randomized, they started with monotherapy or placebo, and a second cohort went to combination therapy with ivacaftor or placebo; and it escalated through to assess what the maximal dose could be tolerated and then what the clinical effects would be. In the interest of time I’m going to skip to the FEV1 data and this looks very similar to the results that we saw in the prospect lumacaftor/ivacaftor trials. This shows absolute change in FEV1 in patients on combination therapy at dose escalation, so there’s a dose dependent increase in FEV1 in the patients treated with the combination arms, particularly with statistical significance at the 100 mg and 150 mg doses of VX-661. This is consistent with an important effect; there’s the washout once the drug is discontinued. This shows the summary statistics, and note that there is a 4.8 percent absolute increase in FEV1 in patients treated at the 100 mg dose and 4.5 percent absolute increase in patients at the 150 mg dose. So this

suggests that this is another effective corrector for patients who have two copies of the delta-F mutation. Another cohort that was part of this trial was patients who were already on ivacaftor in whom we know they have an improvement in CFTR activity and clinical benefit, but they’re not normal yet — can we make them more normal by adding a drug like VX-661, a corrector that may target delta-F? This was the trial. It was a study with patients who were already on ivacaftor who were randomized to either stay on ivacaftor alone with a placebo or stay on ivacaftor and receive VX-661, followed by a one-month washout period. The summary data from this study shows, notably, even though patients improved with ivacaftor alone, we had additional improvement with the addition of VX-661. Focusing on the lung function changes, there’s a 4 to 7 percent increase in absolute and relative FEV1 with the addition of VX-661 to ivacaftor, and that effect washes out during the follow-up period. This is suggestive evidence that corrector therapy can be targeted to single copy of delta-F508. I mentioned that there’s intensive cell biology work that’s gone on over the last decade, trying to identify what the problem is with delta-F. And we now know that there are these complex interface reactions between the transmembrane domain loops shown in dark blue, and the nucleotide binding domain shown

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in green. And the delta-F mutation sits in that pocket there and interacts with those multiple domains. This slide shows in a somewhat complicated but very important way what we understand happens to delta-F508. Delta-F comes through the pathway here in the endoplasmic reticulum. It doesn’t fold normally, and it ends up being targeted in the lower right-hand corner into the proteosome, where it test degraded. So correctors have the challenge of ushering delta-F508 out to the cell surface, where it may be potentiated by a second drug. This slide shows some beautiful work by Lukacs and colleagues, where they used a variety of synthesis models to look at each step along the way of delta-F508 synthesis. They show that in the normal situation there’s an assembly process where the different domains of CFTR are added to make a full-length protein. Delta-F508, shown in the bottom part of this screen, shows instability of these nucleotide-binding domains and that leads to defects in the interfaces with the transmembrane domain. As a result, the protein never folds, as you see schematically; those red subunits don’t aggregate in a normal fashion, so the protein becomes degraded. So understanding that, and then looking at various drug effects, has allowed the identification of different classes of CFTR correctors. These class I correctors affect the protein interactions here, class III affect another area of that interaction,

and then class II direct mutations affect the second nucleotide binding domain. Understanding that and then developing and testing drugs potentially gives you the opportunity to have multiple correctors. This slide shows proof of concept that that’s in fact achievable. So this is an in vitro study where they took organoids, little cells layers of delta-F508 cells that form little spheroids, and exposed them to drug to assess their CFTR activity, and the amount of swelling the organoid has is a reflection of how well CFTR has been restored. In this top green panel, where you add one of each of three classes of correctors, you end up with a dramatic increase in CFTR activity to well over 40 percent increase with addition of those three drugs. Suggesting that this multiple corrector approach is indeed achievable, at least with these sorts of laboratory compounds. If we come back to the pathophysiology, what else is in the pipeline? We focused on drug therapies; what other approaches are being developed? I’d be remiss if I didn’t mention ENaC inhibitors. This strategy has been talked about for many years but hasn’t come to fruition yet. Parion has a compound, P1037, that’s will go into clinical trials very soon. Other companies are developing alternative correctors and potentiators. We focused on lumacaftor, VX-661, and ivacaftor, and others will begin clinical trials this year.

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Finally, gene editing, I want to very quickly run through this and show you conceptually what’s happening with gene therapy and gene editing. Gene therapy was all the rage when I was coming to my first CF meetings in 1992 and 1993. There was proof of concept in the nose, and people thought the cure was right there, and then we went into the dark days where we realized how hard it was going to be. Undaunted, the group at the UK has continued their efforts in gene therapy and they’re now in the process of a yearlong study of monthly administration of a DNA construct that would potentially replace the CFTR gene in airway cells and provide a more enduring response. We look forward to hearing those results from Eric Altman and his colleagues sometime in the next year. Gene editing is a newer kid on the block, and this technology has been proven to work in vitro in cell models, but whether we can translate this into patients or not remains very much to be seen. The approach here is to use homologous recombination, taking a normal gene sequence and basically cutting it out and allowing it to cross over and insert a green normal copy of the sequence in the gene and end up with essentially a completely repaired gene product that then can function. There is proof of concept for that in this sort of study using organoids. They used a variety of these targeting vectors. Where

the slope goes up there’s dramatic organoid swelling after correcting a significant percentage of cells within the organoids. This approach will remain as a nice potential for long-term enduring correction of CFTR mutations. The current generation of corrector editing approaches uses antisense oligonucleotides. I mention this because you may hear about it in the next year because a company has a group of antisense oligonucleotides that will be delivered to patients who have CF mutations in an attempt to see whether they’re safe and whether they’ll achieve CFTR correction. I’m going to close with one thought, and that is, how are we going to apply these drugs, assuming they are developed over the next several years or longer? I like to think of CF now with newborn screening as having several phases. There’s a subclinical phase, the patients I don’t see because I treat adults, where patients are asymptomatic. This is the newborn to early childhood period where you wouldn’t know they have disease if you didn’t have genetic screening. There’s an early clinical phase with normal lung function but intermittent infections and mild bronchiectasis and then a late phase. It’s important to note that most of the drug studies that have been done to date have focused on patients who have chronic infections, reduced lung function, and well-established bronchiectasis.

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We think the future will be very bright as we apply these new therapies to patients with earlier phases of the disease where we can reduce the lung function decline that happens over time and essentially preserve lung function to transform the disease. How is that happening, what is critical to that? Clinical trials are obviously the key. We need patients who allow us to find out what their mutations are. Usually they don’t wear them on their arms, although some of them choose to do that. And we need clinical trials networks. We have a clinical trials network in the United States, we have a clinical trials network in Europe, they work collaboratively, and we have colleagues in Canada and Australia. That lumacaftor/ivacaftor trial was remarkable in how fast it enrolled. That’s a testimony to the commitment of the whole CF research community from providers all the way to basic scientists to translate what we’re learning in the lab to new therapies. So this future gives us all opportunity to enroll and encourage patients to move into clinical trials. To summarize, the opportunity I foresee over the next several years is to take these CFTR correctors and potentiators that now are applicable to maybe 5 to 15 percent of our patients and have opportunities for transformative therapies for those with other mutations. The goal with ivacaftor is to achieve therapy in about 10 to 15 percent

of patients. You may have ivacaftor in a corrector that may help another significant percentage of patients to get you in the 50 percent range, and then you have other patients who have only one copy of delta-F508 or patients who have other non-delta-F508 mutations, with the goal to provide a therapy for 100 percent of patients in the population. I’ll close by saying I think we are making progress, and the future indeed looks bright for a variety of reasons. I appreciate the opportunity to share this with you and will be happy to take questions during the question and answer session.

Segment 6 DR. PATRICK SOSNAY: As we’re submitting any last questions, I’d remind you that the eCysticFibrosis Reviews that are put out online about every six months, Dr. Pileweski has done one, I think all of us have done one of these reviews, but these are available online, interactive learning, you can find those today, there’s information about those at the registration booth. And finally, if you’re interested in more information from this presentation, this Ahead of the Curve Presentation or one of our previous presentations, aotc-cf.org has free resources and educational things to be able to use some of the materials that were used in this session and other sessions for your future learning.

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There’s a good question and this gets to some of the subtleties of clinical trials, so I’m going to ask Dr. Elborn this, that a 3 percent, 3 percent difference or 3 percent improvement in function was not significant in the ataluren trial, but was significant in the lumacaftor and ivacaftor trial, and why do you think that was and how do you relate those two studies? DR. STUART ELBORN: So the response in the combination therapy study was there was an actual improvement over baseline as well as an improvement compared to placebo. In contrast, in the ataluren study, if you remember the curves, both groups actually seemed to go down a little bit during the period of the clinical trials. So there was a difference between the two groups but I think the most important thing about it is that wasn’t clinically terribly relevant. Clinical trials are complex in the way the statistics are done and I think the data is reasonably clear that overall in the ataluren study there wasn’t a significant improvement which is in contrast to the lumacaftor/ivacaftor combination where the change was consistently positive in the two doses over the two studies. SOSNAY: So several of you had questions and I think these were appropriate about we talk about patient adherence but what about physician adherence or insurance company adherence. So Megan, perhaps you can speak to a little bit about your experience, has it been difficult to deal with insurance companies and difficult,

these are expensive drugs and the drugs on the horizon are likely to be expensive, do you have challenging times getting them to pay for them? MEGHAN RAMSAY: Initially, and I have a feeling most centers were probably like this. Initially I feel like most insurance companies approved ivacaftor pretty quickly. And then I feel like when the next month or the three month refill came, they realized the sticker shock and we started to see a lot more prior authorizations and a lot more companies or insurance companies wanted the actual lab report of their genetic testing. We had some that just wanted a clinical note with it, sometimes it was a little more difficult. Some people had been, it was hard to find that report because I deal with adults so it had been many years where they changed centers, but overall, we have been able to get it approved. Sometimes it take a little bit of legwork and other times it was just like refilling any general medicine that you refill that requires no prior authorization. If this gets expanded to more patients with insurances and the cost, that may change, but it wasn’t overall difficult. And considering the value of it to the patients, I wouldn’t say it was difficult at all in that regard. SOSNAY: Several of you had questions along that and I think that differences between the United States and Europe would be grounds for a lot more discussion, so I think that would be an interesting thing if we had more time.

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So finally several people had the question that all these therapies talk about fixing CFTR and Dr. Pilweski, I’d wonder if you’d talk about what do you think, how much do we need to fix CFTR, do you think that there is a threshold at which a dose of, amount of CFTR fixing that will provide a cure or provide a meaningful clinical difference. DR. JOSEPH PILEWSKI: I think we clearly know that 35 percent will have a clinical benefit. So a 35 percent restoration of CFTR activity in the ivacaftor study showed that, improvements in mucociliary clearance, improvements in lung function, potentially a reduction in infections. But what we don’t know about that therapy is if you start that early in life, might you have actually a very different outcome. So maybe if you start early in life, 35 percent would be enough, I think in patients with more advanced disease a higher level of correction is likely to have a greater clinical benefit. So it’s a very hard question, we know at the very minimum if one gets to 50 percent you’re going to reach closer to carrier state and I think those patients, if you got to 50 percent you’d probably have a cure. SOSNAY: Great. There are many more excellent questions that we, I don’t think have time for enough, but I appreciate your questions and I appreciate your making this as interactive of a session as possible. So in closing, I really want to

thank you for coming, thanks to DKBmed, and the technical team here that did a great job, and to our excellent presenters, and thanks you guys for spending your night with us and hopefully we helped teach you a little bit.

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