Welcome, everyone, to our webinar entitled, Can Gene Therapy be the Solutions for Congestive Heart Failure? This program is sponsored by ASBAU, a gene therapy company affiliated with Bayer. My name is Alain Lamontagne, and I have the honor to lead the Global Medical Affairs Organization at ASBAU. Before we start, I would like to share with you some disclosures. First of all, the speakers engaged in this program are either employees or consultants of ASBAU. Secondly, this presentation is not intended to offer legal, regulatory, or medical recommendations. And finally, the product discussed throughout this program and in 101, it's not approved and is currently under clinical development. We have four objectives for this program. First, we're going to provide an overview of congestive gene therapy in CHF. Secondly, we're going to discuss the making of actions of ANN101, which induces the congestive expression of protein phosphatase Aβ1, or I1C. Then we're going to review the initial data from a phase one clinical trial in CHF. And then, which I think is going to be very exciting, we're going to give you a preview of the design of the upcoming phase two gene fit clinical study. Here's the agenda for the next 45 minutes. I'm going to start with a brief overview of ASBAU. Then Dr. Raja Rajar from Flagship Paranay, Cambridge, is going to follow and give us an overview of cardiac gene therapy in CHF. Dr. Rajar will be followed by Dr. Litsa Krenias from the University of Cincinnati. She's an expert in the field and is going to give us an overview of the making of actions of ANN101. Then we're going to follow with Dr. Tim Henry from the Chris Hospital Health Network. Dr. Henry is our principal investigators in a phase one clinical program, and he's going to share with you the initial data of the phase one. Then Dr. Rajar is going to return and share with you exciting information about our upcoming phase two gene fit clinical program. Are you ready? Yes? Let's get started. Who is ASBAU? ASBAU was co-founded in 2001 by Sheila McHale, currently our CEO, and Dr. Jules Sammersky. This slide illustrates exactly where we are as an organization. We're driven by science and a leader in the field of gene therapy. And actually, Dr. Jules Sammersky is celebrating this year 40 years of research dedicated to gene therapy patients. As a company, we're the first company to clone AAV, or adeno-associated virus, for therapeutic purposes. As well, we're the first company to deliver AAV in the spine, and also to treat patients with Duchenne muscular dystrophy, or pumping disease. Interestingly, even 20 years ago, we were the first company to deliver AAV directly to the brain for pediatric patients suffering cannabis disease. We are a fully integrated clinical development organization with six preclinical and clinical programs, including our CHF program. We're dedicated to develop even better gene transfer tools, such as, you know, looking at better capsid, looking at the concept of redosing, or how to enhance on gene editing. There's one thing that is interesting with us, that's very unique to our company, is the fact that we own our own manufacturing capability. With our doggy bone and also protein technologies, we can produce faster gene vector without using human or animal byproducts, setting the bar in the industry. We became an independent affiliate of Bayer at the end of 2020, demonstrating Bayer's commitment to cell and gene therapy. Together, we're going to keep building on Bayer's legacy in the cardiovascular field. We're going to continue to focus on discoveries and bring more asset into the clinic, and hopefully to have a greater impact on our patients. As we're talking about patients, we have our own patient engagement program named Aspers. We're committed as an organization to our patients, their family, caregiver, engaged in throughout each step of clinical development program. If you're a patient, a family, a caregiver, or even, you know, a research coordinator or investigators, please don't hesitate to contact us, to reach out through our program at aspers at asbaro.com. Thank you so much for your attention. And now I'm going to turn over to Dr. Roger Hajjar, who will begin the discussion of cancer gene therapy in CHF. Dr. Hajjar? Thank you, Alaa. I'm Roger Hajjar, and today I will be providing you with an overview of congestive heart failure and introducing the mechanics of cardiac gene therapy. This slide shows my disclosures. So heart failure is a growing health and economic burden for the United States and the European Union. In the U.S., there are about 6 million Americans afflicted with congestive heart failure, and that number is projected to increase by 46% between 2012 and 2030. The five-year prognosis remains quite poor, especially for patients who are in class four and class three congestive heart failure. U.S. hospitalizations for congestive heart failure has increased by 26% between 2013 and 2017. There are about 1.4 million ED visits and about 1.3 million hospital discharges recorded in 2018. Economically, by 2030, the total cost of congestive heart failure will increase by 127% to about $70 billion, or about $244 per U.S. adult. Congestive heart failure has a variable type of progression. Certain patients will have an episode of congestive heart failure and will have a recovery due to treatment or to, most likely, the underlying cause of the congestive heart failure. Certain percentage of patients will have heart failure, acute heart failure, with a very rapid decline and death after that first episode. More commonly, patients with congestive heart failure will have a first episode of decompensation and then will have temporary or partial recovery with oscillations in terms of the number of admissions that they go through throughout their congestive heart failure progression. These recurring acute decompensated heart failure episodes is part of the trajectory of patients with congestive heart failure and the eventuality of death at the very end of that trajectory. For many years, scientists have looked into what are the mechanisms that underlie the status of congestive heart failure. And basically, there are a number of insults and injuries that damage the myocardium within the heart, and that could be coronary artery disease, myocardial infarction, familial and genetic predisposition, hypertension, pregnancy, valvular disease, alcohol abuse, and various toxins. And that can result in damage to the myocardium, whereby the diseased myocardium will be left with myocardial cells that are dying, myocardial cells that are dysfunctional, meaning that they cannot contract and relax appropriately, and completely dead myocardial cells that are now replaced by extracellular matrix fibrosis. In addition, the blood vessels that supply the myocardium in the diseased state are also abnormal, not relaxing and not conducting blood flow in the appropriate fashion. Excitation-contraction coupling governs the contraction and relaxation in cardiac cells. An action potential that depolarizes the membrane induces the entry of a small amount of calcium through the L-type calcium channel, which in turn activates surrounding receptors to release a larger amount of calcium and activate the myofilaments. This induces production of pressure and force. And then during relaxation, the calcium is taken back up into the sarcoplasmic reticulum by a complex known as a circa-2A pump, and the calcium is also extruded outside the cell to the sodium-calcium exchanger. Circa-2A pump is regulated tightly by a small phosphoprotein known as phospholamban or PLN. In its unphosphorylated form, binds to circa-2A and decreases calcium entry and the activity of the circa pump. During phosphorylation, phospholamban inhibition to circa-2A decreases and calcium now can go in at a much higher rate into the sarcoplasmic reticulum. In heart failure, phospholamban is dephosphorylated because protein phosphatase 1, PP1, is highly active and decreases the phosphorylation of phospholamban, making it inhibitory towards circa-2A. In our gene therapy approach, we basically use inhibitor 1 to block protein phosphatase 1 activity. And we've actually made a constituently active form of inhibitor 1 called I1C that blocks protein phosphatase 1. So our target, which is I1C, enhances both the uptake and the release of calcium. And that translates into a better contraction and a faster relaxation, which are very important in the setting of heart failure. Now over the years, we have tried multiple pharmacological treatments to target the circa-2A pump and phospholamban. Those have not been successful. For this reason, we've turned to gene therapy, especially with the advent of adeno-associated vector, which have the advantages that they are non-pathogenic, they have low immunogenicity, and they induce long-term expression, which is very important in heart failure. AAVs have been found to be safe in cardiovascular applications. They are ideal for cardiac gene transfer, able to transfect slowly dividing or non-dividing cells such as cardiomyocytes. And we can use minimally invasive percutaneous approaches that allow access directly to the heart for localized delivery. And these are different modes of myocardial delivery that have been attempted in cardiac gene transfer. And they include retrograde injection through the coronary sinus while the coronary arteries are blocked. They also include surgical myocardial injections, whereby the vectors are injected directly into the ventricle using a percutaneous endocardial approach. There's also antigrade injection, whereby the vector is directly injected in the coronary artery, in the lumen of the coronary artery, without blocking the coronary artery or the coronary sinus. And this has been the method of choice for our gene therapy program because it is relatively safe. It can be done rapidly. And as shown on the right lower panel, this is an injection from a patient where the right coronary artery on the left-hand side is a pacified and the left coronary artery is a pacified on the right-hand side. So the method of choice for myocardial delivery using the AAV vector is an unobstructed delivery through the coronary arteries without blockade. And that injection is done in the cardiac cath lab using a percutaneous approach. During the injections, there are specific factors that increase the vector uptake in the myocardium. And those include coronary flow, increasing coronary flow, and that can be done by enhancing the flow using pharmacological means. There's also permeability factors that can be used to enhance AAV crossing from the capillaries to the myocardium, and that's achieved using drugs such as nitroglycerin. And then we can increase the dwell time by prolonging the injection through the coronary arteries. And that's why the injections that we perform are about 10 minutes and not a straight shot into the coronary arteries. That increased time allows for higher interaction between the vector and the myocardium. One of the novelty that AspBio has been able to produce is an AAV capsid that's novel and re-engineered, and that's known as AAV2I8, which is sort of a mosaic between AAV2 and AAV8. And this synthetic AAV2I8 strain easily traverses a vasculature and selectively transduces cardiac and whole body skeletal muscle tissues with very high efficiency. And unlike other naturally occurring AAVs, AAV2I8 displayed markedly reduced hepatic tropism. So it has a very high level of tropism towards the cardiac muscle, which is exactly what we want, while at the same time having low tropism towards the liver, where most of the toxicity from AAV gene therapy comes in. And what we have manufactured is an AAV2I8 vector with a constitutively active promoter known as CMV and the I1C transgene that I described earlier that induces the expression within the myocardium, thereby improving the function of the cardiac cells. So in summary, heart failure represents a growing health and economic burden for the US and the European Union. Drug and device therapies improve survival, but because underlying pathology persists, patients invariably progress to end-stage disease. Pharmacological strategies for improving calcium handling in the failing heart have remained largely disappointing, and gene therapy is an alternative approach that has been used experimentally and now clinically for proof-of-concept treatment of this devastating disease. Thank you. And now I would like to turn the program over to Professor Liza Kranius, who will discuss the calcium cycling abnormalities in failing heart cells. Dr. Kranius is a pioneer in this field and has discovered many of the abnormalities within the pathways that she will be describing over the last few decades. Thank you, Roger. My name is Liza Kranius, and I am a professor and director of cardiovascular biology at the University of Cincinnati College of Medicine in Cincinnati, Ohio. As Roger mentioned, I will be discussing the aberrant calcium handling in failing myocardium and the potential of NUN-101 to correct this condition in congestive heart failure. Here are my disclosures. On the left here, we have a schematic representation of the sarcoplasmic reticulum membrane. And as Roger said earlier, calcium is transported into the lumen of this membrane by the calcium ATPase to induce relaxation. And this process is regulated by phospholampan. Phospholampan is an inhibitor of the calcium pump in the dephosphorylated state. And phosphorylation relieves this inhibition and allows calcium to transport faster and more efficiently. On the right here, we have a scheme where the phosphorylation of phospholampan is shown during beta-adrenergic stimulation, which is an important process to allow the heart to meet the demands of the periphery under stress conditions. So phospholampan gets phosphorylated at two distinct sites by protein kinase and chemkinase. And those are serine 16 and threonine 17. Notice now that in heart failure, the calcium ATPase levels decrease. These are the pink molecules. And phospholampan is more dephosphorylated, indicating a double insult on the failing heart and the calcium cycling. This dephosphorylation of phospholampan occurs at both the cyclic AMP and the calmodulin dependent sites, serine 16 and threonine 17, as we see on the top in non-failing and failing hearts. The dephosphorylation of phospholampan inhibits calcium transport and calcium cycling and contractility. This is at least partially due to the increased phosphatase activity in human heart failure, which is regulated by an endogenous protein called inhibitor 1, as Roger pointed earlier. The inhibitor 1 is not active, but it becomes active when it is phosphorylated by protein kinase A, and that shuts off the phosphatase to allow phosphorylation of phospholampan and increased calcium cycling. So this inhibitor 1 then, in heart failure, is itself dephosphorylated like phospholamban. So we decided then to generate an active inhibitor molecule and for this we truncated the form to 65 amino acids and converted threonine 35 to aspartic acid to have a constitutively active inhibitor one. And the hypothesis here is that this active inhibitor one would enhance calcium cycling and contractility which could be beneficial in heart failure. In a first set of experiments, we generated an antigenic mouse model expressing the active inhibitor one in the heart and we observed that the phosphatase activity was decreased as expected. This was associated with selective and specific phosphorylation of phospholamban at the two physiologically relevant sites and fractional shortening is significantly increased and the calcium cycling as calcium amplitude is also significantly increased. This model with hyperdynamic function due to the expression of I1C does not have any remodeling fibrosis or hypertrophy that develops through the aging process. The more relevant question is whether I1C would have any benefits in pre-existing heart failure. For this we generated a heart failure model in the red where upon all deconstriction and 18 weeks post-surgery we have heart failure and then we deliver a control gene GFB or I1C. We have three groups as shown on the left, the non-failing, the failing and the I1C. In purple, we see that I1C enhances the phosphorylation of the cyclic AMP protein kinase site. It does not alter the calmodulin kinase phosphorylation because this is highly elevated in heart failure and on the right we see that the contractile parameters both the rate of contraction and relaxation are significantly enhanced by I1C. And then we move to a large and clinically relevant model of heart failure. As we see here, the ejection fraction is significantly improved by I1C. The rates of contraction DBDT are also significantly enhanced. At the bottom we see a stroke work is increased and importantly on the upper right-hand corner we observe that the rates of contraction significantly correlate with the degree of phosphorylampin phosphorylation indicating that this is the major mechanism of the effects of I1C in the heart. So in summary, the type I phosphatase is a negative regulator of calcium cycling and contractility in the heart. This enzyme activity is increased in heart failure and this is partially due to the defective regulation by inhibitor one. Delivery of a constitutively active inhibitor I1C enhances ventricular function in small and large animal models of heart failure. And most importantly, it reverses remodeling and fibrosis in the small models of heart failure. I appreciate you listening. Now, Tim Henry will present some initial human data for non-one-on-one in congestive heart failure. Great, thank you, Dr. Kouranias. My name is Tim Henry and I'm an interventional cardiologist at the Christ Hospital in Cincinnati and director of the Lindner Center. And it's my pleasure to give you the first look at the phase one safety and efficacy in the Anacor 101 patients with class three heart failure trial. In terms of disclosure, I'm a member of the Scientific Advisory Board of Ask Bio and I'm the principal investigator of the phase one trial, but I don't have any ownership or other interests involved in this presentation. So as you just heard, NAN101 is a gene therapy product that's composed of a novel cardiotropic adeno-associated virus vector capid and a gene promoting increased expression of a constitutively active form of inhibitor 1C. The image on the right shows how the AAV218 capsid containing a therapeutic gene enters the nucleus of the cardiomyocyte where the gene is then released to undergo transcription and subsequent RNA translation to produce the inhibitor 1C protein. Now, you discussed this earlier with Dr. Kouranias, but inhibitor 1C mediates a cascade of events in the cardiomyocyte that result in enhanced activity of circuit 2A and calcium cycling. And this leads to improved cardiac contractility. Inhibitor 1C also has direct effects on fibrosis and cardiac hypertrophy has been demonstrated in preclinical models of heart failure. So the primary objective of the phase one study was to evaluate the safety of single antigrade epicardial coronary artery infusion of three dose levels of NAND101 to subject with non ischemic cardiomyopathy and class three heart failure. And then a secondary objective was to explore the biologic activity and efficacy of the intracoronary infusion of NAND101 in order to inform the future clinical trials. So this is the study design of the trial. It was a phase one prospective, multi-center, open label, sequential dose escalation study of two doses of NAND101 delivered by intracoronary infusion. And note that the doses were not weight-based, they're per patient. And the safety evaluations were the key part of this, but we also had an opportunity to observe efficacy with New York Heart Association class, VO2 max, six minute walk test, ejection fraction, and Minnesota Living with Heart Failure questionnaire to give us a spectrum of is there clinical improvement. And patients were evaluated at baseline six months and 12 months following NAND101 infusion. The infusion was done by an antigrade epicardial coronary artery infusion over 10 minutes. 60 cc's were delivered based on the coronary anatomy and it was delivered by just commercially available injection, either femoral or radial through a regular guiding or diagnostic catheter. It's really very straightforward and simple. Patient demographics are shown here and there are some key differences. So first of all, the patients were enrolled were non ischemic cardiomyopathy with ejection fraction less than 35% and greater than 15%. They had to have class three symptoms and that were on appropriate and stable heart failure medication as well as cardiac resynchronization therapy so that their heart therapy had to be optimized before they could get in the trial. And then in the trial, three patients have received treatment with 12 month follow-up in cohort one and five patients have retrieved treatment with a follow-up between six and 12 months in cohort two and we're gonna give you some insights. So first, there are little differences between cohort one and cohort two. Cohort one mean age was 70, cohort two was about 59, a predominant male as with typical and heart failure trials. The mean ejection fraction in cohort one was 33.5 and the mean EF was in cohort two was 19.1. So the cohort two is a little bit sicker. In terms of safety and tolerability, I'm delighted to report there were no adverse events that were directly related to study treatment. Of course, this is a class three heart failure patient population that's really quite sick. So there were some adverse events, but no serious adverse events related to the study treatment and that's encouraging. So the next slide is really, I think, the most important slide of this presentation. And so in cohort one, we did not see any increase in LFTs and no ELLISBOT positivity. So we're not showing those data. So this is data just from cohort two. And as you see that we saw a mild and transient increase in LFTs, both ALT and AST. As expected, these occurred between weeks four and 12 and were transient. But more importantly, on the right hand of the slide, what you can see is that in terms of ELLISBOT activity, which indicates T cell response directly to the AAV capsid and the inhibitor 1C protein. So this is important because the positive ELLISBOT findings demonstrate that the intracoronary administration of the NAM-101 resulted in successful gene transfer into the myocardium. We're very excited about these results. In terms of efficacy, what we did is we looked at what are meaningful clinical endpoint signals for patients who are in heart failure. And these are arbitrary, of course, but just so you know, our definition was for EF, it was a greater than 5% increase. For end systolic volume, it was a 10% decrease. In New York Heart Association class was a 1% decrease. Quality of life was a 10 point decrease. Six minute walk was a 50 meter increase. BNP, 40% decrease. Pro BNT, a 35% decrease. And in terms of VO2 max is 1.5 cc per kilogram per minute increase. So very arbitrary, but you need some kind of a guide of, you know, is there some clinical benefit going on here? And that's not the purpose of the slide, but what you can see on this is in cohort one and moving into the green correlates with what I just talked about is movement into a clinically meaningful benefit. And so I think we are encouraged to see that even in cohort one, we saw movement into what we would say would potentially be clinically meaningful. And then on the next one is cohort two. And remember now, cohort two is still incomplete with only five patients and the fall of still between six and 12 months. And the patient population was a little bit sicker, but what you see to start with is that in general, the same movement improvements in New York Heart Association class, ejection fraction, quality of life, and six minute walk. There wasn't such an improvement in the VO2 max in the second cohort, but again, small numbers, not the point of the trial. The most important part of the trial was to evaluate safety. So in summary, AskBio has developed a AAV capsid targeting delivery of a gene promoting increased expression of inhibitor 1C that has shown promise in animal models and recently entered clinical testing in patients with heart failure. The safety data suggests that nano 101 is well tolerated with encouraging efficacy in both cohort one and two. So really it might be clinically meaningful. And more importantly, we have evidence from the dose escalation that they actually have successful gene transfer. So thank you for your attention. We're excited about this product. And now I'm gonna turn you back to Roger Hajjar, who's gonna tell you more about the plans for phase two. Thank you, Tim. I would like to discuss now our design for the phase two clinical trial and the phase two trial's objectives are to basically evaluate the efficacy and safety of a single antigrade intracoronary infusion of nano 101 versus placebo in subjects with non ischemic cardiomyopathy and New York Heart Association class three heart failure. The secondary objective of this phase two trial will be to evaluate the impact of the single injection versus placebo on cardiac function, exercise capacity, quality of life and biomarkers such as NT-PRO-BNP. The key inclusion criteria for this trial are very typical of a heart failure trial and it includes chronic non ischemic cardiomyopathy. We're targeting adult patients so they will be above the age of 18. The injection fraction is less than 35% but more than 15%. The six minute walk test has to be at least 50 meters and the patients have to be medically stable with class three heart failure for a minimum of four weeks while on guideline based medical therapy. The key exclusion criteria include ischemic cardiomyopathy which is not in the realm of this specific study, presence of neutralizing antibodies of one to five or more, specific types of cardiomyopathy such as restrictive obstructive cardiomyopathies along with infiltrated ones like amyloidosis or pericardial disease, uncorrected thyroid disease, dyskinetic LV aneurysms, cardiac surgeries or cardiac interventions within 30 days of screening, third degree heart block or a clinically significant myocardial infarction within six months of screening. The study design overview is shown here. It's an adaptive, randomized, double blind placebo controlled multicenter trial. Patients are first screened and the neutralizing antibodies are collected at that time. Then if they pass through the screening, randomization occurs and we will have three different arms, a low dose arm, a high dose arm and a placebo arm. And the trial will go on for a 52 week period at which point outcomes will be assessed. There is then a long-term follow-up that occurs after that one years and that long-term follow-up is for four additional years. The primary and secondary endpoints for this trial are based on clinically meaningful heart failure endpoints and we will use a modified win ratio as defined by a hierarchical evaluation of the following assessments in the given order, cardiovascular related deaths, New York Heart Association functional class, left ventricular ejection fraction and a change from baseline, VO2 max change from baseline of at least 1.5 milliliter per kilogram per minute and a six minute walk test change from baseline of more than 30 meters. We will also evaluate safety endpoints such as adverse event and serious adverse event and observe value and change from baseline in clinical laboratories, vital signs and electrocardiograms. Secondary efficacy endpoints will also be evaluated in terms of a more structural assessment using echocardiography, quality of life assessments at baseline, 8, 12, 24, 36 and 52 weeks and other endpoints such as incidence of cardiac transplantation and LVAD implantation, number of heart failure hospitalization and all cause mortality. So in summary, in this phase one trial, all three patients in cohort one at a dose of three times 10 to the 13 viral genome per patient showed clinically meaningful improvements in endpoints such as left ventricular ejection fraction, six minute walk test, VO2 max and New York Heart Association functional class at 12 months compared to baseline. Preliminary results also show that the AV2I8 capsid results in a very high vector genome uptake in the myocardium. These results need to be validated by obtaining further tissue through biopsies from injected patients. Once additional validation that patients are doing well from cohort one of the phase one trial at a dose of three times 10 to 13 viral genome per patient, that dose will constitute one arm of the phase two trial. For the second dose in the gene fit trial, we will await complete data from cohort two in the phase one trial, which has a dose of 10 to the 14 viral genome per patient. And we may also consider a dose slightly lower than 10 to the 14 viral genome per patient to avoid stimulating a T cell response. For more information on this phase two trial, please scan this QR code. If you're interested in becoming an investigator or referring patients for this phase two trial, please scan the following code. So we would like to conclude with the following remarks. Gene therapy for congestive heart failure is a novel therapy that has started to have a foothold in terms of therapeutics for congestive heart failure. NAND101 is a gene therapy product composed of a novel chimeric cardiotropic adeno-associated vector known as AV2I8, containing a therapeutic gene promoting increased expression of a constitutively active form of inhibitor one. In a phase one trial of patients with non ischemic cardiomyopathy, New York Heart Association class three, and ejection fraction between 15 and 35%, the gene therapy product NAND101 was shown to be generally safe and well tolerated. In addition, encouraging and clinically meaningful efficacy endpoints were found in patients following NAND101 administration, including increases in left ventricular ejection fraction, decreases in New York Heart Association functional class, and increases in six minute walk test and peak exercise VO2 max. As we discussed earlier, planning is underway for the initiation of a phase two trial of NAND101 in a similar patient population, and the trial's name is GeneFit. I would like to thank the faculty, Dr. Tim Henry, our lead investigator and the principal investigator for our trial, and Dr. Liza Kranius, a world-renowned expert on calcium hygiene. I would also like to thank our viewers for watching the webinar and learning more about cardiac gene therapy. ♪♪♪

webinar

gene therapy

congestive heart failure

ASBAU

ANN101

clinical trial

efficacy

GeneFit