Well, I get to do the next talk, which is going to be about stem cell for biological pacemakers. Thank you. So I have nothing to disclose. So modern therapy for arrhythmias relied on pharmacotherapy that is not very effective and they can be proarrhythmic, and usually it's not very specific. Catheter ablation can be curative in many instances, but still needs optimization in some of the arrhythmias that we treat. And devices can be lifesaving in multiple conditions, but sometimes they need commitment to multiple procedures and they can have complications. Because of that, sheen therapy and cell therapy, they have been developed or studied by different groups to treat arrhythmias as an alternative. So far, the targets for these conditions, they've been to enhance automaticity to treat bradycardias, to create a bypass tract, something that we reported back in 2014, creating a bypass tract using cells and magnetic beads to be able to restore conduction, to slow conduction in the AV node with a calcium channel blocker by injecting that selectively into the AV node in a large animal, and also for ventricular tachycardia to delay repolarization and accelerate conduction. Today, we're going to focus primarily on enhancing automaticity at different levels to treat bradycardia and creating a biological pacer. As we know, during our lifetime, our heart beats over 3 billion times, and this is due to a highly specialized conduction system that starts into sinus node, there's a decrement and delay in the AV node, and fast conduction through Higgs-Burkinshi system, creating an action potential and spontaneous repolarization. That, it's created by two mechanisms and oscillators, a membrane clock and a calcium clock, that they function in synchrony throughout our life to create spontaneous repolarizations. The membrane clock being primarily by the ACN channel that we can clinically block with the evaporating these days, and the calcium clock by releasing calcium from the intracellular stores. When this system fails, we relied on electronic pacemakers, and they've been around for over 60 years now. This is a picture of the first implanted pacemaker in the United States. However, I have to say that the first one was in 1958 in Sweden. The patient generator lasted only hours and required multiple change-outs. Then in the 60s in the United States, we started doing the picardial pacemakers, and the technology has evolved a lot, as we know, and now we have, since 2016, leadless pacemakers that we can implant without the need to have a generator to decrease the risk of infections. However, leadless pacemakers in the current technology, they don't have the ability to have AV synchrony, so it will be only for pacing the ventricle, so chronic atrial fibrillation without having atrial ventricular asymptomy. There is limitations of pacemakers, as we know. There's no true autonomic response, despite different algorithms to generate or mimic that. You can have dysfunction of the leads or generator, and you can have infections and pace-induced cardiomyopathy that are rare, but they can happen. And they are problematic in the pediatric population and those with fetal heart block because of the size and the growing of this population, and the hardware does not stretch. Infections, as we know, they continue to rise. The incidence is about 4 to 5 percent. They continue to rise in the United States and worldwide, not only because we implant more devices, because of the more resistant organisms that we have. The infections can be arranged from different versions, from a hematoma to erosion to a frank endocarditis, but the common denominator with infections is that when you have a device-related infection, you should expand the device and implant a new system after the infection has been cleared. That can be problematic in pacemaker-dependent patients. That's why you need to put a temporary wire to sustain the rhythm while you're treating the infections, or put an active fixation lead, allowing the patients to have some mobility during the treatment. So the question that we ask ourselves is, can we use a biological pacemaker as an alternative to hardware and devices? And for that, I'd like to review the different approaches that they've been used. The first approach that's been utilized is a functional re-engineer approach. By that approach, you can express one ion channel or a combination of different ion channels to make a normal quiescent myocyte oscillate and create a spontaneous beating. That's been created by, initially, by blocking IK1, KR2.1 mutant, and that was reported back in 2002 by Dr. Marvan's group by creating a dominant negative mutant of KR2.1 and creating a spontaneous depolarizations and beatings that you can see here in guinea pigs, back reported in 2002. So a stem cell approach, which is a topic that we're talking today, it's also being utilized, and here the concept, it's a little bit different. You can use either embryonic stem cells or human iPS, and by doing that, you can create the clusters of beating cells, and you transplant those to create capture of the surrounding myocardium and create spontaneous depolarization. That approach has some difficulties for the maturity of the cells, and also you can have rejection if you are using other genetic cells in that situation. As you can see here, this is in vitro data with human EVs that they've been modified by HCN pacemaker channel, and the heart rate can be affected by that. And this has been also used in animals as well. So in other words, you can use stem cells, but you can also engineer the stem cells to create a faster heart rate if you want to do so. The next approach is a hybrid approach, which cells are actually used to transport the genes, and you can load mesenchymal stem cells, for instance, or fibroblasts, with a pacemaker channel, and by doing that, you can deliver the gene of interest without the need to having a viral vector to do so, okay? That's been reported back by Heechul Cho in 2007, where he loaded a fibroblast with the HCN channel, and by creating fusion, he was able to create the spontaneous depolarizations, you know, in those animals, as opposed to the control where you have to inject current to elicit an action potential. The approach that I'm going to focus most of my time today, it's somatic reprogramming, which involves not a stem cell, but using an embryonic transcription factor to reprogram a cardiomyocyte into a sinoatrial nodal cells. So in this approach, we're not doing a single gene expression of ion channel, we are actually reprogramming the cell and recapitulating many of the issues and features of the sinoatrial nodal cell. Why TBX18? Well, the TBX18, it's involved in the development of the embryonic sinoatrial node, and this is a cartoon from a mouse heart and embryonic week 9.5 to 10.5, and we know that TBX18 overexpression leads to creation of sinoatrial node. So the question that we ask ourselves is, can we overexpress TBX18 and recapitulate those key features of the sinoatrial nodes? And that was initially performed in vitro on small animals, again, you know, work pioneered by H.O. Cho and Eduardo Marvan, and as you can see here on the left, you have a cluster of three sinoatrial nodal myocytes. In the middle, you have a cell that's been reprogrammed by TBX18, and on the far right, you have the classic road-shaped cardiomyocyte that we all are used to seeing. When they isolated those cells, you could see spontaneous depolarizations in the sinoatrial nodal cells. The same could be seen with TBX18 reprogrammed cells, very similar action potential morphology, and in the control groups, you have to trigger the action potential, otherwise the cells would not beat. When they performed the PCR to the transcriptome level, it evaluates many of the features of those cells. They saw that many of the genes involved in the sinoatrial cells were mimicked by TBX18 comparing to the sinoatrial node. So the next thing that we ask ourselves is, well, can we deliver this using the minimally invasive delivery system to make it translatable to the clinics, hopefully someday? And to review that, I want to say that this is a review of some of the most relevant studies that have been done in large animals. Many of them use open chest or arterial catheter delivery system to deliver the biopacer. We are focused in trying to do a less invasive approach by using a venous catheter that we use in electrophysiology, and by doing that, we can advance that catheter and deliver the gene of interest into the region that we want. So this is the catheter that we use, which is known as myostar catheter, which is a unidirectional catheter with a retractable needle that you can inject into the myocardium, so it would be intramyocardial delivery focal. The animals in the initial report, we performed heart block, and we implanted a telemetry to be able to study in real time the dynamics of the pacemaker. And after that, we performed 3D mapping, arrhythmia induction, and histology on the end of the study. As you can see here, the mean heart rate in the study follow-up, this is the initial study two weeks, was higher in TBX18 compared to controls, and the same was true for the maximum heart rate, okay? The backup pacemaker utilization was much lower in TBX18 compared to controls, meaning that the animals, you know, did not require the backup pacing because of the biopacer. They also had autonomic regulation, and we measured that by response to isoproteinol and by heart rate variability analysis compared to the electronic pacemaker animals. To test activity, the animals had an implanted accelerometer to be able to study if these changes in the heart rate, they have any reflection on the animal's activity. And what we saw is that the animals have more activity, were able to move more, and they also, they were able to achieve those levels and sustain those levels for a longer period of time compared to controls. Initially, additionally, when we study reprogramming, we saw that those parameters, you know, were consistent with what was previously reported by Hitchell Cho and Eduardo Morvan in the small animals, and by bio-distribution, we were able to detect levels in the injection site and very low, less than 10 copy numbers elsewhere. There was no prorythmic defect, and we studied that by telemetry. We did not see, excuse me, we did not see any arrhythmias, and we performed program stimulation at the end of the study, and we could not induce arrhythmias in those animals. Additionally, the QTc and the QT and the APD90 measurement of monophasic action potential was not prolonged in this study. The other limitation that I mentioned before, it's a pacing-induced cardiomyopathy, which I'd like to spend, like, a couple of slides on that. And as we know, right ventricular pacing from multiple studies, as this is just some of them, I'm not going to go into details, but I'm going to conclude that right ventricular pacing increases heart failure, mortality, and an atrial fibrillation, and other conditions as well, but we think that we know that this is pretty well established. To overcome that, we come with alternatives, such as CRT, by placing an LVD into a postulateral branch in the coronary sinus, okay? We do know that there's technical difficulties and you need a favorable anatomy, and we also know that over 35% of patients, or close to 35% of patients, I should say, they can be non-responders to this therapy. His bundle pacing, that's been for many years, but now it's reemerged in electrophysiology, it's also a good alternative where you can pace into the his bundle and narrow the QRS, but also has, like, technical difficulties, and we don't have data on long-term outcomes. So the next question that we ask ourselves, and this has been reported and published a couple of weeks ago, is can we treat RV pacing-induced cardiomyopathy by delivering TVX18 into the his bundle region? And to do that, we went back to our swine model, and we implemented two models. The first model, we wanted to prevent that, and the animals were randomized to receive TVX18 or placebo, and follow up with MRI and hemodynamic studies during follow-up. As you can see here, the heart rate was different, and the back-up pacemaker utilization was different as well. But we wanted to answer the most important question, is can we rescue the pacing-induced cardiomyopathy? So rather than prevent it, we induced heart lock, we waited for four weeks, and then we injected TVX18. And you can see here, after injection, there was a difference in the heart rate and decrease in the back-up pacemaker utilization after injection. So here, you can see an MRI of animals injected with TVX18 on the left and on the right, and I can see, I think you can see by eye that you have a synchronous contraction on the left, and on the right, it's not beating, so you cannot see anything, actually. So let me see if it's going to play or not. Well, it's not going to work. So the pool data showed that the ejection fraction was increasing in TVX18 compared to control, and this was primarily due to changes in the end-systolic volume by MRI. And also, when we measured the synchrony by septolateral difference with MRI, we showed that there was septolateral differences that were improved with TVX18. When we did high-definition mapping using the RITMIS system, we saw that the TVX18 activation pattern was faster, and it was activation conduction through KISS-purkinje and activation of both ventricles simultaneously coming from the injection site, as opposed to the IRB case, that as expected, you have the wavefront coming from the injection site, activate the right ventricle, and then transeptally, a delay activation of the left ventricle. Additionally, we checked for the ECC duration and the QRS duration, and we saw that those animals with TVX18, the native QRS was narrower compared to those that they were IRB-based suggestive of electrical remodeling. On the right, you can see that histology show increased fibrosis, changes in connexin 4 and 3, and compensatory hypertrophy in those animals that they were IRB-based, as opposed to the TVX18. So can we do this clinically? Well, in my mind, we need to answer three important questions. Number one is, which is the biological agent that we're going to use from all the ones that we review? Well, I think a cell-based approach is certainly attractive, but I think it has, like, some difficulties with the cell maturation and engraftment that they're probably difficult to overcome. I don't think a functional engineer approach with a single-end channel is going to be able to recapitulate all the features that we need. And I think that somatic reprogramming probably, you know, it's an approach that so far seems to be the most promising one. In the delivery system, I think no one will want open chest of left-sided intra-arterial. I think a right-sided venous approach, it's sufficient and less invasive than the other ones that I described. And I think the patient population is important. I think the device-related infections is a good start to have a temporary biopacer to those conditions. Pacing-induced cardiomyopathy is also an important population that could benefit from this, but we will need a long-term persistent biopacing and not, like, you know, brief duration. So to conclude, I think I showed you that TBX18 pacemaker improved the heart rate and decreased the backup pacemaker utilization and improved the activity in animals. And when we delivered that to the Hispanic region, we were able to both prevent and rescue pace-induced cardiomyopathy. And I think a heart-work-free alternative may be desirable for selected patients. So future and ongoing directions, we have a clinical gray vector, and we are studying efficacy and bio-distribution and toxicology. We did a pre-IND with the FDA, and we are moving forward to follow the IND when we are ready with the toxicology experiment to design the phase one trial with efficacy and safety of biological pacemaker. So I'd like to thank the people who did the work. I'd like to thank Dr. Shane Dawkins and Yufeng Primanti, Eduardo Marvan, Heechul Cho, who is current, and Emery, and did a lot of the work that I showed up here, and the funding agencies. And thank you very much, and I'm happy to take questions. Thanks, that was exciting. Is it clear which population of somatic cells are reprogrammed, and is it potentially reprogramming multiple cell types, including myocytes, fibroblasts, and the like? It is not, I have not looked at directly, but I think you can, you cannot reprogram fibroblasts into sinoatrial null cells. Whether there's changes in other cells to a different morphology, it's possible, but I don't have the data. So the one that we, the data that I showed is with an adenovirus serotype 5, okay? And I think for the infected population, that vector could give you, you know, a good two to three weeks of pacing, and then, as you know, fade away. So that will be the initial one. If we are thinking about chronic, we are exploring other options, including modified RNA to try to do permanent reprogramming. But the initial vector, and the one that we are closer, is adenovirus. Yes? Thank you so much for the beautiful talk. I'm wondering, what is the optimum number of cells that you need to reprogram for long-term sustainment of the pacemaking activities? Yeah, so that's a good question. I don't have the number, and as you know, in gene therapy with adenovirus, those response studies are difficult to perform. We know that in PIX, at least, delivering into Hispokinesia, the efficacy, it's a little better than if we do in the free wall of the ventricle. We are not sure why is that, whether it's because of the insulation of the Hispokinesia system, or because of, like the question that was raised before, some cells are more easy to reprogram than others, but I don't have the exact number of critical mass of cells that we would need. Yeah. Thank you.

stem cell therapy

biological pacemakers

electronic pacemakers

arrhythmias

somatic reprogramming

TBX18 transcription factor