Good morning, everyone Welcome to Cardiology Grand rounds. I'm so pleased to introduce doctor Sylvia Priore, who will be speaking to us about gene therapy for the heart challenges and Opportunities.
Doctor Priore completed medical school at the University of Milan, where she also received an addition to the MD. PH D in cardiac pathophysiology. She's currently a professor of Cardiology as well as molecular. Madison at the University of Povia. In Italy, Dutch Prairie is well recognized in the field of inherited arrhythmia disorders. Her research spans from bench work looking at mechanisms of disease to clinical research with over 440 original publications doctor prayer was the first person to identify the rianna gene.
Receptor mutation as an etiology of CPVT. She served as the president of the European hardware them Association as well as multiple other boards and editorial positions in his one countless awards for her work. Please join me in welcoming doctor, Priore for grand rounds.
Thank you very much and good morning. Everybody so the title of this talk a gene therapy for the heart challenges and opportunity is actually something that there will be reporting. What is an in between topic parter? Is a research project and party is a dream of myself and especially of my group and my group would be acknowledge as we go on because without their help and they're fantastic.
Um willingness to find solutions for our patient, we would not be where we are trying to move forward. These are my disclosures.
And so let's see what that what is gene therapy gene therapy has been a dream for many re investigators an actually started in a very negative way in the 90s where attempts to develop gene therapy resulted in premature death for terrible immunological adverse reaction to a dental virus is that where the vectors used to deliver the nucleic acids that were supposed to be the therapeutic agents, so actually the key concept about gene therapy.
Is that of modifying or interfering with the consequences of alteration that are present in the DNA or modifying the expression of certain proteins that may result accurate if or at least at therapeutically effective in the pathophysiology of diseases that are not primary genetic diseases and I will show some some example of this so in cancer an in genetic diseases is where a lot has been advanced in this field.
And I think that today, we are at a very interesting time that is called the Renaissance of gene therapy thanks to some advancement that we have made in the delivery of the nucleic acids that are used an this renaissance can be summarized in the large number of trials of gene therapy that are going on. If you start following these literature. You will see that almost every week. There are reports of new therapies that have been.
Tested especially in the field as you see here of cancer disease is an monogenic diseases.
And so I think that the is from this field, where we have more results. But definitely there is a long list of other conditions and I would point to ocular diseases that are becoming really another new frontier because the retina diseases can be treated in a very advantages way because they represent a very small localized environment where you can reach the needed concentration for gene therapy to be affective an the Emma Teologi Field.
With the the treatment gene therapy treatment for him. Ophelia is one of the other field that we have a major impact in a very short time because the trial is already pretty advanced neurological diseases also represents a frontier that is being aggressively developed in at this time with a couple of trials going on with very interesting preliminary results and Karyology Nino that Unfortunately we are lagging.
Very much behind for several reasons that are always the one that complicate research in the heart is not a very accessible organ.
The cells are terminally differentiated and of course, you cannot get samples of cells to see what the infection is doing and how they gene therapy is working so for all these 3 reasons. I think that the in our field is more complicated to work and is more complicated to really make rapid advantages so as I said the vectors are the most important challenge in the gene therapy as they were the challenge at the beginning.
So uhm the addendum associated viruses represent now. The Frontier, an other ask the other possible carrier of the DNA of been explored, but none of them of all these other alternative and especially polymers and liposomes that have been attempted you know seems to be good for a place like the heart in particular, so why the virus is an which viruses.
So the the introduction of the iden associated viruses's been something that they've changed the game mainly because these viruses are viruses to which we are various post and this is on one side an advantage on the other side and negative aspects. Negative aspects because being exposed to these viruses 50% of the population as antibodies against this so you're trying to one side to give use them as therapy and on the other side you have antibodies that will attenuate the effect.
And this drives you to go with higher dosages with potential off target effects. But the positive thing about The Associated Virus Is It. They do not elicit at variance with the addendum viruses. This immune response. They were the cause of the problems in the task.
Uh the major limitation of the addon associated viruses is that the amount of DNA that you can put in is limited to 6 case. So let's say you want to put the C DNA of a gene that is a big gene. Let's say their eye on it in Receptor. It would never fit in the capsid of the of the virus. Even if one thing that is done in these viruses is to remove their nucleic acids that control their replication so basically.
We use the virus as a biological syringe, taking advantage of the property of got game going inside the cells, reaching the nucleus and injecting something and that's something we want to be our therapy and not the DNA or RNA of the virus. So the easiest approach for gene therapy. Easiest not be cause. It's easy to do it and easy to be successful, but easy becauses, a logical concept is you think.
Of a clinical condition, you acquired or genetics and then you know the path of Physiology and you say in this disease. This protein is reduced as a consequence of the pathologic process or as the initiation of the process. And if we could replace tinatin amount that is physiologically could be curity or you have a genetic mutation that causes a loss of function so you have either less while type protein or you really.
Protein that is not able to assemble or to go where he should go because of the Mutation and then you use this strategy that is called Gene replacement because what you do before manipulating and put in things in your virus you create what we call it a construct where you have several aspects that make it work in be transcribed in the in the cells like for example, the promoter and then your therapeutic aspect that can be the C DNA of the gene of interest for the protein that you want to replace.
So for example, in in our case wanted to fix the problem over the recessive CPDT in which call sequestering is the mutant gene. We know that there is a loss of function so pulse. Equestrian is dramatically reduced, and then you can think about putting call sequestering here and then put in all these concert in your then associated virus and then delivering it to the mouse model in the future, hopefully to the patient.
And hope that this DNA will be inserted in the nucleus translated transcribed and become aerial protein that is functioning fixing the genetic defect. Obviously all these steps should not be taken for granted. Sometimes you have a nice construct an is not working is not infecting adequately the cells. So the concept might be straightforward, but not the better response of this cells, not necessarily straightforward one.
Full aspects of the addendum associated viruses is that the scientific community is done in an enormous amount of work to try to identify viruses that were maybe not infecting humans, but infecting animals or plans or so that showed a drop is for specific tissues. So all these work of studying different serotypes of a den associated viruses, meaning viruses with different protein in the cops that.
Correct their eyes? What is called the serotype led to this classification of viruses that preferential in fact deliver preferential in fact, the muscles preferential in fact, the heart and so you will see that most of the research in the heart is done using a den associated virus 9:00 and then associated virus 8:00 or 6:00 and this is Becaus. They tend to preferentially go to the heart rather than 2 lungs or testes ovaries brain or other.
Issue where we don't really want to express this proteins.
So we have done a sort of ABC in in understanding of the of the gene therapy. An I want to immediately to show where the best result or the best attempt has been done in the gene therapy for the heart that is. This trial called Cupid and we have added Cupid One Cupid 1A clip in one B and Cupid two and this is the Cupid 2 results. So I am saying something contradictory pivotal contribution or cupid too.
And then I'm also telling you that cupid, too was a negative trial. The idea behind the cupid, too was very ambitious an what the authors wanted to do this work that comes from Mount Sinai. Roger Rogers Group. They started from the evidence that in heart failure. There is a decrease in the circle protein protein that regulates the amount of calcium in the circle. Plasmic reticulum up takes a calcium from the cytosol to the circle plasmic reticulum.
And he thought that correcting this lack of circa could be curatives or therapeutically useful for heart failure patients so they started infecting with they did a lot of preclinical of course in pics models and so on, and then they got approved for a clinical trial. So the first two pilots were positive and they reported an increase in the ejection fraction in the patients after the treatment. But Unfortunately the real trial. You know when they really.
Randomize the patient and so did not show any benefit. However, we still consider these very important contribution because obviously what is the contribution is not about the efficacy or not efficacy of the concept but it has been to set a level of safety for the gym T rapid delivery to the heart and these of course, is a first step that is needed so we're very happy that atrial's been done.
Without reporting any signals of side effects and adverse event in the population is this is the step that then will facilitate introduction of the other gene therapies for the heart in the future. So now we can say that there has been more than other than 60 patients with the first two pilot trials and bigger trials that has been done it was negative with in every one.
Circuit weight so now the A V1.
By Monster investigators were working for gene therapy in the heart is consider it an old virus and most of the people would move either to the aviatour AV 9, but still the idea is that we have a level of infection. An also they showed that it was advantages to deliver these through the coronary arteries rather than injecting Ivy so that is another concept that we have learned from the Q P2.
So obviously our dream would be to cure or approximate cure of long QT syndrome. Brugada syndrome see Pvt. With replacing the function of the jeans an in CPVT or in any other of these conditions. We have either gain of function mutations, meaning mutations in which there is an increase in the function and therefore there is too much sodium current too much potassium current.
With the expected consequences at the level of modification of the action potential or there are lots of function. There goes the opposite way gain and loss of function so how it conceptually you could approach these 2 conditions is pretty clear for loss of function. You can do a gene replacement if the gene is small enough that can fit in the?
India den associated virus an for the gain of function. You can use the RNA interference strategy and try to down regulate. Not all the protein, but specifically the proteins. The mutant proteins. So create an RNA molecules that is specific only for the mutant protein that is present in the patient very difficult and challenging because of course in most of the mutation that we have in this condition, the gain of function results for a change of a single amino acid so.
Having something that is highly selective for a protein that this difference of only one amino acid is quite challenging but this is the concept behind our idea or trying to develop therapy for this condition, so first, the counts. The question was can, we use gene therapy to treat inherited Eritrea's is there a probability that this would work and then the 2nd question is from where what we need to get started so.
The 1st question has been very strong, limiting factors because for a couple of years we have tried to apply to different grant agencies. An we were invariably rejected with the statement that no way the gene therapy would work to treat arrhythmias. Becaus the rationale beyond as stable heart reason is that all the cells are electrophysiological uniform.
And therefore it would not work, then the other. The other concept is when you can test. It gene therapy and obviously you can test. It only when you have a good animal model with multiple readouts and for readouts. You mean end points that you want to see normalized by your therapy. And Unfortunately in all these conditions. We don't have a lot of animal models because eventually you can test them in cells, but eventually you need to go to an animal model.
Or long cutie syndrome, we don't have a good mouse model. We don't have. We have some rabbits, but they are not knocking their transgenic so it's not the same thing. You cannot test you in therapy there. We tried at NYU to make a pig with Brugada syndrome. When we work with Glen Fishman on this project an the pig with a sodium channel. Mutation pathogenic for Brugata in the patients did not develop Ruger that develop conduction disorder, so that was another obstacle and so I think that.
In CPVT, we're very lucky because we have animals models that fit the need of being similar as a favorite Mick. Fennell type at as many other feature as well with what we think is the clinical condition. So we decided to go to treat this disease. So just a couple of words on CPBT is a disease that was considered an abnormal form a strange form of long QT syndrome with normal or borderline cutie because the patients have exercise related arrhythmias.
There it means at variance with long cutie or not or sad, but are this kind of be directionality that can be stable for seconds and then suddenly in one beat degenerates into VF this is one of our patients.
An the outcome of these patients is pretty severe so in an untreated conditions. This patients become you know symptomatic now. 80% of them by 4 age 40, so very early on, and you see how steep and the slope is not changing much overtime patients are currently treated that predominantly with beta blockers with the high recurrence rate. As you see here. Our data in the Japanese group data pretty similar within showing lot of recurrences.
Even with treatment of full doors of an add a lan part could be compliance part could be really no response. Now there is flecken eyed as an additional therapy, but still, we know that also with Flecken Eyed. We have recurrences, not at this rate, but we may have and Furthermore what we know is that the CPVT patients respond less well to the defibrillator and a lot of a lot. Some definitely more than one group reported shocks.
Inappropriate I mean, appropriate shocks that were ineffective in terminating the CPVT that exceeds the success that is usually present in terminating other types of greatness so let's start from the recessive formal CPVT that I anticipate it could be the low hanging fruit in the sense that we could just just try to replace the missing prote A little bit of Arctic architecture of the area of the heart in the structure that we're working on.
This is a teachable's the imagination of the membrane of a cardiac myocytes in the teachables in this part. There are aligned. The calcium channels in front at the 20 nanometer distance from the teachables is the junctional Sr and the junctional. Hazare is the home of the reality. Receptor and the cardiac Elsa question so the Physiology goes then, when the action potential gets the depolarized potential where the channels.
The calcium channel can open the calcium channel open and a little bit of calcium. Not much meaning that this is not the calcium that activates contraction is a signaling calcium reaches the reality in receptor that is very regularly aligned to maximize the opening of the channel that these vestibular of the channel to get as much calcium as possible to signal that it's time to release calcium from the junction alissar so from this area.
Of the Sr and these half moons in pink is Casa question that is uh composed. This protein composer polymer and the polymer is a buffer for costume. This is the traditional concept cancel question is a buffering molecule, but now we know thanks to the work of Wayne Chan and others that in Thunder Gorky as well that this protein is also working as a modulator of the function of the Rye or knitting receptors. So when we in is a stabalizer of the reality receptive.
So when we have a loss of Couse Equestrian. We're losing, something that stabilize the closing states of their own. Iti receptor so pray dispose into the opening so this is the reason why 'cause the question cause is the same thing that Mutation in the reality in receptor that make the channel prone to opens in diastole are doing because is stabilized. So when we lower the Castle question our cells become prone to developer retinas, so here is.
Homemade cartoon that intend to show you you know what happens this is the Teachables. It read the calcium channel. These are the reality and this is the junction alissar so calcium comes in through the calcium channel triggers release from the Sr then here is a circle. The protein that is been singing our failure that Raja Raja was trying to increase in the Q P2 so circle brings this back to allow this release and re uptake.
And then here we start having too much calcium that is released when there is a mutation in the costume is not released only during systole, but is released also during diastole, So what happened is that these excess of calcium get extruded from the Sr with a stick geometry that one costume with 2 positive charges goes out and sodium with 3 positive charges is exchanged and therefore generates positive current that depolarized the resting membrane potential and if.
This is a strong amount of the polarization. It will trigger the beats so all this story to explain to you. The molecular mechanism of the originals in the CPBT so now we think that if we would be able to stabilize their eye on it in Receptor by put him back. The Casa question that is missing in the negative. The Recessive form, then we would be able to interrupt this circle. Similarly, for the dominant form if we could.
Silence the muton channels that open during diastole inappropriately we might be able to cure this disease. And here you see when you record from the mice with Pvt that you see exactly this kind of phenomena. So this hump is like this one and these bits at the end of pacing that the Red Arrows are the pacing you see that these are triggered beats so this is triggered activity.
So here is the concert that I showed you before, and here instead of DNA of your choice. We have the DNA. The represent the sequence of the Kelsey question. This is Marco Denegri, who did a lot of work and made this project successful publishing a couple of papers that were our first attempt to develop the gene therapy. Then you take the virus. You injected in a human actually we were injecting in mice. This stage it will go in the nucleus.
Use the protein to rescue the fennel type, hopefully so I said that we had different readouts. So one of course, the most important one would be to see if there might stop having the be directional. Vt that they develop when we stimulate with catecholamine. These mutant mice, but another interesting. One is that there is a mysterious phenomena mysterious because we don't really know how it happens. Now we are studying these and we're getting more sense of it, but that premature to talk about that.
When there is a mutation in the Casa question the amount of Casa question is reduced probably because these cannot assemble the well and therefore is illuminated by the cells in the cells because it's just not a physiologic protein able to do the job. But then other proteins are also diminished jumped in and try adding so-called sequestering jumping and try adding are the triad of protein that works and regulates the director needing receptors. So when I said the concepts in modulate the function of reality in Receptor.
Is actually in partnership with these other two protein and there's Tacoma trees always aligned so if this one goes down all the other go down but also, if I reduce try. Adding all the protein will go down in the same way. So obviously the objective now is to see that when we put back the cow say question, we will abolish the original clear, but we will also increase the cost requesting an.
Change this and here you see that actually when we infected them eyes. We were able to rescue call sequestering that goes back to the level of Walt iPad also jumped in and try adding and here you see also at the western blood level that we're able, you know if this is the normal level. When we put back Tulsa question. You know, we go back to values that are close to the to the normal for the different proteins.
And other aspects that is important for fixing all the problems created by the lack of Tulsa question is that when we have a mute on animals. They instead of having spontaneous calcium waves that may occur rarely in the wild type what we see is that we get a lot of Sparks that do not really did organize the in becoming waves, but they can predisposed to delayed after the polarization. Zander it neos anyhow so.
We see that when we put back the Casa question, we normalize the behavior and these are 2 different experiments. So we are active not only at the level of the proteins level, but the consequences of the protein level in the Castle Physiology. And here is what we really want to see that when we inject these protein in our minds with the directional. VT's after epinephrine. We do not see arrhythmias anymore. If we put back the calci question so The Cask.
R33Q almost Tigers are infected as opposed to the Cal sequestering almost I guess not being infected and having the original and here you see the dramatic effect that is really, really powerful.
So now.
The question that is interesting is since we have had all these scientists telling us know. You cannot be funded because you need to have a uniform electricity with makes sense. But now why is going to be affective so actually we started discussing this with the group of Jim Wise, who had for many years studied triggered activity at the fundamental level than what they demonstrated in silico very interesting experiment.
If the fact that.
I show you this waves this depolarisation that are called delayed after the polarization's. But since the cells are all coupled Adele aid after the polarization by itself is unlikely to propagate becaus the negative resting potential of the adjacent cells will not allow the propagation of a positive currently is so insignificant as compared to the driving force to staying stable of the neighboring cells So what it really, really takes.
To propagate is that many cells are coupled to each other and they oscillating synchrony, so that the delayed after the polarization developing multiple cells at the same time, so here they show that in silico if they have 79 cells that are coupled and oscillate in center in synchrony is not enough to propagate this. But if you have 80 cells is the threshold for the experiment that they were doing to propagate.
So basically this model before we explored in our in ourselves was a demonstration that block the IDs. You just need to uncouple some of the cell that stop oscillating because they've been rescued in terms of the normal Physiology and then you still have some cell oscillating, but this will not propagate and therefore will not create a richness in that issue so this when the reviewer asked explain us? What is the mechanism we gave this mechanism?
An now is this is considered the reason why maybe triggered activity is particularly susceptible to gene therapy interestingly triggered activity mediated by the ad is also considered the prevailing mechanisms for original Genesis in our failure and this creates a nice loop with the attempt of Roger where maybe circle is not there, I way to replace an 8:00 and prevent their it means in heart failure or even worse is not the right 12.
Prove a contractor function, but in terms of everything going to an idea that probably targeting triggered activities. A good objective for gene therapy in cardiac arrhythmias is true. Now we're trying to study a new model that we have developed for seen whether these holes through also in long. QT syndrome where the mechanism of arrhythmias is bit more debated so now if we have a favorable mechanism in CPV T4.
Target in it with gene therapy? How can we do with dominant CPVT with the RNA interference that I mentioned to you so this is the cartoon just to show you that what you can do is to identify this molecule that will be able to lead to degradation. The M RNA that is the target so in our case is to promote the degradation of the M, RNA for the mutant ryanodine receptor that is present in the patient so before going to our objectives. I just want to acknowledge the work of others who have attempted in other models also to do our any interfere.
Is for arrhythmias this is a interesting? I I will be showing data from an IP S cardiac myocytes of long QT syndrome. Type 2 patients. Of course, is not just studying bit in vivo is in vitro, but very interesting to show the model So what they did. They first group first started the Act 293 as pressing I heard Mutation and showing the ability of leading to degradation of the mutant RNA an.
Trying to reduce the effect that on the abnormal current property that were present in the mutant heard her give Gene and then in the induced pluripotent stem cells where you can get a bit more hints of the final type and in particular, you have an action potential that you can record an if you get these cells from Al Cutie 2 patients like they did it in this work. You see prolongation and even development of an early after the polarization up here plus.
The reduction of the trafficking of the IKR channel so the channel is not there to activate the repolarizing current and potential start oscillating and this is one of their parties on there is magentic mechanism in long cutie syndrome. So when they did. The allele specific silencing they were able to reduce the transcript. With the mutation repressed innate. The traffic in of the protein an shorten the action potential in ventricular sell it your cells and pacemaker cells.
So this was a good proof of counts have that, if you work hard to develop your RNA that is specific only for the mutant you can have a final type at the level of the action. Potential Christine Seidemann attempted these inv evil in a model of Hypertrophic Cardiomyopathy. This is the common mutation that is present in this process. Kamerik protein and the key aspect is that with this relatively standard. Those now for doing gene therapy in the minds.
They were able to have the success of inhibiting these mutation get into the reduction of the mutant transcript and have some changes that were very positive. Indeed, hypertrophic cardiomyopathy that was here and then you know decreased an almost normalized the size of the of the benefit the problem with these experiments is that Unfortunately after 9 months. The M RNA molecule that they had produced.
Was silent in the mutant transcript stopped working an this was something that has been anticipated as a problem for the use of RNA interference as a strategy for for gene therapy. So when we started testing in developing our molecule. We were encouraged by in vitro data a little bit discouraged by these short-term efficacy that was shown in this model, but also encouraged by the fact that they got.
A result in the entire heart. So when we started and Rosanna in the lab was the person who spent one year in trying to identify these RNA that was able to be selective, so she made a big screening using a car sector, with the excellence of the of the reality and Receptor That Harbor, the mutation winters that is this mutation that was the first one, that we identify when we discover at this gene and so she tried to get something that's selectively will block.
This one without interfering with the wild type and discuss sets were tagged with different fluorescent proteins. So that we could really see when the one color was going down because we were silencing and not producing that that protein anymore or not so this is the experiments in neonatal mice infected and to go to the bottom line. You know, and avoiding all the other factor that I've shown to you in terms of what we can get as other.
End points in the in this in these models. This also was very, very, very effective as you see here in reducing that the mutant protein and therefore represe nating. The resistance to exposure twice approach here and also the animals did not develop the The Bidirectionality. So what we observed is that what happened was not totale silence in the mutant but changing the ratio between the wild type an the mutant protein that is still there.
But there is less so why is this ratio so important an uh? I don't know if this would be equally important in other proteins that are not. I own channels, but foreign I own channel that is a tetrameric structure. Like the reality in Receptor. This is actually something that is likely to be very important so if you think of Annette arose iagos condition where we have normal and abnormal protein your ion channels that are in the junction ellasar.
Will be a mixture of a binomial distribution of channels with 4 normal and channels with for abnormal subunits. So you are the mix. Normal protein bad protein and it distributes with a binomial way. This is the theory for all the tetrameric ion channel. So you have few channels that are completely good and few that are completely bad, then you have some that are.
A bit more that are with 3 wild type and one sick and the opposite 3 second one wild type and the majority are 5050.
If you change their amount of the protein and is no longer the 5050 that you normally have? What you see here. The bad stuff. That is the yellow and the purple gets less than you have here.
And the good one increases as you see the blue that goes from here to here after the infection. So the more you're able to push the ratio between the mutant and the wild type in favor of the wild type. You have an amplification of the effect because you actually modify the number of healthy channels that you have in the membrane and that you know is probably the reason why you don't need to 0. The mutant protein, but is enough that you change the ratio to affect electrically in a favorable way.
The cocktail that is affected so now Sippy VTS become and I'm very happy about that. You know the preferred model to study many different other gene therapy. So this is a study by Thunder variance who took another approach so you hear about gene editing and gene editing is now the best therapy that everybody's been dreaming because rather than using the indirect approach is that we have selected as press more one protein or decrease the expression of a protein.
With the gene editing the idea is that we could be able to remove by a model with recombination. The mutant segment of the Jinan put back a copy of the normal one that we are also in Co injecting the big, big problem is that the heart being a tough stuff for tough people. That's why we are working on this on the heart cannot be amended mended using the gene editing.
Because for gene editing to work you need cells that are in the replicating things their hearts is terminally differentiated. So no way that we can use it. But so in fact, there are a lot of experiments, saying we have developed a good therapy of gene editing for the heart. But Unfortunately is done in. I PS cells that are replicating so you could envision doing that in an embryo, but not in Neo Nate or adult individual.
So was under is done, however, has been to use something that happens as a side effects of gene editing that you can use to your advantage So what this results is that there is a random insertion insertion, not to tell you random random near the mutations of the cassette that you prepared and these instead of being analogous recombination that take out a session and put a session back aligned so that the frame of the DNA does not change.
This create a frameshift is a damage an results in an allele silencing through creating a stop codon basically in the in the mutant protein and not in the wild have so is another way of doing what we have done that may be risky because you don't control 100% this, but conceptually is a very clean experiment that shows that no matter how you reduce the mutant allele. Even with another strategy you end up having the same result.
Alpha protection of the heart from Derek news as you can see here that they should results very similar to ours in terms of the effectiveness of reducing the mutant allele expression an very, very recently, a couple of weeks ago. Another gene therapy strategies for CPVT emerge and I'm very happy about this because targeting calmodulin kinase. Two that is one of the forceful relating enzymes so that our first relating the reality in Receptor together with PK.
And if you follow this feel that there's been like 15 years of fight home. Weather is more important PK or weather is more important companies. So it's like a soccer game. Whether you are from Milan or Inter soccer team. My soccer team is complaining so I think that sometimes, this is very, very important. I love complainers and with a fantastic Chinese fellow work form with me for 8 years.
We demonstrated that actually if you use a complainers In addition, with K and 93 and we tried. Many other inhibitors you're really, really able to abolish all these triggered arrhythmias and delayed after the polarization. You get only you know, these things that are so small that they do not trigger beats and so we propose in 2011 that target income kindness would be a good approach and again. We got a lot of treaties in that is impossible to target companies because this ubiquitous.
But now come Chinese is seen at the level of farm a lot of interest and I'm aware at least 3 companies that are developing cardioselective or brain selective tomki nizhny bitter so this might be at the pharmacological level. A new trend. But someone got a very bright idea. There was 2 S Presser, an inhibitory peptide and this is the paper that just came out that is very fascinating an over express that they come Tyner inhibitor by gene therapy.
Anne what you see here is that they demonstrated that if you take a wild type and you give this peptide nothing happened. No arrhythmias here and no arrhythmias after you inject this. But if you take the mice with a mutation an you express the peptide that Inhibits companies is very effective, so this is strong point that supports you know the factor that here is another evidence of the Vt that develops before you inject only GSP in the a dental virus or?
If you inject GFP tags inhibitor peptide for campiness so whether the field will go in inhibiting companies with gene therapy or with molecules is unknown, probably if molecules are very selective. It will be easier to test molecules, but definitely you know this is another very important proof of concept.
And now I just want to close with something that is at the same time, fantastic ING scary. An just to remind you the ethical issues that come with working in manipulation of cells and manipulation of human beings at the end, that when this will be translated to the cleaning so this is just the schematics for the gene editing. You know, saying that yes, you can put back a normal sequence where you have a mutant sequence if the cell C replicating.
So for many years, this issue of in no replicating tissues. The brain the heart. Maybe this should be done in embryos has been said.
But not done.
And obviously I think that the 2:00 concepts are important first manipulation of embryo is sensitive for obvious reason, but among the obvious reason of ethics of changing the embryo.
Is also that well if I in fact an adult or a child? I'm changing something but when the child as a baby the baby will not receive.
They changed the material if you in fact, an embryo. This will go to all the cells and will be also in the reproductive cells. So will be transmitted to other generations. So potentially is a dramatic technology and so the field has been discussing this forever.
And one day we opened science and is there someone has done it and someone is publish it and who is has been absolutely shocking to the field that science decided to publish and that someone decided to do. The experiment not the most friendly face. I would say for guy came out very badly in this picture has been going on around the world. I don't know if on purpose because of the discomfort of these aspects oh.
There has been enormous amount of discussion. Weather was good to publish and the argument of science was we have the technology someone was going to do it, it has been done.
And we have to report this, but it also generated some positive reaction on the scientific community, calling for a auto imposed so as scientists. We want to regulate and we want to limit the manipulation of the embryo. The thing that is very striking about this is that I would have thought someone will do it to cure a terrible disease. You know, and there are so many terrible diseases in the world that.
I would like to think that if there is a a muscular dystrophy that puts people in a wheelchair or SM ES that is a disease neurological disease that kills infants and by the age of 6 month or so.
I would understand the pressure for that, but actually this.
In utero that was not done in Utoro, but opens to the in utero gene therapy. This was done using embryo that were not used for in vitro fertilization and after they did. The gene therapy. That was it. You know they didn't put it in neutral, but is actually opening to the next step. That is applying these since he was successfully neutral but look at what they did it. You know they accepted this therapy to treat and Hypertrophic Cardiomyopathy, which I think you know it's a.
It's a breathtaking concept because we know that Hypertrophic Cardiomyopathy's cardiologist is a severe disease can lead to death can lead to that is not a disease that would kill beside very rare mutation or double mutational cases. So we have not been my first peak to test this one. So we have also to think you know that when we're thinking about gene therapy in even in my perception is.
Something that is far away and then you know the technology is going so fast that this is really coming so in response to this paper, the community that people more active in the gene therapy for dramatic diseases as posed moratoria that was published in science. After this was published whereby for the next 5 years. There is a commitment of the scientific group to try to limit limit.
The experiments on embryos but I think that is very important that a scientist. We are all aware that this is happening. Ann is all the advancement in technology is up to us to make it a good thing or a bad thing. Unfortunately, we know that very often when technologies available. Most of the people will use it for good, but someone may have bad thought and use it for bed uses that we can, we can imagine and we can think. So where are we moving forward?
Gene therapy has been proven safe in different fields and also in the heart maybe even if we have only one trials. Our trials for other assessive kalsa question has been approved by FDA Ann. I just need to get the courage to do it and the money by the way but that is a detail. I'm more concerned about doing it in a patient even if I have patience really want to do it, but you know it's a tough choice if they say. No, you cannot do it, you can say complaining.
Yeah, I would have done it, but you know, but now it's up to us so it's very.
It's very difficult to think of what to do, and you know that the technology is much far advanced is running sorry was nature on our side is the technology is going really, really fast ahead of us and they had what we are ready, emotionally and psychological to take but it's good because eventually you know, we will have a step that is. Maybe not so far away to think that we could eradicate this disease is from the planet because if you think about gene therapy in utero.
It means eradicating these diseases, so this is a fantastic step. But we have to go slowly and carefully and keep ethical concern. A and ethical standards. Very high and just not to say where we are used. Now we can manipulate whatever we want. Thank you very much for your attention.
Thank you.
Recently, about uniformity of transfection and have you incorporated things like reporter probes in particular imageable reporter probe into your technology in vivo no?
Probably just for you know the lack of real need to track track. The distribution in the nice because they might have a small heart is so things look pretty uniform. At least when you do RNA by sampling different by office. But definitely this could be a very important point our that we're moving to test a gene therapy in large mammals hard. So it would be interesting to know more of what you have because one problem.
Is a GSP dazed?
No.
So OK.
OK, that that could be a good approach because actually one of the problem is that the GFP for example, in the peaks is very toxic and gives seizures to them when it goes also to the brain that is part of where you get so I might be interesting to talk to you at the end and and learn more what you have.
At this pain lines.
Necessarily just related to iron channels and sometimes but it has to do with fibrosis.
2.
Yeah.
There is an why I'm starting from the channelopathies is that these are conditions in which from what we know from patients study. MRI study in the patients and so they don't have fibros is associated so they have a normal heart and is really pure electrical abnormality. I think that already does step up the hypertrophic cardiomyopathy may be different OK in embryo because then you prevent from the beginning, but once the diseases developed the fibrose is I think that it would be very.
Very difficult to simply modify a protein and revert what has already occured so I wonder that people who are working in the in the like. The side man in the gene therapy for the cardiomyopathies you know how they think to address that whether they are thinking of neonatal diagnose is an and treatment. At that stage because few years, 10 years of age 15 years of age may already start having this and you know the problem that we have with the gene therapy that.
From how we see it now if you have to do one shot. One shot, meaning one protein one target. It's OK. But if you need to say OK. I I change. The mutant but then I also give something else that is another gene therapy for preventing the fibrosis. Then it becomes something that probably is more country indication that then the drugs.
One of the Top Gun so my understanding is the episode. Although it can stay in a search for like couple months, but it's not life or lasting construct so changing editing another way. Maybe it's a better way because it created permanently change, so my first question is so how do you address the transient effect of LV the 2nd question is that for the email for the gene editing that create start column.
That will create couple insufficiency weather CPT patient will happen insufficiency may still have disease so all good points. So first of all. I think that the episomal problem is not a point of 4 months because there are now human data. They show that patients infected for gene therapy still have an active episomal localization. That is still producing the protein after 8 years. This is the highest that is being reported.
And I would feel very comfortable if my patients would have no side effects and then episomal that trust even 5 years because the real problem is if you have a 6 month and you have to re do you still have a high level antibodies and that would be really a problem you know because when you do the gene therapy. You elicit with the high dose if you have to give you an antibody response. So you cannot do if another therapy after 5 months. But if you have something that is affected for 5 years I think that this would be absolutely enough.
The gene editing I'm I'm a bit cautious with the crisper cast 9 because uh a crisper. Caroline has a lot of off target effects and so it might be beautiful. But you know the minute that the account through goes in the just step back. So we've been very careful in the field of gene therapy to try to avoid the viruses that insert the construct that you produce in regions of the chromosomes because you are never sure that is only.
The saved harbor that you are targeting it may be somewhere else. Maybe in a non coding region that down. The line would be cancer, inducing or things like that, so the school of thought that tries to avoid the insertion is also a bit sceptical of the effect of the crisper cast 9. O molecules recombination because you know it may act as a non amalgams recombination somewhere else sing start, creating stops of protein or just you know insertion of abnormal.
Sequences in there beside in the heart is not an issue because you can really not get we've tried so much. We started with all the possible technologies. You know from the same fingers 10 years ago. All the way to talent and now crisper cast 9 and we don't get more than 2% of the green fluorescent that suggest that there has been a molecules recombination, so I agree with you that test that.
Using it for non homologous recombination can be very risky because if there is enough target effect. You can truncate other proteins. So yeah, so I still think the nip is is is a safe approach.
So that's pioneering work and wonderful luxury.
Arnad delivered an at least in some advanced diseases. It looks favorable at this point, so I wondered if you could just come with.
That could perhaps it in December.
Yeah, the problem the nectar and a delivery It doesn't last for a long time, so you don't get to the point that you have a permanent cure and a permanent solution. The other point in that in our hand. Neither next delivery nucleate acid deliver nor the plasmid delivery in even the X is on delivery has been successful an I don't know.
Anyone that is being successfully in the heart.
To get that.
And I have no idea how long that would remain but clearly we need to get out of the virus. You know it's like the Ablation. We keep burning and saying it cannot be the final destination is a period and maybe the periods last a lot on a many years. You know in the in in science, but I am sure that we cannot continue with the viruses so.
There should be other way of synthetic virus like structure that can target.
Yeah, or chemical in modified? Yeah.
Yeah.
Where did you?
I'm sure you thought allies you stand and think about making this big step study.
What is the population of your thinking of launching is this?
In this field in China, where should the first steps, yeah, I think that the Recessive. Stupidity is a disease that is very, very rare being very, very rare. We know it means that is very, very little and these patients if they miss couple of those is they are really at high risk of sudden death more than the rayon it in Receptor patient that we already consider patients at high risk.
Uh a large trial would not be possible. This trial is anticipated with nine patients, as a pilot study. I think that there are patients. In this population that are good candidates for this because they don't respond to any therapy. I have a 25 years old woman who has had a lot of sharks and a lot of events and she's really calling as every six month and saying.
Is the trial starting is the trial starting because she just cannot leave knowing the sooner or later she will have another one, so there are patient that can be good candidate for that. Another group of patients are patients where the compliance is not good for example, there is a cluster of these patients in Israel, from bad win family, where they actually discovered the gene that keep marrying within the family keep not treating the children and so.
You know, maybe conditions that are extreme, but still you know, these people cannot follow a medical practice on in contact with clear. Gibson, who you know beside being a scientist was also doing gene therapy is heading. The Cardiology, where these patients are followed sort of say so. Maybe you know, some of these people with the stream conditions might be persons who may benefit of such a therapy for me is really important to have a proof of concept where we have good rationale for for scene.
Efficacy an also to test you know how much we should give to this patient and sound so as just because I'm also thinking that this is a optimization of a therapy that we still do not know how to handle.
I'm I've not started yet so I think that I would like to see that this therapy is going uniformly to a big heart and so to do some mice experiments, but consider that I'm struggling now because finally my studies are funded by European grant that as as a mission to find a high risk research. So we are in the high reso despite they gave us a lot of money.
Producing the virus that there is needed for one injection in the pig is more than 100,000 euros.
So it's not that you can really play a lot with these and say today. I test is one hunt tomorrow. I test his other one.
An so I think that may be an optimization or a possibility of assessing the distribution could be very helpful.
Thanks so much for a great life.
