 Okay. I think we are live and I wanted to begin the meeting. My name is Charles Venditti. I'm a senior investigator in the NHGRI. It's my pleasure to provide a few opening remarks this morning to talk about the genesis of our meeting and acknowledge the participants and talk a little bit about the logistics. I would first like to mention how this meeting evolved. As many of the audience and listeners know, the field of organic asademy is in homelessness and yours is enormous and vast. And this meeting actually derived from a plan meeting that we were going to have in person before COVID with the organic asademy association, the PAF and HCU. And during the course of the pandemic, this has obviously shifted to the virtual format and the agenda was therefore somewhat related to the patient meeting that we were planning over almost two years ago now. I also want to acknowledge all the organizers on the team here at NHGRI, particularly Dr. Irene Manoli, Jennifer Salon and Oleg Shilachov, who really have led this effort. And I want to also mention that all the speakers were very gracious to provide their acceptances nearly immediately upon invitation. And I want to also mention that we had 100% acceptance rate for all of our invitations. So I think there's a tremendous interest in this meeting and what we're going to talk about during the next two days. In terms of the logistics, what we have done with the help of the unbelievable NHGRI team of Gerald Somani, Alvaro Encina, and William Mays is to create a series of video archived talks, which we will then play and then have after the talks as availability of the speakers will permit a short Q&A session. There will be no formal introductions of anybody because on the website, there are extensive speaker bios. So I think I'm probably going to close my remarks in a minute here to just again thank everybody that's been involved with this meeting. And thank all of you for your time. I know people are joining from all around the world. We have almost 500 people that are signed up for the meeting. Again, the way the format of the meeting will work is the video will play. There will be a short Q&A session, and then it'll move to the next video. And then at the end of the day today we will have a larger panel discussion and you can look online to see the exact agenda and the timeline. So without any further ado, I would like to again thank you all on behalf of the NHGRI and on NCATS who's co-sponsoring this meeting with our team to a very exciting two days. Thank you. Good morning. My name is Charles Bindini and I'm the senior investigator and head of the organic acid research section. And I'm delighted to be able to speak today on genomic therapies for methamerotic and propionic acid. I have some disclosures related to research support from biopharmaceutical partners, particularly past partners, current partners, and there are a number of patents held by the NIH on behalf of our team related to some of the material which I will present today. I want to acknowledge my outstanding section. I'm delighted and honored to work with all these members every day. And together we have made great progress in the study of MMA and PA at the NIH. The names that are highlighted in blue are trainees of laboratory and clinical trainees, and we're particularly proud and honored by our trainees who have done great things and are going to some of them will speak during this symposium. Today I would like to speak about two main objectives. One is to understand the rationale for genomic therapies to treat MMA and PA. And this will involve a deep exploration of pathophysiology and a review of path of, excuse me, a proof of concept studies. I would then like to overview briefly the new genomic therapies that are in development and or in the clinic. This includes mRNA therapy, genome editing, and gene addition therapy. We have a few remarks about the MMA and PA syndromes. This will be covered extensively during this symposium. These disorders are a group of invoices and branch gene amino acid metabolism and vitamin D12 metabolism that were first described in the 1960s. The patients have absolutely massive accumulation and excretion of disease-related metabolites, including methameronic acid, two-methocetrate, and C3 species, an appropriate carnitine. In the United States, babies with these disorders are detected by newborn screening, and we believe the incidence approximates 1 in 50 to 1 in 80,000. The prognosis in general for these conditions is guarded, and this has motivated the search and assessment of new treatments for the patients. This slide is an overview of the pathway of branch gene amino acid and organic acid metabolism. It shows the main precursors of the compounds that enter into the mitochondrial matrix and eventually will be converted into propranoconzoma. This, in turn, shuttles through a series of reactions. We need it by the propranocoy carboxyloxanazone, which includes the PCC-A and B genes slash subunits. There's a racemization step. We need it by the epimerase reaction. You can see MMA being generated here by the action of the team-ethanomic cohydrolase. There is then a racemization step where the L-methyloxanazone cohydrolase is summarized into succyl-cohydrolase by the action of the mutate enzyme, M-mute here. This enzyme requires a denosocobalamin, which is the activated form of vitamin D12, and found in the mitochondrial matrix, which, in turn, depends on its formation through the action of a number of other enzymes, including the enzyme M-mab. The cherish on cobalamin B is the complementation class designation. M-mab deficiency and M-mute deficiency are severe forms of M-mab, most often are non-vitamin D12 response. The bottom half of the slide shows the remethylation of the pathway, providing the B12 metabolism, intersex, and participates in homocysteine remethylation. I will not discuss this. This is going to focus on the entire set talks tomorrow. The end result of this produces ATP, water, and CO2 through the prep cycle, and then energy. So this is sort of a very brief overview, a basic overview of the pathway. Some people I know are parents, but this is what we, when we talk about the enzymes and genes, this is where they fall in the pathway for mutate and tabules. Now, what happens to patients when they have M-mab and PA? There's recognizable clinical phenotypes associated with the disorder, and they're very, very, not every patient experiences every symptom assigned. For example, not all patients have an intellectual impairment. Not all patients were developed based on ganglion stroke. Many have a dependency to do so, and well, many have had strokes, but it is not a uniform feature to condition. Chronic renal therapy is something that we see in most patients with MMA as the age, and it's also being noted as a complication of propionic acidemia. Then you can see here the other symptoms and signs on our list. Note that in propionic acidemia, there's more of a dominant cardiac phenotype, and that's unknown why that happens, but also a targeted therapy. All the patients do share one thing, and that is a phenotype under duress of metabolic instability. And that is variable. Some patients are much more stable than others, and many times this can be associated with hyperamemia and encephalopathy and big doubt. And it is this current metabolic instability that drives the patients and our care providers to see treatments that can completely block this. And for this reason, transplantation has arisen. That's going to be the summary to the second talk. But this is something we want to block with all of our new therapies. I wanted to just make a few comments about early observations from the 1960s and 70s about the patients with MMA in PA, because they're still relevant. One is that these conditions as a crew were called ketonic hyperbolic anemones before they were recognized to have organic acids at the fundamental basis of their biochemistry. It is still unknown precisely why this occurs. What is the basis of this association? That will be a talk later today by Dr. Pence, our head. The early clinical research on MMA and propionic acidemia did know the features that we still see today in the patients, including the intermittent lethal instability, in the patient's right manifestations on many organ systems in the body, multiple pathways being affected and intermediate in tablets, and then in somewhat specific effects perhaps in some of the patients, which PA patients were noted early on to have movement problems, and there was kidney disease noted in the very first patient with MMA. Early clinical research also noted that the management of these disorders was very difficult with disease progression and death in some cases despite biochemical response to protein-restricted diets. And this is an important lesson that we should continue to remember as we think about how we will enable new treatments in the patients. It's telling us that we cannot simply rely on circulating metabolites. The patients in this study studies have biochemical responses and poor outcomes. So we have to think about this as we think about how are we going to determine whether or not our intervention is producing a clinically meaningful outcome. Can we just rely on metabolites? This is a good question for the Q&A. Early observations saying no. Okay, I'm going to go ahead and now talk about the enabling studies that came from mass models to get us to the treatments that are gene-based. I'm going to talk about a study we performed many years ago. We expressed the methamethanolcone mutase enzyme in yeast, and the reason this is done is because yeast can process the metabolites of mass and human enzymes, and then you can make active enzyme. You don't have to worry about folding. All you have to do then is add a dinosaur involvement in the dark and get active mutase enzymes. Super active. So people in biochemical oriented, this is a great system to do an enzymology. We used our enzyme to go ahead and make polyclonal antibodies. And then we were able to determine the expression of the mutase enzyme in the various tissues of the mice, and this is what we expected in some ways that this is expressed in the liver and the kidney and throughout the body. And again, this goes along with the fact that this pathway is probably active in many, many tissues in the body. And the question then becomes, what tissue do you have to correct to what level to see phenotypic benefit? Now, when we first made the knockout mice, and other groups have made knockout mice, and we all saw the same thing, just severe immediate lethality in the full knockout mice on the black six background. We learned a lot from these mice. They actually recapitulate the severe human phenotype and that biochemical parameters are very close to what are seen in very, very sick human patients early in life. They're difficult to study, because they almost always perish by day of life, by the first day of life. And when we hybridized the FBBN background onto these mice, we obtained rare survivor animals that were sporadic that could live to weaning. They had severe MMA that no enzyme activity, but they can live to weaning. And as soon as they were to mean to regular chow and experiencing stress, they died right away. These mice were very useful because from these mice, we learned a lot about the secondary mitochondria of the human. What I'm showing you here is an electron microscope picture from this mouse that died the next day after this photo was taken. And those are the liver mitochondria. They're massively swollen and distended. The matrix is pale. You can maybe make out the pristine. And then we saw these in the mice and the tubules, the proximal tubules in parts of the pancreas and in the liver, not in the heart and not in the skeletal muscle. Then the question became, is this what you see in patients? And the answer is yes. We're able to obtain liver samples at transplant from some of our patients, a precious donation that we are very appreciative of our patient population for donating these organs and studying them extensively and show they had the exact same phenotype biochemically morphological end of EM as mice did. This really established the secondary mitochondria as part of the pathophysiology of M&A. This is very important as we think about when we're fixing something in a patient, we need to fix this. So the next thing we did was to make transgenic mice doing an add back with germline transients. And the way this is done is by configuring a gene to express in a specific cell type and making a transgenic animal with that and crossing that to a carrier mouse. And then by breeding, producing mice that are knocked out at the methamethyl metal coagulatase locus with a rescue transfer. And you asked the question, was the phenotype was a rescue? So again, guided by patient observations by the way, this is the publisher. People want to read the paper on this. What you can see just in the mice, and here's a rare mouse that lived to day 32, it had some FEDN genes in it. You can see it's not that healthy. Convectin glutamate, which you can imagine a mouse that's either going to be dead or might look like this when it has the transgene, it looks like this. You almost can't tell the difference between the dead and the littermate. And when we looked at the survival phenotypes of these mice, indeed they survive. Here's an example of the mice that don't have a transgene, 100% death on day of life one to two. There's mice that have a germline transgene or a largely rescued and they look basically normal. They're slightly small. They have very high levels of metabolites in the blood, urine, and body for this. And one of the first things we did with these mice to ask the question, how much protection can the parasite mediated expression we taste confirmed is to challenge the mice. And the way you do this is you put them on a high protein diet. This is a 70% of rich casein diet. Here's an example of the heterozygous mouse showing you the electron microscope picture of its litter. Those are normal mitochondria. And you want to compare that to the littermate now that's going to be the knockout mouse for the rescue transgene. And as we expected to our delight, we saw this normal mitochondria. And these mice that had massive elevations of circulating metabolites. Over 2 million mold in their blood, which is what you'd see in a patient in renal therapy. This is a thousand times a lever you'd see in a normal mouse. Yet these mitochondria were completely normal. The cells were completely protected by a very low level expression of the mutants. And we measured the ETC of these mice as well. And it was totally normal. So they don't have a secondary defect that we could only measure as a matter of these mice are completely protected in their parasites. Now what happens to the kidneys? Well, in the same mice, I'm showing you now the EM of the proximal tube. Here's the brush border. And there's the EM of the proximal tube in the control mouse. And then there's the littermate. That's the mutant mouse that gets the high protein diet. And you can see it's completely abnormal. There's a mitochondropathy and a tubulopathy in this mouse, which we extensively studied in this paper, including performing single nephron GFR measurements. So this tells us a bit about autonomous. You must protect the liver. You must correct the liver. But we have to be mindful that there's other organs that can still be affected. So we have to consider this when we think about treatments. And that's a blow up of what those mitochondrial remnants look like in the tubules. Grossly abnormal. That same mouse had normal liver, hepatic mitochondria. So in an extension of the autonomy model, we made another mouse model the same way, with add-back of by-transgene into the germline of an transgene that expresses only the skeletal muscle. So I should say largely in the skeletal muscle. This is the mouse-creating kinase converter. It's insulated. It's the same strategy. What one does is produce knockout mice that have the transgene and ask the question, what's the phenotype? Well, there's rescue from lethality. And there's tissue-specific expression of the enzyme. And what I'm showing you here is this only expression of mutants and enzyme is skeletal muscle extracts. And the mice have a severe phenotype. And here's an example of mice that have fed a high-fat, high-protein, excuse me, high-calorie diet to their own lives. There's the mutant mouse. And there's its obese littermate. They seem to be protected from obesity. They're eating. And it's not that they're running around the cages with hyperactivity. They're not. This is part of the physiology. They're runted by patients that have growth problems. The mice have the same thing. And in the same set of experiments, we were able to find FGF-21. It was a very important and predictive biomarker of the hepatic function of the hepatic mitochondrotherapy of MMA. And what I'm showing you here is that this paradoxical response is what was further studied in this paper, where when you take a heterozygic animal, fast the mouse, the normal response is to have an increase of FGF-21 from low levels to higher levels. And in these mice, they have massive elevations at baseline. And then when they fast, instead of going up, they go down. That's a paradoxical response. So using this approach, we identified a number of hepatic biomarkers and validated them in patient population, in the interpatient population. So with that as a background of the models, I'm just going to go ahead and quickly now review where some of the studies have been performed with mRNA therapy. I'm mentioning this one here for protein coaxial loxate deficiency. I'm not going to talk about the details of this study, but there's an open-label study of this. There's the clinicaltrials.gov entry. The next one that it was active is clinicaltrials.gov entry for genome editing using AV, which I'll talk a bit about. And then the last one is systemic mRNA therapy for mRNA. This just became active. There was a previous clinical trials entry. It was entered. Then it was inactivated. And now there's a new name of the RNA. It had a different number. So we don't know exactly what the differences between this RNA and what was published, but there's now this site that you can see. It's recently active in clinicaltrials.gov. OK, let's start off with the mRNA therapy since those were the ones, papers that came out first. The way this works is a lipid nanoparticle is formulated in a proprietary fashion. And inside of it is packaged in RNA, which has a 5-prime untranslated region and then the orph of interest. And then there's a 3-prime region, which has regulatory elements, polyethanolation syndrome. This then is formulated into these lipid nanoparticles, which are then injected into the mice by systemic delivery. And the question is, what is the effect on phenotype? In the first studies, we were performing Moderna. We used MCK mice. Those ones I talked about that I have a muscle transient. And I'll show you just a little bit. Data on those mice in a second. The follow-up studies that the company did with two different models are mildly affected in the MAMOS model and the more severe model, the MCK model. And there was yet another study which I'll now review using a dual mRNA approach to treat very mildly-affected mice apropos on chastendemia. And that was published recently as well. And I've provided a citation here for people who want to look at that paper and move it. I'm going to give you a brief overview of the results of this study. I'm going to give you an example of the study that we performed that we knew right away that there was biologic effects of the mRNA therapy in our MCK mice. And the way this worked was we had a small number of mice. We gave them a reasonable dose of the mRNA therapy by retroorbital root, which is a systemic delivery method. And what I'm showing you here is a Western model from these mice after 72 hours. And normally there would be absolute no-ends on expressed hair in the liver. And you can see how these mice have quite a bit of auto-intake syndrome. Within a short amount of time, they had a first biochemical response. The serum mRNA went down almost in order of magnitude. And they had an oxidative response as well. So what this does is it measures the ability of the animal to oxidize C13 propionic acid into C13 CO2. This is a very physiologic assessment of liver hepatic mutants function. What you can see here is after therapy, only a few days after therapy, the mice now are oxidizing much more than they did at baseline. Not quite as much as a normal mouse, but they have really improved oxidation. So there were lots of other studies done in this publication during those studies looking at the dosing intervals and doses needed to treat the mice. It's been published, but to just show you an example of the reason I'm going through this is the power of the mouse models. I'll figure this out quickly. Is it going to have an effect? So you can see the strong effect in the mice. There's also a weight gain effect on the mice. Now I'm going to move on now to talk about the AV mediated nucleus free genome editing for mud animal. Again, this is a technique that uses AV and the natural cellular machinery to insert a therapeutic transition at the very end, right before the stop codon of albumin. So what happens here is the AV comes in as a donor cassette. Models recombination then leads to the in the cell. This is editing. So this is a permanent correction to that. What happens then is at the end, the last exon of albumin then produces a fusion RNA that includes a 2A peptide which allows a ribosome skip from the end of the stop codon of albumin to the beginning of the mutate enzyme. It adds a proline on by the way. It's not always remembered by everybody, but in this case that is removed upon importation of the protein into the mitochondria. So what's left here is a mark. This is a permanent mark where albumin now has 2A on it. This remnant from the virus, but it's a circulating biomole, and then the mutate enzyme will then be released to the mitochondria and it will function. So we're very excited in early days because we looked at mice that were treated as neonates and saw this pattern. So you see here this is a modest that's 15 on surveillance. Ground patches are all patches that edited. The reason we're excited is because we looked at the wild type control. We saw a very limited expansion of the cells which is what you this is what you would expect. This suggests integration of growth advantage due to correction. This is exactly what we ended up confirming by many, many other studies in the MCK mouse mode. And what I'm showing you has a picture from that hepatology paper we recently published that shows over time this is the ground patches over here. These are corrected cells. Increase in the number of cells over time as the mice age. And that's a company buy-in signal with the protein as well. So this really shows over time you're getting more and more cells corrected. And this could help with a editing approach that without nucleases is very inefficient. The other thing that is useful about this approach as I alluded to is you can use the 2A peptide circuiting biomarker to tell you about correction. You can see what's happening in these mice that are corrected over time. They're increasing the levels of the 2A. Now it's only a small percent of the total amount of human, but it does raise the question of what could that do. We could talk about the Q&A perhaps. So that's where this is coming from. So the last group of therapies I'm going to talk about are the ones that are the most effective in the animal models. These are the therapies that can rescue the sickest mice quickly and have durable effects. And Dr. Chandler, a member of the team that's going to talk more about this later, he really produced a number of very fine papers showing this really, really is effective in very sick mouse models of M&M, full-mock almost. And that really, I can just show you a picture of this. This is sort of what it looks like. Here's one of that sick mouse I showed and made today 32, some VFB engines in this mouse. But when you compare the way I say this mouse would look to a mouse that was received AB as a neonate, it was very exciting. These mice almost look normal. They're slightly smaller than what it makes. They're very high circadian tablets. They're incredibly robust in terms of phenotypic correction. And this is what has constantly led us back to canonical AB as a, what we think a very effective treatment for patients. It's very, very potent mice. So I'm not going to review all the studies, but to show where we're sort of the current studies and some of the more recent ones that are in process publication. There are different capsits, either ADA capsiders or caps called ANK80 and Cessro Serotype 80 and made either liver specific or ubiquitous promoted cassettes and given those to the mice mouse models I talked to you about earlier. And in the case of the MCK mouse model, it's very effective. What we can see here is we treat these mice with a reasonable dose. It's a 5V12 genome copies per K in the mice, which is a modest dose, lower than some patients are getting. For example, current gene therapies and a durable response with them, serum MMA being really well controlled and also the response of the biomarker FGF 21. In these mice, we can also look at the durable response of appropriate observation, which what I'm showing you here is label recovery and a bunch of mice that knock out mice that don't have any AB, excuse me, controls compared to mice that don't get an AB and then sort of this group of mouse mice compared to mice that were treated with AB at the same time, at 12 days with either AB, ADR and GATING or even as much as a year. They have almost normal appropriate oxidation. That's the physiologic measure of the product, my function. And you can see when we take their livers out, it makes sense. They have a lot of reactivants even as long as one year after a single dose of AB and as much as a dose of juveniles. The last thing I'll mention very quickly because there will be another talk on this is the PAGE team effort, which we're delighted to be working with in a joint endeavor with NCATS. And through this endeavor, we've developed treatments for PCCA and are working on gene therapy for limit B deficiency. These will be covered later on in the symposium. But again, it's using an AED9 vector. So I will close with this one table summary for the group and the audience, which summarizes the newest therapies. There's mRNA therapy for mMUT deficiency and PCCA deficiency. It's an IV delivery route. It's an IV infusions, like it depends on replacement therapy. It's a nanoparticle mediated delivery approach. And it is mainly dominated by the effects of this particle in the liver. Are there side effects? Could there be? Yes. The approach of gene-ride, again, is an AED approach using the LK03 serotype. It's a one-time infusion. There's targeted integration of the mUTase enzyme into the albumin locus with HR. A patient has been treated a few weeks ago. All we know is the name, some basic information about the patient. We don't have any other information other than the patient has to retaste efficiency. Could there be side effects from this treatment? Yes. The last group of treatments that is not there yet, but will be coming, I predict, use canonical AED, different serotypes. It will be delivered by IV. It's going to be a one-time infusion. It's a gene addition mechanism. We don't know how the AVs can figure this could express in other tissues, like the muscle and the heart. Maybe even the kidney. Maybe even the brain. So that's a real advantage of this. Could there be side effects of this? This type of treatment? Yes. I would suggest that maybe in the Q&A, we continue to explore these treatments, the pros and cons for possible side effects could be. That's part of the discussion that the audience was interested in. With that, I will close. I will show a picture of my wonderful colleagues who this picture, believe it or not, was taken in 2019 late fall, which is the last time we got together in some ways as a group before COVID. And again, lots of our trainees. This is Dr. Pam Sart-Hedge. She's going to be here later. And with that, I would thank you all for your attention, and I look forward to the Q&A session. Hi, Jack. So we have a couple of questions. Targeting the liver, only all these therapies that we presented are targeting on the liver and will have a partial correction. Is that going to be enough for clinical improvements? And how about the exahepatic organ complications? Right. So that's an excellent question. Just to rephrase, is correcting the liver enough? Yeah. And there will be a sort of a discussion of this as well for obviously the use of a liver transplant. What we know from the mouse models and from the clinical phenotypes is that the liver dominates the phenotype. So certainly, hepatic correction is absolutely essential for any new gene-based treatment. What we know from the mouse model studies, we have a model of what we call conditional autonomy. So it could be, and this remains as an unknown, that there is a threshold effect where a certain level of metabolites in the tissue that's deficient of the enzyme is needed to produce pathophysiology in that tissue. For example, in the proximal tubule. We know this in the mice because when mice, again, in the mouse models, at lower levels, without a dietary stress, there's a mild change of the kidney, but it's, you know, it looks nothing like occurs when the mice get challenged. And again, the extension to the patient population would be, and again, this topic might come up with Dr. Volkley's talk, liberalization of diet after transplant. We certainly think this is something that has to be very, we have to be very cautious about for a number of reasons. So can we just correct the liver and fix the condition totally? No. Can we correct the liver and provide a huge therapeutic effect overall to the patients, potentially with their quality of life, diminished crises, frequencies, possibly slightly better growth in other aspects of, you know, health? Yes. And so, again, without the need for medicines, this is what we think, the post-liver transplant state, and again, this is a talk that's coming up next, is influenced by all the patients I know, substantial need for immune suppression and a lifelong regimen of immune meds with risks that are associated with those. So just as a, that doesn't fully speak to the comment of, maybe my short answer. Another quick question, maybe, can the mRNA therapy be a bridge before the liver transplant? Yes, so this would be a theoretical question about whether or not a patient that needs a liver transplant as an infant is very sick. Could they get mRNA therapy and then switch over to another, you know, either a liver transplant or something else? Theoretically, I think the answer to that is yes. I think we should also be cognizant of this in our discussions of side effects because a patient who has immune naive to the enzyme and receives a large amount of active enzyme in the form of mRNA, could that patient then be set up to have an immune response to the transgene? That is the mutase enzyme. And so, akin to the lessons from ERT, would this be something that could sensitize a patient, make them prone to an immune reaction? Against, again, maybe an organ that expresses or a gene therapy. The answer is unknown because in the mouse models, the mice always had expression of some residual enzyme either in their muscle or as a transgene. So, the mice in some ways would not be able to recapitulate this immune effect. But whether it could be done in theory, I believe the answer is yes. The practical side related to the immune response and the clinical responses, that is going to remain under investigation. And I think we are it's time to move maybe to the following speaker and we have more questions for later in the panel discussion. We will hear next from Dr. Vokli, the chief of medical genetics and director of the Center for Disease in the Children's Hospital of Pittsburgh who will be talking on the role of liver transplantation in the treatment of methamalonic and progenic acidemia. Thanks for the introduction and I'm glad to be able to do this virtually. Hopefully the next time we have one of these meetings, we'll be able to do it in person. But as noted, I'm going to be talking about liver transplants for inborn areas of metabolism and I'll focus in the last half of the talk specifically on PA and MMA, which is a relatively newer use of the technology. Just to start off with my disclosures, research funding and consulting for a variety of biotech companies and biopharma companies. And well, let's start off by asking some questions and this will really frame the discussion for today. First, are non-life-threatening diseases candidates for transplant or do you really need to literally be doing them for lifesaving purposes? Can a transplant be considered a legitimate partial treatment rather than a cure? If you don't fix everything, is it still okay to think about it? How should we best determine optimal timing for a transplant? Infancy, early childhood, adulthood, and when does the benefit outweigh the risk? That ultimately, of course, is the driving decision-making calculus for doing a liver transplant. As we look at liver transplant, let's take a step back and talk about options for metabolic therapy in general. First of all, augmenting a missing enzyme is the goal of liver transplant, but it's also the goal of gene or cellular therapy. It is accomplished through enzyme replacement therapy or enzyme substitution therapy and small molecule chaperonins can allow mutant proteins to fold and, to some extent, restore enzyme activity. You can reduce the substrate going through the enzyme so that you don't accumulate the toxic compounds as a result of the enzyme block. The classic way of doing this is dietary or environmental manipulation, but there are a number of molecules in understudy and one in clinical trials that allow this for PA and MMA. And then, of course, you can get rid of the things that are toxic, and we don't have really good ways to do that for MMA and PA or most organic acidinias. We use carnitine and isoballaric acidemia. Glycine is an option. And in ureocycle defects, ammonia conjugating agents are useful. Not so much in organic acidinias because of the mechanism of generation of the ammonia really responds to other therapeutic interventions. So why a liver transplant? Well, in some disorders a liver transplant really is life-saving. In ureocycle defects, some of the most severe forms of those, children just can't survive much beyond a year of age without a liver transplant. It certainly is life-altering, so even if the disease might not be compatible with life with significant therapeutic interventions, those interventions oftentimes are very difficult to follow, and so having a liver transplant can relieve that. And then the end result of that is that it's an improved life. So let's talk about the things that we should consider in the context of a liver transplant. Obviously mortality and survival, if the risk of a liver transplant outweighs, or as less I guess is a better way of saying that, then the risk of dying from your disease, then a liver transplant is a good option. But as we've already started to discuss, if the morbidity of the disease is high and the liver transplant reduces that, it's worth thinking about. Similarly, if any non-transplant therapy that you might use has morbidities associated with it, then maybe the liver transplant is better than that. The effectiveness of non-transplant therapy is really probably at the crux of most of what we're going to be talking about here. And so if the liver transplant does a better job than standard care, another reason to think about the liver transplant. And ultimately all of these issues related to the morbidity of the disease and therapies impact the quality of life. And so the bottom line for liver transplant is if we think it's going to improve the quality of life, then it's worth considering. The liver transplants were first used for urea cycle defects among the inborn areas of metabolism. And that was because, as I mentioned, it was really impossible for children with ornithine transcarbamylase deficiency or very severe carnitine, carbonylphosphate, synthase deficiency to survive much beyond 6 to 12 months of age with medical therapy. And so liver transplant was life saving in those individuals. However, probably 20, maybe even 25 years ago now, our first began looking at the possibility of doing a transplant for patients with maple syrup urine disease. And the in conjunction with the clinic for special children and Lancaster, the recognition was starting to be made that these patients, even though they were on metabolic therapy, dietary therapy that kept them from the worst of their metabolic symptoms, they weren't doing as well as we would like. So several years ago now, probably 10 yikes, it's a long time, our transplant group looked at MSUD patients and many of these were, as I mentioned, were in conjunction with the Center for Special Children in Lancaster. And was able to show that the majority of those patients had IQs or adaptive measurements that were lower than normal. So anything in this quadrant here is below the mean and anything in this quadrant is considered intellectually disabled. And even down here, these individuals have pretty significant intellectual challenges. It really didn't, so our standard of care therapy wasn't doing what we had hoped it would for these patients. And so liver transplant was started. One of the things that was important to recognize and was obvious in these 37 patients was that it really didn't matter what age you were transplanted at, the opportunity for doing that was unrelated to either the IQ or the adaptive score. Those individuals fell across the spectrum. And that over time, here one in three-year follow-up, individuals with MSUD who had a transplant were, had their cognition stabilized. And so the idea that doing this sooner rather than later rather than waiting for damage to occur was probably a good thing. Now I mentioned ureocycle disorders and this comes from the ureocycle consortium in an article published by Kravitzky in pediatric research in 2009. And looking at the natural history of the patients accumulated in their registry, what you can see as individuals with early onset disease, the vast majority of them had some intellectual challenges. Later on set disease, they did better, but there's still about a third of them had difficulties broken down by disorder because patients with OTC deficiency were getting early transplants, they seem to do relatively well though they also had some challenges because of early onset episodes of hyperaminemia. Whereas some of the later onset diseases had more intellectual disability largely because we weren't transplanting those. So this actually led a relative change in the field to considering transplanting these patients which we thought were better able to be managed medically and instead go ahead and transplant them. So the question really where you have to ask in this setting is a liver transplant inevitably all or nothing. It's either life-saving or you don't do it. And I think you could probably guess by most of my introductory comments that I don't think that's the case. I'm going to show you some data focusing on the morbidity related to liver transplant and the outcomes in specifically our patients with PNMMA that will come around to the end and give you some I hope a better understanding of how to make a decision on that. So if you look worldwide from about the mid-80s until we're near the end of the 80s to the end of the second decade of the 2000s the worldwide there were almost 6,000 patients with what were described as metabolic liver diseases transplanted. Now some of these fall into what we would normally consider metabolic but some of them are things that we don't really usually think about as metabolic. But you can see here for organic acidemias overall in that what is that a 40-year time frame 129 patients were transplanted with organic acidemias. Of those the vast majority 120 were children. This is the scorecard at children's here in Pittsburgh from a similar range a little bit longer range and you can see that in that time frame we transplanted 283 patients the definitions of the liver disease were the same as in that previous chart. What you can see is that in that time frame we transplanted 7 with propionic acidemia and 9 with methylmalonic acidemia. That was really all probably within the last 10 years. There may have been somebody very early on in that but this is a relatively new phenomenon for those two disorders. Now what's the outcome in those liver transplants? Because that really drives what we are going to recommend in terms of liver transplant for the various disorders. You'll see very different data if you look at adult liver transplant and even some of the non-metabolic liver transplant data in kids. And that is that the survival care overall in kids for example is it's made worse if you have systemic disease. So things outside the liver. That's not too surprising. And then if you have either don't have systemic disease and it's more you have some limited extra hepatic symptoms the survival is much higher. And this is the majority of our patients. And I'm going to show you this number here which looks like maybe 80-90%. And then compare that to our numbers. This is a slightly different age time range than I showed you before. We have about 270 patients. But ours look pretty similar to that overall. 80-90%. But what you have to keep in mind is that in the early days of liver transplant not too surprising here in the blue line in the 1980s if we break our liver transplants of children's down by decade what you can see is that the mortality that in the 1990s and since the turn of the century really liver transplant the long-term survival is now really closer to 95% and maybe even 97% when you take into account children who come in and are not acutely ill for some reason. In other words their disease has progressed to the point where that transplant is being done in a semi-urgent or emergent fashion. Let us look at MMA and PA specifically. So we know that the natural history of this disease has significant morbidity and mortality. What does liver transplant offer? Well this jumps to the end of this section and I'm going to put these up here first so that you can take a look at them as we go through the data. The benefits it does decrease the frequency of metabolic episodes. I would say that metabolic episodes are rare with a liver transplant as opposed to relatively common without it. In propionic acidemia it improves cardiomyopathy. There often is residual disease but that disease is much better and while we don't know how quickly it's going to progress most of our patients though not all have not experienced significant worsening of their heart disease post liver transplant. It improves protein tolerance and there does appear to be neuro protection. We all worry about an MMA and PA of the metabolic strokes and there have been isolated case reports of that happening post transplant but the incidence is much less than before liver transplant. There are still some residual problems. We do end up recommending some protein restriction for most of our patients however I'll say that not all of them listen and so we do have patients who are on a completely normal diet and doing fine. I mentioned the continued risk for metabolic stroke though it's low probably a percent or two kind of maximum and there have been some reports of pancreatitis recurring in patients post transplant probably because of local toxicity as opposed to systemic toxin circulation. Let's look at the biochemical parameters in PA and MMA. First of all in our cohort of patients with MMA and these were published back in 2018 you can see that in general the serum glycine level an indicator of methamelonic acid drops significantly as does the serum methamelonic acid it doesn't go down to normal but in general it drops by two orders of magnitude so usually 50 to 100 fold lower in after liver transplant. Amonia is not a big problem in a lot of patients but it is in some and so it tightens up around the normal range after transplant in PA and in MMA and if you have a bunch of them all together you can see that the changes indeed statistically significant and not all patients with PNMMA have elevated lactic acid prior to transplant but if you do it tends to normalize so you can see here it's lower in patients with late propionic acidemia than it is in individuals with early and if you bunch them all together post transplant it tends to drop. This is a table from that same paper that I mentioned from 2018 that just summarized the then experience with PA and MMA and you can see this is a lower number than I mentioned in the previous chart and you can look at these and see that there were relatively few surgical problems here that everybody has a little bit of rejection that's sort of standard and wasn't any worse in the PNMMA patients the echocardiogram in our PA patients one of them completely normalized three of them still had some residual disease but it was much better than it was before no problems with kidney disease these were isolated liver not liver kidney and while everybody here is listed as being on dietary restriction actually some of these patients had stopped it on their own this is one of those charts you're not really meant to be able to read because it's too small but it's just there to remind me to talk to you about living related donors so you know for as long as liver transplant has been out there we've used a cataviric donors and now we're moving to living related donors and in many cases that is we have to make sure that the donors are not carriers for the disease i.e. the parents are not candidates but in the case of MMA and PA it actually turns out that that's okay and we've done a number of parental to child donations in PA and MMA and it seems to work okay here you can see that the heterozygous donors have been used domino transplants are also used in this disorder you can take for example the liver from an MSUD patient and give it to a patient with MMA or PA actually blocks the flux of amino acids isoleucine and leucine through the pathway and some of our best patients post transplant were in this category there are a number of other diseases this has been done with but I'm not going to talk about those and here you can see a report of one PA patient who the isoleucine and leucine levels one of the unveiling the amino acids that are usually high in patients with MSUD or in the normal range in the PA and MMA patients they have the normal metabolism of these amino acids in the rest of their body so they can handle those amino acids just fine so some of the additional considerations when we think about liver transplant in PA and MMA is are we doing isolated liver or do we need to do a liver kidney transplant this is in MMA because PA you don't really have kidney disease and so really the definition here or the decision here is based on whether there is current kidney disease usually you would include a liver kidney and do a liver kidney transplant or if it's predicted to be developing liver disease in the near future talking about living related donors I still think that non-carriers are best but parents a carrier transplants have been have worked out just fine as have the domino transplant and then just to say one more time transplant in this disease is not a cure you may need diet we still have to watch out for your symptoms and many of those symptoms may be released and may be improved with other adjunct therapies so keep an eye out for clinical trials in that setting you'll hear about gene therapy elsewhere in this session or in this meeting so I'm not going to talk anything about that so where do we go next well we have more than one option and that's a good thing so you should be talking to your metabolic physicians whether transplant is right for you dietary therapy is better or some of the other gene therapy or small molecule therapies that are coming down the down the pike bottom line is that in PA and MMA we're really rethinking the paradigm liver transplant helps it doesn't completely fix but that's okay and may be good enough for your needs or your child's needs for this disease so with that I'm just going to thank my lab and my clinical research team and you for listening and I think we'll be coming back live as soon as this video is over for some questions thank you very much again unfortunately Dr. Vokli had an emergency this morning that kept him from being able to join us live for this short Q&A so I will move on with our next speaker Patrick Forney he is a clinical research fellow at the division of Metabolism in the Children's Hospital of Zurich in Switzerland with a long-standing focused interest in MMA and he will present on the updated clinical guidelines for the management of mythymalonic and propionic Asidemia and as well as his latest work on some omics approaches to identify novel therapeutic targets so we'll hear from Patrick next I would like to thank the organisers for giving me the opportunity to present some of our work today in the first half of my talk I'm going to talk about the first guideline revision on metabolonic Asidoria and propionic Asidemia and in the second half of my talk I'm going to present a current study we're performing on MMA when presenting this first revision of the guidelines I will give you an insight into the process of how this development runs I won't be able to go into the details of every single recommendation but I'll provide some more maybe controversial recommendations which will be the basis of some discussion later on I have no conflicts of interest to disclose and I'm starting with this very basic slide which we put in the guidelines revision which depicts the intercellular cabalamin pathway and I'm sure you're very much familiar with it I just wanted to illustrate that the pathway of cabalamin actually runs from the gut uptake into the bloodstream and then into the cell along a lot of different proteins and genes which are involved until the cabalamin molecule actually enters the mitochondria where it serves as a cofactor of the methylmolyl-coe mutase enzyme which is defective in the case of MMA and just the reaction once the proximal to that is defective in the case of propionic acidemia when we look at this pathway a little bit more in detail we see again here the cabalamin arriving into the mitochondria and then when we look a little bit more in detail at this prokinate pathway we see that it starts with certain amino acids from propionate derived from gut bacteria and other sources and that there is an accumulation of certain metabolites here in case of a block at the pathway at this stage, in the case of PA or when there is a block at this stage of the pathway in the case of MMA so what's the basis of our vision there was an original guideline published in 2014 by Bangor Adal and since then six to seven years ago newly published evidence has become available and we tried to make sure to include all this novel evidence and I'm going to point out a few important things which we've included now in this revision the whole aim and purpose of these guidelines is of course to enable well-informed decisions in the context of MMA and PA patient care and management we had a panel of 21 experts and we've tried to be as inclusive as possible but it ended up to be quite Europe and US centric but still we had quite a diverse panel also background wise so we had medical doctors but we also had biochemists and dietitians and psychologists and we also had external reviewers so during the process of the guidelines reviewed by seven external experts and very importantly also two patient representatives to include the very important patient perspective I briefly would like to touch on the new evidence so only a few examples on what evidence is actually brand new and was included in these guidelines and there are two aspects which I would like to point out there's other new evidence of course experimental evidence which was not very much included we focused on the evidence which is directly patient related and I will come to that a little bit later but the first aspect the first bulk of information which had a lot of novel evidence was the aspect of transplantation outcomes in MMA and PA patients just two examples of quite fresh papers and secondly I want to point out the impact of using precursor-free methods where we had some new papers coming out as well and I'm just giving three examples here in terms of methodology what is new there previously in the initial guidelines 2014 the approach sign the sign method was used for evidence evaluation while we have now switched over to the grade methodology without going into the details of these acronyms we need to understand that this is a structured formal process how to approach a bulk of literature and evidence and how to evaluate it and grade it so because we used the new method we did a complete reanalysis of the whole body of evidence so we did not only include the literature from 2014 up until the start of the guidelines revision but we actually included all the evidence which is available from the beginning and then what is new method wise as well this is part of the grade methodology is that it is outcome oriented and I would like to show you how we did that with review, continuous review of the patient representatives we have developed a list of patient relevant outcomes and they are listed here in this diagram and they were also rated by the panelists, by the expert panel but also by the patient representatives in terms of how important they are to the patients and as you can see we tried to group the outcomes into meaningful entities and to then also structure the guideline paper according to these patient relevant outcomes, you can see that survival was rated very highly and some others were rated like intermediary and the least important outcomes were hematologic complications and we also looked at the analysis of the outcome and how it was important for the patient health but it was important for us to see that the guideline panels or the experts saw the importance of most of these outcome parameters in a similar way as in dark blue the patient representatives how did we approach the literature evaluation then as the next step so we performed appointment research and several detailed steps of filtering those papers so first we filtered for only papers on MMA and PA then for papers with clinical data then for patients which or for papers which had at least three patients included then only papers which were outcome relevant and some papers didn't have enough information in there but they were excluded as well and then these were put into these different groups according to the outcomes and then we had working groups working on these specific outcomes with the literature which is derived from here some papers of course were in several groups at the same time and they have been evaluated according to the great method the aim was then for these working groups to come out with recommendations and they were structured according to these outcomes and we finally came up in the guidelines with only 21 recommendations which is about a third of the initial 60 statements which was in the original guidelines and the reason is we only formulated recommendations if there is enough supporting evidence and if there is a consensus among the panelists this doesn't mean we did not include certain controversial points but we then discussed them in the text of the guidelines so we should not only look at the recommendations there but also at the text of the brief glimpse at the guidelines with two examples one example is early diagnosis as an outcome and there I'm going to talk briefly about newborn screening and then another second very important outcome was metabolic stability and there I'm going to touch on working transplantation so newborn screening what is there in terms of new evidence there are some technical findings which were new but more importantly some studies investigated the benefits of newborn screening and came up with two main points that pre-syptomatic diagnosis of late onset patients is possible in newborn screening and potentially newborn screening can prevent unital mortality on the other hand other studies found that it still may be too late for you to avoid metabolic decompensation and there is no improvement of important outcomes for example cognitive development so this was a very controversial discussion among the panelists as well and that's why there is no specific recommendation but a long discussion in the text about the pros and cons regarding newborn screening and I briefly like to talk about metabolic stability this is defined in our work because it's used very often but we have to define it very specifically as the absence of hospitalization and the exacerbation of disease signs and symptoms especially metabolic acidosis and hypermonemia and the opposite term would be metabolic decompensation and there we recommend very basic things such as we recommend avoiding catabolic metabolism of course to improve metabolic stability which was not controversial and had very strong evidence and then we also discussed organ transplantation to give you a first impression which organs are most frequently transplanted in MMA and PA the liver is quite frequently transplanted in MMA and PA a combination in MMA and very rarely only kidney in MMA benefits of organ transplantation is decrease of metabolic decompensations and hospital admissions improvement of biochemical markers disadvantages are neurological complications cannot be avoided in all cases and there is still the possibility of lethal metabolic decompensations so overall this topic was quite controversial but we still came up with a recommendation because the benefits seem to be very clearly there from the evidence and we recommended to consider liver transplantation in MMA and PA and combined liver kidney transplantation in MMA to improve metabolic stability if you would like to read more about the discussions we had and about the recommendations I would like to point you to the actual paper which came out earlier this year and it's open access and everybody is free to look at it. In the second part of my presentation I would like to give you an insight how we move on to find novel evidence in terms of disease mechanisms and then potentially novel targets as well because based on the revision of the guidelines it was very clear that there are still big holes in terms of knowledge and big holes in terms of sufficient patient management strategies so we are performing a project at the moment which is entitled Integrated Transomic Analysis of a Rare Inborn Error of Metabolism which is MMA. I'm going to start with a study overview so we have a group of 80 controlled individuals and fibroblast cells and we have 150 patients and their fibroblasts based on this material and this information we generated different omics layers and we started off by whole genome sequencing to generate genomics data added transcriptomics and proteomics layers and for a subset of cells we also performed metabolomics and we combined these big data sets with the already available clinical information as well as biochemical information which I'd like to show you here briefly the pathway again with which you're very familiar with but this becomes important later on in this presentation and then the aim of the study is to integrate all these different data layers by mostly bioinformatics method I'm just giving a few examples here they should be just there as an example we performed many other things as well and the aim is to identify dysregulated pathways in MMA which will improve disease understanding and potentially offer novel therapeutic targets the first layer we looked at was of course the genomics layer the first name was just to confirm that there are biolealic and disease causing variants in the mutase gene and as you can see we solved almost all of them for some we even combined the transcriptomic layer already with the genomics layer and we came up with this distribution of all the mutations on the mutase gene many of these mutations are well known and well characterized as for example this one and some others are less well known and less frequent as well but this was not the aim of the study of course but this was kind of the starting point we then went on to combine the genomics layer with transcriptomics and proteomics in an analysis which is called multi-omics factor analysis and there you can find factors and then identify certain pathways which are driving a certain factor and then go back to the initial data sets and look where are these pathways which are driving this factor and let me look at this something very interesting stands out and that is that the TCA cycle comes up as dysregulated many many times the transcriptomics data set as well as in the proteomics data set where also respiratory electron transport and ATP synthesis dysregulated so we have first hint and this is of course expected if you know the MMA pathway but even with this untargeted unsupervised method the results were pointing towards dysregulated TCA so in a very naive way we first looked at TCA proteins and genes and looked if they're dysregulated just by naively looking at them individually and this is a very simple plot just depicting a few of the TCA and associated proteins and genes there are many more but I'm just showing you this snapshot and in mutase you see very clearly that there is a tendency that controls have higher transcript levels as well as higher protein levels compared to the mutase deficient samples here in bread so we hope to find similar obvious dysregulations or disturbances in the other proteins and genes but it did not so it's very clear that there is no clear tendency to see which protein or gene is driving the TCA cycle changes with other untargeted methods such as PCA analysis for example there is no grouping in the transcriptomics data set and proteomics data set which made us clear that the data set is actually quite homogeneous it might be due to the use of large cells but we could also discuss that of course and then with other untargeted analysis just by looking at the protein correlation plot here on the left you see again the mutase deficient samples versus the control samples there is no clear grouping no clear tendency when you look at relative protein expression when you look at absolute protein expression when you for example also look at peptide length etc so we tried to find certain driving factors to see whether there is any separation of these two groups but there was not so we had to find a way to look at theomics data in a more sophisticated or a more specific and detailed way and the obvious data layer to go back to to find such driving markers is of course the phenotypic layer so within our data set we have a lot of markers from the phenotype so we have over 100 markers and I'm just showing in this graph a few examples and we went to see whether we can identify one of these markers as a disease correlate and as a marker of disease severity and this marker could then be used again to compare to theomics data set so the first thing we did is just an overall correlation plot and you can already see that some of these phenotypic markers cluster very positively together or some of them go negatively together and we were hoping to find one single marker which correlates well with many many of these other phenotypic variables to sort of find a representative marker and we did this by exactly doing this namely compare every single variable against all the other variables calculate correlations and then plot the p-values in these histograms and I'm going to explain it by pointing to this positive control so this is a clinical score we've made up based on some of these phenotypic traits so this one is expected to be very strongly correlated with all these other variables on the x-axis you see the p-values so on the left hand side you see the p-values which are very strongly significant and here we have a lot of significant p-values because on the y-axis you see the count so whenever you had a certain p-value it was falling into one of these bins and when a bin is very high then it means a lot of p-values are in this bin so this is the positive control as expected showing a lot of very strongly significant p-values when we look at these other variables then none of them had a similar picture except for this one and this is an assay which we use in the lab which is called PI assay, propionate incorporation assay and I'm briefly going to explain what this assay exactly measures and why it might be a very good disease correlate this brings me to the pathway slide and I've introduced it when I talked about the guidelines so I'm not going to spend a lot of time here but of course you see again the mutase enzyme which is defective in these 150 patients which requires the deniesal cobalamin and the fact that they derived from intracellular cobalamin but very importantly the propionate pathway or the propionate pathway funnels into the TCA cycle it's very closely related to the TCA cycle so what happens in this assay the PI assay we use Fortin-C labeled propionate which funnels into this pathway and the Fortin-C atoms actually go through this pathway overcome the mutase reaction into the TCA cycle then leave the TCA cycle again via catapherotic reaction and finally get incorporated into Fortin-C labeled proteins the overall reaction is expressed as PI activity so propionate incorporation activity and this measure was used in our further analysis to investigate how these TCA proteins and genes maybe can be specifically identified and point us towards specific dysregulated aspects in MMA first of all I just would like to show you a nice illustration how we compared the propionate incorporation activity to the other phenotypic traits and this is shown on this slide with a volcano plot it's actually rather simple on the right-hand side you see phenotypic traits which are high in methanolic aciduria on the left-hand side you see traits which are low in methanolic aciduria and this is based on using the pathway activity in a regression model and I just would like to point out a few things which you're very familiar with if you're managing these patients but it's just nicely that almost all these variables actually run as expected patients with MMA more frequently had acidosis while they had low pH and low base success they had a low GFR where they had high MMA plasma levels and here also I would like to point out that the protein mutase level also was low in MMA as well as the transcript level of the mutase gene so all these phenotypic variables they run more or less to our expectation and we then used this parameter to combine the information from the PI activity assay in a differential expression analysis with the other omics layers and there finally we could find not only a broad indication that TCA cycle associated or direct TCA cycle proteins might be dysregulated but actually we found specific players by using the PI activity as a phenotypic trait here again things on the right-hand side proteins on the right-hand side are high in MMA, proteins on the left are low in MMA and you see as a control the mutase protein which is low as shown before in this DEA and then finally we find for example OGDH which is strongly dysregulated as well as GLUT1 we were of course very excited to see these TCA cycle proteins very strongly dysregulated but then of course the question is where are these proteins situated and this brings me to the summary slide and on this summary slide maybe you were wondering before why I was already drawing the GLUTAMIN anaplerotic pathway here and this is of course because these two proteins we've just identified they're strongly dysregulated according to our analysis in MMA this is maybe something we also partly expect from other studies performed in patients and also common knowledge where GLUTAMIN levels are often low especially in a crisis of an MMA patient but the question is how do we validate this data now so we only saw that they're dysregulated but we want to see whether these enzymes are actually changed and whether the flux is changed so follow up studies include direct enzymatic assessment of these two proteins in cell based models and then also to perform metabolic flux studies by labeled GLUTAMIN to see how much is the contribution of this pathway when we compare disease versus non-disease and potentially then come up with this pathway as a novel target I briefly like to acknowledge all the people involved in this big work which includes a group of distinguished analysts who helped me with this this work and then several centers distributed in Switzerland mostly in Zurich but as well as in Geneva who helped with mostly data generation as well as analysis I would like to thank you for your attention and I'm very happy to take your questions and discuss aspects of our work thank you that was a wonderful presentation many of the questions here refer to the transplant talk so but we'll give them for the panel discussion I think one that is maybe relevant with the guidelines in talking about avoiding catabolism in MMA or PA patients is the one asking about how sensitive or worried are we in MMA patients for the catabolic so steroid therapy would you want to make a comment on that this was even explicitly mentioned the steroid therapy aspect in the first guidelines since then there is not much novel evidence on this topic and I think we did not include any certainly not a recommendation and we did not discuss this at length in the new guidelines revision and there is just not a lot of data on this subject in the literature thank you then I was we were surprised to see this this agreement between the panelists and the patient representatives and the importance of early diagnosis and a recommendation about newborn screening having met many patients with very devastating presentations of cobalamin A, B or milder mild MMA I was wondering about that discussion the discussion was indeed quite controversial we felt as an overall panel that potentially the late onset patients might even benefit the most because the very severely early presenting patients for them the newborn screening results might be available too late but for the late onset patients to avoid complications in their future that might be the biggest benefit of newborn screening but the discussion was too controversial to finally come up with a smooth recommendation and clear recommendation also the literature is not that clear on newborn screening outcomes and also the nice table with protein and energy requirements was removed from this updated guidelines I guess new information was even more confusing or to make a clear commit to a certain amount of protein or use of special formulas to recommend yes absolutely I mean we at some points refer back to the initial guidelines if there hasn't been any changes and the reason we did not come up with a very specific mandatory management plan is again the evidence is not clear enough we know certain aspects which need to be considered including the use of precursor free amino acid mixtures which we discussed quite at length in the guidelines but to then really recommend a general scheme in terms of protein intake I think that was not possible we opted for the recommendation to tailor it individually to the patients needs and to the patients biochemical surveillance parameters and clinical parameters so that was sort of the in between we chose with dietary recommendations you will see there are some additional questions and that in the chat maybe we can answer type and answer life you want to comment on this the club cycle regulation that comes up in the mouse models cell studies you validated this now with this great data set of human cell lines do we use an aplerotic agents and citrate is that commonly or recommended no it's not commonly used there has been a study a couple of years ago which trialled three different anaplerotic substances including citrate as you mentioned I think the effects on the clinical phenotype are not clear yet this needs to be seen in potential future studies but as you say the biochemical and sort of scientific evidence of several models point towards a disturbed TCA cycle including anaplerotic reactions so this is a potential target we think there was a question about the checking B12 responsiveness in patients I know you have done a lot of the molecular work in the variants and their response so there is a protocol of how to test responsiveness yes there is a protocol which has been suggested already 2008 and we recapitulate and summarize the protocol to test patients for B12 responsiveness also in the guidelines revision and we think regardless of the molecular diagnosis like the detected variants I think every patient should still be evaluated for B12 responsiveness clinically it is a more specific question about the TCA regulation does it act as a regulated operon with mRNA as well as protein reduced yeah the TCA this regulation we're just about to explore in more detail we coming back to the question whether how the mechanism of regulation takes place and also maybe even more basic where does the regulation happen like on the transcriptome or the proteome level or only in terms of regulation of enzymatic activity we actually don't know consistently we don't even find consistent findings throughout the different data layers which is just reflecting how complex biology is but we certainly find with different orthogonal methods that the TCA cycle and anaplerotic reactions are clearly affected in MMA thank you Patrick again we can type in maybe some answers in the Q&A so that we address the audience questions and with that I will introduce our next the last figure of this morning session Alessandro Luciani who is a senior scientist in the Institute of Physiology and at the University of Zürich Switzerland again and he will present his work on mitophagy the mitochondrial self-eating process in the pathophysiology of MMA renal disease so we'll hear from Alessandro next thank you good afternoon to everyone thank you so much to the organizer and it's a really privilege to be here I'm going to talk to you in the next 25 minutes about the rule of mitophagy methylmalonic asidemia mitochondria are intracellular organisms that play a key role in the production of ATP in the regulation of diverse anabolic catabolic process and in maintenance of several carcinoma rod redox homeostasis they also establish a network of interaction with other intracellular organelles where exchange of ions, metabolites and other macromolecules take place yet they also serve as central hoops coordinate signaling cascade that toggle the balance between cell survival and death pathway therefore, maintaining mitochondria integrity and function is crucial for cellular and organism homeostasis the dysregulation of mitochondria network may therefore confer a potential devastating vulnerability to many cell type contributing to a broad spectrum of human disease such mitochondria dysfunction can stem from inherited defects in the mitochondria localized protein and enzyme as exemplify by methylmalonic asidemia as many of you may know this disease is caused by loss of function mutation in the mute gene coding for mitochondria methylmalonic coenzyme amutase that is involved in the bridge amino acid and certain lipid metabolism the loss the complete loss or partial loss of mute enzyme function leads to an accumulation of toxic metabolite within the matrix of mitochondria network leading to morphological and functional alteration in mitochondria ultimately causing severe organ dysfunction that affect primarily brain, liver and kidney as a mute enzyme is rubesly expressed within the mitochondria of kidney to both cells with this and be our group working on kidney disease we decide to investigate the functional property of mitochondria in kidney to both cells derived from urine of either healthy or MMM patient cells as you can see here those MMM cells perfectly recapitulate the metabolic signature associated with methylmalonic acidemia as you can see by increasing level of toxic metabolites transmission electron microscopy analysis revealed that mitochondria which appear as an interconnecting and elongating mesh work of organes where fragmented and characterised by role-like shape with perturbed crystal organization in MMM cells and confocal imaging of mitochondria target green fluorescent proteins and semi-automated image analysis confer the morphological abnormal mitochondria in MMM cells this was a structural change where parallel binding increase in the numbers of mitochondria as visualized by monoprote analysis of overall mitochondria proteins and by quantified ratio between mitochondria DNA and nuclear DNA and by measuring the numbers of ATP phi be a flag in mitochondria suggesting that the multiplicity leads to accumulation of MMM affected mitochondria consistent with increased number of abnormal mitochondria membrane potential of these organes was drastically collapsed as testified by the mitochondrial dye tetramethyrodamine that really accumulate within functional mitochondria COS metabolic flux analysis measuring the consumption of oxygen confirmed impairment in the mitochondria are a based biometric profile as show by decreased basal respiration mitochondria derived ATP production and maxima respiration and these cellular defects were complimented by a major production of mitochondrial stress as identified by life cell imaging of a bona fide mitochondria a bona fide reporter of mitochondria Ross and this change ultimately leads to an increased production of lipokalin a protein that is released by kidney cells following oxygen stress and several damage analysis to human cells we were able to observe similar defects such as metabolic mitochondria dysfunction in mouse keenness and the right proximal to the cells carrying a knock in a lead knock in a lead corresponding to a normal patient mutation and knock out the patient on a secondary to further explore the consequence of mood deficiency we generated the first zebrafish model of mood deficiency by using criss-cast gene editing technology we obtained one mutant zebrafish carrying an 11 base pair criss-cast induced deletion that result in a primal to stop codon within the exon 3 that leads to a truncated protein deprive of enzymatic activity the homozygous mutant zebrafish which looks normal display no obvious development defects display an accumulation of MNM metabolites which were planted by introducing the wild type enzyme in the liver so supporting the concept of the efficiency of the gene deletion strategy similar to several defects seen in patient cells and mouse keenness of no-kino-cout mice both liver and key name of mutant zebrafish display abnormal mitochondria characterized by an increase of circularity and perturbed crystal organization these cellular chains were parallel by in pain mitochondria driving by energetics as testified by reflux analysis in mutant zebrafish and those chains were parallel by a major production of mitochondrial stress as shown by in vivo imaging and a raciometric flashy microscopy based analysis of gluten to redox fluorescent signaling in mitochondria of liver mutant zebrafish so suggesting the evolutionary conservation of this connection furthermore the mutant zebrafish swim over shorter distance and display excessive mortality which were planted by using the mutant zebrafish with the low molecular with the low protein diet and by expressing the what type enzyme in the liver of the mutant zebrafish we came to the conclusion that the absence of the mutant enzyme in the resulting accumulation of toxic metabolites super mitochondrial or network anesthesia function leading to epithelial stress and tissue damage the question remains how mechanistically the absence of mutant enzyme trigger the mitochondrists function and hence the tissue damage recent studies have brought forward the idea that the other process could lead to autophagy can selectively remove damaged mitochondria thereby acting as a cytoprotective system and how the autophagy maintains the quality control of cells and organelles we put the size that mitochondria function induced by the absence of the mutant enzyme may reflect any change of the autophagy pathway so therefore we decided to measure autophagy by quantified the conversion of non-lippidated form of lc3-1 to the autophagosom associated form of lc3-2 and by quantified the number of lc3 flag autophagosomes in cells treated with a buffalo myosin a well-established lysosome based inhibitor that block the degradation of autophagosom that subsequently accumulate compared to control cells MMSS display an increase of lc3-1 to lc3-2 conversion and then high numbers of lc3-flaggy mitochondria autophagosom sorry furthermore treatment with with a buffalo myosin increase the level of lc3-2 and high numbers of lc3 decorated autophagosome at two different time points where any change reflect alteration in autophagosome per genesis it has been shown that any intracellular and extracellular cues damage the mitochondria leading to fragmentation of the tubular networks after that mitophagy receptors or ubiquitin autophagy adapters are recruited and activated surface of the mitochondria damage the mitochondria and this leads to subsequent recruitment of other autophagic proteins that in it say the formation of phagophore organelle that starts surrounding the damaged mitochondria once the phagophore close to format of phagosome the damaged mitochondria are engulfed and targeted to the lysosome for the final degradation so given the persistence of fragmented mitochondria high numbers of autophagosome in patient cells we hypothesize that the absence of mutants may therefore skew the discredition of MMA damage the mitochondria to verify our hypothesis we treated the both healthy and patient cells with sublital concentration to damage mitochondria and selectively activate the mitochondria the mitophagy driving the degradation of damaged mitochondria and as you can see in panel A and B after 24 hours of treatment with the routinon control cells display decreased number of mitochondria and decreased ratio between mitochondria DNA and nuclear DNA Y both parameters were retained in MMA cells suggesting that the muti-efficiency may skew the degradation of MMA damaged mitochondria in MMA cells we went a first step further and utilized a raciometric pH-sensitive imaging to follow the delivery of mitochematagin mitochondria to autophagy, lysosome the relatively compartment so when dysfunctional mitochondria are engulfed within autolysosome agreeing to redshift in mitochema proteins occur loving to loving to low pH in this degraded compartment so we similarly expressed mitochematagin in both alpha embedded cells and treat those cells with sublital concentration of routinon to follow up the delivery of mitochondria to the lysosome after four hours of treatment control cells explicitly display a red green to red shift as you can see here in the micrograph image and as you can see also here by quantification of mitochema red-green fluorescence ratio this shift was surprising blocked in MMA cells suggesting that mutificences cure the delivery of damage to mitochondria to lysosome it tells me sure that in mammaliasis damage to mitochondria lead to the stabilization of the protein kinase is called pink one to damage surface of mitochondria once there this protein can decrease another protein called which starts other mitochondria proteins at surface of other membranes of mitochondria that culminate with engulfment of damaged mitochondria within out of a gel lysosomal degradative compartment and given the reduced of damage to mitochondria towards degraded compartment we hypothesize that mute deficiency may compromise the pink parking priming mechanisms to verify this hypothesis we label the mitochondria of both healthy patient cells with green fluorescent proteins and expose those cells to to score the translocation of parking to Jeff P. flagged mitochondria as I have told you before the translocation of parking to Jeff P. flagged mitochondria is a bona fide reporter of pink parking priming mechanism activation after 4 hours of treatment with the root and on control cells show an increase in parking cluster and translocation of parking to damaged mitochondria while the same treatment did not increase the number of parking structure nor the translocation of parking to damaged mitochondria this change were complimented by the lack of the engulfment of damaged mitochondria within electron microscopy structure compatible with autophagy vesicles suggestive of defective marking of diseased mitochondria to autophagy lysosomal degraded compartments and performed gain and loss of function intervention that demonstrate that restoring pink one out surface of mitochondria increase the cluster parking positive cluster and the translocation of parking to MMA damaged mitochondria inducing their delivery and degradation by autophagy lysosomal degraded compartments as testify by Mitochema reporters say by monoplot analysis of overall mitochondria proteins in parallel the same gain of function protocol improve the mitochondria function and also their biometric profile as you can see here by Sears metal body flux analysis to demonstrate the link between mutifistancy defect mitophagy and mitochondria functioning and epithelial stress and serial damage we decide to derive approximate tool cells from flux mouse kidney and transduce those cells with an adenovirus express a critical conditional inactivate the function mutigene in in vitro the deletion of mutant enzyme which was reflected by an increase level in MMA metabolize leads to reduce degradation of damaged mitochondria as you can see here by Mitochema reporters say and those change were paralleled by generation an increased generation of mitochondria stress and overproduction of the lipochallin 2 which were blue planted by restoring being parked in direct mitophagy on the surface of damaged mitochondria in deleted kidney cells we came to the conclusion that the absence of mutant enzyme and the resulting accumulation of toxin metabolite impaired the pink induced translocation of parking thereby alting the delivery of MMA damaged mitochondria to autophagylosomal compartments this in turn leads to an accumulation of damaged mitochondria that trigger epithelial stress and kidney damage if mitochondria dysfunction promoted the accumulation of damaged mitochondria and several stress and kidney damage and intervention that repair mitochondria function will subsequently restore seromestasis and prevent damage in MMA cells so to explore the translational potential of this concept we decided to treat both patients and the mutant zebrafish with the mitochondria target Roscaventus which are tested tested recently in other disease associated with the mitochondria dysfunction and interesting enough the treatment with the mitotempo which blocks the production of mitochondria oxidized stress in MSLs improve mitochondria morphology and their pyrogyetic profiles and lower the overproduction of lipocalin true in MSLs despite any change in MMA metabolites similarly mitoq treatment of cow zebrafish with mitoq which blocks the production of mitochondria oxidized stress improve behavioral phenotypes and planted the excessive mortality in cow zebrafish independently on any change occurring in MMA metabolites so if I have to summarize my talk in just one slide I will say that we have identified an overlink between primary gene deficiency, mitophagin dysfunction and mitochondria alteration and epithelial stress and kidney damage this finding substantiated role of ping and park mitophagin maintenance of mitochondria network hence its role in homostasis and function of specialized epithelial cells we have showed that mitoq zebrafish which Gikapiturin mitochondrial pathology phenotypes associated with MMA represent a powerful tool for drug discovery and we have also demonstrated that targeting damaged mitochondria targeted antioxidant repair the homostasis of mitochondria and prevent damage in preclinical models of MMA and in perspective our findings suggest that timing half mitophagin may serve as novel therapeutic strategy in MMA disease and with that I would like to stop here my talk and thank all the contributor in Davos lab national international contributors and our funding agency nothing was possible without these great collaborators and I thank you so much for your attention thank you Alessandro that was a very nice presentation and a novel mechanism targeted for therapies from the submitted questions there was a question on rapamycin that affects autophagy and would it affect mitophagy in the same way through the pink parking signaling yeah so this is a very very very good question well I did not have time to show you our complete autophagy characterization of MMA cells but in our settings I mean in our MMA cells we did not find any dysregulation or signaling pathway so this suggesting that probably instead of looking to rapamycin we should look for other more specific compounds that are able to selectively regulate the mitocondrial degradation so for instance recently it has been show that naturally occurring compounds like spermidine for instance reservil atrole for instance again and also ureliting A are able to selectively induce mitophagy in different diseases associated with defective mitophagy or with defective mitocondrial and they are also able to prevent some disease phenotype in preclinical models for instance if you take cancer if you take neurodegeneration for instance if you give these compounds to these animal models you are able to rescue some disease phenotype so just come back to your question yes rapamycin but better if you use more selective compounds acting on mitophagy so we have seen this increase mitocondrial mass in many tissues humans and mice your mice and zebra fish and all of that there is a balance between degradation through the autophagy versus mitocondrial biogenesis is the biogenesis pathway upregulated or downregulated in your systems this is a very very good question yes the pool of mitocondrial within cells is created by a balance between degradation and biogenesis and several studies now start pointing to the fundamental role of pink and parking not only for the degraded pathway but also for biogenic pathway it has been sure that parking as well pink is able to regulate a repressor of PGC one alpha called Paris and once parking is downregulated so the Paris protein is up and is acting on PGF alpha blocking the the biogenesis and the function of mitocondrial so this homostatic link suggest once again that actinomitophagy and specifically actinom pink and parking direct mitophagy is important only for maintaining the quality control of mitocondrial but is also crucial for maintaining the the biogenesis at the renew of mitocondrial then reviewing mitophagy and the reporter mice mito QC mice they have increased mitophagy in proximal versus this that huge which goes well with our observations in which the cell autonomy of the proximal cubil versus other parts of the nephrodon being affected but they also have a lot of heart involvement mitophagy is very important in the cardiomyocytes it has always puzzled us this more normal heart physiology in MMA and the insights from the mitophagy work so within the mitophagy within the mitophagy field for instance if you take animals with deletion of pink and parking and check for defects of mitocondrial in cardiac or muscle cells they have problems because this is a general pathway working in mitocondrial maintenance so if you take pink one or parking knockout mice they have problem with heart yes they show cardiomyopathy they have yes problem with the heart yes I think we have reached our lunchtime and I think thank you all for this nice first opening session today we all go for a lunch break and come back at 12.30 thank you all