In the first half of the pre-recorded session and interviews at EADV 2023, Dr Alexander Nyström (University of Freiburg, Germany) and Dr Su Lwin (Guy’s and St Thomas’ NHS Foundation Trust, King’s College London, UK) discuss experimental data and investigational therapies for epidermolysis bullosa (EB).
You have to be very careful what we can say from a preclinical model, right? A preclinical model is often an ideal setting that you have, you have very controlled environment. Genetically the models are very, very controlled and there's not too much diversity. you don't get the external contributions that you would get in a normal setting, in a clinical cohort. So they are much more homogeneous, which often makes it quite challenging to translate. So I think the broader strokes, the broader concept you can translate, but then in order to understand how you can treat something and how effective and when to treat, this is something that you can basically only test clinically and you have to be able to understand this. And it's important to allow development of the drugs also occur through clinical trials and through treatments. Try to optimise this and that, again, knowledge. It's not reasonable to think that okay, you can directly translate what we've seen in mouse one-to-one in a human settings. But you have to have clinical signs occurring later to educate yourself, educate this, and allow them to optimise treatment.
Dr Nyström discusses the rationale behind his research on recessive dystrophic EB (RDEB), how results from these studies can inform clinical studies, and why understanding such experimental data is useful for clinicians. He also describes expectations for other subtypes of EB.
Dr Nyström started the session discussing drivers of EB, covering preclinical research into the role of collagen VII and fibrosis in RDEB and mechanisms that support tumour initiation and progression in the microenvironment of RDEB skin. Genetic loss of collagen VII causes RDEB and data from a recent animal model study show collagen VII reduces fibrosis in RDEB6,7 .
Why investigate collagen VII and fibrosis in RDEB? – Why is it useful to understand experimental data? – What do we know about other EB subtypes?
My background is, I'm a biochemist by training. So when I was doing my PhD studies, we worked with extracellular matrix and I'm primarily in this extracellular matrix. And back then the extracellular matrix was considered to be more of a structure, a scaffold of things. But the matrix that we were working on is where the base membrane and ligands, which are signalling molecules. We are addressing them the signalling aspects of the matrix. So that during those studies we established the signalling concepts of extracellular matrix. And further on then, I want to deploy this knowledge more clinically and access if this dystrophic epidermolysis bullosa, a very important in disease in this context. And it's a genetic disease of extracellular matrix that allows us to address those questions in a clinically-relevant setting. And when you look at the disease, you obviously see skin blistering, which you think is due to the structural effects. But then we see a lot of other additions in the disease pathology, which is more prompted us to think that this is more instructive. And that sort of triggered the sense that the matrix is instructive, and in this sense, of course it's a very rewarding model to study because you can then suppose structural concepts and the instructive concepts in this. And within this it is clear that the manifestations are, you get inflammation and you get subsequent fibrosis, there's a link, right? And this is very subtle. It is important, the concept that we bring up is important to consider for healthcare professionals in the sense that we show the complexity of the disease. We show different mechanisms and perhaps we sometimes challenge dogmas that are maybe not well established in the EB field, but in terms of other fibrotic inflammatory diseases. What we learn in our preclinical models is a little bit different than what you might expect, which for sure that there's a high degree of complexity in disease progression, and this is very important, consider for clinical or healthcare professions. This is a bit more challenging to answer, and one advantage of recessive dystrophic epidermolysis bullosa, we have had very good preclinical models for this. We have developed, for example, a mouse once, we also have sporadic models of the disease, but they're not so severe. So this allows us to study the disease in a more advanced setting, whereas when it comes to other diseases like the junction epidermolysis bullosa, often those models are not as good for these kinds of studies. They're often quite severe, if you knock out of course, that's a little phenotype. So it makes it a bit more challenging. I think in terms of similarities and differences, there are some similarities. Of course, they all arise in a similar context. You get injury, even blistering of the skin and all that that comes with that, you get an acute injury response, for example. But then there are also differences in terms of where the injury occurs in the skin. In dystrophic epidermolysis bullosa, it's a little bit deeper, probably get more inflammation. In the other subtypes, the blistering might be more superficial. So that's a different response in terms of that. And there are additional things that we have to consider. We have to consider that the proteins are not only expressed in the skin, but you have extracellular manifestations as well. And they're not always expressed in the same ways in the body. So you get additional things that contributes to the pathology. So I think we are quite advanced in understanding dystrophic epidermolysis bullosa. We not to a level at all or where we would like to be in terms of specific treatments and try to understand the stratified patients, et cetera and so on. But in junctional epidermolysis bullosa, primarily I think, which is also might also be effective with fibrosis, we're not really there yet, we don't really understand exactly how the diseases even be. We might postulate things and we see that some drugs are working but we also see that some drugs are not working. So we need to have better models and we need to have better analysis in a sense.
Dr Nyström presents the highlights from his pre-recorded presentation of the session at EADV 2023.
Introduction – Introduction to collagen VII – Recessive dystrophic EB – Collagen VII and severe fibrosis – Collagen VII reduces fibrosis in EB – Microenvironment in RDEB skin
I will talk about drivers of disease advancement in dystrophic epidermolysis bullosa. So dystrophic epidermolysis is caused by genetic deficiency of collagen VII. Collagen VII is the largest mammalian collagen. And in the skin, it's produced by both basal epidermal keratinocytes and dermal papillary fibroblasts. Collagen VII, outside the skin or axillary to collagen VII molecules assembles into antiparallel dimers, which then further laterally aggregates to form anchoring fibrils, which attach the epidermal basement membrane to the underlying papillary interstitial extracellular matrix. Loss of collagen VII causes a skin that upon minor frictional challenges, such as you can see here, exemplified by this recessive epidermolysis bullosa donor skin, blisters at the level just below the epidermal basement membrane. And a consequence of this injury that occurs, which is a little bit deep in the skin, is also that it's often associated with heavy inflammation. So a primary manifestation of recessive dystrophic epidermolysis bullosa, which is the most severe form of dystrophic, or generally most severe form of dystrophic epidermolysis bullosa, is skin blistering. But the disease is a very rapidly progressive disease, so it quite soon advances into a more generalised wound healing pathology, with slow healing wounds, occurrence of chronic wounds, and progressive fibrosis. This progressive fibrosis is here illustrated by the formation of a mutilating mitten deformity in the hand of this boy here. You can see that the formation is very rapid occurring, over only a few years. And this mitten deformity, the webbing of fingers and the contractures are driven by fibrosis, and as such very heavily fibrotic, stiffened, chronically inflamed and chronically injured sites. A microenvironment that both supports the initiation as well as the progression of lethal cutaneous chromosomal carcinomas is initiated or is established. So in addition to such cutaneous manifestation, recessive dystrophic epidermolysis bullosa is often associated with extracutaneous manifestation. You have involvement of eye, oesophagus, even hearts and kidneys can be involved. And to some extent, these manifestations are due to also important functions of extracutaneous collagen VII, having specific functions in those tissues. So we have also seen that collagen VII may not only function as an anchor, but in a wider function, it might also have more instructive functions on tissue homeostasis. But it's important to say that collagen VII deficiency by itself is not sufficient to trigger massive or full-blown fibrosis or full-blown TGFp activation. As you can see here in the mouse model, where we have taken away collagen VII later on in life, and we don't see even after prolonged times, with absence of collagen VII, we don't see any major microscopical or histological evidence of fibrosis in this skin. So there are other factors that promotes fibrosis in recessive dystrophic epidermolysis bullosa. And to address this, Rocco Bernasconi, a former PhD student in the lab, took a mass spectrometry based proteomics approach, and this allows us to look at multiple proteins at the same time. And what we could see from these proteomics analysis was that there are relatively few changes when we look at mice of different genotypes with the same age. Most changes occurred between different ages rather than between different genotypes. Suggesting that going from such a normal state to such a heavily fibrotic state, requires only changes in a few selected proteins. And one thing, one driver that we discovered then of this fibrosis was that when we looked in the dataset from the proteomics, was that we could align many processes to proinflammatory immunity, so we could drive the disease in the recessive dystrophic epidermolysis bullosa occurring through heightened exchange between cellular and structural immunity disease between inflammatory cells and fibroblasts, this creates a loop that perpetuates and drives fibrosis. When we relieve this communication by targeting for example, inflammatory cells and fibroblast activity using natural peptides, angiotensin- , which is part of the running angiotensin system, we can see that dual targeting of inflammatory cells and fibroblasts leads to attenuation of fibrosis quite effectively. We can also see that when we just do, we have also tried the prospect of systemic protein replacement therapies for recessive dystrophic epidermolysis bullosa mice as a disease modulating treatment. So we injected our recessive dystrophic epidermolysis bullosa mice with high concentrations of collagen VII for seven weeks. And what we could see was that this injection protected from development of fibrosis here. The mice displayed a lesser level of mutilating mitten deformities is being formed, as well as this was correlated with lower abundance of profibrotic proteins, TGF-Beta signalling and pure tenascin-C as a marker of dermal fibrosis in this context. So high doses of collagen VII in vivo seems to downmodulate the prefibrotic activities naturally occurring in recessive dystrophic epidermolysis bullosa So we can conclude that there are many mechanisms that contribute to the formation of a profibrotic microenvironment in recessive dystrophic epidermolysis bullosa and establishment of this profibrotic, heavily inflamed microenvironment, promotes then both the initiation and progression of lethal, cutaneous carcinomas. So collagen VII we have previously shown is important for skin wound healing, it promotes reepithelialization and dermal healing as I was discussing, and its loss leads to reduction of, or altered activity of epidermal signalling as well as enhanced inflammation. Subsequently, when we expose skin to continuous damages, this inflammation then leads to a response that leads to production of a profibrotic matrix, a stiffening of the matrix, and creation of a stiffened microenvironment, which is heavily inflamed, that promotes cancer cell invasion. Systemically, extracutaneous expressions of collagen VII, upholds antibacterial immunity, systemically. And when you break this access, then you get a skin that is more susceptible to bacterial colonisation. It to a lesser extent, regulates bacterial colonisation, the skin becomes hyper-colonized and this is also not a trigger of inflammation. And altogether, this then creates, together with ability of collagen VII to normally quench TGF-β activity, creates a microenvironment that is chronically inflamed, promotes tissue stiffening, and progression of tumours.
Future EB treatment landscape – Assessing data in treatment developments
It's really very exciting at the moment, as you may be aware, there is, well, no puns intended, but inflammation is a hot topic in EB. And of course, along with that emerging interest around, you know, what is going on in chronic wound healing and inflammation, there comes the understanding of the mechanisms involved in that stage of that wound healing as Alex mentioned earlier. So that opens up a whole new opportunity in terms of drug repurposing. So at the moment we've got so many different drugs such as biologics or small molecules being used to treat common diseases such as eczema, psoriasis. And, you know, these are approved treatments, they've already gone through the R&D process, they've gone through the initial stage of safety assessments and they are well recognised and used by clinicians around the world. So why not just repurpose for the use in EB? So you then cut a lot of costs and that's made it much more available and easily accessible for patients around the world. And I think we do need to start to think about that cultural change, that yes, it is important and we are learning still so much from the severe end of the disease spectrum, but as Alex said earlier, we need to start thinking about other EB subtypes and also about our colleagues and the patients in low and middle income countries such as Burma, Armenia, and, you know, how about them, how about drug repurposing, which is making it much more accessible to that population with EB. So it is exciting, and there are of course not just the drug repurposing, but hopefully, you know, at some stage we would understand the genetic profile, the clinical profile, but also the immune profile of these patients. So we can tailor different treatments such as gene therapy with anti-inflammatory, or cell therapy with new topical gene therapy. So a more personalised approach to treatment. That is a challenge, isn't it? Because it's such a rapidly growing field and there are so many different treatments emerging, you know, from advanced therapeutics to repurposed drugs, to new molecules and new treatments such as birch bark extract, which is, you know, another EMA, you know, approved drug for treatment in partial thickness wounds in EB patients. So that's all really exciting. But I think as a clinician looking after EB patients, when you are assessing the data, always bear in mind about the patient you're trying to treat. Because these different treatments have different mechanisms. So for instance, if I have to use an example of repurposed drug, gentamicin. So gentamicin is an antibiotic and it's a nonsense read-through drug. So what it means is that it can restore the missing protein, but only in patients with nonsense mutations. So by knowing that, you can then select, right, okay, that's for the nonsense mutations patients with junctional or recessive, whatever. And also, you know, other therapeutics such as this B-VEC gene therapy.
Dr Lwin discusses translational research and how different investigational therapies, including gene therapy, cell therapy and repurposed or new drugs, aim to address wound healing. For example, systemic allogeneic mesenchymal stromal cells (MSCs) are currently being investigated in clinical trials for use in EB for the treatment of RDEB in children in the MissionEB trial8-10 .
Dr Lwin discusses the potential role of cell therapies and gives her view of the potential challenges associated with future developments in EB treatment.
Incorporating B-VEC into practice – Potential role of investigational cell therapies
We're all very excited about the event because as you know, it's the first ever FDA-approved topical gene therapy. And you know, that is actually putting EB on the world stage as the disease that is actually driving the advanced therapeutics as a clinical use, so that's all very exciting. Now the issue is that, or rather the caveat is that it would require repeated application, because the type of gene therapy technology doesn't allow the permanent gene correction. So, which is, you know, fine, but the other, you know, along with that there comes the cost of, you know, how does one continue applying this? At the moment, I think it's more than half a million pounds per patient per year. So it is considerable, and we are not quite there yet for implementation in, for instance, in the NHS as per rooting treatment. That said, it is exciting and also it is something that is available, so that will hopefully encourage more competition in the area, encourage other scientists and researchers to get involved in this area of research and hopefully competition will bring down the cost in the future. Cell therapy, of course, is another sort of a passion of mine and I've been fortunate enough to be part of the preceding trials leading onto Mission EB. These were the EBSTEM trial and the ADSTEM trial. So these are the allogeneic MSC trials in children and adults and youth as you know. So the significance of Mission EB is that it is funded by NHS England. So the aim is that ultimately, once this placebo-controlled, randomised trial is proven to be efficacious for use of allogeneic systemic MSCs in children, then that will be hopefully commissioned for use in the NHS, and that means it will be transformational, transformational for our patients. But of course it is tested in recessive dystrophic because that's the most severe end of the disease, but it's not limited to that. So hopefully that would actually open up a whole new arena. So, you know, to be considered for junctional or other severe simplex subtypes and so on in the future. But for sure, that would add to the armamentarium of treatments for EB in the first instance.
Dr Lwin and Dr Nyström discuss their opinion on potential clinical developments for EB in the next 10 years.
Where do you think, clinically, we will be in 10 years?
In 10 years. Really great question. I mean, we all think about it collectively. Yes, absolutely. So, as I mentioned earlier, I'm a genodermatology registrar. So the training is such that I have to do different subspecialties within dermatology. And of course, you know I was in Professor McGrath's and lab for four years. And of course, having seen those patients with them as well, just understanding that emotive need and the desperate need to find effective therapeutics is really urgent. Now, for me, you know, I know I've spoken about this a lot, but it really is about applying other expertise. That now that I have seen how other diseases are managed, I'm thinking "how can we actually apply what's being learned in other diseases, in treatment?" And I think that, collectively, you know you have seen it yourself. In the past 10 years, we've had so many therapeutics emerging, and it's really very exciting. 2010, the first paper of cell therapy.
Right, I know.
Bone marrow transplant. You know, with high mortality. And now we are talking about topical gene therapy. And I do hope, and I do believe that in 10 years time, if we are standing here talking about this again, hopefully, we are gonna have this personalised approach of treatments for patients and families. And also bringing that cost down for advanced therapeutics to make it much more accessible, much more widely. And again, applying more of repurposed therapeutics there, as well. I think that's what. What do you think? What thinking we should be?
I think it's, yeah. I mean, so we've been basically here before. Similar times, right? I have maybe senior, more years experience than you. But it's been, development have been massive. It's amazing! It's, the track is just, exponential.
Exponential.
Yeah.
Absolutely.
So it's really, really exciting.
Really it's, yeah.
Yeah.
Yeah.
And now, as I said before when the topic of gene therapy, really put EB on the map.
Yeah, yeah.
As the diseases were in forefront of genetic diseases for therapies.
Definitely.
Yeah.
Definitely. And the other thing, you know, the other piece of information I want to share is that EB, it has absolutely been at the forefront of bringing advanced therapeutics into dermatology. And I'm not talking about cancer, which is of course really advanced with CAR T-cell therapy, et cetera. But within, you know, the field of genetic skin disease. And also inflammatory skin disease. You know, we've got advanced therapeutics. But now, you know, part of the other vision that I have is how can we apply the learnings, from EB to common skin diseases, too?
Yeah.
So, we've recently treated psoriasis patients using allogeneic mesenchymal stromal cells. And that's been really quite exciting. Though, for me it is about that intersection.
Yeah.
Of rare to common, common to rare, advanced repurpose therapeutic. I think that intersection is really very exciting.
Dr Lwin presents the highlights from her pre-recorded presentation of the session at EADV 2023.
Introduction to wound healing – Wound healing in EB – Developing therapies for EB –Gene therapy – Cell therapy – Repurposed and new drugs
So I am following on from Alex Nyström's talk about the drivers of the disease mechanisms in EB. So wound healing is a normal physiological process. It is a complex process that is tightly coordinated by four different stages. So after the initial skin damage, you have the homeostasis that allows the blood clot formation so that the innate and adaptive immune cells can fight off any potential infections in the wound area. But then each stage needs to come to an end to allow the following stage to take place. So after the inflammatory process, you have the regenerative process, and then the longer term tissue remodelling will take place. In acute wound healing, this all sounds fairly tightly controlled so that each stage will naturally come to an end. However, in chronic wound healing, these different stages don't quite work very well. So what happens is that there may be a recurrent infection, and leading to chronic inflammation and also defective wound healing. There is skin fragility in EB because of the same genetic mutations. And as you know, EB, there are four major types in EB, so simplex, junctional, dystrophic and Kindler. In all four types, there is certainly fragility of skin. So you've got that perpetual repeated skin damage with the slightest mechanical trauma. So it's a little bit like you've got your foot on the accelerator when you're driving. So you cannot take that foot off.
Therefore, you've got that repeated attempt at wound healing, ultimately leading to chronic inflammation. And therefore, you cannot have the regenerative process to take place effectively, enabling recurrent wound infection, and therefore, chronic wounds ensue. Now, in terms of targeting all these challenges posed by wound healing in EB, there have been developments in different therapies, such as gene, protein and cell therapies, so these are advanced therapeutics, but also more accessible means, such as repurpose and new drugs targeting different aspects of wound healing, as stated here. Now, in terms of the recurrent infection, there are dressings and antibiotics as well as microbiome-based therapeutics being developed. So gene therapy. Of course, EB is a single gene disease. And therefore, with the advances in biotechnology, a lot of groups have focused on replacing the defective gene with a functional copy of the gene. So that's a gene replacement therapy, often using viral vectors to transfer the gene into the host cells. And, or you could actually cut and paste the defective gene at the DNA level. Or you can use RNA-based therapeutics to try and skip over the mistakes in the genetics. Or there is also a form of natural gene therapy. Now, these different types of gene therapies can be delivered in different formats, such as the epidermal graft, skin equivalent graft, topical therapies or even intradermal injections. But the aim of the gene therapy is to address the primary pathology in the genetic defect so that you can aim for a potential cure, at least. Now, the very, very, very exciting time at the moment because we have this treatment called B-VEC, by Krystal Biotech, and led by Professor Marinkovic from Stanford. This is the first ever FDA-approved topical gene therapy for dystrophic subtype of EB, utilising herpes simplex viral vector. And it carries two functional copies of the Col7a1 gene, which is the defective gene in dystrophic EB. So the HSV then allows the functional copies of the gene to be transferred into patients' skin cells directly in patients' skin. So based on the phase III trial data, the FDA has approved B-VEC to be used in dystrophic EB for the first time. Now, the data demonstrates that 46% of the wounds treated with B-VEC achieved complete wound healing at six months. So this is really very exciting. So we are talking about replacing the missing protein in dystrophic EB. However, the caveat is that it doesn't incorporate into the host genome completely because it's epidermal. So you have to keep applying the cream over time. And we still don't quite know in the longer term how that might affect in terms of toxicity. Now, again, and similarly, but using a different viral vector, self-inactivating retroviral vector, Professor Hovnanian and group have led this gene-modified skin equivalent. So using not just keratinocyte sheets, but also fibroblasts as a skin equivalent, gene-corrective skin equivalents to be grafted back in recessive dystrophic EB patients. So watch that space, and hopefully, in a few years' time, you should see the results of this trial. But I'll move on to cell therapy because these are the types of therapies that can potentially be utilised as allogeneic.
So it's from somebody with, from normal humans, from healthy human cells, and this is more off the shelf. So in terms of manufacturing process, it's much more viable and less expensive form. So there are so many different types of cell therapies that are being developed, but I'm going to focus on mesenchymal stromal cells, their subpopulations, the stem cell, hematopoietic stem cells, and also keratinocytes. So mesenchymal stromal cells, or MSCs, have made most of the advances within the field of EB in terms of advanced therapeutic treatments. So our group has treated children and adults with recessive dystrophic EB using allogeneic MSCs, and they were given intravenously, so systemic effect, essentially. So that means that you heard from Alex that types of collagen, in RDEB, you can have chronic wounds affecting mucosa as well as oesophagus and so on. So this is a very attractive form of therapeutic development. So in children, the response was much more marked compared to adults. We also learned that not everybody responds to MSCs. So there is a lot around the patient's immune profile, as well as what types of patients may respond better to MSCs and which patients may not do. So there's a lot of work that needs to be done in terms of patient stratification. So at the moment, based on these really exciting data, our colleagues at Great Ormond Street have been leading the MissionEB trial, which is a double-blind placebo-controlled trial with a view to having a licence use of MSCs in the NHS proven efficacy. So I've spoken about MSCs as a whole population, but these, MSCs are sort of mixed bags. So you can imagine a bowl of mixed fruits, but sometimes you might wanna pick out just the cherries on top. So, for instance, these subpopulations of MSCs, called ABCB5-positive MSCs, these are dermal-derived MSCs with ability to express types of collagen, they tend to home better to the skin wounds as well. So these are very, very attractive subpopulations. So clinical trial data are quite promising, and there was a significant improvement in not just the wound healing, but also itch, pain and the overall disease activity. So in terms of recurrence, it's a little bit more prolonged, and also the wounds are less severe overall.
Now, another subpopulation is called muse cells. So these are multilineage-differentiating stress-enduring cells, with particular ability to last a little bit longer within the skin site. However, the response has been a little bit less impressive than ABCB5 cells. Now, the last form of cell therapy that I will talk to you about is the bone marrow stem cell transplant, which, about 20 years ago now, the parents of the children with RDEB donated the hematopoietic stem cells to the children. And that resulted in very impressive efficacy, but also with high mortality, due to quite aggressive chemotherapeutic regime. The same group later on then harvested the epidermal graft from the same donor and transplanted back in the children who received the bone marrow transplant from the parent. So that demonstrated really quite impressive wound healing. Particularly in terms of the mitten deformities, they were able to release the digits and also transplanted back with these epidermal grafts. But the caveat here is that they needed to have had the bone marrow transplant beforehand. Now, I'm going to move on to the repurposed and new drugs here. I'll touch on three key repurposed drugs, Gentamicin, losartan and biologics, and one new drug, which is the birch triterpenes. Now, gentamicin, we know that it's an aminoglycoside, used as an antibiotic, but it also has the ability to read through any nonsense mutations. Particularly in dystrophic and junctional EB, that's been demonstrated to be efficacious. So the group that treated the RDEB patients using topical and intradermal injections with gentamicin demonstrated restoration of the missing protein. And also intravenously, it restored the missing laminin 332 protein in junctional EB patients. Losartan, you've heard from Alex's talk about the fibrosis, how certain drugs can actually inhibit the fibrotic disease progression. So losartan is an angiotensin II receptor antagonist that's been widely used, of course, for centuries to treat hypertension. It suppresses the TGF-beta that you heard about earlier and also other inflammatory cytokines. So clinical trial data is emerging, and some promising results, but the issue here is that fibrosis is very difficult to measure. And also, in order to reverse fibrosis, whether or not losartan can provide that effect. Dupilumab is a very effective biologic, Th2 targeted biologic treatment used in eczema, but recently, it has been repurposed to treat very itchy form of EB called EV pruriginosa. And you can see quite remarkable clinical improvement in these patients. The larger clinical trials are required to provide efficacy here. So now, finally, and very excitingly, this is the new drug that has been approved as treatment for partial thickness wounds in dystrophic and junctional EB in Europe for patients from the age of six months. So oleogel-S10 contains birch bark extract with anti-inflammatory and tissue-regenerative properties. So the EASE trial has been the largest randomised controlled phase III study in history for EB, treating 223 patients from 26 countries, covering 49 sites. And the data has been really quite promising.
