 So, my talk will be a bit different because I think we're going from ideas and concepts to practical applications, I think that's the final objective. Here I'm going to go and end up with the final objective and actually come back and reflect on the path that we took to get to that, to where we were. So, we started off and one of the first things I did in my scientific career was actually work out the enzymology of protein prenatalization. And we worked on the farnesylation of RAS and then we moved and we discovered this pathway that was dedicated to RAV-GTPA's lipid modification that included an enzyme itself but also an additional component that we call RAV escort protein or RAP that is actually related to RAV-GTI for those who know about RAV-GTPA's and have heard of this protein. And the first surprise that we had was that really early on and out of the blue we realized that the mutation in RAP-1 originated a human disease and this was just really serendipitous because this disease is a retinal disease, is a next-linked disease and the reason why there was this connection in 1992 was because this gene had been one of the first ones that had been cloned by positional cloning. So the databases then were not very big and yet we found a match and we were very excited about this. What is this disease? It's a next-linked disease, a slow degeneration of the retina that eventually leads to blindness and this blindness has a certain characteristics that are exactly like retinitis pigmentosa, which was the subject of a previous talk here. And unlike retinitis pigmentosa, Croidremia has enough phenotypic characteristics to be called one single entity caused by one single gene, the Croidremia gene or RAV-S-Cord protein 1. And just so that you may not be so familiar with the retina and retina is a complicated structure, it's a series of cells and this disease involves progressive degeneration of three layers in the retina. The photoreceptor cells, the retina pigment epithelium and the coroid, which is this vascular layer. And altogether they are compromised and give rise to this unique distinct entity that the doctors called Croidremia, actually for absence of coroid, that's where the name comes from. So I want to reflect a little bit on what we did until we got to a treatment, which I'm also going to tell you about. And the first part in the 90s, we were very much concerned about function and biology of the system. We're still uncovering this and this was the most part of our studies were about the biology. And then the first question was why wasn't this lethal? Because RAVs are very, very important, as you know, and this meeting, that was very obvious. And if RAVs are not lipidated, they're not going into the membrane, they're completely inactive, so why wasn't this disease lethal? And really quite early on, we realized that there was redundancy and that this protein activity was able to be compensated for by a related protein called Rep2. So that explained why the disease was a disease at all and not a lethal phenotype. And then the other thing that became rather obvious was that the defect was not a direct consequence of the loss of this protein, but a defect in the factors, the RAV GTPAs and all the functions that they provide. So the idea that emerged early on was that there was this function in the pre-nulation of a RAV, one or more RAVs that led to a deficit in some step in membrane traffic that would then lead to retinal degeneration of these characteristics. And so really the first summarizing, a lot of data and a lot of work on this, really chrodremia is a strange disease like most diseases, because it's not even obvious. First of all, it's a systemic disease. Every cell of the body has this process and yet the degeneration only occurs in these three layers of the retina. And the patients really have no other phenotypic abnormality, although they do have a lot of abnormalities in the cells. The cells are not normal, but it's a subclinical defect that they observed. Then the presence of a compensatory protein, Rep2, prevents lethality and the disease itself is a result of dysfunction of the substrate to the RAV, GTPases. So what could be wrong in membrane traffic and ID generation, or in this case, RPE degeneration or photoreceptive generation, lots of things. And here are just some ideas of things that could be controlled by RAV, GTPases, that could be important for this phenotype. So we went on with a biochemical approach and we figured that the way we could crack this pathogenesis would be to try to identify which RAVs or RAVs that was unprinulated or not particularly lipid modified in chrodremia cells. So chrodremia cells are predicted to have a pool of RAVs or RAVs or single RAVs that is selectively unprinulated. And then we could identify this RAV by subjecting lysates from these cells to in vitro-prinulation with radio-labeled lipid. And then we could observe that while in the normal cells we had no phenotype. This is what we did, and we were extremely excited a long time ago. It's one of the very first experiments that we did in my independent lab now. And as we predicted, normal cells have no perinillation substrates available. So the cells efficiently perinulate their substrates. And if you shut down the mevalonate pathway, then all RAVs become, before the cells die, we can harvest them. And then we can perinulate many, many different RAVs. So this is all selective, all the RAVs that these particular cells are expressing. I have to say this is 1990-something, and what we had access was lymphoblastoid cell lines, EBV transformed lymphoblastoid cells. We had also some access to fibroblasts, but these ones grew much better, so we could do a lot of purification. And then we were very excited because when we found this protein that we thought was really, really key to the pathogenesis of this disease, because it was selectively unprinulated in the carotid remia cells and could be pre-nulated in vitro only, preferentially by the addition of recombinant RAP1 and not RAP2. So this is exactly what you would predict that to be the right target. So and then we identified this protein. We purified it on the basis of hydrophobicity. So the protein was initially hydrophilic, and we removed all the hydrophobic proteins. Then we pre-nullated in vitro, and then we tried to enrich for hydrophobic proteins, and we could get enough enrichment to get this purification of this protein. It turned out to be RAP27 because it was the next one that had not been named. It was just RAP26 was the last one, and this was RAP27. But the story became very disappointing because eventually we realized that RAP27 had nothing to do with carotid remia, at least apparently, because later on, much later on, we realized that RAP27A knockouts have normal retinas, at least morphologically. They have one phenotype in terms of melanosome motility, but that's sort of something that is not really creating the problems that we're seeing. And we thought maybe this is an isoform and compensation, but double knockouts also have normal retinas. So the question became, could this disease be due to dysfunction of other retinal specific rubs? Because I told you we use lymphoblastic cells, maybe in the retina there is this magic substrate that is very important, this mysterious rub, or is there a more general effect of partial dysfunction of many rubs? And I won't have time to go very much into the detail, and I'm mixing up a little bit going back and forwards in time. But what we were able to do was then later on, and I'll explain to you that, we created coradremia mouse models, and we were able to actually try to figure out what was going on in the retinal pigment epithelium, and that's just because it's a layer of cells that we handle a little bit better in vitro. And so we saw that there was phagosome accumulation. One important function of these cells is to eat other segments, photoreceptor other segments in large quantities every day. And so these had a phagocytic effect essentially, these cells. They also accumulate lipofucine and melanolipofucine, which in the same way, young mice from coradremia have accumulated rapidly lipofucine in the same way that older mice do. So these are older mice, and these are the coradremia mice. These are very old mice. And so then what we saw morphologically was that there was a number of defects that were similar to what we see, or perhaps even more exuberant than what we see in aged mice. And in age-associated diseases like macular degeneration, which is where these characteristic defects, this is the retinal pigment epithelium. The photoreceptors are here. This is the choroid. And at the interface of the RPE and choroid, there are the posits and malformations like these ones. There's thickening of this membrane called the Brooks membrane that separates the RPE from this is an endothelium here, and this is the basal infoldings of the basal side of the RPE cell. And what we see is a thickening, exactly like we see in age-related macular degeneration. So just to go quite a bit fast here, the conclusions are that definitely RPE is a disease of memory, coradremia is a disease of membrane traffic. However, we think it's not due to a single RAB or a single pathway. It's more like an age-related change, a change in homeostasis that is driven by the partial dysfunctions of several RABs over time that give more of an aging phenotype. So a lot more complicated than we expected when we started this project. So let me change gears more into practical applications. And one of the things that the human geneticists done then over the years was to characterize once the gene was known, then it was possible to characterize the mutations. And here we were rather lucky because all the known mutations are loss-of-function mutations. So there's been a lot of many different types of mutations, long deletions to small-point mutations, but the end result is loss-of-function mutations. This obviously led us even in the late 90s to propose that we generated a good monoclonal antibody and we had a practical diagnostic test for the disease using just blood cells, a Buffy coat, and then do a simple Western blood. And this is an example of a Western blood where you have reactivity in a normal individual and no reactivity for RAP1 in Trudremia patient. And this had been used for a long time until now all the DNA sequencing methods are now a lot cheaper and a lot simpler, but for a long time doing a simple Western blood is much better than sequencing 15 or I don't know how many exons and introns and everything else. It's a big gene. So we moved into what was critical to provide possible treatment was to mimic this mutation in a minimal model and the mouse has been, as you know, a very commonly used and useful model. And the main question that we have for therapy purposes was really which layer is the origin of the disease because this becomes very important in tissue pathogenesis because you want to know which cells you need to target to treat because there's no point in targeting photoreceptors if the disease originates in the core, there's no point in treating the RPE if the disease is all in the photoreceptors and it's all what's primary, what's secondary, that's a very important thing. And we had an initial drawback and in fact it was not our group but the group of cramers and collaborators, the geneticists that actually had isolated the gene in the Netherlands, stumbled on something that happens, actually I wouldn't say often but sometimes, which is that the phenotype in mice is different from the phenotype in humans and in this case was a much more severe phenotype in that there was lethality. So the female carriers can never transmit the mutant chromosome and this was explained by their group and I think we have now our work also suggested the same that in fact female carriers can never transmit the mutant chromosome and the explanation for this is that in mice there's a more strict pattern of X in activation and that the maternal X is important for extrambionic tissue formation and what happened in these embryos is that rep1 activity was very important for and crucial in the mouse for extrambionic well probably in humans too but in humans it's compensated by the chimeric, by the random nature of the X in activation so there's enough activity there to keep the cells growing but in an environment where all the cells are mutant then placenta does not develop. So we had to revert to conditional knockouts and I think we had already several examples of conditional knockouts here in this meeting and I was a bit worried about the mixed audience and understanding a conditional knockout but essentially what you have is that you can induce the mutation in a spatially restricted manner or in a temporal restricted manner. Spatial restricted manner you put tissue-specific promoter with a recombinase and a flux mouse and then depending on the tissue specificity then you get a specific mutation in a certain layer of cells or tissue and then in the temporal one we use that to induce males, flux males, induce them that induction with the drug actually led to germinal cells also recombining and then the mouse, the male mouse could be mated with a wild type female and give rise to carriers, female carriers. So we started with female carriers and we were really happy to see that we could reproduce in the carriers features of the Croid Remia disease namely photoreceptive degeneration and photoreceptive degeneration is quite, it's the easiest thing to describe because you see the thickness of this, the outer nuclear layer is just a combination of nuclei of photoreceptors and they have typically 10 rows in a wild type mouse and then what you see here is an eight month old mouse had already quite a reduction in the number of nuclei rows and in some places given the mosaicism of these carriers you can actually get much more severe in some areas the retina was actually severely degenerated already at eight months but even at eight months this is reflecting the slow onset degeneration that we see in the humans and then we created tissue specific knockouts and just to summarize a lot of data what we observed is and we haven't been able to put the Coroid in this we've been really we've been doing work essentially with photoreceptors and RPE because people believe that the Coroidal degeneration is secondary to RPE degeneration and it's a more difficult for us it was a bit more difficult to tackle that problem so we stuck to photoreceptors and RPE and what we observed was that if we did the photoreceptor specific knockout of the Coroid Remia gene the photoreceptor degenerated but degenerated very very slow and much slower, much much slower than what we observed for the carriers that I showed you and also if we did RPE specific knockouts then we had degeneration and abnormalities of the RPE the kinds of the things that I showed you before and others and actually cell death in the RPE and then we crossed these two mice and then what we observed was that in the double knockouts of the photoreceptors and the RPE then the photoreceptor degeneration was highly enhanced so the defect in the RPE although it's so the idea is that disease is cell autonomous but manifests itself in the RPE and the photoreceptors independently however we think the RPE plays a major role in this pathogenesis because this defect in the RPE greatly accelerates the degeneration of photoreceptors and for gene therapy purposes some obvious conclusions that you should treat both layers if you can but even if you don't then focus on the RPE and if you treat the RPE you could already have some improvement so the next steps were really to try to provide preclinical data to support a clinical trial and here again I think we were a bit lucky because the eye is in terms of solid tissues perhaps the best model for these pioneering studies in either gene therapy or regenerative medicine for the reasons that I explain here I mean this is in addition to a matopoietic system which has been obviously has advantages and will always have the advantage but in terms of solid tissues the eye is very appealing first because it's small and you may think this is an insignificant thing this is probably the most important characteristics is that these treatments are extremely expensive and and the ability to transduce cells the number of cells is a very important limiting factor so if you're talking about a few thousand cells or a few tens of thousand cells is a completely different proposition than trillions of cells in the liver or trillions of cells in the in the in the brain for example or in the muscle okay so this is or in the lung so this is a completely different ball game basically then it's accessible now there's amazing techniques OCT where it's like a laser confocal microscopy in vivo where you see all the layers of the retina in vivo in patients and you can repeatedly do this over the course so you know exactly how about retinal structure in a lot of detail with these accessible techniques then there's some immune privilege in the eye and and last but not least it's an important organ but not a vital organ so the patient will not die the worst that can happen is that it will get blind which is very dramatic of course but it's not the same as the patient actually dying and as a bonus it's a symmetric organ so you have a control in in your experiments so what we did was really something that was not particularly innovative because I think that one of the things that we're used as basic scientists is to always look for the very novel things and and I think for technology for applications it's actually the plus is on the beaten track because there's so much more confidence than out there for a certain product for a certain utilization or even for licensing and things like that for going through gene therapy regulatory committees the more it's known about these things the better so we stuck with with what was well known and we knew that standard AAV2s would affect RPE and photoreceptors they provide efficient gene expression and they are safer than other the other viral vectors because they're non-integrating in the genome so what is it what is the purpose of this the purpose of this is to do a sub-retinal injection and what is a sub-retinal injection is an injection where you put in the virus between the photoreceptors and the RPE you create an artificial retinal detachment that will reabsorb within a few days and and in fact before we got into humans we had to do this in mice and in mice it's a lot more problematic because the eye is so small so i'm going to show you here the technique so the first thing is you dilate the the iris of the mouse it's and then here was just for visualization and once the cover slip is there and you focus you can see the fundus yeah you see the fundus of the of the eye and then you go in with the smallest needle available but it's huge for the mice and you have to find the sub-retinal space we added a dye so that we could control the injection and see that we actually were injecting in the right place and and here this is a huge sub-retinal injection about half the eye has been detached and and used in this and and and transduced with virus you won't do this in humans necessarily but but but this is possible to do in the mouse and so we did the the obvious experiments we showed first of all that that there was expression and and we could see we could show actually these are not bi-cystronic vectors we use the GFP as a surrogate for the rep one so these are GFP viruses that affect the the RPE and the photoreceptors and in a higher magnification here clearly nuclei from photoreceptors and and this is very much what has been described for other genes there's variability the photoreceptors take up some the RPE takes very well and this this viral vector and but also we showed with the real vectors that you could get expression and we could also show activity of this of this of this newly introduced gene inside the retina this is also something we were able to do once we were collaborating with off with an ophthalmologist because this is a human retinal X plant that drive that was obtained from a surgery normally you don't remove a retina in a surgery but this patient had a special disease that had to actually remove the the the retina and instead of throwing it to the to the bin we used it as and add our viruses to this and did some experiments to show and we show that in the human retina we could see similarities in terms of transduction of the viral of the virus then we observed in the mouse which is always an interesting thing that to add so AV rep one sub retinal injection leads to production of active protein in RPE and photoreceptors and and the function analysis was a bit more tricky i mean i could show you but i don't even i don't understand them very well it's electro retinograms and and and vision physiological assays and you see small differences but but the mice are not heavily affected in the first place so it's a bit hard to show actually the the the differences between the two but but but we can the experts said that that there were noticeable improvements in those it's just i cannot explain them very well so i pass on that and believe me believe me when i say that so actually around 1990 around 2010 we were now ready to embark on a human clinical trials and at this point really and this is a reflection so i have a really a confection a confession to make is that the the mouse studies that we did that we thought would be absolutely critical and very important were actually not that important and and i mean they were not irrelevant but what the people in the committees were worried about was about safety and things that we are actually think are really trivial like a degree of overexpression in mammalian cells and tissues and possible deleterious effects and things like that these are the things that really the the regulatory committees were really on us so we could actually progress in a lot of this in the preparation of the clinical trial even before we have full data of course we have some data but for example functional testing mice were not necessary and they never ask for that because the phase one clinical trials are all about safety anyway not about efficacy so and then they say well the mouse is not that's how the ophthalmology itself but mouse mouse they don't even have a macular and then what's that they're not they're not humans the real proof is human so i think that we really need to talk to other people because sometimes the path is simpler than we expected that that there you'll be so these are the kinds of things they really like to see is activity lots and show that the activity was there levels of overexpression were very important because there was this precedent with cystic fibrosis that the overexpression of the cystic fibrosis gene was also toxic to cells so there was a problem with under expression but it was also problem with overexpression in this case we were able to show very clearly that even if we wanted we could never really push the expression very high on this on the cells had controls on how much protein they make and there was no obvious deleterious effects on the on the expression of the protein so we were ready for a clinical trial and then and you know took quite a while to get through the regulatory committees and also for the funding because it's not trivial to get AAV viruses produced in GMP facilities and in quality for for for human use so that was all that was all and all needed to be sorted out and the initial trial was actually quite low-key I mean there were 12 patients with actually several then this was a little bit changed there were several doses of the vectors and the ophthalmologist was very concerned and so he was very conservative in the amount of virus that you put in and he would rather put less and then increase than the other way around and having problems with excess virus so what we found is that he started off with smaller amounts and then and then as he titrated and put more there was a bit more effect so so it was it was worthwhile to proceed and and the summaries of these are you know very specialist things and of tomology types of tests in patients but I just wanted to highlight that in in these cases here in in this patient there was a great increase in visual acuity okay there's some effect on the non-treated eye and that seems to be some sort of reaction cerebral reaction to seeing better in this eye but but still it was significantly different this is letters read on the ophthalmologist okay so you go there and say a b c d e and this and they can go and see another 21 letters that's a huge improvement on on this and then this patient also had an improvement of plus 11 letters and and the the treated eye continued to degrade a little bit over the course of these studies but but to summarize this and was that the two most advanced patients had substantial gains in visual activity and then most of them have improvement the maximum retinal sensitivity in the treated eyes but the important thing here was that if you that it was this very advanced cases these are patients that have very very very very little retinal left and in those cases when you have very little retinal left you're close to blindness so and in this case you could see definitely the patients and if I have time actually have a movie of a patient describing how he feels that there were there were amazing improvements of function this is not the expected thing in in in in a gene therapy but actually proof that this had happened this improving function came from studies like this where this was a this is a variable fixation study so these dots that you see here in in blue are are parts of the retina that are active and are transducing electrical signals essentially okay and then what does this is the pre-surgery of that retina that is really heavily compromised this is the macular here and even the macular has very little retinal left there is a little island of retinal left here and what happened post-surgery is that this thing has shifted into an area that was previously not really responsive and so what what we think has happened in this case is that is that the rescue of the of the of the with the with the addition of the chrydremia protein leads to rescue of these cells that are alive but but inactive so this is almost like a resuscitation of cells you know cells that are but they're not dead because they can recover function but they were already so compromised that they would not be working on on photo transduction and that's probably that was the most rewarding part of it the other rewarding part was that in the rest of the patients there was a tendency for the treated eye in most cases the treated eye stopped degenerating as we predicted while the control eye continued to degenerate slowly as as predicted and these patients have now been followed for five years and especially in these cases of of severe loss these two patients they continue to to do well and they have not degraded further from that initial improvement of the of the of vision so what we have here is really 25 years from discovery to treatment where we spent like 15 years in fundamental research a lot of it actually quite irrelevant for the final for the final result we spent some time in translational research and now since 2011 in clinical research which is been in the hands of investigator led so this is this was us on the phase one trial there's now four investigator led independent clinical trials in four different countries in the UK in Germany in the US and in Canada and but now the the the next step is definitely with a company with the companies and because the the next step is to license this this this this treatment and just this week I just it was just this week the company that was spent out of this study it's called nightstar and the reason why it's called nightstar is because one of the patients described after the trial that he had seen stars at night and that was something that he hadn't seen for years and that it was an an amateur astronomer and he really was in love with stars and that they could never see this and and what we they just announced on monday with the beginning of this meeting I think it was good armor that they announced initiation of a phase three trial for for quadrimia so I'm pretty confident that if things go well as they have in the phase one two trials that have been that this will be an off-the-shelf curative treatment for these patients in the in in maybe three years okay and then we have 30 years to licensing and from a gene identification so let me just finish with with two sort of off-sides and we're a bit I was fine I was not interrupted so no you're not allowed to interrupt me yeah go ahead so what's the negative control and something that you know it's now working isn't that an ethical have a negative control yeah the negative control the negative controls is the non-treated eye but that's an ethical you should give vision to both eyes no no no no that's not no no no no no no on the contrary that's not an no it's not at all actually if something goes wrong you only screw up one eye no no no it's it's a lot safer of course of course these patients came out of the trial and they said please do me the other one fast before I lose the eye but that's not actually what I can make up on I have vision in only one eye partial vision on the other and I'm very sensitive to that matter so I'm not sure that I would agree with that well it's not me it's the regulatory committees and what's sort of standard ethics so we can we can discuss that but but but it is it is a safety precaution that and now of course these patients are number one to get the treatment on the second eye and yet that's probably also unethical or not and not unethical but not fair because why would three patients in the world get the full recovery while thousands of other ones are still waiting for the for the trial so at least you know at least you you have one eye going for better for but we can discuss that let me just finish with two sort of off the wall but we're in a nice environment here so I thought I bring this one is this aspect it's this disease was not really fundable by the main funders and the and what drove a lot of this research were the patients themselves so in this case you could say that the patients themselves changed their own future because these associations this is a more well known and bigger foundation that supported us in the beginning but these foundations and this one is also a UK based foundation but we have a specific family that uses fight for sight and says and donates this money specifically for Croydremia research in in my lab but the Croydremia research foundation is a is a is a new well it's it was begun about 15 years ago and has increasingly increasing fundraising power and and and really they can do a lot of things and then with the success of the trials they're even more excited and more they were already quite active now they're super active so let me just finish by by acknowledging one very talented postdoc that did absolutely almost 90 percent of this work Tanya Tomachoma and she was in my lab for 17 years it should be unusual she's a senior postdoc she never wanted to leave and she never wanted to be independent so she stayed and this was the end result thank you very much which we are using which type because you might be able to increase efficiency because the av there are different types yeah which are specific for different cells yes so what yeah we yeah we tried av 22 and 25 and and and now there's all series of new avs and all that the trade-off as I explained was in terms of speed you want to piggyback on things that are well covered and and so because every time you change something you have to start from scratch and you have to do a lot of work so we compromise them perhaps a slightly lower efficiency with photoreceptors but we had enough there in terms of the photoreceptors because we think they aren't be easy even more critical and that is more easy to transduce and so we didn't think that the trade-off was to use that one and speed up the process because we could piggyback on a lot of other work maybe not so important but do you know why red 2 cannot complement red one well yeah we we we know why he does not complement yeah no it's still a mystery so it's expressed in part 2 it's again rep 2 is expressed yeah yeah rep 2 is expressed but it's expressed in most in most other places in in in all other places as we know they both ubiquitous proteins and why the degeneration of the red that's a very good question I need another 20 years to come back and and tell you that we don't know it's still a mystery from what I mean this is a good I think the first talk this morning was quite interesting in that I think Rudy was was referring to the same thing the complexity and the prediction you cannot predict I would have predicted that you know a single rub would be retinal specific rub was very important in the retina and nowhere else and that was a beautiful hypothesis but it's completely wrong it's just so much more complex and difficult than than than we expected that that is still a mystery so the virus is replicating in this no no no it's not but integrate actually in a specific site yeah on the dna specific site on the dna it doesn't really integrate right it does it does actually a v integrate yeah otherwise you will not get expression for a long time nobody you have this no no it has an integrated it has a site specific site where it integrates yeah so so the trick the trick here in this that's why it's it's hard to make if if the virus was replicating it would be very easy to produce because the virus itself would be millions of copies of this and it would be no problem but what you do is you remove the replicative genes and in that tight space you put in your transgene okay so you use that space to put your transgene and that creates a much safer virus but that creates problems in producing the virus because they're not replicating so you have to be always transforming cells so when the virus was injected is it a very I mean it's a very local injection or is it a local injection it's not propagating to the whole retina no no it's just on the on the side it depends on the size of the retinal detachment so it depends on the size of the fluid on the on the volume of the fluid that you inject so if you inject the larger fluid you create a larger detachment if you if you inject less fluid you create less detachment but actually there are a number of issues here because a lot of these retinas are fibrotic because they are completely degenerative so one of the major problems with the surgery was are these retinas going to break they're not elastic enough and and and and so the surgeons the surgeon McLaren is really testing that and and he's so far he's seen no problem so are there viruses these viruses specific for these cells I mean no no no no no not at all no no no they're quite widely used different types some type works better for certain cells than others so that's the reason I asked the question because you can find maybe type of av that I think av8 works better for photoreceptor for example you know there are some studies these ones are quite widely have a white tropism and and they can be can can affect many cells yeah the the other issue now is with patients is how many of these treatments are you going to make because everyone is a surgery a major surgery yeah so uh how much of a detachment and how worldly do you go so that's a real consideration that's what I was asking if you're going to a further development if you would choose avars that has decent specificity for but the but the rp is fine the rp is fine no no no but I meant you could then put it in the blood in the circulation ah no no no uh no because then you would risk a lot of problems uh because the eye circumscribe so you even so the patients get anti-inflammatory they get cortisone to prevent and and one or two patients actually develop a bit of an inflammatory disease so the more constrained you shouldn't be if you don't have to then you shouldn't be putting this thing on the blood on the bloodstream you know it's not yeah so I was wondering what your ideas are and why what would make this more effective um and uh also is there a stem cell population that um would regenerate the retina and is there are there any attempts going on to use that kind of strategy stem cell strategy so I I think this is not the end of it because I think for all these practical reasons and costs and and all the rest we haven't even touched the cost uh but that's another important thing but but in a situation like this if the effect is really for 20 30 years the economic benefit is is there so you just invest initially but then that that patient is not a patient it becomes a person and uh is continues to be active and productive so that's there's a benefit there but this is probably never going to be a situation where you absolutely prevent any degeneration and these people have a completely normal vision okay and that is not going to happen also you have the cases of those that have already gone too far and and that gene therapy is not is not an option so obviously retinal um uh cell therapy uh regenerative therapies are obviously uh there and and there's been even some RPE transplants for macular degeneration which is a much more common disease and and the RPE there seems to be the key um layer involved and there so you create a monolayer of RPE on a little scaffold and then you patch it somehow uh surgically and uh remove the the disease the RPE and you put that patch in and the the Japanese have already been doing that and there are other trials planned uh but not photoreceptors so far uh the photoreceptors are much more difficult people are doing some cavalier experiments of trying to put some progenitors and things but that's not been like in many other situations they've not been uh effective is the eye immunologically privileged i mean with every with every rejection i assume that i don't know where you're getting these well there is some immune privilege um and i nobody knows uh at least it's a little bit protected uh from from rejection more than other more than other tissues again one of the advantages of the eye what we are uh quite interested is in the now in the mechanisms of cell death um because nobody really understands uh further about this matter i mean along the lines of what Bruno was asking but more specifically how the cells die and what are the pathways that are activated because what we could is potentially consider perhaps some pharmacological treatments that would delay now i mean it's all about the delaying game right and trying to get more delayed while at the same time permitting the the gene therapy to to keep a pool of cells in in good condition so so there's a combination of things that there are still very much possible this is not the end of the story but what it is is that uh is you have to realize that that these people are diagnosed when they're 10 years old and they're told that you're going to be blind and there's nothing we can do and and that story changed and fortunately that's that's a good thing