Confirmation Number:371001
Event Started: 6/16/2005
And I should comment for the record, Dr -- who is not in attendance this morning are in conflict for the discussion and review of this protocol.
Thank you very much. I want to my gratitude to all the folks that are here to look at our proposal and especially to OBA for allowing this discussion to occur. The title is phase I single dose of sub -- adeno-associated virus or rAAV-RPE65 which we define if a moment in patients with labor -- due to RP65 mutation. And I want to acknowledge that long, hard suffering work that has gone on to bring this presentation to your attention over many, many years through the major efforts of many of the people listed here and many more, Dr. Jean Bennett and Dr. Albert McGwire, Arthur and Tomas, Dr. Ake Lund. Margaret Humphrey and Edwin stone. And many others. We worked for many, many years with the hope of eventually helping patients with a specific form of serious visual disability and this is a start. The talk today will try to give you some background of human retinal disease and mention -- try to mention briefly the -- concept studies which encouraged us to move forward then the step backward we took to examine whether these successful treatments in models and that's preempting the result actually have relevance to the human beings that are being modeled and then go through some of the preclinical safety studies which some were answered and the insightful questions by the reviewers but some of which we promise to elaborate on here. So first some background. What is this retinal disease that we are talking about? It's a group of diseases calls -- untreatable at present. Incurrable. It's very easterly onset in infancy or early childhood most of the time and heterogenius clinical diagnosis that causes severe visual loss. is a patient I have known for many years, obviously 14 or 15 years who came at three years of age and had is inning itmous which is instability of thize suggesting inability to focus or fix. And this was a problem that had been there since infancy. Her mother clearly and the mother got worried and diagnostics were performed. Among the many historical issues here, one important feature was that the electro retinalgram which was the diagnostic test used to make sure this is not a cerebral condition or optic nerve condition but a retinal condition the layer inside the eye and that was not detective. -- detectable so that meant the photo receptors were not signalling at an early stage ever vision and the diagnosis with that complex of problems was labor congenital am rosesis and the inheritance is recessive which it often is. Four years later she was worse and we were and the vision was big E vision. She retained the same diagnosis and slowly in the year 2000 we became more sophisticated and it wasn't just labor gonegental amoroussis and bad vision and seriously impaired visual fields and the stigma of this disease, but it became a mutation in RP56 we had seen her subsequently. The view of the eye inside of the eye or the fundus is shown here in that particular patient and it's not very scarred or pigmented. The blood vessels eminating from the optic nerve are somewhat thin and pigmentary changes in the periphery in early ages and then another patient can have more pigmentation and another patient can have even some scarring. So the fundus features of the exam in patients with this type of labor congenital amoroussis may not be necessarily as revealing as some clinical features of some diseases. Over the years a more recent years we recognize that the clinical diagnosis that we find -- that we can make for some of these patients to define their problem or at least localize their problems have many molecular causes and actually many molecular causes and one of them is this RP65. And in an article by Franz Kramer so if we look at this line here, if it's visible, we can see that the mutation frequency of this particular gene which is on 1P31 is anywhere in different series, anywhere from 3 to 13.6% so roughly 10% of the population are labor congenmoussis patients have this as their molecular cause. And the retinal pigmentitousa and other diseases something that presents clinically is not something that is molecularly homegenious and is not homegenious to the single cells involved. This is a complex pathway. This is the outer retina depicted here. The sclera is down here and the vitreous is up here and cornea is several floors above us. And the -- receptor complex is shown here with all the different gene causes of labor -- and RP56 gene has sitz effect of the retinal -- what is the role of RP65 finally defined here? The specific protein of 65 -- this particular gene encodes a 533 amino acid protein that is exists not RP cells and is very critical and exact -- critical to the vitamin A cycle that changes the form of vitamin A that is released from the photo receptor opts that's hit by light and sent to the pigment openthalium to be regenerated into a form that it can be used again. So some of this work was done at NIH fundamental wonderful work by Dr. Redmond defining the fact in the RP65 knockout mice that the 11 vitamin A, and retinal visual cycle is truly necessary and obviously and RP56 is necessary to make it. And this article here by Dr.S Thompson and gal show these in a depicted way with my favorite types of colors. The photo receptors down here now, pigment openthalium here, blood supply up here and foeten of line cycling around and RP56 being part of the cycle of vision to regenerate the visual pigment so another light foeten or particle of light can hit this elegant molecule and cause us to see. So what kind of proof of concept studies have we performed that convinced us that we should be wasting your time and thinking about a clinical trial? Well, we performed ocular subretinal injections and RP65 -- actually adeno-associated virus RP65 in a naturally occurring dog with RP56 mutation that has -- was discovered by doctors many years ago and Michael Redmonds RP65 and mice now with another mutation being worked on by doctors. And in all these different models, large and small, vision has been restored. And it all started in the several years ago with three dogs where this was material with the canine RP65 was injected under the retina in certain locations of the RP65 mutant dogs and we monitored the effects of this and remarkably vision was restored or at least retinal function was and we proved that vision was actually also restored. And you will see a lot of this coming up and this is the electro retinal gram the signal that is elicited from the eye with a flash of light. A photo receptor component to this down going part of the signal and an up going part of the signal that's from the by polars or the second -- bi-polars or second order nurens. A slide that will focus you to this one part of it which shows the down going or photo receptor response in normals, the photo receptor response in untreated RP65 mutant dog or could be dogs received the vector not under the receipt new but the vitreous or a dog the treatment and this is between the retina and the pigment openthalgram and -- openthalium and delivery with the elegant techingue of our doctor to -- Dr. to put it in a certain reelinal region and there was remarkable improvement, not perfect, but certainly much better than that and it worked for a cone specific or a daytime vision receptor population, the above one was for rods or night vision.
We have done amazing number of study subsequently and was submitted as man Vice-President by Dr. Ake Lund -- as a man Vice-President by Dr. Akeland. We can look down here at rod function and cone function to aptitude for rod ERGs and coney ERG and compared to normal and compared to no treatment or interveteral treatment and using a very strict criteria in restandard deviations 23 out of 26 animals that had the subretinal injections with a different variety of vectors some AB2 and some AB1 with promoters but all in all 23 out of 26 animals had success by this definition. And very important question is that something that lasts for a week or two or three or whenever you deem to record it and the answer is, no, it lasts. And again focussing on this particular part of this graph here we can look at this and this is a pretreatment of CRDs and post treatment will ignore the red ones at the moment but post treatment we get a response and the response persists for years. Here plotting the intravitryial eyes but no treatment but subretinal treated eyes seem to be stable and that's remarkable to us. All of these are remarkable to us. We weren't expecting any of this. And moving to a model that happened to be heroic experiment with inutero gene therapy ands we were participants as well. Again, it was remarkable treatment success in a mouse model to validate the type of behavior and pattern we were showing. Does that mean my time is up? Or somebody is trying to reach their mother or father.
Cell phone.
Now, a very important question that at least I had to ask as the clinician here was even though we participated and were stunned by the success having spent many, many, many years seeing failure in similar experiments, it wasn't all success, by any means. Was there any evidence that the particular unique paren Pat Northwestern these RP65 mutant animals, the dog and mouse, did they have some similarity to the human patient which would be the obvious target of benefit from all this work? And so some very important feature of the animals that were treated and that is they had a disproportionate loss of function compared to structure. They retained a lot of their structure in their eye and it wasn't a degeneration as much as it was a dysfunction. This was a biochemical blockade, it was missing the RP65 protein which allowed the psych tole continue. The structure, the photo receptors, the pigment openthalium was not normal but they weren't gone. We asked that question and we recently put out those results to ask the specific question and that is in their there is a relationship? And we used sort of modern technology imaging of the retina looking at the layers of the retina and ask whether the components of the retina are actual three in these patients are gone. If they are gone, it's really criminal to consider treating these patients. So here is cross section of a retina of post moredom donor. Here is a premoredom donor, mainly my eye for comparison of similar age. And just to show that the dark areas are cellular layers and shown here as a way form and the other layers are sigh npsis or photo acceptter outer segments or inner segments. And the way we portray this, this is a -- the way the eye doctor looks at the retina and here is some topographies, some mountains and valleys of the same picture. The yellow and orange warmer colorize higher levels of elevation and the thinner retina is green or blue and this is normal. So let's look at patients with RP65. This our normal. Here is a patient who looks normal. Here a patient that looks quite normal. This person is 21. This person is 41. Some people are thinner than normal, but the statistical definition of thinness for normal is here. Maybe many of these patients will fall into the range of thickness. But retinal thickness total is not with a we are looking for. We started that way. We are looking for thickness of the photo receptor layer which is what we can measure. Can't measure RP cells this with technique. We were looking at this dark layer here and this is a cut, a horizontal section of the -- outer who disblntally and up superiorly and we can see that the dark layer is present in these patients at different ages and different severities. There are actual bumps of photo receptor layer and we dare to actually quantify this. The grey here is the normal range and the line is the mean of the normal range and this 11 patients jumbled together and we can see that in the center many of the patients had actually normal thickness of their outer nuclear layer, their photo receptor nuclear which will mean their Connecticut. Other patients in various regions may be four to six millimeters out showed some normal, some less than normal but more nuclear layer like this person here is showing. At a distance. That happens to be the distance where most of the rod photso receptors mount up in the so-called rod rich ring. So then we went further and plotted the thickness of the outer nuclear and we used some modeling here to define what would expect if it was just degeneration losing function from losing structure which is from the literature on animals and a wonderful paper that went through this several years ago and these lines here define with the model where from the normal here who would have normal thickness and high sensitivity of their retina, what actually would be the air where we would expect normal behavior to occur? And these are not all RP65 patients. took patients without RP56 and asked if they were different. And the difference we were looking for would be the difference that would relate to the models, if there would be more structure than function. The answer was complex. Some patients definitely had more structure than function and some didn't. Some had severe vision where they would not see anything in the area we were testing. They were essentially blind and they would get these diamonds here, diamonds here in different other areas and these other folks had some vision and when they had the vision, many of them would actually have less vision than they had structure. They had more structure than vision. That was a key point and the key point was that it wasn't there everywhere. And to further question going back to the animals who went and collaborated to jean Bennett and asked whether -- retinal degeneration compounding the dysfunction in patients. And that relates to one of the questions that the astute reviewer asked, how did degeneration or patients relate to the animals, so hence the burdening with you all of this. All in all, I guess one could say that looking at outer nuclear layer of thickness in the animals, these are the RP56 mice it deteriorates with time and layers get thinner and so we raised mice for two years until they were severely degenerate by most people's estimates and then treated them and asked whether they would respond to subretinal gene therapy and rather than amazing 80% rate that we were getting at three month old animals, two or three month old animals it dropped to maybe 15 to 20%. And when we looked who the treatment successes logically they were the animalswho had more structure. So let's get on to safety phase which I believe we should have gotten on to first. We have done safety studies. We have done safety studies nonGLP safety studies in RP65 mutant dogs and let's go through that quickly first. This was the vector used in all the safety studies. And they are 15 dogs that were used in the safety studies. They were all at age to begin with between something like two or three months and seven months of age. And they had subretinal, they were intended to have subretinal vector injections and they had different series of -- they were four eyes. Bi-lateral injections at the same visit. Via control and then a range of doses going up to what is 3X and 1X is in vector genomes through micro liter. And the normal controls were injected in these locations, these are the four eyes and the locations of the subretinal injections. More into -- retina and abductor and McGwire did these injections than the nonto pdle retina injections this is divided by eyes. And this is dose ranging data which I thought would be important as we move toward thinking about doses and dose escalation. Here plotted again are ERGs in all of these animals that underwent this and on this axis we are looking at normal and ERG aptitude dark adapted and light adapted to different parameters that are used in humans. Normals are sitting up at a high level of size or amplitude for their way forms both these two signals, untreated animals are here. Control animals the four eyes that had the subretinal injections of vehicle are down here. And these are all the vector injected eyes. And after a certain point all the eyes responded. Most of the eyes responded. I won't say all of the eyes responded. But there was no real easy function, a nice signal to plot, but more a step than anything else. But there was definitely a response occurring at and beyond .1 -- and other vectors rather than the one intended for human use. So to take you through a little of the wording descriptions of the safety studies-- the deaths, or this is the three month dog study still. Clinical pathology, details in the letter, there were no changes at least in hematology and clinical parameters that were tested. The clinical eye exams are very important and were commented on when presented in the letters and these were all performed by doctors at the RDS facility in Pennsylvania. They were graded levels of severity of -- mainly inflammation of the Kong -- the findings in a moment. There was inflammatory changes noted. Both in vehicle control and -- main vit Reyes side. By one month the Kong tiffa had almost entirely appears but cell leather moderate -- cellularity and most of that had cleared except for five eyes one which had a vit Reyes hemorrhage and a couple cases of mild poseterier sub Cappsler cataract. Graph that in this way with vector tighter going from high to low for each category and these are the one week results, one month, two month, three months and the feeling for this data there are more mild and moderate changes at one week and far less at three months. Some persistence of changes as I just mentioned, but most of the eyes were quiet and normal looking. Clinically opt mossoscopy by my colleagues, by three months the retinas had become flat and there is definitely linear and curve linear pigmented scars that were most obvious in the peeda retina. Surgical sequelae and one of the dogs that had the inferior retinalology had depigment near the site of the region of the injections and three animals were noted to have thinning nearby the retinalomy and one animal ask the intrato pdling injection had marked pigmentary change. It was not intended injection into the to pdam but that had a reaction. And there wasn't an obvious vector dose related set of complications. Now most moretime findings, the most moredom findings for mainly no neck Pattersony finding of termination of other organs were described independently as those would be seen in canine toxicology studies and no vector related severity of incidence of any particular hisso president findings and morph leal they were intact. The ocular hiss of the -- was performed and independently again and these are just excerpts from her very extensive notes taken on these animals which can become available to the group and I think it's an excellent study of these eyes. There were drawmatic retinal leakses attributable to the surgical intervention described as retinal perforation, retinal disruption, fragmentation, atropical wave very much athere havey. There was some inflammatory lesion one which was a vehicle control and low dose animal with some aexume ligs of in the vitreous and macrophages and leukocytes and the lesions attributable to the lesions themselves were notable to the doctor and now being reported independently by Dr. Ake Lund and Gary and all of us in the collaborative paper. And here is the is work on biodistribution that was mentioned but not tabulated for you in this way. And there were definitely vector sequences detective in different organs in the diaphragm in some animals and in Brackets here are the actual numbers rather than positive or negative and whether you can see this or not there are mutations whether it was in a replicate sample or not. And here is an opt kyism in one of the 1X eyes. And an optic nerve detection in one of the .1 X animals. So there were detections and we can talk about this. It isn't seem at least from this type of approach to be very clearly vector related. But there are positive results. And the human -- nonhuman primate GOP studies were performed at an organization and they were done in proper GOP fashion. There was a one week study and ongoing three month study which is coming up. For the one week study, only six animals involved. Then we used two highest dose as we could to actually elicit toxicity. And here they are. The right eyes were all performed and left eyes were not and they were equal numbers of females and males and the neck roips occurred one week -- necropsy occurred post injection and 11 further animals had other injections like these animals are on going and that particular study is due to be completed in about a month. And these are the sites of the subretinal injections in the right eye and the control group and in the vehicle -- not the vehicle control group, in the vector delivered group. Generally the poseterier retina aiming superior than inferior in the primate eye. And to review the findings to date, which are compiled as a draft document. I have the draft document. There are things left to do, but in general it's near completion. But the end life observation showed no early death and body weights did not change. The clinical pathology no test -- related changes in the hematology or clinical chemistry. On the clinical ocular examinations, I and Dr. Shaw showed causal exam in the animals at different times, sometimes both together and sometimes independently but there was also the GOP facility had their own veterinary ophthalmologist doing their exam and doing these. These are the informal ones. So preliminary findings to date via the informal route is that one day and one week there were mild inflammatory changes and this is done as if it were human beings. -- was transported to the facility and so microscopy was done and tomorrowgraphy was done by the veterinarian of the institution -- mild inflammatory changes after the surgery at one day and one week. And the one month data are now actually now from the on going study because these animals most of these are six of these are gone. The one month animals 13X animal showed anterior lens changes which may have been due to the parent senditoussis or the needle in at the time to take out flewous for anterior chamber analysis, fluid analysis and another 3X animal had cellularity but this is not there at two months. Two months was essentially last week. And I thought it was important to tell you how things are going and I was gratified but not invested in it but found there were no abnormality in the Kongtivea -- Kong of ita or nuclear at two moneys and we dared to actually graph this and where there was evidence of abnormality at one day and one week and these two 3X animals I mentioned all that clears by two months. Now looking at the eyes, is there examinations by myself and the doctor looking through our eyes and the eyes of the mopingies. And the one day -- monkeys. And the one day the overnight exams showed the retina was elevated as one would expect. Small little hemorrhages within the retina with some pigment epithelial changes near or at the receipt in themy site and this was consistent with -- one week the retina seemed flatter, less prominent elevations and apparent demark cgs lines between the elevated retina and the surrounding retina. You saw those little blue circles that were drawn to depict that. One month and two months exams, the retinas were flat but there were RP changes. There was scarring at or near the receipt in themy sites and the demarcation lines persisted but this is a clear view and the are avages of surgery are there -- ravages of surgery are there but localized and we plan to another exam in three weeks prior to necropsy.
Post moredom, no gross findings. No microscopic lesions. Ocular hiss pathology was performed by Dr. This tissue was taken for two purposes and so unfortunately the doctor received a Hemi second eye which certainly dissorted the hissology that she is used to doing in a careful way her estimate what was going on there was to distinct pathology in the right eye which were the injected eyes versus post moretime changes which were mild in the left eye. Retinal detouchment was near the injection site and we saw clinically not saying mysis opt andic view but would be consistent but she saw what we couldn't see-- photo receptor nuke lecry resulting from the retinal detachment surgery despite the exceeding care and elegant technique of Dr. Ma gyre. There was no necrosis or extensive hemorrhage that she noted. And here is a possibly too busy slide of the biodistribution to date. Some of these are being rechecked but there is a message here. are the organs and the cerebellum other than the eye, other than the visual pathway that are depicted on here and there are isolated findings in the 1X animals here we can see in muscles of the thigh muscle there was so much detention and the Perry retinal node had some vector sequences and here is the left eye which is the uninjected eye and when there was a sample, there was no detectable sequences. In the right eye, this is going from anterior chamber fluid through to the vet Reyes to the retina to the optic nerve and the uninjected orbit tissue. And we can see here there Issacharable -- is considerable in the vector sequence in the right retina and the eyes that received that. And I assumed that's confirmation that we did the surgery or at least Dr. Ma gyre -- McGwire did. And the vitreous had some vector and there was some in the right optic nerve far left. None in the orbit that was detectable. And if we go down further through the visual pathway on the kyism, there were three examples, one in the vehicle control. We will have to think about that one. That showed some small sequence numbers here positive. And the visual cortex on the left and one of the 3X animals had a positive result and in the 3X animal here, the could Lake Cookious did and this was the right side the brain and there were examples here of optic track and LGN in the 1X and 1 -- the 3X and 1X animal. From the god in analysis in the three females and the three males of study one, there were no detected vector sequences on both replicas. I wanted to all for listening to this. And express just think gratitude to our patients who have known about all of these preliminary studies for a long time have sustained hope based on them are not invested in them because I don't invest my patients in any of this. They listen carefully. We talk about possibilities in the future and these long consultation sessions I have with them. No promises. But they do trust and they do tolerate all of our attempts to actually get this particular therapeutic concept Iker it at the time correct before applying to actually perform it at the ultimate level which is to help these patients. Thank you.
Dr. Jay somebodycon thank you very much for a thorough and very interesting presentation. But we will next do to go reviewer by reviewer and I will be asking the reviewers to restate the concerns that they wrote in their original reviews and then to comment on whether their original concern is now satisfied. And if not, ask them to have a brief discussion with you to attempt to satisfy the concern in this setting. And so if you could stay at the podium that would be very helpful for us. Dr. Csaky, we hope that's the correct pronunsation, we generally start with the ad hoc reviewer and I hope you will begin for us.
Soy guess I will start by just restating what I wrote in my review and I guess we can discuss
Right. You don't need to read the entire concern and give us a synopsis and if it's satisfied already by the discussion, we don't need to further discuss.
Absolutely. I guess the first thing would like to say is I want to commend all of the investigators. I have been following this work for many years and have been involved in the periphery and it should be tremendous kind of gratefulness to the investigators for doing a thorough job in trying to explore this realm and I guess really the issues that I raise were the obviously -- between as much as you can do in animals and -- and being that this is the first AE be trial and injecting I think it's reasonable to say let's be overly cautious and do as much as we can initially to ensure that nothing slips through the cracks because as we get further on into other trials or either as this trial progresses, it's better to have been sure that we explored all the various possibilities and so I guess my concerns were really more directed towards ensuring that in the clinical trial issues that were raised here today would be addressed. And I guess as you re-iterated in your last slide the issue of biodistribution of the AP -- vector and within the LGN and the issue of what that means in patients and how we would go about exploring any possible toxicity that might represent to a patient clearly there are limitations to any of the animal models, both the dog, monkey and the mice, that are not totally representative of the human situation especially when it comes to immune systems and vector immunity as well. So what were your approaches going to be then for the patients? I had raised the question about scanning other studies to look at is there any possible toxicity in the optic nerve or optic kyism once they receive a fairly substantial dose of the vector.
Well, we were going to look first of all very carefully at the three month animals and see if there is any evidence there that would heighten our concern even more. We have planned neurological exams, but I think in light of your comment maybe we should have a neurologist on board to look at the patients in of that. The issue of MRIs was a possibility as an option.
The reason I bring this up is typically when we look at damage to the optic nerve of kyism we think of visual field and --
Very hards in the function is already severely -- even if there was toxicity to the optic nerve or kyism we wouldn't be able to pick it up by our routine what we normally do in a study which is look for visual field defect. That's why I raise the issue of doing additional tests, especially at this early stage in the first groups of patients because like I said, these things could easily fall through the cracks because what we normally would see is patients would complain of visual field defects or other things these patients won't be complaining of visual field defects. Because how do you exam the optic nerve or kyism in a patient who has severely impaired visual function already.
Gaining some experience in that already independently. One of the other issues we were concerned about and Dr. Geare at Penn have been investigating along with Dr. in the dogs is what is the MRI in congeneral blinding diseases. There is literature out there but not specifically in these diseases. We have looked at that and we were developing base line data on a bunch of the LCA patients and it would not be the relationship has been developed with Dr. Geare, the neurologist to look into that and his high resolution Kansas of the optic nerve for example are spectacular for the resolution that we were asking. That's not an impossible question to ask at the moment and I think it's a concern that should be addressed rather than ignored.
Again, what is your -- in terms getting to a second concern of the potential for a sustained ocular inflammation? So again
Excuse me. Dr. Csaky are you comfortable with that response then?
Yes.
So the question would be this issue again where the animal models have their limitations and primarily with the immune response and the issue or not these eyes especially in patients would be predisposed to a more severe inflammatory response either with the vector itself or with the fact that it's I think unless there was a known mutation I would be surprised that there would be a auto immune response and more concerned about the amount of vector that's being injected in an eye which has already a compromised blood retinal barrier. And so had you thought about issues of pretreating for instance in a patient -- before surgery we will premedicate the patients with antiinflammatories to prevent any kind of inflammatory response that you can't get ahold of afterwards. Had there been discussion about what specific surgical approach would be taken regarding these patients versus the anples which clear -- the animals, the dog has a mild form of the disease so some concern that it's really more clinically relevant here.
That's a good question. These interaction of the -- in Dr. McGwire and -- has been tried to iron out a real protocol about the surgery now that we have these data on what the human disease in detail really looks like. And I don't know if this would be answering your question or not, but there is plan to use Perry ocular steroids and perry ocular steroids and I we are seeing in the dogs whatever we feel about that in relationship to humans we are seeing an untreated mild inflammatory response. I don't think my surgical colleagues would let that go. They would treat it. Whether to pretreat meaning before surgery even is a good question. And we have not addressed that. Specifically. But it's worth the concern and I think Albert and I have to huddle those two with this question and come up with a protocol that actually specifically addresses it to ward off any inflammatory response rather than try to deal with it later.
Right. And then there was an excellent discussion on this issue of the variability of LCA as are all aware of and this really as you elegantly put this discrepancy between a structure and function and the again getting to the issue of the human disease that is the surgical intervention which is I think should be emphasized as fairly traumatic surgical intervention doing a retinal attachment can be itself visually compromising in the long term that is once we detach the retina sometimes the function will not come back completely to normal. Especially patients with compromises function and again the problem with the dog model I think should be emphasized it is a very mild form of the disease and so what we potential issues that we do not see in the dog may not be relevant to the humans. And so making sure that the patients are aware they have 20/200 vision that the surgical intervention could be severely compromising for their vision and whether you have any plans you have out lined this idea of using the OCT to detect some type of outer retinal layer, are you going to simply only take patients who have cones with cone function? Or I wasn't clear how exactly you basically showed that all the patients have loss of outer retinal layers in the periphery if you get toward the center they have some maintenance but what is -- I'm not yet clear on how you were going to select the patients based on the OCT findings mutationle findings how you will Corlett those two together.
They have to have obviously some molecular evidence of RP65 mutation that is credible. And in terms of the sicko physics and imaging criteria, that isn't 100% solid yet but results are relatively new, but the thought process as it evolves is that it may be less than sensible to put to make a receipt in themy scar in the middle of the only photo receptor group that we can detect and because that would be at least from the current hisso palology coming from the eyes ruin us to the visual vision that we are trying to at least test the safety of if not to possibly help. And the thought process is that we may do better by putting the receipt in themy scar in the area with less photo receptor or no photo receptor nearby and lift only a part of the retinal elevation reduced by the injection, a smaller blend than possibly the 150 micro liter bleb that is created in the animals adjacent rather than directly targeting the -- those patches of cells. Certainly if there is absolutely no evidence of photo receptors in the eye of one of the patients I think it would be both inappropriate, unethical and cruel to enter them.
Does that directly answer your concerns?
Yes.
About the variable feigno type.
These are unknowable in some points
And
These are a leap of faith and more of a matter of bringing into kind of record that these are things that the investigators will be reviewing and discussing. It's really more these are things that are at some point unknowable and I think as I said at the beginning investigators have done all they can in the realm of understandable knowledge that -- which you have to go in and I think -- and that's really it. So I'm comfortable at this point.
very much.
Thank you. I would like to thank the investigators for their very careful discussion of the questions and it's clear this is a protocol that has undergon a lot of thought and consideration. For the purposes of the record I will elaborate the questions that I asked many of those have been answered and a couple of questions remain which I will elaborate on. So I think the first point for me is a member of the RAC is the nature of the disease. It's not a fatal disease unlike a lot of the protocols that we reviewed here. Clearly had a huge impact on life so I'm not engaged in that anyway but perhaps that changes the risk benefiting assessments which have to be made. So my review was principally based on the aspects of safety of the protocol, perhaps from three different aspects. The first is toxicity as it regards to local injections surgical procedure that delivery of the vector not specific to expression of the transgene itself. The second is local toxicity which might be caused by the specific expression of the transgene. And then the third consideration is systemic effect through vector distribution expression of transgene and possible integration of the vector. Those were the general terms of reference of the questions that I put and as I say many of those were answered by your client, by the presentation this morning. My first point was related to the outcome of the ocular hiss pathology in the monkeys and that's being presented. And I -- superior knowledge in the clinical field as to whether these findings of inflammation itself will have an effect on the patients in terms of making the sight actually worse than it is. What I don't understand is whether locally inflammation will lead to destruction of vision over and above what is already present. Far more qualified to understand those questions than I am. I guess.
It can. And that's why we were -- we had two purposes to device this relatively sensitive measurement of vision that we will be using as a secondary outcome at least in the safety study to determine what the threshold or sensitivity is in these visually insensitive patients to get a series of base lines on them that would allow us to determine whether those base lines change negatively in the safety trial everyone thinks of it as looking for efficacy, but the primary concern was to have something to measure that would allow us to determine any negative effects, specifically those. So my second point I raised was the ocular hisso pathology from the three month trial which is on going coming July, clearly investigators place a lot of importance on that as I think the RAC should in terms seeing what those results are and the investigators are proposing to present those to us. So I think that was
I won't Hemi sect the eyes. I promise. The rest of my questions really relate to the expression of the transgene and perhaps some of the imnothing from a personal bias I should focus on some of these questions I don't have necessarily been answered but again maybe they are not necessarily answerable. So the next two points were really together. Will be the long term consequences of expression of a foreign gene in the contact of these eyes? So it's unclear to me as to the mutations which these patients will have whether they will affect for instance the single amino acid in a otherwise fully functional protein, immanology will have relative effect on a tolerance mechanism of this protein. On the other hand the mutations of patients are such that there are no protein so the patients have never seen RPE65 you might propose expressing fully functional RPE65 in the eyes might cause, might be seen as a completely foreign protein. The question is on whether there are any available studies perhaps with GFP or perhaps clearly blatantly followed if that is expressed from the AE vector in the contact of the retinal openthalium, does that cause an auto immune reactivity? Does it cause increased inflammation? The response which investigators gave was that they put human RPE65 into more than 28 dogs and they have not seen any antibody to RPE65. Again, this is ignorance on my part. I'm not sure whether how close human and dog protein is. So would you get the same sort of review activity against the human RPE65 in a dog, what is the mutation of the dog? are sort of very theoretically immanological aspects and if there was a point to make about protocol, perhaps the imknowology of the aspects maybe you have im--ology
I would have to defer to my colleagues who have done experiments which we didn't include in the reply because I thought it was better to discuss them rather than document some of them which are more anecdotal than not. It is a vast experience and some are graphed and some are worth mentioning and looking toward my colleagues for help. Dr. Bennett
I'm Jean Bennett and we think that's an important question as well. First, there is very high hemology between the canine sequence and the human sequence of RP65 both on the nukely youked it level andup amino acid level and so we think some of the data are extrapolatable. In our large number of dogs we injected AB in some dogs which had canine RP65-CDNA and other dogs human RP65-CD ta and looked at antibodies to RP56 protein in all those dogs. We looked very carefully and in fact we saw none. That's the best evidence that we can give far as what mutations in the human patients are equivalent to the ones in dogs, we don't know. We think that the mutations in the dogs and the mice are null although there may be some protein present that we can't detect and the challenge with the humans is that there is a wide number of different mutations which can lead to the disease and no two patients will be similar so we can only do the experiment, I think, in humans.
Have you detected well as antibody do you detect T-cell mediated reactor, do you have assays for this?
Using the dogs, there are no good reagent to do Ellis spot assays for example so we haven't done that. The studies can be done in monkeys. But we have not seen any evidence of cell muted response in terms of the hisso pathology infiltrate, for example. Dr. Vile, are you comfortable with those responses?
Yes, I am. I think it's clear that the investigators are aware of these issues and again it's difficult to know how to address those but aware I think it would be fort going in the trial and discussion with the patients -- and knocking on that door a little more, I wonders -- my next question is whether the investigators have tried intentionally to raise auto immune activity and by direct vaccination with approaching or virus expressing protein as a very worst case scenario to see auto immune activity can be raised and the response was clearly that they are thinking along these lines recombinant adeno virus and we wish them well in those studies. The next set of questions really related to the third toxicity and concept of distribution of the vector whether that vector integrates and what the of the transgene may be if there were integration. The biodistribution studies have been presented again and very carefully, very thoroughly and honestly sometimes you see positive with the vehicle control and not sure what is going on. From the protocol and perhaps from the response I was slightly unclear as to whether there was feeling this vector disseminates anywhere other than the eye. And in the description that was written there were these sporadic positive detection events which were considered to be not significant and again it was just little bit close to what a significant detection is and nonsignificant detection is. Whether you had a view or whether you think there is systemic distribution.
I heard my colleagues give views and maybe they should be the ones to discuss that. If they know first hand.
My name is bill houseworth. The word sporadic was used because the positives we found outside of the injected eye and the optic nerve in that eye were not consistent among animals at that organ site and were not clearly dose related. They were sporadic in that sense and as they displayed in the table they were sporadic. So we felt we can't fully explain why we saw those positives. Most those positives were barely above the detectable limit of 100 copies per microgram. So we felt that they were -- not Signet sense that they indicated a reproducible reliable measure of bio-distribution. Usually one time only and one animal and not necessarily the highest dose or dose related.
Provide distribution as you look by PCR for vector have you looked for protein anywhere? I mean, that would be one way if you see these sporadic events, can you have see that on --
Maybe Jean or Gus -- I don't know beyond the retinal sections by antibody hyper cation. I don't know if you can go back in the dogs and it would be the only.
We have done extensive immuno-- and protein.
And somewhere
No. The answer is no. Not in those distant organs. Thank you.
So Dr. Vile, is there something in addition that you would recommend for the biodistribution studies?
I guess again possibly hypo critical. One of the concerns have I is what happens if this virus were to get to the liver by some mistake or something bizarre happened or to some other organ and maybe intravenous injection of the virus to see where it distributes and so on and then the consequences were over expression in this gene and those other sites might be the worst case scenario experiment to make sure there is no adverse effect from there. I can't answer obviously. We looked at those organs but there is one cell type that receives probably as much vector as the RPE cell and that's the photo receptors which are right next to it. This vector would express just as well in photo receptors and we can't detect the protein in that cell. We don't know the guess is either there is a post transcriptionle difference or -- and or the protein is unstable outside its natural celled it, the thought is even if you had missed expression at distant sites the protein would not be stable. The evidence is by extraping will by receptors where we can't find it and there are plenty of gene copies there.
And then the other couple points was really the integration of the vector either within a target cells or in other cells and the possible role of the transgene on the target cells or possibly other cells so the -- my question -- direct question was the data to show integration within the target cells the response to that was that it's difficult to study integration directly in these populations. And other data have shown a profinancetive for nonintegration, was there any further information on that? I mean, there are methods to detect integration and so on and relatively small numbers of cell. I wonder if there was any additional information.
We provided some of the supporting data from other sites of injection that indicates that as a general rule -- integrated low frequency less than 5%. In the contact of the retina of the RPE, you have the point there is obviously insufficient DNA for -- behinderization cells are not able to be cycled. So it's not applicable. The other technique I would be a PC R based method and that has not been done. It is fiestble to do it, but it's been somewhat more difficult technically with AAV than with some retro virus because of GC rich regions and the ITRs but it could have been done but no one has devoted the effort to rather -- try to do that in these samples. That's the answer. Thank you.
This not a direct measurement of the vector that we intend on using. But a few years ago in antic dotele experiment we attempted to measure integration with another vector. GNP containing vector and we couldn't find a signal. That that doesn't mean it doesn't exist at some level. That's the best I could do experimental.
If you don't get a signal.
What does that mean, yes.
And related to that is I asked whether the RPE65 protein fully functional could in anyway be thought to have some effect on promoting growth in the trans-- cells or any other cell type putting those two questions together as sort of scenario that I'm trying to look at if the vector which integrate into either the target cells or perhaps into for example an immune cell which is infiltrating the result of inflammation is there any reason to believe that the combination of integration plus expression of the RPE65 may provide some sort of transforming event either for the target cell or some other cell. The response was I think good as they could be that this protein is an enzyme does not appear to have any relation to cell growth and therefore it's difficult to see that this have a real okaygenic transforming capabilities. And all of my issues have been addressed. If we come back just very quickly to the summary and safety issues and the toxicity and local issues, I think there is clearly evidence there is that local damage. I'm not qualified to say what that inflammation may do to the residual vision in these patients and think that's something that is under consideration and should remain under consideration. The local toxicity of expression of this transgene I think those issues have been addressed. I don't see there was any cause for concern there. And systemic effects due to vector distribution transgene expression and vector gene integration I think investigators clearly had these issues in mind and will follow those.
Thank very much. Dr. Heslop. I would like to thank the investigators presentation for -- my first question was about additional data of biodistribution which provide -- inaudible. I have questions about the direction of transdata I have serious questions about the auto immune which have been answered and to clarified in the by Dr. Vile. I did ask this in detail about the degree of vision impairment, the patient's role and criteria gene mutation but doesn't specify criteria for visual function and I think that response to the previous questions. I also asked about measuring decline in visual function because table that was under the present protocol doesn't address this issue and I written response there which has been discussed today. My next question was about the informed conson process and that these patients -- and how will that be undertaken and I had a written response that displayed how that would be done and my final question was there long-term follow-up -- inaudible.
you very much. Ms. Kwan. Thank you. In my review of the document I neglected to write in my critique that I thought the informed consent document was unusually well written given the many informed consent documents that we have looked at. I think there was very careful use of language so as not to suggest benefit that was not likely to be the case. And I think also in the presentation that was consistent with the presentation when you spoke about how you had been working with your presentation populations. And I just want to have in the record that it's very gratifying to see this type of informed consent. My comments had a lot to do with the fact that this was population that was not facing nonfatal disease and that people as young as 18 years of age could be potentially enrolled in it it was difficult and there was not really a response in either written form or in the presentation to address the issue because I'm not sure that it can be. I guess my concern is in dealing with a population as young as 18, net likely not like to see a benefit because it is Phase 1 safety more for the sake of the investigators themselves and the integrity of the experiment it's really necessary to be very careful in selecting the potential participants that they have a good understanding of their responsibilities in participating and that it is likely that they will continue to keep in contact and provide follow-up data in the ensuing 12 to 15 years. And also they would be fully understanding and cooperative with regard to the fact that they should not engage in sexual activity while the experiment is going on and at 18 years of age I think you are talking about a population that may very well be sexually active. So I guess my suggestion here is in the process of recruiting that you might want to engage the assistance of a psychology or social worker. That can help you evaluate the likelihood of the participant to be able to fill the obligations long term as well as short-term in enrolling in it.
And absolutely stellar suggestion. As I think we mentioned in the replies this has been a concern for us. And we try to know everyone as best as possible. And we also believe it or not encourage despite our enthusiasm to hopefully help these patients, we encourage them to continue their life paths as they will and they should not wait and I actually find I am very encouraging of them to find alternate sources of help. Do not inhibit their progress through life if the they are young and I won't say force upon them but encourage them to get guide dogs if they haven't gotten to that point for some social reason. And increase their independence -- independent of the prospect of any future therapeutic interessential or safety study. So these are great burdens. And I think you bring them out very poignantly. I think an addition of a psychology would be a wonderful thing to the team. It's a brilliant suggestion and I will follow it. Questions, comments from other members of the RAC? Yes?
You have in the statement three week at least three weeks before the dose escalation and the next one. Three weeks adequate to fully assess the safety in terms of inflammation and retinal detachment and other adverse --
Well, the Perry operative complications that can occur should be very obvious within that period of time. We chose three weeks not any fixed rule but it seems like a reasonable amount of time. I think longer could be equally acceptable. I'm not just benting to question but those details are not set in stone just yet. Certainly it won't be less. As we went through the safety data on mainly the monkey study, I assumed the end that we would review that and ask ourselves if there are late occurring complications that at least of surgery that we would need to consider when booking patients for next event. So three weeks seem to be a time that would cover those, but more may be indicated based on reviewing some of these at the moment maybe some of these link evering little responses in the monkeys that at one month may force us between one and two months.
If you will do one eye so if there is a difference in visual acuity or visual function between the two eyes is it the best eye or worst eye?
The intention was to pick the worst eye, assuming the worst eye was worst bio-chemistry, assuming bio-chemistry rather than total loss of outer nuclear layer or photo receptor components. So if one eye had one -- I think it would be cruel to use that eye. Because it would be no -- wouldn't be a safety -- even a safety drop. The intention was to use the worst eye.
Dr
Yes, have a couple points. I'm sorry I can't talk to you and use the microphone. But tremendous potential in this protocol and I congratulate you on such nice data. Just two points of clarification. One, you showed in a couple of monkeys that you had specific expression or vector in areas of the optic trend. So it's based not normally -- transported how is it getting to the couldlicilous and LGN and then visual cortex? And the second question still not clear to me which -- what are the important elements of this important information whether it's surgical or the concentration and volume of the vector injection. I'm wondering if you are controlling from percent of package in unpackaged particles in the vector which could influence the degree host reaction to the --
The only part I can answer and my feeling of presence from the data and from most of the viewed inflammation is surgical rather than vector related. But maybe the doctors or doctor Bennett have information on the information to answer your question of the packaging. As far as response to the --
Could you give your name.
Bill houseworth. far as the response to the caps that the -- it just came in yesterday. I'm sorry about this. These are -- these are the serum from the last group of dogs preand post samples -- pretreatment serums and then the serums at three months post treatment at termination. They are basically no rise in any of the AAP2 antibody. If anything they go down. You probably can't see. t they are near level of detection anyway. We aren't raising a capsid --
I'm thinking more local reaction. AAG and CNS the higher degree of packaged particles, the less reaction and the matter of the infection.
Locally, Jean, anything done in dogs? Jean tells me in the dogs she has looked at there is no local inflammation she can measure. That's an tearier chamber fluid.
Jean Bennett. We haven't done hisso pathology immediately after injection. We condition answer that from a his toelogical basis. The doctor presented there is some information shortly after injection in going for in some cases up to two weeks or maybe longer but it does subside. In response to the other question about finding evidence of vector in targets of gangian cell Axsons. Lateral jolicilous and -- I would like you to remind you that the access of the gang Leon cells terminate on the superior, et cetera, and so it would be natural to if there was virus in the gang Leon cells to see it in those organs. It should be noted the virus was not detected in other organs which would have secondary Axsonle contacts, for example, visual cortex so the data made sense.
I thought it showed visual cortex perhaps it's my mistake.
I think it was one.
One monkey.
What is the degree of packaged particles in the vector.
Looking back over particularly the dogs which we have been studying for the last five years now, we really -- purified -- we used two kinds of purification protocols the first one was which will give you 90% geneam containing particles and more recently we were doing a -- protocol because that's consistent and actually parallels the GOP production of the vector. It has more empties than fulls and in neither prep do we see a difference or clear immune response.
Don't see a difference even in the percent of RPE cells infected?
No. I must say we haven't done dosing -- a dosing study with the desk knoll prep. We were above the threshold for we were well above thar erapeutic threshold. Any difference you might have seen is inapparent at the dose we are using typically. Thank you.
Dr -- does that answer you questions? Thank you. Dr.
I want to thank the investigators for a very careful thoughtful presentation and your attention to the complicated scientific issues. I wanted to follow up on a question raised earlier about the long-term consequences of the subretinal -- surgical procedure and to ask having had this subretinal injection and retinal surgery, are these sub -- now at greater risk for long term adverse events such as post Cappsler cataracts, retinal detachment and retinal hemorrhage because they had the surgical disruption? Is that a possibility?
That's a good question and complex question. Patients with these retinal degenerative diseases are prone to sub Cappsler cataract by the basic disease process whether they are accelerated by the intervention is a good question. We don't know. Assume there was no evidence in the monkeys that at least two months that was occurring but that's two months. That's not type of long term you are talking about. Sorting out the the appearance of a poseterier sub Cappsler cataract in a patient with retinal degeneration as it relates to the treatment or introduction of the vector, will not be easy, but there will be a time line that will be and there may very well be any surgical procedure in the vit Reyes and the retina ap increased chance of cataract. We listed that as one of the concerns and this as an adverse event and categorized it. Whether the retinal detach chronically is an important question. The tether of the retinal openthalium to the photo acceptters will be disrupted. And it will be disrupted in a very diseased eye. We will pick and choose locations that will be less diseased for the advantage of the patient to try to circumvent that problem but chronic retinal detachment in a diseased eye hasn't -- this anecdote, I apologize, but retinal surgery is not done on retinal degeneration patients in this form. So subretinal injections are not the usual. Cataract surgery is performed and would solve the other problem. But subretinal surgery is usually for retinas that are diseased in other ways. There is no vast experience. Retinal detachment is a very unusual phenomenon spontaneously in retinal degeneration patients and when I had patients with small retinal detachments, they had resolved but those are unusual patients. There is no clinical background to answer your question here. There a chance that it would enhance and that it was part of our adverse event concerns. But no strong clinical or other evidence to support that. And certainly in the animals there was no very obvious retinal detachments other than in the initial monkey one week study but in the long term dogs there wasn't long term retinal detachments of significance.
Greg akeland. I think we should mention in the dog studies we had several followed up from a minimum of six months to now four, pushing five years. And four years, sorry. And there have been no detachments in any of those dogs. One Doug is still alive now. Four years post therapy, 4 1/2 years post therapy and eye is fine and some have been necropsied at times from minimum of three months to period of years and no was there evidence of -- or detachment at hiss pathology.
I would like to offer a suggestion. I want to first start by concurring that I thought the attention you gave the process was exemplary and I applaud the consent form and your plans to really speak with the participants and assess their comprehension.
Just one second with apologies. I wanted to extend extreme gratitude to someone who isn't here, Mrs. Sharon swarz and her collaborator, miss Margaret Humphrey who did most of the work on the consent form and truly the most decent and careful people that I ever worked with. Sorry. The unsung heroes.
As I read through the replies -- revised consent form that you gave us with your -- packet today. It struck me there was an emphasis or I inferred were short term, risk of complications of procedure. Suggesting a sentence or two about the possibility of long term reasons including long term -- can't anticipate because some the patients will be quite yuck and it really is a lifelong disease. People who otherwise have fairly long life expectancy.
We will could that.
Dr. Csaky.
I overlooked one question and actually was back to this question of the infectious -- ratio. I wasn't clear and I overlooked this when you were in terms of the injections you are talking about injecting a certain number of infectious particles per patient, is that correct?
Well, that's a question I ask Bill all the time.
Vector genomes.
Okay, so will there be some limit when you do your purification as painful as it would be after doing a GMP run when you find there is not a reasonable ratio of empty capside to genome containing particles there is some ratio you would say this is unacceptable and you wouldn't use it for the clinical trial.
I don't think there is really evidence to support a specific criteria on that. I mean, there is institutional history of the proportion of a fulls to empties from these preps and relatively small amount of variance. But we have a product reviewer here from FDA who might be able to comment on that. But lack of any specific evidence that there is an issue.
I guess I'm referring to the fact that if you will be doing the trial and you find that you have an unusually high but let's say a rather high run of empty capCID you would be injecting proportionately more capsid which could be potentially toxsic and could be concern with these being unknowable issues and in a human patient you might want to orb trearl set for your -- arbitrarily set for yourself criteria this will be unacceptable.
I don't think it's unknowable to say that within a dose that is well in excess of the per-patient dose that it is not in fact the highly inflammatory or highly inflammation-ogenic or the term you used so I don't think there is evidence the capsid are highly pro inflammatory or direct kind of response. Your argument is empty capsids are toxic impurity but I don't think the evidence directly supports that.
We just don't know that in the subretinal space and that's my point. The tissues in the human patient in present --
I think to kind of explain it a different way, I think the approach is that trek P proportionate -- through this methodology has a fairly safe -- safety profile in preclinical data that you see before you. So what the attempt is to be consistent with the same purification methodology and presumably have the same approximately the same product in the patient as was in the preclinical approach. Now, if the course of those multi-different preps and the preclinical data there were some that were much more toxic and those had a property of a higher ratio or low ratio fulls to empties, however you want to phrase that, then I think what you are saying would fit with the data. But right now I don't think there is data to say that if it's 10% full, it's good. If it's 3% full it's no good. So I mean, that's why I said I don't know that there is data on which to base a specific lot release criteria based on empties to fulls.
Help us out?
The one thing is leading out here there is is quality control and how these are manufactured. So certainly the doctor has considerable A experience so these locks, there is not that large of variance. In this. And there is also lot release as he mentioned and so you do keep track of that. But were not talking about making one lot that has one to ten and possibly another lot that has one to 2000. There isn't that kind of variability.
Right, that's the point -- the historical consistency there is. This procedure appears to be making something relatively similar in its behavior over its period of time. I don't know if that answers your question exactly the way you want it.
Dr --
I want to expound a little more on this point and it seems to me that it would be interesting to have data in mouse. The goal is to have your maximum efficiency of infecting RPE cells where you have a minimum number of cells left so the question is, is that a function of percent of package and unpackaged particles? In mouse you have 90% packaged particles do you get many more RPE cells? In affect in mer vector dose than with --
It's an interesting question. I don't think the data supports a big difference. When you dose based on genomes I believe the dose in the retina bill correct me if I'm wrong, doesn't seem to grossly affect the proportion. You would have to be in a lowcally near saturation for that receptors for be the case.
I think as I said before in mice we haven't done this kind of dosing that would directly answer the question. But as I said before, vectors made two different ways. One would racksmise the ratio one which would min merchandise it and we don't see a difference at a relatively high dose. Vector genome we have. And the other -- even at the 3X dose to certain number of vector particles we only see a modest immune response, very modest and transient. I can't tell you exactly what our highest dose in the clinical trial will be, but we were centering on something about ten fold lower than that. Even a very extreme prep of the GMP vector has this ten-fold advantage that we will be lower vector genomes than highest coast we currently use.
I couldn't see the eye test Dr. Jacobs gave us with respect to the biodistribution. We understand that there is one monkey out of about ten that vector in the visual cortex with that -- was there any quantification of the numbers of genomes there where there many genomes viewed? And was there any examination of distribution of vectors of the genomes in that region of the brain and in that particular monkey?
Bill Houseworth, the copy number was in the 3X one in the 2X animal and it's 200. It's just above the minimal reportable copy number.
Sensitivity is about 200 genomes.
Sensitivity is 100.
And in that animal just for comparison, the number of copies in the retina is 470,000 vit Reyes and 91,000 in the injected eye.
Was any hisso chemistry done or distribution studies looking at cells in the --
No, there wasn't any -- there was no insight to behinderization of those Tishers.
Of the many exemplary things in this study the collaboration among investigators is tremendous. Just a question. Am I correct in understanding the study will be done in Florida but you will be doing the examination in Florida, is this correct?
The design of the study is evolving and will be probably coming back with a evolved version. A current plan is a two center trial that will occur at the university of Pennsylvania and at the university of Florida. There are advantages of both settings and that has evolved over the last month or two. And we were actually waiting for an NIH grant that was submitted recently to permit that to actually occur.
Any further questions from members of the RAC? Then doctor?
I would like to follow up on one of Dr. Heslop's questions about the informed consent process in individuals who are -- read the inform could be isn't document. I would recommend that you give the subjects perhaps like a cassette tape and make sure they have some way to listen to the tape so they can actually review the information when they go home and even after the study starts. Or ensure that they have some way to read the informed consent document or provide it in Braille if they are able to read Braille so they can have again not be dependent on other people to read this document to them when they need to refer to it.
I think that's an excellent idea. Thought of u enumerable number of responsibilities in that regard and even test run that type of situation with one of the patients for another purpose just for a phlebotomy sample by borrowing my daughter's iPod and had a little microphone attached to it. So there is certainly the small cassettes I think are less expensive and possibly a better version of that idea. But thank very much. That is a big concern to us that they listen to it and understand it.
Other comments from the public? All right, I have a few concerns that remain which I will read to you. And please feel comfortable correcting me. And or adding. Especially you, Dr. Vile, I'm not sure I caught exactly what should be reflected in this document. As everybody has commented on, this is the first AAV vector trial with AAV injected into the eye and plan for subjects with a nonfatal disease. Therefore our interest in this protocol. The terms of the preclinical data I had two remaining concerns for the group. The first it is important to carefully review the ocular hisso pathology from treated monkeys at three months. The group still remaining. To utilize the fort coming information in the design of the clinical trial. Obvious. The potential newly expressed protein into the human eye should be fully considered. Dr. Vile, does that capture your issues?
That's good.
Clinical. Biodistribution of vector to the optic nerve or kyism in the dog model mandates the very careful clinical assessment of subjects careful definition of potential toxicity in man which cannot be assessed by visual field testing leads us to suggest MRI using high resolution scans of the optic nerve. Next, ocular inflammation UV-its was observed in the animal model observed in perry ocular steroids to modulate in man and addition consideration should be given to pretreating subject with steroids. That is prior to surgery. Next, the protocol includes a three-way ky yaitous between the accrual of a subject at one dose level and initiating the next cohort. The RAC encourages the investigators to carefully review the full set of animal toxicity as well as consider potential toxicity in man to redefine this time interval. And then I move to inform consent. Is there anything I have not included that is clinical or preclinical that you all would like included?
Dr. Vile?
I come back to the issue we discussed about whether the vector is integrated or not. I think it might be nice to put in the recommendation that perhaps consideration to those studies should be given in the consequence of some adverse event occurring whether this vector is integrated or maintained, I think it would be important information to look at.
And you would do that in the animal model?
Yes.
Consideration of evaluating model integration and the animal model.
I have to say I don't really agree with that recommendation the event is so rare and AAV has been used in so many clinical trials that I would hate to hold this up for that type of information.
Okay. Thank you.
I will take that out.
I just have one point on that. For the risk to when we -- I think it's important since in essentially the investigators are in a bine and are taking patients that have some function because they feel those are the patients that would have some degree of success, but those will patients that have the potential for losing the remaining vision from that procedure itself. So I think it's very for the patient's sake that they understand we are not taking completely blind patients. We are taking patients that are functional with central islands of visions of 22 hub they can ambulate and do a lot of things and you have the potential of albeit it's not supported by the preclinical data but obviously given the limitations of those studies we have the potential for taking away that remaining vision and I think that needs to be emphasized so that these patients who understand that how heroic they truly are in going through and basically undergoing this experiment.
And so if I -- I haven't done the informed consent yet section. So let me try to integrate your comment there. I think that's where it belongs. So informed consent one as young as 18 years of age are likely to see who are unlikely to see direct benefit will be enrolled. We encourage the investigators to engage the assistance of a psychologist or social worker to help evaluate the capacity of the potential subjects to adhere to the protocol and to participate in long-term follow-up. Two, the informed consent should explicitly state that the risk exists. That their remaining vision will diminish because of the surgical procedure?
Diminish or be lost. Not just diminished. There is a potential they could go to complete blindness with this procedure and I think the risks should not be minimized.
Three, consider revising the risk section of the informed consent document to include a section on the potential long-term risks following the retinal injection of product including retinal detachment as well as those that may not yet be anticipated. It is suggested that the informed consent be provided by cassette or in Braille so that the individual subject with severe visual impairment can review their own document. And those president four IC comments that I took. And anything else that people would like added? All right, may I have a motion for approval then? so moved. Second, Dr. Lo. And we will take a vote beginning with Dr. Vial, Dr. Webber, Heslop, powers -- is in recused. Barkley, Rosenberg, Lo, somery, Wara, yes, DeLuca, bone, due hares, Ms. Kwan. Thank you all very much. We will take a brief break. We were scheduled to regroup at 10:15 for a brief presentation on biological containment. Noncontempt rather strains of influenza if people could return to the room at ten after 10:00. Thank you. Test test test test test
If everybody could please steak -- take their seats, we'll reconvene. We're going begin. We're going to begin this last session of the hundredth meeting of the RAC. With a brief presentation on the biological containment for work with noncontemporary of influenza. Dr. Kathryn Harris from OBA will introduce doctor Jacqueline Katz, an ad hoc presenter from the CDC, and I understand that Dr. Katz, then, will explain the contemp rapeeous guidelines. Dr. Harris.
Good morning, everyone. In this session, we will be discussing the recent changes to the recommended biological containment level, we'll work with noncontemporary of of influenza. We'll intriek -- describe how the classification of the strains is being revised in the 97 guidelines to reflect -- 97 -- NIH guidelines to reflect them. I would like to introduce Jacqueline Katz, the chief of the immunology environmental pathogen enflewenza yank -- branch at the centers for disease control and prevention. Dr. -- katz will discuss the changes for work involving noncontemporary of sties A. and also discuss the rationale behind the changes. -- involves noncontemporary of of influenza and also discuss the rationale behind the changes.
Thank you for the opportunity to address the committee today. I am just going to go briefly over the, over the process and the rationale for updating the recommendations for working safely with influenza viruses and in the laboratory. As you all know, this is part of a process of updating the manual, the biosafety and microbiological and biomedical laboratories, the department of health human services publication, which provides AND NIH GUIDELINES FOR THE safe handling of the infectations in the laboratory. The update is done roughly every five years. now up the fifth edition which, is expected be published in the fall this year, and my role was as a subject matter expert on the committee of subcommittee that was formed to provide guidance for the updating of the agent summary statement for influenza, and, of course, this manual provides recommendations that are advisory in nature and they're really, they are there to provide a basis for individual laboratory risk assessment by principle investigators and their institutional biosafety offices. So this process started just a little over a year ago when the subcommittee of subject matter experts for inflewenza were formd and this committee involved myself and another colleague from CDC CRPZ -- and a number of ideas -- individuals from NIH, USDA personnel with expertise in avian and influenza and NIH grantees from the community. And our role was to review the status of knowledge of influenza viruses and provide guidance to the steering committee and then provide a peer of youth for the preparation of the document. So that's not -- now being 99 -- done, and we have a penultimate version of the agent summary statement, and that's being taken to the steering committee, which is -- consists of both CDC and NIH representatives and they make the final recommendations and provide general oversight for the revision process. So, we finalized the document just a few months ago and due to the inadvertent release of h2-n-2 virus as a pro-- virus in a proficiency testing kit, which I am sure many of you heard about, CDC and NIH decided it was -- that we should post interim guidelines and this is now on the CDC website with excerptd the essential biosafety recommendations from the draft, and it can be design -- scene at that website there, and we expect that there will be only minor changes, editorial changes and so forth before the publication in the fall, but this, these recommendations have been reviewed both at CDC and have been approved all -- up through the office of the director of NIH. So just this is just a highlight of the key changes that have occurred. The fourth edition recommendations really addressed influenza viruses as bsl-2 agents in recommended facilities and practices that were acceptable for working with influenza viruses in the laboratory, and this did include noncontemporary human strains such as the h-2/n, two influenza avererous and didn't address with working with the avian enflipenza viruses and didn't address working with viruses that contained some jeeps or all Gene -- in genes or all genes of a 1918 virus, and of course, these are more reconvenient recent issue that have come up in the last several years, so therefore, the new commendations had to address all of these issues. So, what will be in the 5th edition is that BSL-2 is still recommended for contemporary human strains, the currently circulating human influenza viruses of the h-1 and h-3 subtype and type b viruses and low patho genicity influenza viruses of most subtypes excluding h-5 and h-7. Noncontemporary human vine -- strains like the wild-type, human h-2/n-2 vis I haveerouses, it's recommended they should be worked with at BSL 3, or animal BSL 3 w protection, and I'll go through in detail all the different categories, and likewise, BL 3 or animal BSL 3 with protection and shower out for personel is recommended for any work with viruses possessing one or more Genes of the 1918 influenza virus, or the highly pathogenic avian influenza viruses. The key laboratory safety issues for influenza is generally inHalation of virus from aerosols. And this -- there's really been little documented information about lab-associated infections and any information is generally anecdotal. There have been rare literature reports of laboratory infections with novel strains such as avian influenza viruses such as the h-7 viruses, but in the laboratory, the main hazard, as I said, is inhalation of the virus from aerosols that are generated whole either infecting animals or processing infected tissues or manipulating high concentrations of viruses and processes such as sent -- centrifugation there. Is a possible risk of inoculating mucous membranes if you have virus contaminating your glofd hand. -- gloved hand; how far, now that reverse genetics is an approved process in the laboratory, we need to understand any has the potential to alter the host range and the pathogenis etand the aspect genic composition of the -- antigenic composition of the virus. of all, the rationale for elevating the recommended biosafety levels for the noncontemporary human h-2/n-2 viruses, and we're -- here, we're considering wild-type viruses. These viruses we know can infect hows but have not circulated in the population since early 1968, and so a large proportion of the population, including graduate students and post-DOCs of that age have no immunity to this, to this virus, and, therefore, we must ear consider that there is some pandemic potential with this virus. However, like all other influenza viruses, the h-2/n-2 viserouses are sensitive to two inflewenza antiviral drugs. The inhibitors and there is a therapy for treatment -- treatment with these, for infection of these viruses, and therefore this is categorized as this virus as a bsl-3 agent. Recently, we also found out that the vaccine seeds docs that were used for production of the vaccine back in the late '50s and early '60s against h-2, n-2 virus. It's still in existence so a vaccine could be made; therefore, the recommendations are now that work with these wild-type h-2/n-2 viruses be conducted at bsl-3 level, or animal bsl-3 willful using bsl-3 facilities and practices and procedures, and it's also recommendd that additional protection, another from negative pressure hepa-filtered respirators or positive--- respirators be used and recommended a complete clothing change. That means changing out of street clothes and into a -- into scrubs and using disposable surface PPE, no shower out for this, for this virus, and although it's in the current draft that on the CDC website, we are working on putting in an exception statement to exclude viruses that are attenuated, and is particularly for the cold adapted live attenuated h-2/n-2 vaccine strains that are now used for manufacturing the flu mist live attenuated vaccine, and this is a particular exception because these had many, many years of safety testing, and we know that these a 1088d. So we believe that they can stavely handled under bsl-2 and we're working on adding a statement to exclude that particular category of viruses. The low pathogenicity avian viruses, avian and all other animal viruses are regulated by the USDA, so there are particular permit requirements for working with an Avenueian inflewenza virus in the laboratory, that rush -- usually requires a laboratory in-- inspection as L. so, bsl-2 is considered appropriate for the low path avian strains of influenza a viruses of h-1 to h-4, h-6 and h-8 currently, the draft said h-15, but recently in the literature and I -- a new subtype, h-16 was documented so that will be amended. Now, it will be h-8 through h-16 with a low pathogenicity strains, and then depending on USDA approval and the permit to work we -- with these agents, there mean additional aphous requirementings. You -- requirements. Can you see we have excluded low pathogenicity h-5 and h-7, this is again USDA-driven, because the viruses have the potential, once they're manipulated in the laboratory to become highly pathogenic, the USDA is particularly concerned about work with the viruses and so additional permit-driven containment requirements might elevate the level of containment for these viruses, that's the USDA concern and not a CDC-NIH concern. The highly pathogenic avian strains, again, are regulated by the USDA and there is a particular concern of the agricultural and economic implications where these viruses were -- to be released to the environment, and, again, aphous permits are required. These I have rues are agriculture-select agents, so there is additional biometric security issues involved with handling these viruses. Some strains, as we know, clearly pose a risk to human health and then, therefore, also risk to the laboratory worker. But, again, they're sensitive to at least one of the influenza antivirals and near -- epHectors and there are numerous vaccines that have been under -- under clinical e valuation, including one ongoing now. Again this agent, there is therapy for the agent, the bsl-3 is the recommended containment level and with rigorous adherence to additional or spiritually protection and clothing change and a shower-out protocol is recommended and may even be required through USDA permit needs. The recommendation is, again, for use of negative pressure hepa-filtered regulators or pathers, and this, there might be a need for hepa highlight filtration of laboratory exhaust air, depending on the particular facilities, the USDA may also roar additional, may also have -- require additional, may also have additional requirements for approval of the permit to work with the strains. And finally, a new category have had to consider because the genome of the 1918 viruses have now been almost fully elucidated and so there is a number of laboratories working with reassortments that contain either one or more of the 1918 viruses and very soon it might be possible to actually fully reconstruct the entire genome of the 1918 virus. And really the the laboratory worker is unknown, but we have to assume that there is a potential for enhanced vir lens based virulence based on our understandings and the documentation of the pandemic of 1918. It's not really known the extent of protection the immunity in the population. There were studies in animals that suggest current influenza vaccines, which contain a related subtype, the h-1/n-1 virus, that might be some level of protection, but that's still not clear and additional risk assessment data is desirable, but, nevertheless, we must assume there might be some pandemic potential associated with viruses as a of the population may lack immunity to this early h-1/n-1 virus. But, again, we know from studies in animals that these viruses are sensitive to both of the influenza antiviral agents and it's quite possible it's being under discussion that recombant -- recombinant vaccines could be produced. Again, the subcommittee consensus is that this should be a biosafety level 3 agent, and so those recommendations have been made using bsl-3 facilities, practices, and procedures and particularly for large laboratory animals, they need to be housed in a barrier system within the bisafety level 3 suite, and rigorous adherence to the spiritually protection and clothing change protocols are required, well as a shower out prior to exiting the laboratory. Again, the recommendation for negative pressure hepa-filtered respirators or pathers and treatment of the exhaust air by hepa filtration. So, for any work with influenza viruses, and particularly, we felt the, the committee felt it couldn't address every individual situation and so with these main recommendations for these categories of viruses, it's possible for institutions and PIs to develop their own risk assessment and additional guidance is given to should be considered when you're thinking about the biosafety level for working with a recombinant or reassortment of the viruses containing one or more Genes first different viruses I have just discussed. And so the Gene constellation that is use side critical. Obviously if you have surface glico proteins of the virus and they're not currently circulating in humans, then that is -- there is a definite risk for having's suset -- susceptible population, and generally if, if there are two parental strains that I use to generate a reassortant virus, the biocontainment level of the parent, which is higher, should be used as a issue of measure of safety and a conservative evaluation of the risk of infection with these viruses. One should also consider evidence of clonal purity and the stability of the reassort entthat is being produced, and then for lowering the biosafety containment level, clear evidence of attenuation of a strain should be determined in an appropriate animal model, in a model where you know you can reflect the virulence of the virus, and in that model, you should demonstrate reduced virus replication compared with a wild type parent. Finally, for work with many of these viruses, the committee felt that it was very prudent to include recommendations for developing occupational health considerations, so for work with highly pathogenic avian influenza trains, other strains that have infected humans, the noncontemporary strains like the wild-type h-2/n-2 viruses, or any 1918 viruses, their reassortants, one really should set up a medical surveillance and response plan for the employees and the first thing, and I know this might be controversial in some institutions, but the recommendation, there is a strong recommendation to store baseline serum. In some cases, that's the only way you're going be able to tell if you have, if somebody has been infected. Also, annual vaccination should be recommended and should be available to employees working with these strains. The employees should be councild on knowing how to -- counseled in knowing how to recognize the potential influenza infection, the fever and should be a plan how to monitor symptoms in an individual, and also a plan, defined protocol for how to respond to a suspected lab acquired infection, and that needs involvement from the occupational clinic at the institution, it may also need as much detail as knowing where you would hospitalize or isolate a patient, were they to be, to have a laboratory applied infection. And finally, there should be available antiverule -- antivirals for post exposure proofo lactics and treatment, and so all of that nods to be built on into a comprehensive medical response surveillance and response plan for working with any of these agents an institution. So, I'm going to finish there and just acknowledge Dr. Deborah Wilson and Dr. they were the -- from NIH. Deborah Wilson is also on the steering and she took it upon herself to really put together the final document, which was reviewd and modified by other members of the influenza subcommittee. Thank you. very much. I would like to defer questions for just one moment. Dr. Harris, I understand you have final comments.
Thank you, Dr. Katz that presentation. I will make the short presentation on the revisions to the NIH guidelines which, are being made in response to those recommendations. As of you know, appendix b of the NIH guidelines classifies microbiological agents into risk groups according to their pathogenicity for healthy adults. There are four risk-group cat doory -- categories. At the lower end are risk group one agents, agents-generally not associated with disease and healthy adults. At the highest end of the spectrum are the risk of four agents and they're agents likely to serious disease for which preventive-or--- or therapeutic interventions are not usually available. The NIH guidelines also assign containment levels for certain of experiments involving the risk group specific agents. And these containment levels are often equivalent to the risk group but be raised or lowered with institutional biosafety committee approval depending on the comprehensive risk assessment. Appendix b of the NIH guidelines currently lists all the influenza viruses as risk group 2 agents, generally would correspond to a starting point of-- biosafety level 2 of agents. The NIH guidelines don't differentiate between various influenza strains and the risk group classification listing. Now, even though the guidelines state that containment levels higher than the risk group classification might be appropriate, we want to avoid the potential for any confusion or the presumption that all influenza work can safely be conducted at biosafety level 2. We want to make it readily apparent that biosafety level 3 containment is appropriate for research with the subset of the epflew evidencea viruses, that is the non--- noncontemporary strains T. that being a risk assessment should be conducted when working with any agents to determine the specific appropriate containment level for the activity being performed. And as you heard from Dr. Katz, V -- CDC CRPZ CRPZ ANNOUNCED THE -- announced to -- from biosafety level 2 to biosafety level 3, and that decision was made in collaboration with components of the NIH after taking into consideration the pandemic poterm of these strains and the need to provide an extra margin of lab worker and public safety. In addition to the announcement, of course, the upcoming new edition of the bmbl will provide similar guidance with respit to the non--- noncontemporary human strains research involving reverse genetics involving 1918 influenza and the highly pathogenic influenza, h-5, n-1. So, in response -- response to these new recommending as, NIH is revising and clearifying the NIH guidelines risk group classification for the non--- noncontemporary strains of all the mix of viruses. We'll be specifying that the risk group classification for the strains is risk group 2. This includes h-2,/-2,- h-1 hie -- highly pathogenic influenza and 1918 virus. The contemporary strains of the veus havees will obtain their classification as risk group 2 agents. We will also be providing specific biosafety recommendations for the research with the noncontemporary strains that are in with the recently publicized recommendations that Dr. Katz just discuss. We consider the changes to be necessary to Harmonize the NIH guidelines with the other federal guidances to avoid confusion in the biosafety community and with investigators. So, specifically, the changes to the text of appendix b of the NIH guidelines, up --um, are shown here highlighted in blue. Again, the contemporary strips will ob17 their classification as risk group two agents and the ex~ exexception coos exceptions will be listed as the noncontemporary strains and the highly booth -- pathogenic influenza, avian. They will be specified in the a pentics b-2-b as risk group 3 agents, this is the text added to that section. Text will also be added to six -- section 2-a-3 to highlight the importance of considering a number of factors when working with these strains. These factors include those discussed by Dr. Katz, which include the number of years since an antigenetic virus last circulated, where the pandemic potential is significant and whether the risk assessment data is available. So, in addition to stating that non-tim strain -- non stem strain, practices, procedures and facilities in level 3, a number of other malicious, including vigorous adherence to clothing change protocols and the use of hepa fit operation for exhaust air will be recommended. So, to sum up, the NIH guidelines, the NIH is revising the NIH guidelines, classification for the noncontemporary strains of influenza to reflect the new guidance, regarding the recommended containment for these agents. The classification for the noncontemporary strain of influenza in appendix b will be risk group three, specific biosafety recommendations for the research with the strains will be added to section of the guidelines and contemporary of influenza will con-- obtain their classification as a risk group 2 agents. Thank you.
very much, Dr. Harris as well as thank you, Dr. Katz, and I would like to open both your presentations up to questions or requests for clarification. Dr. of all, I really appreciated the cloveds from both of you. I wanted to make a request if we could post those on the website we're using to post this. Dr. Katz is in particular, very detaild and has useful information to IBC. I want to talk about the question between the harmonization of the NIH guidelines and the bmbl. The material we received relating to the rewrite of the NIH guidelines is much less nuanced than the presentation. Dr. -- Katz -- differentiation between strains and the differences in terms, for example, the shower requirement which gets that facilities instructd, and I guess I wanted to request the NIH guidelines to reflect that level of nuance, quite important.
Right, I agree. I think we will need to look at the, particularly the shower disterchgz. I don't think that was very clear in the language that we have proposed. , that obviously, we will specify that that is for the avian enflew apse and the 1918 influenza, rather than the h-2, rather than the noncontemporary human strains. That's something we should look at.
Thank you.
The other question, I guess, or clear -- clarification, I wondered, what exactly is a noncontemporary strain? We heard today that 1968 is considered noncontemporary. Is there, and I realize this is probably a very arbitrary distinction, but there a magic time period that we're thinking of here that would determine something to be considered noncontemporary?
I'll turn it over to Dr.
Non--- yeah. That's a very good question. Clearly the h-2, n-2 viruses that circulated between 1957 and circulating to 1968 1968 are the classic example because we know that there is a population born after 1968 that has no immunity, and that's really what we're trying to really cover. But you could argue by the same token that in 1968 when the h-3, n-2 virus emerged and it's currently circulating today, we know there is being tremendous antigenic variation there, and we, we expect that the, the individuals that have had recent exposure to either h-3 or h-1, and that's a majority of the population will have some level of immunity even to those earlier strains, but what level of i mine -- immunity, if not very well-defined or understood. So, we're really categorizing non-conTim -- noncontemporary as categories that didn't circulate, and 1918 falls into the category. It's an h-1, n-1 virus, we currently have h-1, n-1 virus circulating. It's not clear toda what extent, the immunity of current strains, the strains of the last 10 or 20 years would protected -- protect against a 1918 virus, so I think clearly the way we have categorized it is the human h-2, n-2 subtype. We would also consider avian influenza viruses, although they're circulating in avian species worldwide, they're not in the human population. Does that answer your question some knows?
Dr. Barkley? Dra.
I'm certainly pleased to see the harmonization of these two sets of guidelines. I think that's very, very useful, and I really commend, really taking the initiative to see that was done very, very effectively. I also like the fact that you -- that in their efforts to do this -- they stay within the general context of the guidelines, which I think is important, although I think, I do agree with Steve's comment, there is some disconnect and how people might interpret the two, so I think this is -- I time and approach for goodlines, and there might clearly be a reference to the CDC documentation and as a principle source for carrying out risk assessment, so I think it would be helpful to de -- delineate that in the guidance. That's the h-2, n-2, strains as a lesser risk than that of the others, and I think that would help emphasize that. I was also pleased that you found a way to alter the guidelines under the minor changeegate -- category, which excelling -- accelerates this guidance at this time. Had you not done that, we may have been asked to look at it more restrictively by the congress or some other entity. So I'm pleased with the Indiscernible ] With which you proceeded with this. Thank you, and we will look at getting some language in that to cliniate between the h-2s, n-2s and the other strains on the consideration.
Dr.
Yes, I think the most important aspect of this guide and seizure for the majority of the work, which will be making reassortent reassortants between highly pathogenic avian flu or 1918 and contemporary strains of flu and, um, I believe in the, in Dr. Katz's presentation, at one point early on, it stated that if one Gene from one of these more pathogenics were included into a reassortant that it be done at bsl-3 -- later on, there was a risk assessment. I think -- is it your intention to say okay, that's done it, bsl-3 until there is -- .
I thank would be appropriate. I that's the way year -- we're handling it now and other institutions. The cdc is handling it that way in the work that we do there will be situations where one Gene on the background and the contemporary have us have going to pose a very high risk and because we can't, the bmb can't address every situation, that's what the guidelines at the end of, for the particular institution to develop their own rationale for lowering it and, again, we would advise -- advise a risk assessment would be done and some data be provided -- not highly virulent in a animal model. From the standpoint of ibcs, it would recommend it's clearly spelled out in both the cdc documentation and then in the NIH guidelines, that yes, this is -- you unusual -- initially started this higher level of containment but it can be lowered pending a protocol-driven risk assessment.
Right. Right.
Okay, otherwise if it's a standalone thing, this is done at bsl-3, ibcs come in all flavors and different people see that and they say this is it. This is going be done at bsl-3.
As I said, these are only recommending as and the best that we can do is provide the general categories for bsl-2 or 3 and there's going to be a myriad of situations that fall somewhere between and it's the whole idea of the bmbl document is to provide guidance for a proitol -- protocol-driven risk assessment at the institutional levels.
I wanted to make sure that that was, I think that's the most important thing to make clear because that's the thing going to happen most often.
Right. And cause the greatest amount of confusion.
Right, and the latter part of the agent summary statement addresses those issues.
Dr. M u
I would echo Dr. De Luca's comments. They attempt not to attempt their own personal risk assessment assessment, for one thing. Know you -- knowing you can do that, even if they --y that outsourced to another agency, it was helpful to the local investigators. The other thing I wanted to revisit was the contemporary-noncontemporary issue. Somebody sets on my local IBC, that's going to be confusing. I was wondering if there was any reason, given that we're talking, I guess, about a limited number of strains here over the last, since the beginning in the 1900s, there is there any reason why we could not have a supplement -- supplementary take table in one the documents, listing what your consensus is as a contemporary-noncontemporary strains. Again, this is the local IBCs.
Real -- really, unfortunately, the non--- noncontemporary term has sort of taken a life of its own, and in this document, we really just refer to the noncontemporary h-2/n-2 virus, and the other categories are 1918, which, obviously, is noncontemporary, but the 1918 virus itself, but as a subtype that subtype is currently circulating. As I said earlier, it's without information of knowing the level of immunity in the population, it's difficult to categorize things. And so I think the, I think the, just ge-- defining the different particular strains or subtypes that we're dealing with is the best we can do at this point. I mean we deal with all the subtypes, so we deal with the subtype, the human subtype that is currently not circulating in humans, and then we deal with the specific examples of the other human subtype, which is the h-1/n-1, 1918 and we deal with all the avian subtype. They're all there, it's just It's a very gray zone as to whether an early h-1, or early h-3 is really's noncontemporary or not.
You know, this might be one of those cases where you just want to stop making it gray and make a decision because this is going to be the source of confusion for the local IBCs, I predict.
Dr. De Luca.
I agree 100s for 1 pawn 100% with Dr. Muzyczka. I think it's yourp tepz not to include h-1, n-1 in the category of noncontemporary.
It's under a different cath gory, under a highly pathogenicp flewenza. -- I misspoke. The noncontemporary should to human strains. All the avian strains are -- we brought them into the agent summary statement because, obviously, there is a need to be more comprehensive in the update, but all of those subtypes are a real -- really under USDA guidelines, so they're referring to human subtypes and clearly the h-2, n-2 is the only one strictly speaking noncontemporary.
Okay, I think the jargon always causes confusion, I think, and if that's the cas, then h-2, n-2 substitute that for noncontemporary.
So just take out noncontemporary.
That's what I'm hearing.
Okay Indiscernible ]
Then you have to cover the h-1 Indiscernible ]
Uh-huh.
There is one more.
I mean the -- then the point is that any human virus that hasn't circulated for a period of time, you need to consider that in your risk assessment, and that's -- that's what we're trying to get at with the term "noncontemporary."
But what you just said is very clear and so hearing the need for specificity, you can modify the specificity and then by adding that simple statement. And that would work. Miss kwan.
Having served, um -- being on several IBCs and also attending some meetings that include biosafety officers and IBC members, I think this just emphasizes, again, the need for institutions that are ingaging in research engaging in research to provide more resources to their IBCs to make it more attractive for people to serve on them and to gettrand to understand -- get trained to understand their responsibilities. Because the frustration of scientists is clear when the IBCs only look at things and, you know, test it against black and white and don't think about it. other hand, one has to be kind of grateful for that if, in fact, they don't know what they're doing. And, I think a lot of this is reflected in how well the institution itself supports itself IBC.
Any other comments from members of the RAC? Yes?
The, which I agree with. It was a recommendation for providing influenza vaccine to people working with the viruses, of course, we had a shortage and as I recollect, the CDC priority groups lab work were not listed. They were those priority groups were written into North Carolina regulations, so actually to have violated those for a violation of state law. If we have a vaccine shortage again this year, lab -- will lab work be included in the priority group?
I can't address that. That's an independent body at CDC that makes those guidelines, but I think it would, that would certainly be something that would need be to be considered.
All right, I would like to thank both of you very much for your presenting as. Dr. Harris, Dr. thank you for joining us and for updating us. We're going to move now to our final discussion protocol for this section, protocol 707, dose finding and safety study of an opco litic polio rhinovirus against malignant glioma. I understand the presenter will be Dr. Bigner from Duke University.
Thank you. Good mornning. I'm not Dr. Bigner. Dr. will be the holder of the IND for this projected trial. My name is Matthias Gromeier. Thank you, sir. I apologize. Thank you. I want to thank you all for hearing our case and I want to point out before going into the science that's major and very appropriate criticism that was identified in the initial RAC review was the insufficient information on the toxicology plan and in accordance, the plan dosing that we have here present, our colleagues from NCI ray, Dr. Muzyczka and their -- and Dr. Cripe, their colleagues are morably to -- I also, I had planned to take with me the surgeon in change of this project, Dr. John Sampson. Very difficult to schedule brain surgeons for anything, and he couldn't make it because he has a study section. Finally, I would -- I want to apologize to Dr. Indiscernible ] She had to lessen to all of this a few weeks ago at our meeting. The talk I prepare side basic and I am afraid it will probably exceed the time a lotment was given of 15 minutes. I felt because this agent is very now and has never been -- very new and has never been considered, the micromechanisms I'm going to introduce to you are completely new and poorly understood and I thought this was it was necessary to give a better understanding where -- why we're doing this and why we think that it's a safe and potentially efcrashes strategy. This -- efficacious strategy. This idea to use polio virus for the use I intend, I believe is very different from the other strategies to use viruses for similar purposes, and you will see why that is. The first difference is that we never planned to do this. The sperms, the -- experiments, the signs done were seren dip us to. At the time I first performed the first experiments in animal tumor models, hi no idea this was the opportunity that other people were approaching similar opportunities with other viruses. And I was as a scientist, my training and my interests lies entirely in the molecular mechanism of polio virus neuropathogenesis. Poliovirus spiral -- spinal chord meeto motor neurons, you see here for the transgenic, polio virus transient isor, c boo, cd 165, they're selectively targeted by the virus, destroyed and this load leads to the characteristic pathogmomec features of pathopole Joe Indiscernible ] There were no infectious agents aurguably -- arguably that can induce anything at this level of specificity. There is no target of the virus at these cells and don't know what is responsibility or more -- most people think we don't know what is responsibility for this level of specificity. The next closest thing to polio myelitis are chronic degenerative diseases of the cns. Now, in describing to you the science we have carried out, which leads to the oncolytic virus strategy we're pursuing, I would like to distinguish three parameters we used to describe polio virus neuropathogenesis, and those are tropism, which we define narrowly, according to the entomology of the Greek tropis, meaning strictly to lean toward, and we define tropis as a self-surface molecule expressed on cells that mediates virus entry and replication, potential replication. We distinguish this from virulens, which we define as influencing propication and and spread. The third is the rel -- I. relevant for this strategy. The plan is to inject the oncolytic agent and question directly into the brain so there is no place for circumstance or condition of the host. So, I would focus on these two elements, tropism, mediated by the recenter and cell internal factors that determine virus prep, intercellular prop -- propagation spread, and I will try to show you how we will utilize these two factors to direct polio virus specifically toward malignancy a rising in the cns. The first question, of course, is that of tropism. As I told you, we define this as the ability of the virus to recognize self-surface molecules to serve as a receptor for verule viral entry. It's a molecule of the globe ulin family known as cd-165, modified 1959. The polio revent -- receptor. There is no yews physical role for this molecule. you gage the critical contribution of cd-155 to the pathogenesis of paralitic polio. When you compare mice transjepic for the cd-155, human cd-1 3EU6 Gene infected with polio vees -- virus and wild-type mice infected with the intra-- a distantd to grow in the rodent cns. You can immediately see the kind of clean appearance of selective eradication of motor inureons, gives rise to -- neurons, gives rise to encephalomyelitis, caused in the absence of the cd-155 receptor. If you take away the specific determinant of trip onism for motor neurons in the cns, polio virus k by using alternative receptor cause a much more diffuse type of cns infection which, is more characteristic for verule viral encephalitis. This experiment demonstrates how polio virus tropism for motor inureons must depend at least in part on the reventor cd--- receptor cd-155. We believe we have come close, at least, to provide more definite proof for this by making a mouse, transgenic mouse that expresses the report of beta galactic Indiscernible ] In control of the cd-155 sequences. In these mice, when you look at the developing spinal chord, you see the test -- activity of the cd-155 promoters in three structures. The motor chord, the antearior horn of the spinal chord itself. were delight delighted to find this. It's these instruct ours known to reduce maturation of motor neurons. We had correlation of overlap of activity of cd-155 promoter elements, with the Pugh Tateive -- putative tread of structures that give rise to motor neurons in the cns. Interestingly, this pattern of cd-155 activity is a perfect match to the expression and the activity of the well-known is any widely-studied sonic hedgehog, also expressd in motor chord and floor play in the temperal overlap to cd-155 promoter activation. So, by finding this overlap, we had, of course, we had to check whether sonic hedgehog, which is a very potent Indiscernible ] Involving a variety of signalling pathways and important embryonic development and cancer, we had to test with us the signalling pathway. Has something to do with cd-155 Gene activation. Sonic hedgehog through the interninal soliable, can patch receptors on the cells and illicits a signalling cascade that is in a simplified manner consists of activation of gli factors, gli because they were isolated for manighing Lantz -- malignant myem myel willoma and transferred to the nucleus and turn on sonic himhog responsive Genes and I don't want to go into too much details. We found the cd-155 jeep is responsive to Sonic hedgehog itself and also to the downstrain regulators, the gle, the trans-- transcription factors of the gli family. In addition to the connection to the motor neuron developing the motor neural system, we found an association for cd-155 with signalling cascades induced by sonic hedgehog and most importantly, with cancer. Because sonic hedge-- hedgehog now is a major, considered to be a major factor behind the genesis and homeostasis of mean many tumor types and they are, sonic hedgehog inhibitors in clinical trials and various stages. Now, our experiment provided a direct line of evidence to investigate whether cd-155 actually is associated with cancers. And we did this in breast cancer, for example, here. These are -- breast cancer cell lines that are available through atcc. We developed an antigen captured, very sensitivant -- captured essay to -- assay to detect cd-155 and we could find it on the breast cancer cell lines, albeit, a different concentrations. The human explant million -- manary epithelial cells, there is hardly any cd-1 3EU6 detectible. In accordingance, we determine susceptibility. For example, the high-expressing cell, was with the virus 12 hours after infection, where some lower-expressing cell lines, for example, this one can't be entirely liced. They're cells with 30 to 50%, even after 24 hours after infection, given the polio virus, induces cell death, defect of cell death two to four hours after infection. This must mean are cells in these cell lines that are resistent to polio virus. They're not clonal cell lines, there is no explanation for this. The fact remains there are differences according to cd-155 expression, the breast cancer cell lines. A similar difference of express levels we do see in actual tumors on patients. These are breast cancers. We have no information on histological or anything at all. We're exempt from irb review, but can you see that we see also various levels of cd-155 expression in breast cancers, please note that normal breast epithelium doesn't appear to contain appreciable levels of cd-155 in contrast. The most solid correlation of cd-155s expression and we did see in malignant glioma, amongst the tumors we tested, and this is just a part of the studies, and we took here six cases, these are all patients diagnosed with a recurrent gbm grade 4 tumor, we took primary cultures from these patients, we infected them with polio virus. They succumbed to infection six to 12 hours after infection. The virus application was efficient in all the cell lines and we did see that cd-155 expression in the primary cultures established from the tumors correlated to the expression levels in the actual time -- tumor tissue. The tumors in the cultures from patients correlated very well-- with expression levels in established atcca tapable brain gbm cell lines that are also very susceptible to polio virus, and we did see far higher levels of cd-155 and then in lumbar spinal chord that contains motor neuroopposite, which we assume express cd-155 or normal cortex, normal human brain cultures can't be affected by the same manner they resist infection. So, this concludes the tropism part of my presentation, I just want out believe a failure do need the molecules and that the receptor molecules for tropism a major problem in any attempt to harness viruses therapeutic purposes, and this is identified a a a virus receptor that can be cancer specifically. Also, it us to test prior to therapeutic intervention and are -- for receptor expression. It would make no sense to target a cancer like this with the polio-virus-based therapeutic agent. The tropism is an important part of our project, and actuallyd motivation to consider pol Joe -- polio virus for the purposes we untend are not based the virus but the receptor cd-155. We got unexpected help because there is now an increasing realization that cd-155 does play a role in tumor cell biologist. I can tell you it's difficult to talk to cancer specialists about a molecule whose only known function is binding to polio virus. They won't be interested, but this is now slowly changing and there was came out the study by an industry consortium with some academic investigators, and I can tell you anything about their approach, it's a very complicated screen, but they're -- they'vive -- identified cd-155 as a determine cell motility and cell metastasis and tumors, thoroughly confirmed our characterizations of the association of cd-155 with cancer. Now, this is all find -- fine and good, but what good is it, is it to have a cell, a cellular receptive virus that is a deadly killer. So, we had to think of a mechanism, how to restrict polio virus replication and now this is the second parameter we are very -- considering with regard to pathogenesis. The viu virulence, the internal cellular part. We had to Indiscernible ] In neuroops. This is really the only pathogenic target for polio virus in the cn -- cns arguably in the human organism. The mechanism I'm going to tell you about has to do with translation. Polio virus is unique in, uses a very rare mechanism of trnz -- translation that has to do with fact that the polio virus genome, which is being a plus in rna virus, functions as an mrna, doesn't carry a cap modification, a universal cap modification, and instead has a genome link protein vpg. So, the virus utilizes an alternative mechanism of translation initiation known as internal Indiscernible ] Entry mediated by the entuner internal entry sight Iris, the structure located in its untranslated region. The region -- reason why we became interested in the Iris was a simple experiment I did as a naive post-doc, not too long after the Iris had been characterized and we didn't know what to do with it. So I took polio virus and I swapped out the Iris element of polio virus with that of a relative, the human rhinovirus type ii. Vineo -- Reno virus utilize the same translation strategies as polio virus does but don't cause neurological disease, of course. They utilize a different receptor, and we were very surprised to find first that this virus replicated with wild type kinetics in malignant cells we use in the lab to grow polio virus but that this virus, de-- despite being apparently without growth impediment in these crime these cells h a very strong neuron-specific growth that was evident of massive teeters into cd-155 transgenic mice, which didn't develop any damage to motor neurons of or poll -- polio myel itis, it was not able to replicate in the police, it was days after infection and in contrast, it's -- with the strain of mice enneckd 200 pfu, of wild-type polio mice to kill a mouse and eradicate all motor neurons and viral replication isn't the spinal chord of wild-type infected mice. By exchanging the Iris element of polio, we have achieved complete inability of the virus to keep -- replicate in cells of neuronal origin.
Yes.
For a clarification -- the coding sequence.
Yes.
That's from the Indiscernible ]
The actual product, yes.
In this very experiment, it's not. This is world type mahoney.
In our original paper.
This doesn't -- didn't off as an oncoletic virus. Most of the science done was not done with that in mind. So, I want to briefly explain to you how translation at the Iris works, and I think we have a very good idea what have the mechanism behind this might be. So, in ordinary, cellular mrnas, again, the universal cap modification, translation occurs upon the formation of the ribo nuclear protein network to that consists of a series of factors led by the cap-binding eia-4-e. It binds to ei 4-g and which then binds to pollo Indiscernible ] Binding protein forming a circular temp late, really, by providing a ribo nuclear bridge. This is what is needed to for the subunit that then brings in the initiator trna met to the ier iniation chord on the scanning and the translation can ensue. In polio virus, this is consider is -- very different there. Is no coop -- cap. E has no binding partner, ef 4 g, discovered is cleaved early after polio virus infection and the same fate is bestowd on the poly-8 binding protein Indiscernible ] So as an end-result, we have degredation of the main mediator of the protein synthesis in mrnas, and instead, the virus utilizing the highly complexd structure to recruit the subunits internally that then can initiate translation at the initiation chord on. No -- now, the neuron-specific deficit that we have described in transjepic mice the virus we like to call pbripo, the polio virus containing a rhinovirus. At this point, it's irrelevant what the Capsid is derived from. We can describe this phenotype invitero in cell cultures and I'm showing you three culture lines, skmc neuroblastoma cells and 293, hhk, 293 kidney cells. Polio -- polio virus grow list in each one of them. The traditional classic for polio virus neuronal models has been Indiscernible ] And we use them quite a bit ourselves. They're very unsatisfactory model because neuroblastoma cells spontaneously shuttle between a neuroon like and fibro bust like phenotype. Depending on the passage of the day or the character investigatoringses -- investigator, the experiments will vary with the kills. The results will vary. So, I want to tell you an anecdote relating to the 293 cells. I think that sleds very interesting light about the, the agent. Because we were, we never were really quite interested in -- to use them in our research. The ran why we stumbled over them was I approached Dr. Muzyczka with our concerns where to produce the agent pb ribo. The first choice would be viro cells. That's where most viral agents are and the polio vaccine virus is grown in. But they're seemy in origin and monkeys don't naturally support rhinovirus infection, and we were concerned that by growing a virus that contains a regulatory element of arino -- Reno virus in it nvero cells, we could select for adaptation events in simian cells. I asked Dr. Muzyczk, what other human -- human cells we could use to produce the agent. She said only wise choice is 293 cells. I said great, they're kidney origin like veral cells. We got a batch of gm 3 grade 293 cells from the nce, and we began experiments and what we saw was this. Our virus did not grow in 293 cells at all. It was dead the tracks. That's in sharp contrast to wild-type polio virus. We had no explanation for this. These are kidney cells and we know of polio virus grows, the receptor must be expressed on them. Why would there be an a resistance to allow of this virus be a kidney origin. And I can tell you we treed everything. We -- passaged the stuff, we used mois of a hundred, we did our infections, my post-op was ready to quiet after months when we had a visit from top Shenk -- from Tom Shenk, himself, a reputable adeno expert. And Tom told me matthias, 293 cells are neuronal. Indeed, a painer from graham who made it in the '60s originally, showed that. 293 cells of neuronal origin. There are neuroblastic cells in human embryonic tissue and sharedm brieonec dna cells use for unknown reasons, immortalize cells of neuronal origin. We published a paper confirming the results and showed expression of neuroownal Marcus in 293 cells and of that. We had, I thought, generated the perfect neuronal model the purposes. 293 cells are not a naturally-occurring cancer. Can you see the neuron-specific growth deficit of our virus is far more pronounced than 293 cells than neuroblastoma cells, which are somewhat inter3450ED@. In these -- intermediate. In these cells, we see a specific expression in our cells, with wild-type polio virus, very meager Gene expression in the n, uron-like neuroblastoma cells. There is no viral Gene expression in two hours n 293 cells in correlation with the lack of fired propagation in these cells. So, we speculate that this ribo nuclear protein network in uca ritic mrmas let -- leading to template circulation is at -- the genomes that lack at least one anchor for this network, the cap, replaced by non-kananotical non protein network, and such protein interactions can either favor translation by forming the correct cirquela tempt plate or may inhibit translation by somehow interfering with the correct arrangement of such a configuration. And we went ahead and we looked whether we could find factors in neuronal cells that would somehow -- somehow interfere with the correct configuration of tempt late and thereby, repress translation. [pausing to switch captioners]
Occasionally we see neurons or normal glioma cellspersist in these cultures.When we treat with PVS agent 48 hours after infection,the tumor is sh rap nel and neurons are normally cellsin these cultures live quite happily there after so wehope something like this can be achieved in an in VIVOsituation.So we believe that tropism is mediated by selectiveexpression of the CD 51 receptor in mortar neurons.A topic expression widespread epitopic expressionconfers natural toppism to -- genetic features of poliovirus but not of rhino virus permit replication.Condition not applicable if you inject the virus intothe brain.So this is my lab.I guess it's not.I want to acknowledge my -- my advisor, most of theresearch was hatched in his laboratory at the universityat the FDA who has helped us concerning SAs and alsoanother in Tokyo who has done primary experiments.Thank you very much for your attention and I'll takequestions.
Thank you very much for a very elegant scientificpresentation.I believe there are still some questions and commentsremaining for the formal RAC reviewers and I'd like youto stay at the podium if you don't mind, and we'll takefolks reviewer by reviewer, ask them to brieflysummarize for the record, not reread, but summarizetheir initial concerns, then state whether the concernhave been addressed fully, if not, try to addressthem in this setting so that we can put everything torest.I'm going to begin with Dr. DeLuca who will alsosummarize Dr. Johnson's comments.Dr. DeLuca?Oh.And I should say then Dr. Aaron felled if we could moveto your comments and after that, Dr. Lo and myself.Dr. DeLuca?
I'd like to thank you.That was a beautiful presentation.
Thank you.
Okay. -- this protocol obviously addresses a veryimportant disease and while replicating viruses from virus families have been previously applied thisis the first application of polio virus.The protocol advised a very clever strategy which isbased on various sound basic science and is very clearin its description.I had at the time of the review some safety issues to bediscussed and I'll just go over them.Most of them if not all of them have been addressed inthe written response.Okay.The first -- the first thing I wanted to ask andDr. Johnson also had this was the status of nonhumanprimate studies.There is -- it's stated that they're proposed and Iguess you've added that these are going to be conductedwith material produced from an NCI raid -- raid grantand those studies will begin the end of the year.Is that correct?
Yeah.My colleague from NCI would want to comment on that.
Hello.My name is Karen and I'm the toxicologist for theproject.That's correct.We can start the monkey study at end of the year.Since we do have test article right now.
Could you just briefly describe what those are goingtoWhat your to bes plan is?
Sure.The TOX plan has -- if you'd allow me just a second toWe have four monkeys an equal number of males andfemales for each dose group and we have three dosegroups a vehicle control and then one times ten to the6th platforming units per monkey and then one times tento the ninth in your protocols that you received it saysone times 10 to the 8th but we certainly agree that weshould increase that dose to one times ten and the 9thand we should not have a problem with that.So we have two sacrifice times day 29 and day 58.In the interim between those sacrifice times and beforethose sacrifice times we'll be doing clean path drawsand we'll also be taking blood for antibody testing.
Given that change, do you propose to change yourclinical doses from ten to the 6th to ten to the 8th?That's what you currently have, half log from 10 to the6th to 10 to the 8th.Are you also now going to change the clinical doses?You've changed your doses that you're testing in themonkeys.Is it still your intention to keep the same --
The clinical dose will depend on the outcome of thetoxicology trial and the design of the toxicology trial.Since this was not set in stone, I -- I did notintend -- what I put in the clinical protocol was notmeant to be a final proposal.This is up for discussion, of course.We have to reconsider this in conjunction with thedesign and outcome of the toxicology trial, so --
This is direct cerebral injection?
I'm sorry, it is.
For anyone that's interested those studies areoutlined on page 29 or 30 of the clinical protocol.29 to 30 of the clinical protocol.Now, the -- the other question I had -- I started to asksome questions about comparing this to the saven Istrain.Since the background of this is the saven I strain and sure there have been plusser studies on nonhumanpray mites prior to the use of the vaccine strain withthe -- with the experience of that was and -- and Dr. GoMeyer provided a lot of information.What I was trying to get at is compared to the dose thatyou proposed to give the monkeys, what were the effectsof the saven I -- just the saven I vaccine strain atthat same dose in monkeys just to get a handle --
Yes, that's -- that's a little bit difficult toanswer.
Okay.
A lot of the experiment everyday that exists ishistorical so in those days they had different -- therewas a different level of scientific rigor and it's hardto -- to gauge -- or to get a comparative picture plus alot of the evidence was obtain in chifrps which weprobably couldn't replicate here.I just want to quickly point out here in this -- we hadincluded in the study the type one saben virus isconsiderably less attenwaytive at least in culture thanthe agent we are testing here so I don't want to suggestthat our virus is better than the saben strain but I'mconvinced that it has a higher level of andcertainly a higher level of genetics ability.We would have to do experimentally if we wanted to dothis.The release of the vaccine is somewhat subjective.People go actually and look at spinal cord sections andto do this correctly, you would have had to -- you wouldhave have to have an expert who goes section by sectioncomparatively to conduct this study.This would be the only rigorous way to answer yourquestion.
I'm not suggesting that you do a side by sidecomparison.I was just trying to get an idea perhaps at presencesince the monkey study hasn't been done with your agent,what would comparative of the amounts of the saben beand you've satisfied my question and can't really --okay.You'll get the information with your -- with your agent.
Yes.
Okay.The -- the other question that I had was again relatedto the saben vaccine strain that it can mu state at somefrequency to more pathogenic form and I was wondering towhat extent you've explored the possibility that uponpassage, your virus can MUTATE to a morepathic form.
It would be preposterous to claim with any RNA virusthat it has a stable phenotype, there's no such thing. of course all RNA viruses are capable and will --will do under certain circumstances, this one includedbe able to adapt to a mar pathogenic phenotype.I can only tell -- that's why I told you the sillyanecdote about the 293 cells because we really wanted itto grow, believe me, and we couldn't get it.We -- we tried serial passages to obtain viruses withimproved phenotypes and we were unable to a matter of fact, virus fizzled out over time.We lost the input tighter.So while we cannot categorically exclude such events tohappen it's just impossible with this class of viruses,we have done the sensible studies in CD 155 mice incells which are used for the production of this agent,in cancer cells, these are established GBM lines toexclude adaptation events in susceptible cells.In the 93 cells which is all neuronAL model and we didnot see adaptation to the phenotype or adaptation to types in any of these assays so we would loveto make a recombinant that had lost its ability to adaptbut this is just not possible with these viruses.There will always be an unknown.
I can maybe shed some light on that.I'm Karen with the NCI raid program and we've done twopreclinical safety studies to address the issue ofaversion to a phenotype. of them was conducted by Dr. Womener at stony brookand he took the PDS rip po and injected it into thebrain and then harvested it from the brain and spinalcord at different time points and the intention was toisolate any neural that came out of thissequence and find out where the mutation was and whatthe rate of reversion was but he was never able torecover any neural variance over a six-week period. conclusion was that the virus is growing very slowlyin the central nervous system of the mice and he alsoshowed that it's cleared from the central nervous systemafter about four weeks and we also did a neural virplent study in mice with the FDA and we sent pilotmaterial made using our production process to the FDA attwo passage numbers P 2 which is where we would harvestthe clinical lot and P 5 which was just to push the test is -- was conducted using the WHO protocolthey used to release vaccine lot and none of the micethat received showed any virulence.Infrequently experience some paralysis and or death at alow rate but this is sometimes accepted in terms of thevaccine.We've concluded based on these two studies that PVSripPO is actually more stable than the saben strain.
Thank you.Very nice.And I had another question with the comparison.How do the proposed clinical doses of your agent compareto the vaccine p found in other doses because this is --then you might expect and -- and you -- you oo*if givenme some information there and it looks like the clinicaldoses are of the -- of the vaccine doses are on par withthe lower proposed clinical dose.You describe the work in the CD 55, 155 tri genic mousevery nicely.I also ask, is it possible to comment on why the iris --the difference between the function of the two I restand nondividing cells and you gave a very elegantpresentation today describing that.Incidentally Dr. Johnson also had that -- also had thatquestion as well, and that's no longer a question.Another comment I had was this 293 thing.And I brought that up and I didn't think it was in theneuronAL line.No one ever describes it as such, and you went throughthat in great detail.
I'm beginning to get accepted, I can tell here.
I have to see if my herPES colleagues --
Can I just ask a question about the 293.The argument is that this is actually a neuronAL line.It does not replicate.But 293 is a tumor cell line transformed.
Well, it's a lab generated immortalized.I make a distinction between a naturally occurringcancer and a lab generated immortalized cell.I would not consider 293 to be tumorous.They're permanent, they're immortal, but I do notconsider this to be a cancerous cell, per se, becausethe origin Honor mall embryonic human cells.They were not malignant.
But they grow forever in --
Exactly immortalized but I would like to draw adistinction.See, intuitively it's unsatisfactory to test a virusthat's supposedly has a tumor specific growth phenotypeas a -- in a tumor cell line as a normal model that wasnot very appealing to me intellectually.We prefer the 293 cells which are immortal ietzed as youpointed out.They do not originate from a human cancer.
Right.
It's an academic distinction.
It's a bit of semantics.Some people would say that you use 293 as a transformcell line and a model of something which is fullytransformed and lives forever.
But see, I -- I don't have the whole gamut on thecytology of 293 cells so don't you agree that some ofthe alterations of the chromosome mall rearrangementsare more likely to occur in 293 cells?
I agree you know the whole of tumor biology and it'scertainly unclear.The virus does not grow in that.In my mind, maybe it's a semantic.In my mind, it seems closer to be a transformedcancerous cell line than a normal cell line and yet thevirus is not growing.
It's cancerous I agree.
If you strike cancerous I agree.
That's an ap demic -- that's a definition question.Basically we don't know what -- what I can tell you is293 cells we just published this express a whole rangeof neuronAL markers and they do replicate the pheno --the pathogenic phenotypes of our polio virus constructsin animals.So we can -- based on our experience we can say the 293cells are by far the most faithsful subculture model forthe pathogenic model.And I'd like the herPES colleagues to test this.It's very unsatisfactory to model anything unpathogenicin cell culture so it's a very primitive model, but withthe -- with existing limitations, this one came theclosest providing us some answers.
Yes.
If I may make a couple of comments.The 293 cell line was
Could you introduce yourself?
I'm job on staff at OBA. that time he was doing research on adenovirus and hewas -- he was looking for essentially segments withinadenoviral DNA that -- that complimenting with -- withnaturally occurring adenoviruses that these could thengrow.So what he did is he basically took human embryo kidneycells and he transformed them with sheared adenovirus a bunch of cell lines and so the 293 cell is one I think he kept alive for a couple of yearsto -- maybe even three years if I -- it's been a whilesince I read the papers, so these cells essentially,they've over gone their natural crisis which is why theyare transformed and no longer contact inhibited but theyalso contain the genome, the exact map has not beenestablished probably because there are multiple copiesand in some reports there are also -- there have beenpapers that have suggested that maybe the three primeend of the adenovirus might actually be in there aswell.So that's -- that's the origin of the cell line.
Thank you.
Tom, the graduate student tried to repeat thisexperiment and couldn't and the reason is he believes,that he used embryonic kidney slightly older than thosethat frank Graham had used and at that stage theneuroblasts in kidney cells had disappeared, havedisappeared so that was another hint of the neuralorigin of 293.
Okay.
Shall I continue?
Please do.
Okay. last question was one of dosing, and that's --that's been addressed.Now, I want to quickly turn to Dr. Johnson's concerns orquestions.He's not here today.We've addressed the -- we've addressed the need for thenonhuman primate study.He's asking, how was the number of animals to be studiedchosen, and you said --
not a toxicologist so Dr. Swiek ert wouldprobably better know how to answer that.
Typically, we use 4 to 6 animals per dose group.In this case we chose 4 simply because currently there'squite a shortage of nonhuman primates.
Okay.All right.The other thing was why was the maximum dose in thestudy the same of the highest dose in the human trialand guess that's where your change in the dosingoccurred.The -- the next question different regions of the brainbe injected since GBM is not likely to be confined andyou say that you're -- this merits some consideration.
Yeah.I -- I just want to point out that duke university braintumor center is the largest special ietzed center fortreatment diagnosis and research on CNS malignancies inthe world.There's no place where clinical trials more invest gaytovrtive procedures are carried out than at duke.One of the large leaders -- really designed to maximizeexposure of a wide region in 2 CNS.We have extensively discussed these issues.Since polio virus is naturally endowed with thecapability to penetrate CNS tissue there's not an urgentneed to consider this but we have done some researchthat did suggest that CED did not add the benefit to theefficacy of this agent at least in rats but the lastword on this is not out.We have a meeting with chief brain surgeons at dukeafter this review, and we will look at all options, howthis should be best administered.This -- we followed the -- our advice from theherpesvirus field because this how it wasadministered in the first phase 1 trial and we felt itwas compelling to use a very simple straightforwardmeans to deliver this agent.I'm not against this idea of having a more widespreaddistribution.That's an excellent idea but I'm just wondering what'slogistically the best approach and we will address thisamongst ourselves.
Okay. you.And the last question had to do with why does --
Excuse me.As I read Dr. Johnson's comment though, he -- he isasking, shouldn't different regions of the nonhumanprimate be injected in the NCI propoetzed study.
Oh, that's not how I -- I didn't considerThat could be.We probably should ask him.
Yes.
Well, with -- with -- with regard to thatpossibility, I can say that in the old days, it wasquite common to test polio virus vaccines or strains andstudies by the intraceramic route so that wouldprobably -- this is kind of the default injection site,I assume, for such studies at least at that time and ifone would repeat that, one could at least compare thetoxicology results for some studies.I don't know.We should ask our brain surgeon colleagues.
We're always planning to do a study with the clinicalmaterial, a neurovirulent study in monkeys and that's anintraneural spinal injection.That's the standard WHO protocol for releasing Thank you for the clarification.The question, then, I'm sorry, Dr. Johnson isn't here,but the question is then, not quite consistent with theproposed TOX study.What I you say is you're going to injectintraspinally.
No, that's not -- that's a safety study.
All right.Thank you.
Okay.And lastly, there was the question about why the ISfunction is only dividing cells and you've shown thatit's a neuronAL cell and you've answered that verynicely.
Thank you.
Dr. Orren
As he indicated I had some opportunity to have somelong discussions with him about a week ago so most of myquestions were answered before I even had to ask them.I did have a comment, which I think maybe for the recordwould be good to include.Which is the fact that there is a requirement already inthe study that patients have humoral immunity forinclusion in the study and I think as people worry aboutpossible reversions and reversion rates and so on, thefact that all patients will in fact already haveprotective immunity to this agent, I think reallydiminishes the concern about reversion of this agentwhich certainly with this mutation which -- which may --we hear be even more stable to begin with, but if youadd that to the other determinants of the saben virus Ithink there's no question about improved safety and ifall the patients are already immune, I -- I think -- socomments I had in that regard, I think have been clearlysatisfied. did have one question about whether or not familymembers care providers should be boosted with saltvaccine just as a precaution.And the response was that they would actually maybechange the instructions on the consent form orrecommending the people -- at least people should be of this but I'm not sure why he wouldn't do --
I've learned with members of my lab, I'm alwaysdiscouraged by my institution to do that.So they don't feel this is necessary. This isdefinitely a possibility -- it should at least beoffered, maybe.
I --
Yeah.
To me it just seems like a safety thing to do, butthis is certainly not my area of expertise.
Yeah, I know, but I think excellent ideaand it can certainly be included in the consent formthat this is a recommendation.
Thank you very much.Dr. Lo? Yeah.I want to start by -- by complimenting the investigatorsfor a very thoughtful and innovative approach to a -- aterrible disease and they've thought very carefullyabout the -- the risks inherent in doing such aninnovative study.My concerns really centered around the -- the sort ofnovel issue here that some of the risks in the phase onestudy are not to the participants in the trial but tothird parties that might come in contact with this PVS virus and I had a series of questions aboutpotential risks to operating room staff, nursing andcustodial to the hospital and family members andhousehold contacts at home.And the investigators actually annalsed my questionsvery nicely in terms of -- in some instances requiringof a history of immunization.It doesn't quite get at Dr. Aaron felled's question ofwhether a boost is needed but they are planning to that staff in the hospital have been immunizedand they also have plans for family members andhousehold contacts who might have immunodeficiency toavoid contact.I obviously don't know the clinical idea aspects of whena boost and the saben -- of the sulfa vaccination wouldbe useful or not.I also had some questions about possible prolongedshedding of virus and they showed data that's veryunlikely to happen so my concerns I thought were verynicely addressed.
Thank you, Dr. Lo. concerns too have been almost fully addressed and Itoo want to compliment you on a very elegant scientificpresentation of your own work leading to potentialeffective gene transfer in a disastrous disease.My first questions were similar to Dr. Johnson's and andasked why the proposed NCI studies consideredreconsideration of dose and of product for thepropoetzed NCI studies, and that's certainly beenaddressed.I had a clinical question regarding the -- the protocolwhich states quite strongly that routine biopsy of at recurrence will be done in all of theproposed subjects and the investigators and I questionedwhether biopsy was routine in that setting and whethersome subjects would undergo a nonroutine biopsy, if youwill, for purposes of the study.The investigators have assured us that at theirinstitution, duke, which is what's critical to thisprotocol, all subjects with glioblastoma who recurundergo biopsy and they further have stated that that isnot necessarily true at other institutions which istrue, and my question is fully answered.
Thank you.
I was concerned obviously, about the possibility ofthe additional risk of rebiopsy.I am a pediatric immunologist who has taken care ofchildren with primary immu know deficiency which hasdeveloped polio myolie tis from saben and whose patients to Congress and were instrumental in changing ourvaccine laws in this country so that led to my nextquestion, which is -- just to put some context to it.I was -- oh, and I've also taken care of twoglioblastoma patients with pneumocyst I knewmenia.That led to the immu know deficiency in glioblastomapatients and perhaps an underestimated risk of yourproposed product of polio and you have fully satisfiedmy concern.That is, all patients with glioblastoma have some degreeof imewe know deficiency but the proposed product ismost unlikely to cause any form of polio in thesepatients.
Yeah.
Is that a fair --
Yes.Well, I couldn't write all this up.It was getting a little bit too long, but we have someexperience as you pointed out with immune suppressedchildren.Either inherited or acquired, that occasionally harborpersistent polio virus infection of the GI track. to be said that in these cases so aftervaccination there are astoundingly long times of poliopersistent and occasionally very long intervals untilneurologic deficits appear and those always involvegenetic adaptation of the virus, so I -- my -- myintuitive answer would be that I believe in the GI tractof an immune know deficient child with a very longpersistence interval the virus is given an excellentchance to adapt.I don't think with the form of administration in thepatient population we're targeting, this -- thisNow, I just reviewed a case in Taiwan where there wasthis six-year interval between vaccination and therecurrence polio if we have that problem in GBMpatients this group.We are very lucky.
Agreed.So my questions which emanate from two separateexperiences, that primary immune no deficiency andthat from caring for glioblastoma patients withopportunistic infection, you have beautifully satisfiedthem.Thank you.My next question regarded unanticipated risk to familymembers and care providers and I -- that whole issue hasbeen nicely resolved and I very much like the idea of atleast offering booster to these individuals.It's very simple and literally risk free.Finally, I did have a question about the consent and Iwant to be very direct about it.I asked, should the consent include a statementregarding the possible death of the subject fromBecause are going to be intracerebral injectionswhen they occur, and the response was that the consentalready did include this language.I really meant should there be a very direct statementrather there is a risk of this and a risk of that astatement of it is possible.
Yeah.
-- I would be that direct.
I wrote it that way and my RIB told me to --
Took it out? I -- it was very blunt and they didn't like that, butI can -- I can -- I can tell them about your concernsand I can certainly prevail, I think, but that was theirstyle.
Well, you might be helped then by a comment from theRAC stating that, you know, this is -- although yourpresentation was exquisite, because of the proceduresinvolved, I think there is a -- there is a risk, be itremote --
Oh, absolutely.
Of death.
Absolutely.
From participating in this study.
Yes.But I have been fairly -- I haven't been too concernedabout the consent because I -- I anticipate that Iwill -- I have talked to I can't tell you how manypatients already, and I've -- I anticipate to continueto do so and certainly with potential participants inthe study, so I think due to the unusual origin ofthis -- this agent and the unusual nature of this I think it's necessary to give every potentialparticipant the opportunity to engage in a discussionwith people who can explain the risks.I think there's no way to pit this appropriately in aconsent form.And I don't know the rules, whether I can put inthe consent form that there's an obligatory conversationwith the PIF of the study or something of that nature,but it will -- I planned that.I don't see any alternative to that and so --
Well, Ms. Kwan may have some suggestions.
No, I just wanted to point out that in past whenwe have had this conflict between what general agreementbetween the PIs and what we're suggesting and the IRBsthat a clear statement by the RAC has often been ofbenefit to the PIs.
So my comments are satisfied.Are there other questions or requests for clarificationby RAC members?Dr. Gelehrter?
I just have a quick historical question that relatesto the new knowledge for me, that -- that thepart of the nervous system.And that is in ander's work on the growth what were the cells he used?I thought they were kidney but I don't recall for sure.
They probably were
So were those human kidney cells prior to the 293they were prior to 293.
Oh, yes and I think those were primary cultures.
kidney?
I don't know -- don't know with embryonic.
I guess it relates to the question of why did thevirus grow in those cells?
Well, this was wild type polio.
And it didn't very well.I mean, it was blind past and forced past in order tofinally --
Dr. Vile has a comment.
I may have missed this.Have you got any data where you've cured gliomas inmice? Sure. can cure -- I don't like sdeen graft studies.We prefer to do primary cultures but we have conductedsigne graft studies and those are published in mice, inrats, subcutaneous, intercerebral, it's all done,certainly.
And the CD 155.
We can cure every tumor in every mouse, no problem.That's not a question.
Thank you.
Dr. Bowen.
Maybe you just answered this but I had a question onthe basic science of testing for to bes sisty in primaryneurons in VITRO.I appreciate those cell lines and one slide you showedof the tumors where there seemed to be a survivingneuron but have you done studies of primary neurons fromrat or, you know, you Even prepare them from humantissue?
Yeah. had planned of course -- it's very attractive becausethere are some pretty good models out there and we hadplanned this.I must tell you, this is not something trivial you do,you know, spontaneous in the lab.You really have to prepare for this and create a setupand I've been unable to secure funding by the NIH.They always say why don't you use SK and MC and be donewith it.I had a whole specific aim on this and I was forced totake that out.We're very interested in some very interesting modelsbut they're very difficult to work with and I can't dothis -- I would have to hire a special staff and thingslike that so this is not something that can be done.So what we're doing is a compromise.It's certainly much easier and it gives us, I believe,the answers we need, but you're absolutely correct.It would be the very satisfying to use such improvedmodels. Sounds like you need a collaborator.
Exactly.
I appreciate the need for -- or the concern aboutimmunizing or boosting your subjects or family members.What makes you think that when you -- when these peopleare immune to polio virus that your virus will have anyeffect anymore?
Yes.So -- oh, you mean in patients?
Correct.
In patients?So the available evidence suggests and this has beenstudied as historical in chimpanzees is thatthe to polio virus by oral administration ofthe vaccine are some kind of protective agent will -- will, of course, generate neutralizingantibodies circulating neutralizing antibodies but theimmunities can be circumvented.So I think saben did this actually.He nietzed chimpanzees and when you inject thevirus into the -- which suggests with neurologicalsymptoms do actually have protective immunity thatextends to the CNS.
I would suggest you might want to study this a littlemore closely.What they're doing in those exPierce peerments isinjecting a whopping dose, if you will and so they cangenerate some -- some things.What you want to do is inject into the brain andactually get this virus to continue to replicate for awhile, it seems to me, to get the oncolytic effect thatyou're looking for.I don't think you're necessarily going to see that ifyou have circulating antibodies and this is something --this is a different situation.I think you might want to check that.And it may very well be a function of how much of theantibody is still circulating with the age of yourpatient is and so forth.The background of this is that virtually every othervector that's been looked at in the CNS now, it's becomeclear that if you have circulating immunity, peripheralimmunity you get very little transduction in brain.
So, I have a question.How would one do that outside of a primate study?Could you do that in a small animal study?
I would guess you could do that in the -- in themouse line the transitic mouse line that has yourreceptor.
He did that and did get excellent oncolysis.I provide you with the details but he did this ina -- he's collaborating with a group in Japan that'sconsidering polio virus for a different type of tumor.He did this in mice.I apologize that I don't have the -- he was traveling alot and so was I, so I couldn't -- but if you areinterested, I can get this.You probably talk to himent every now and then.You can just ask him, but I can provide this in a moreformal form, but they have looked at this.My on this, I believe if we didn't have vaccineagainst polio virus or if there was no vaccine coverageit would certainly add an element of risk to thisproject.It is probably very difficult to model.I mean, we could -- you can immunize monkeys nicely butwe don't have a tumor model in those and you cancertainly induce tumors in mice but you can't immunizethem.The tumors we have do not permit gas ro intestinalreplication so we can't immunize these mice by oraladministration.So whatever model we would create would be highlyartificial and -- but it's not a bad idea certainly.I find out from ebbing ert what he did exactly.
Thank you very much.Comments the
I had a couple of comments on the informed consentdocument.One was that the document does contain some complexlanguage that would not be understandable to allsubjects.I also noticed in the most recent version that I have,which is the April version, in the procedure for virusadministration there's a statement that once in thebrain, the agent makes its way into the tumor to killcancer cells and my concern is that that implies that itworks, and that -- that we know the agent will killcancer cells so just a suggestion that that -- you know,something we hope it will make its way into the cancercells to kill them or something.Except that this is talking about what we're going to doto you, I think.
Okay.I noticed that you proposed that health care workers orclinicians who are compromised would be excluded fromtaking care of these patients.You can certainly inform them of the risk and if theywant to be voluntarily reassigned you can do that butyou can't exclude them because of an fi discriminationlaws.
Okay.On oo On that note, thank you very much.That's actually a very important comment.I'm left after this interesting discussion and beautifulpresentation with just a few comments.Preclinical, 1 is an obvious.Nonmu happen primate studies -- and it doses up to tento the 9th will be conducted through the NCI.The bio distribution and toxicty results should be to guide the proposed study design withglioblastoma. that's in response to your comment that the clinicalprotocol is still in progress.
It -- unfortunately, Dr. Safrpson is not here.Surgeons only get really serious about something whenit's this close to reality, so I have the feeling wehave a giant meeting scheduled and I have the feelingthat there will be some tweaking here or there.The basic stand, I mean, so I don't think we will implyCED, that we will follow the herpesvirus guide --guidance that we have obtained and model this accordingto them, and there will certainly be a lot of input withthe FDA with regard to that.You know, what's neurosurgically possible.
And taking at face value Dr. Johnson's comment andnumber two consideration should be given to injectingmultiple areas of the brain in the nonhuman subjectsplanned by the NCI.Three, consideration should be given to evaluating thecapacity of the polio/rhino virus for replication ofneural cells in the presence of specific antibody.Is that what you intended?
Yeah.I wouldn't even consider that adding that as a safetyissue.I don't think it's a safety issue.
Okay.It's an efficacy issue.
So it shouldn't actually read neuronAL.
In glee cells?
Right.
All right.Thank you.Clinical 1 consideration should be given to offering theboost -- IPB boosting of family members and careproviders with vaccine.In other words, offer, but don't man date.That seems reasonable and regarding the informed consentdocument, one, the RAC reck menlds inclusion of thisstatement regarding the possible death of the subjectfrom participation in the study.It could even read possible but unlikely death butsomething that's -- that is con freet.2, recommend simplification of IC language to increaseunderstandability of the document and three, remove isIC document to eliminate any overstatement of benefit. else, members of the RAC would like to add?Any modifications?
Yes.
Did you have in there that the RAC would like toreview the results of the primate study?
I did not.So the RAC would like to review?I think that's fair, the primate study results.Other thoughts?All right.May I have a motion then?
I move
Dr. Bowen so moved.Dr. Lo second.Dr. Vile.
Yes.
Dr. Webber, Dr. He is lip, powers,
Yes?
Bark lee, yes
Lo,
Yes.WarA yes.
Bone
Do Hurst?Ms. Kwan?Yes.
Al bell da
Yes
And nem ro.Yes
This concludes the 100th RAC meeting and thank youall for your active participation over the last twodays.Also, a final good-bye to the half dozen RAC members whohave served on this committee for the last four to fiveyears and been a pleasure working with everyone.I expect to see you all back in the fall as add HOCs. you.
I just wanted to thank Dr. Paterson and her staff.They have done a amount of work that hasallowed us to have a very productive two-day meeting andit's very much appreciated.