Dr. Sahay, an MIT alumnus, is one of the world's leading scientists in pharmaceutical formulations for vaccines and therapeutics. Gaurav Sahay is an Associate Professor in the Department of Pharmaceutical Sciences, College of Pharmacy at Oregon State University. Dr. Sahay's lab is developing novel nanotechnology-based platforms, including lipid-based nanoparticles for effective delivery of messenger RNA therapeutics to treat cystic fibrosis, retinal degeneration, and against SARS-CoV2. He has done pioneering work to dissect the intracellular transport essential for nucleic acid delivery to the cytosol and developed methods to overcome endosomal barriers. He has more than 48-peer-reviewed publications in top-tier journals, including Science Advances, Nature, Nature Communications, Nature Biotechnology, Nature Nanotechnology, Journal of Controlled Release, Nano Letters, etc.

He is the winner of a 2013 American Association of Pharmaceutical Scientists (AAPS) Postdoctoral Fellow Award, the 2015 Controlled Release Society (CRS) T. Nagai Award, a 2016 American Association of Colleges of Pharmacy (AACP) New Investigator Award, a 2019 Oregon Health & Sciences University (OHSU) Distinguished Faculty Senate Award for Collaboration, 2020 Phi Kappa Phi OSU Emerging Scholar Award and 2020 CMBE Young Innovator Award. He serves as the Principal Investigator on awards funded through the National Institutes of Health, Cystic Fibrosis Foundation, and in the past through Moderna Therapeutics, MRF New Investigator, and OSU.

Dr. Sahay serves as a consultant and scientific advisory board member to several biotech and venture capital firms. He was the Chair of the 2018 NanoMedicine and Drug Delivery Symposium (NanoDDS, Portland, OR). Dr. Sahay completed his postdoctoral research with Prof. Robert Langer and Prof. Daniel Anderson at the Koch Institute for Integrative Cancer Research at MIT and received his Ph.D. from the University of Nebraska Medical Center under the mentorship of Prof. Alexander Kabanov.

T&T Scientific, Dr. Gaurav Sahay: Lipid Nanoparticles for Delivery of mRNA Therapeutics and Vaccines 

[00:00:01]

Nima Tamaddoni, PhD

Hi, everybody. Hi, Dr. Sahay, thank you so much for joining and the pleasure to see you again after, I think three years, we were in Oregon and had a pleasure to meet you. I think we have 170 people in the lobby and waiting for more people. Right now, we have 20, 25 people in the room. I wanted to get a very brief introduction, so it does not take a minute, so we, the audience, can take the most advantage of your expertise, and we are very humble, truly humbled to have you here. So, I started the introduction, Dr. Sahay is one of the world's most influential people in the COVID vaccine. He has been at MIT before joining the Oregon State University as a professor. His group has been working with mRNA, LNP, lipid Nanoparticles for years before the COVID vaccines. He has contributed significantly to the COVID vaccines; we truly have a pleasure to learn more; you do not need more introductions, people definitely know you here, and so I passed the mic. Thank you again for being here.

 

[00:01:18]

Dr. Gaurav Sahay

Thank you so much, Nima Tamaddoni, PhD. Thank you for that very kind introduction. I still remember three years ago you coming to Nano DDS; we did not really do much in regards to the COVID vaccine; those are Moderna and Pfizer have developed those. We have been working on the field, and then we are very excited to find that these technologies that we and like many others and many pioneers before us have worked have reached the clinic. So, I am really very humbled by your introduction, but I also wanted to correct that, you know, we have not really been primarily involved but have been working in this field for a while, and we are closely watching, and it is a very surreal moment for all of us as these technologies get, you know, injected and a lot of us… And so today, what I thought was I would talk about these technologies those liquid nanoparticles give the audiences a brief taste of what kind of different technologies exist and then our work in the field of mRNA-based therapeutics and where I think the field would go next after this year of 2020 where we have seen such remarkable progress. So, mRNA-based therapeutics and vaccines, especially vaccines, are moving from a field of renaissance to an industrial revolution. We basically saw how quickly Moderna started its clinical trial, and then, you know. Also, Pfizer with BioNTech and Acutas started to use the technology.

They had developed to move into the space very quickly as well, and everything played out in front of the New York Times and here are some of the clippings and I think historically 2020 will go down and as a, you know, breakthrough year and this is a fascinating period for, you know, many of us working in the field of control release drug delivery and some of the key aspects as you think about it in pharmaceutical sciences which are, you know, not in the academic field looked upon that much but industry looks very carefully at it. But, it makes it clear how important those aspects are. One of them is how to ship those vaccines at minus 80 degrees. So, I think two young audiences out there, the field has done a lot of exciting stuff on lipid based technologies, but at the industrial front also there has been a lot of innovation that has occurred, and that is sort of is a key moment to realize, and it is a very interesting time to be in the field to start using certain technologies, some of the technologies developed by TNT for formulations of lipid nanoparticles, and so I think it is a key moment for young scientists to really get involved and see the impact and this is so real because at the end of it you got injected with one of these vaccines as well and I was, you know, it showed how involved I am with lipid nanoparticles that I was actually dreaming of LNPs, the whole night, waking up and then it was a recurring dream.

So, I think that reality catches it. But we are moving from a renaissance period from 20, you know, before COVID to an industrial sort of revolution, and I will explain a little more as we go along. So, pre-COVID, we used to talk about what the potential of mRNA-based technologies could be. First of all, there is no risk of genomic integration here; it does not need to enter the nucleus, it is highly efficient, it has a rapid effect, and it is transient as well as regulatable. So, for vaccines, this is the key element because I think for therapeutics, you need to deliver mRNA for a long time, and I think companies have been working on that; our lab has also been working on that for a long time, but it is sort of a little high bar because you have to keep on injecting or through other routes deliver the RNA again and against and maintain that level, but with a vaccine, it is a one-shot, you know, or a two-shot treatment where the immune system takes care of the rest and all you need is the antigen that is formed and then it goes away.

So far as an application, it was a great application, and before COVID, again there was a lot of talk about why, you know, we are moving into vaccine area in this field, why the companies are investing in this field, and I think now it looks more precise that there was a lot of effort done in industry as well as academia to develop some of these tools because once the pandemic hit, it helped, it move pretty fast. So, a lot of mRNA modifications which were pioneered by Drew Wiseman and Katie Crico were then also worked more by industries like BioNTech and Moderna and many others. So that became a key element of modification and that also brings us to the point that you need to modify an mRNA and have selective sequences, for example for the COVID vaccines you had these two proline modifications that were introduced. So a lot has been done in that regard. But, the challenges with these technologies they are very negatively charged, they are unable to enter cells, and they are highly susceptible to degradation. So, the other unsung hero in this story is the lipid-based nanoparticles, these LNPs are multi-component structures, they have an ionizable lipid that basically interacts with RNA, that is the hero of the story, and then there are supporting actors like the structural lipid, the peg lipid, the cholesterol which help in the formation of these lipids and a lot has been done into understanding the structure but we still do not know what exactly where these lipids are within the formulations.

The hope or the thought is that the lipid interacts because of its positive charge with the RNA, encapsulates the RNA, and then the peg lipid helps in the circulation, stability and formation of these lipid nanoparticles and then cholesterol and structural lipids like DSPC help in stability as well as endosomal escape and you can use different devices like microfluidic devices or TNT based devices to actually formulate these LNPs. So what is the main goal? One main goal is to package and protect, and you will find a lot of ionizable lipids that can package and protect the nucleic acids. But the challenge and I will talk to you about it there are only few that can actually deliver this vivo and cause something called the endosomal escape and I will talk about that as well as we go ahead. There are lower in immunogenicity, the other really cool part of these particles are their large capacity. So, for example adreno associated viruses cannot carry above 5 KB of genetic components and a lot of these genes like cystic fibrosis, they are more than 5 KB, some are like 10 KB in size. They can be made biodegradable. You have a redosing capacity, so for AAV's, you have just one shot, one and done but with these technologies you can do a re-administration if need be and then there is modularity, by which I mean that a lot of different cargos can be added or included in these LNPs.

For example the first SRNA that got clinically approved on patro is an LNP based system, you can basically use the same LNPs to encapsulate mRNA, you can add crisper cas9 agents and Italia had a recent paper that they had a phase one clinical trial where they have given a crisper cas9 components and caused gene editing in human liver and knocked down this protein called TTR, and it is like one and done kind of strategy and finally scalability, I think scalability has got a new definition now. I mean these were scalable but now they are being scaled to be, you know, injected in billions of people and what that does is that allows us to basically have the scalability problem resolved. So, if you figure out tomorrow a lipid nanoparticle that can deliver something to the eye, to the brain, to the lung, there is the capacity to actually make billions of this and if it is a rare disorder, you can provide these for the lifetime of a patient, these kinds of technologies. So, I think what this has done is caused an industrial revolution because of their modularity and scalable nature, LNP sort of can be used for different modalities and it is possible that, you know, a lot of different diseases would be cured because of sort of how fast things moved in the clinic for the vaccine, and how these things can be easily used for other technologies that can treat other diseases.

So, I think it is a very interesting time for us. So, the main actors in this story is the ionizable lipid, these are the previous version DLN-MC3-DMA is approved for siRNA delivery, there are lipidiods based system and then there are the next generation lipids that were developed to deliver mRNA based technologies. So, the first product that got approved in 2018 was Onpattro, again using an MC3 ionizable lipid in an LNP to deliver against siRNA, against the TTR and this was an IV based administration. For the mRNA vaccines, we have a different form of lipids, so if you look here, these are the ionizable lipids here that are published now. And, you know, they are very similar sort of in nature, there is a difference in the peg lipid, there is slight difference in the ionizable lipids as well. At the end of the day I think the body, once you are giving an mRNA which actually is making a spike protein is seeing sort of a similar product with different dose components and slight differences in the peg and the ionizable lipid and so, the other aspect is how are these vaccines being manufactured, we are basically at the lab scale, do all these processes but what I want to say from this slide is that, you know, from making the pure DNA, then to linearizing it, then growing it into bacteria, all need GMP scale manufacturing, and I think you guys have put a wonderful program, and the next sessions you would be learning from core leaders in this field. But what I want to say here is that it is possible that these things, 20 years from now or even five years from now will say that this was a primitive way of manufacturing these mRNA and these vaccines and because then you have to package them in like these lipid components and that sort of is also a rate limiting step when you start brewing this together.

So, I think there is a lot to be done in the vaccine manufacturing space as well where this could be a much more rapid fast way and a lot of innovations are going to happen in this space as well and as I said before, you know, this has been a global sort of effort, this again was on the front pages of New York Times of how Pfizer is making these vaccines at different places. So, again once you have the correct lipid and if you could do delivery to other sites then suddenly you will have the possibility of treating lot of other diseases. So, once the vaccines are made, they are then injected intramuscularly, they go in the muscle cells, to the lymphoid region and into the antigen presenting cells where the antigen, in this case the spike protein with these two pre-proline modifications is made and then that activates different kind of immune response and again, I am not a immunology expert but this sort of gives you a taste of how the activity of delivery to selective immune populations and the muscle is happening, that further leads to a response which at a clinical trial has been amazing, there has been a 95 percent efficacy of these vaccines with, you know, all packaged, as I said before with lipid-based nanoparticles and having mRNA with these two pre-proline modifications.

So where do we go next? I think it is great now we can hit the immune population; we have a siRNA drug that can hit liver after an intramuscular delivery. But I think where the field at the level of academia and then potentially at the level of industries, moving is in the realm of extra hepatic delivery. I will talk a little bit about our work in the space of lung and the eye but a lot of people are working in the space of brain localized delivery and so each of these organs, if we are able to target these particles tissue and cells selectively, I think that would open up a lot of new areas and I said before we have already taken care sort of… as a field has been taken care of the manufacturing issues. So, I think the next steps then are in line and that can be transformative. So, let us begin with first understanding how these lipid nanoparticles work, so you took all these components, mixed it in a in a device, form these nice looking liquid nanoparticles. Once you inject them IV, the peg lipid of these LNPs sort of falls off, and there is a protein in the cinema called ApoE, that protein binds to the surface of the lipid nanoparticles which then is taken up in the hepatocytes by the LDL receptor.

The LDL receptor then internalizes it, the particle moves into an early endosome, where it starts to disintegrate and then the ionizable lipid which is positively in charge interacts with these endosomal membranes and you see a release and one of the key problems for the technology is that the escape is relatively low even though these things are in clinic and they are very potent materials. But I think we can make them better and therefore it would drop considerably the manufacturing costs related to it. If we are able to make particles that have a better escape efficiency, the mRNA based escape I think is around like 15 percent and what we found when I was a postdoc at MIT that these particles are also recycled out and there is a cholesterol transporter called Neman pig type C one that refluxes some of these particles outside the cells. So during, you know, so from 2015, onwards till 2019 we had the chance of working with Moderna and what we did there was that we basically utilized different cholesterol derivatives, so because these lipid nanoparticles have a cholesterol component, and I said before that there is a cholesterol transporter which is recycling these particles, we hypothesize that is it possible that using cholesterol, so replacing cholesterol with its derivatives would cause an improvement in mRNA based gene transfection. So, we group these cholesterol derivatives based on the structure of cholesterol.

So, some of them did not have the same body structure, different tails and different head groups and then replaced cholesterol and started making LNPs with these derivatives. What we found here and which is also very interesting is that the C24 alkyl derivative of cholesterol which is beta cytosterol and the groups formed particles which were very similar to the typical LNP; they had the similar encapsulation efficiency and size. But, if you look at the transfection efficiency it was around 204 better in heater cells and depending on cell types it could be 222 or 200 fold difference, which was very interesting to us because, you know, the field has focused on ionizable lipids as the key components and it was interesting to find that cholesterol also has a key response in how it enhances delivery inside the cells. So, we then tested two lipids, one is the DLN-MC3-DMA and then lipid-9 was the gift from Moderna to us, and we looked at the encapsulation efficiency, they were very similar, the size was a little bit higher and what we find was that in cytosterol containing LNPs and what I would term them as ELNPs or enhance LNPs, you see these polyhedral shapes, so this is the typical LNP, the different ionizable lipids in it, and all the other components. The only difference is the cholesterol has been replaced with cytosterol and what you are seeing here is these polyhedral shape structures. If you look at the internal structure of cholesterol, it is pretty much the same but it is just the external structure which was different and what we also found was that if you use these analogues of C24 alkyl derivatives, you again would see differences and how the shape of these structures was but most commonly that these were polyhedral in shape.

So, you can again look at the same sort of, you know, morphology changes that are happening and we think because of these structural variations this leads to differences in trafficking of these particles. So, this is another video showing 3D multi-resolution imaging, we took with a collaborator, we did this sort of like a high resolution imaging where you could visualize these lipid nanoparticles as they enter cells in 3D and with LNPs, you see that they are basically bumping across the endosomal membrane and sketching it and are lacking that escape factor but with these ELNPs or enhanced LNPs, you see this very linear movement within the cell which we think is possibly because of a better escape or also because of a better diffusivity inside the cells. Furthermore, what we did next was we wanted to know whether this actually was an escape parameter. So, we made a cell line that was galectin-8 based, so for endosomal escape there are certain cytosolic proteins, if an endosome sort of becomes damaged, galactin8 binds to its surface and what you will find is these punctate structures that form that are indicative of the escape that is happening. So what you are seeing here is, in green cells that have been stably transfected with galectin8GFP. All you see here is the nucleus and the cytosolic nature of this protein. Once you add cholesterol, LNP, which is again unlabeled. You start to see these small punctate or dot structures.

All those events are rare events that show escape of these particles. As I said before the escape is relatively low and we, you know, we still want to figure out method that can prove it. But, once you add the ELNP that is contained in cytosterol, you see this massive appearance of galectinate which was cytosolic here. Now, you start to see a lot of endosomal vesicles that are being formed, indicating that you are seeing a very high escape that is happening within the cell. So, if you then quantify it, this is basically the cholesterol escape, and with cytosterol you start to see this very high escape that happens as indicated by this connecting aid recruitment assay. So, we are forming lipid nanoparticles with cholesterol derivative beta cytosterol or its analogs that leads to a better diffusivity inside the cell. Somehow there is disintegration of the particles and in a better way with the conventional LNP and the ionizable lipid that then interacts much more efficiently with the endosomal membrane causing better escape and that leads to sort of a damaged endosome where then galactinate binds and you can visualize it. So, this gave us one more hint of how you can improve the endosomal escape. So, when the COVID pandemic hit, we thought, you know, we will also contribute in our own way in the field and, you know, industry was already working on the vaccine front. So, we thought why not we work on the strategy to prevent or basically an antivirus strategy of a therapeutic and using mRNA based technology. So, in this case what we did was, so SARS-CoV-2 enters the lungs using the ACE2 receptor which is also an enzyme, there is also a soluble form of ACE-2 which is very slightly expressed in the body. We thought if we engineer the soluble form of ACE2 as an mRNA, deliver it inside after an IV injection or inhalation, this will lead to secretion of the soluble form of ACE2 which will act as a decoy and prevent infection from happening.

So, we engineered the soluble form of ACE2 where we made a variant which did not have the Trans membrane domain of ACE2 and we had a V5 tag just to quantify once us transfect cells. So, in Calu-3 lung cells with our ELNP formulation what we find is in the culture media you see a huge amount of solver less too. If you transfect the cells and do a western blot analysis, again you see a lot of protein production. So, the protein soluble ACE2 is being formed by the cell and then it is secreted out in the media. Once you instill these particles in the lung because lung are the primary site where the virus basically acts with the reporter assay, you could see that the expression after nasal installation is primarily in the lung, and in the bronchial alveolar lavage fluid of the lung, you start to see secretion of ACE2, within 24 to 48 hours and we also did an IV injection and you could see as early as two hours, you start to see secretion of ACE2 in the serum. So, what we did next was we made a SARS-CoV-2 pseudo virus, which had the spike protein on it and luciferase gene, this interacts with the ACE2 receptor and then you see luciferase activity.

So, what you can see here is that when you over express ACE2, you start to see that the spike protein actually starts to bind and transduce the cells. If there is no ACE2, it does not work at all. VSV, which is a glycoprotein that is independent of ACE2, can infect cells, independent of their availability of ACE2 in them. So, once you have this ACE working at that time because we published this in July of last year, you know, it took us some time to like figure out how to make this work but once you then treat the lipid nanoparticles that had the soluble form of ACE2, this is the spike pseudo virus infection in ACE2 over expressing cells. Once you add the soluble ACE2, you see 90 percent inhibition of transduction with this pseudo virus. VSV has a control independent of whether or not there was soluble ACE2 still can transduce the cell. So, currently we are working with some of our collaborators to do these studies in live animal models and hopefully, you know, next time I will have some data on that end as well. The other thing, pre-COVID we used to work on and that basically helped us move into the field of lung delivery pretty fast was cystic fibrosis. CF is a lung disorder; there is an absence of CFTR which is an ion channel that secretes chloride. So, if there is an absence of chloride, the mucus sort of thickens and then there are secondary infections that cause fatality. Vertex has come up with some small molecules that have been really transformative for patients with CF, but there are a lot of mutations amongst CFTR and so, if we can come up with a strategy that can give back CFTR gene, it is possible that independent of the patient genotype, you will be able to resolve this problem. So, in this regard, we again engineered a CFTR-mRNA, we made the lipid nanoparticles, and then transfected the cells and how you test CF patients even in the clinic is doing a nasal potential difference because the airway epithelium is very similar to the lung epithelium. You will see that the chloride channel is not working. So, using the nasal potential difference, we again quantified our ability to deliver the LNPs with CFDR-mRNA and what we found is that once you give the CFTR-mRNA, the chloride secretion comes back and, you know, stays there for around 14 days.

So, currently what we are doing is making nebulized particles, so that we can do a repeat administration to the lung with CFTR-mRNA. The other thing which we also did was we said can we use another target in the CF. So, another target in the CF is a sodium channel, over expression of this sodium channel causes sodium to influx in more, and there are studies which suggest that if there is no CFTR, the sodium enact channel is hyperactive. Wherever sodium goes water goes and therefore the mucus becomes very thick. So, companies have tried to make a siRNA against the sodium channel and so we thought rather than the siRNA strategy why do not we make an mRNA based strategy where you could make a mutant of one of the subunits of ENaC called the alpha ENaC and this subunit is the key element, so in the timer of alpha, beta, gamma, alpha is always needed, alpha and beta can work, and alpha and gamma can work and the other great thing with this technology is that, this is sort of cell selective because the channel would only work where the other subunits are also getting expressed.

If you give the alpha subunit tone cell types that do not have the other subunits, it would not work at all. So, once we made these mutants, we transfected the cells and what we find is that the sodium current basically sort of decreases after the transfection as compared to the untreated version. So, this is the untreated version, once you start treating it with the alpha mutant, the sodium channel sort of becomes less… has a less influx of sodium inside the cells. So, our current strategy now is that if we can give the CFTR-mRNA with the ENaC mutant, take combined that might be a better strategy against the CF lungs. So, currently the idea now is to nebulize the particles and do a sort of a dose titration where we do different doses but also dose it for different times and maintain the function of these target proteins. As I said for therapeutics it is much more difficult because you have to redose the animal multiple times and the issue with nebulization is that the particles, basically they are very soft material, so they can break apart. In that case you need a stable particle that can disintegrate in the cells and very recently a grad student of mine has made a certain formulations that can take our typical LNP. And, you know, with some formulation changes in it, we can nebulize these particles, and they transfect the mouse along pretty well. So, currently we are working with using the CFTR mRNA or even the ACE2 strategy and delivering a nebulized particle with the similar composition of an LNP and as I said before, if we start delivering things to the lung, to the heart, to the other organs, it is transformative because there are so many other gene targets that you can go after, you can go with gene editing of some of these.

For example for CFTR because they are modular in nature, so once you become successful here it just opens your doorways to a lot of other diseases as well, and other different types of cargos as well. Finally, the last place where I think we are moving in, and I think for extra hepatic delivery is the localized delivery to the eye. The eye is, you know, the visual acuity in the eye is due to the retina which is not present in the back of the eye, so the light basically enters and goes to the retina, and so if you look here it is basically going from this side here from the ganglionic cell layer towards the photoreceptors and photoreceptors are where the light is then converted into an electrical signal which then goes back and is transmitted to the brain. If there is degeneration of the photoreceptors it causes blindness. So, a lot of inherited retinal degenerations are caused because the photoreceptors degenerate and slowly, you know, you could see a child as they grow into an adult, they can turn blind. The other cell type that is critical in inherited retinal degeneration is the RPE; RPE is a pigmented epithelial cell, so these are all neuronal cells here. RPE is the epithelial cell, which helps in cleaning up the photoreceptor outer segment, so every morning when we wake up these outer segments basically are chopped off, they are regenerated back and the chopped out of outer segments of these photoreceptors are engulfed and degraded. So, if RPE goes bad, the outer segments sort of start depositing on the RPE and that also causes degeneration.

So, mutations in RPE and the photoreceptors cause, you know, these commonly lead to an inherited retinal degenerations and retinitis pigmentosa. One therapy that has been approved is an AAV therapy that gives back a gene called RPE-65 and that is a one shot sort of a treatment but there are a lot of genes that are bigger in size and if you want to do anything it is better to deliver some of these genomic components like the cas9 as a nuclease rather than having it stably transfect the cell. So, we thought can we develop LNPs that can deliver to the back of the eye, especially the photoreceptors and the RPE. The challenge is the delivery. So, the route through which you can really transfect the back of the eye is a sub retinal bleb. So what you do is you take a needle and you go to the back of the retina behind the sub RPE and behind this region here and inject your particles here. So, the good news here is that it is much closer to the proximity of the cell type. The challenge is that you have to go all the way back up, you need like very highly screened skill trained clinicians to do this, and it is a onetime shot, you cannot do a re-injection. However, the other way you can deliver to the back of the eye can be intravitreal injection things like VEGF are given monthly as an intravitreal injection, there is higher patient compliance, no damage to the retina but vitreous is very viscous and there is low tissue penetration due to this. So, for non-viral as well as viral vectors delivery after an intravetrial ejection is a huge issue and so, we make a lipid nanoparticle and this basically shows the problem. So, you can do a back injection, use an LNP, you do see the transfection of the RPE, so not the photoreceptors the LNPs are typical LNPs, just inject the back of the eye those epithelial cells, which again if you can treat some of the diseases of… which are due to the mutations of RPE, it could be transformative as well.

As we have seen with luxtarna which is an AAV based method. But, if you give an intravitreal injection then what you will find is that after an intravetrial injection you would have transfection just on the periphery. So, there is a small membrane called the inner limiting membrane that prevents anything that can go past and reach to the back of the eye. So, the cell types of our interest are here, this is the vitreous chamber where you are injecting and the stuff, the nanoparticles are basically getting stuck in this region. So what we want to develop is methods that can lead the LNPs to cross these barriers, reach to the back of the eye, and deliver drugs. So what we did previously was that we made, we screened different cationic lipids, and we found that among them MC3 was the best one to hit the RPE, very little to nothing in the photoreceptors, then we also screened other components. So, we modified the cholesterol, peg is, you know, causes stability as well as like hype regulation can lead to a better penetration into the vitreous. So, we wanted to test that as well. What we use are Ai9 mice, which have a log speed stop site with 3D tomato to it. So, if you give a three mRNA, it does cause editing and because of that anything you can visualize 3D tomatoes.

So, once you inject that into the eye, after a sub retinal injection we started to see using fundus imaging, so this is in a live animal, you can just take an instrument and look into the back of the eye and the red signal is suggesting that there has been editing that has happened because you have given lipid nanoparticles with three mRNA. After an intravitreal injection as you can see here the transfection is relatively very low and once you cut the sections you realize most of the transfection is in the RPE and this is basically here it is in the inner limiting membrane and nothing really reaches to the back of the eye. So, we also looked at what are the mechanisms by which delivery is happening. In this case we give an LNP with a mCherry to it. You again see an RPE getting transduced or transfected. In ApoE knockout mice, if you take ApoE knockout mice, deliver LNP's IV, you would not see any liver delivery but in case of the eye you do see a very good delivery in the back. So that means that ApoE is not the primary sort of endogenous ligand that is helping the particles go into the RPE. We are had another phagocytic receptor called MertK and again you still see a very good transduction because of that. So what we have now done is we are actually using different technologies to hit these LNPs to the photoreceptors, and we have made some success there. But also have had some success in making the particles and engineering them that they can cross and reach to the back of the eye and hopefully I will be able to discuss those as we publish them in like a couple of months' time.

So, the next time I am here I can talk about how these we have engineered these technologies that they can actually cross through and hit different cell types, especially the photoreceptors and the RPE and I think that opens a lot of doors for not just gene delivery but I think I am excited about genetic, you know, gene based editing of a lot of different mutations in the RPE or the photoreceptors because it would be like a one shot treatment. So, this is some of the images of our sort of unpublished data where we have figured out methods to engineer these lipid nanoparticles that you can inject intravenously and they start to transduce a different cell population, especially the photoreceptors in the eye and I think that is very exciting for us. So, finally, I think where the field is moving is extra hepatic delivery, there was evidence and when I was a, you know, a post doc at the Langer Anderson lab, we actually had injected some particles to the brain and you start to see expression in four hours.

Again, the thing here is that it would be limited for some time only; intramuscular delivery now is possible, liver delivery is possible. But, I think we are slowly moving in the phase of localized delivery. I showed you some examples of eye-based delivery, nebulization based lung delivery, but there are also intravenous approaches that are helping extra hepatic delivery. For example Dan Siegert published his work using a technology called sort that can hit lung and you can use it for gene editing or gene delivery. There is evidence of using polymers for intraperitoneal injection that can hit macrophages. I think the field would also move into the area of cell type specific and this is not something that is discovered recently, sometimes I talk to folks and it is not that the field was not working in this area. But I think now with these advents of new technologies and how the pandemic has played, there is so much interest that we might be able to solve these long term challenges that we had in the field.

The DNA barcode technology, I think that is another great way of screening a lot of these lipid nanoparticles and that can also help enable delivery, like finding if a lipid nanoparticle is hitting different cell types in the organs, other high throughput strategies of making these materials will still be in the way, new materials and chemistry is are important. But, I think this would be like a cyclic thing where we are doing some things that are based on making particles, and then just testing them, but then trying to fundamentally understand them. Like in the case of ApoE, based like LNP delivery that can help us engineer better and better materials and then go back to the cycle and in sort of evolve these materials to their next generation and I think that is where we are where it is likely that, you know, in five or ten years, we would or even faster given the interest on the field that we will be able to devise newer materials that are the next generation materials that can hit different targets and once that happens, you could deliver different cargos because of modularity of these materials and once you can deliver different cargos it, you know, it can silence a gene, it can give packaging, it can edit a gene and I think that would be transformative for the field, and that is why this is an industrial revolution. Finally, I would like to thank my current members in the lab and the past members, collaborators and also the CF foundation and the NIH for funding and with that I can take any of your questions and again thanks a lot for this very kind invitation, it is been a pleasure. 

[00:45:53]

Nima Tamaddoni, PhD

Of course! Thank you so much, Dr. Sahay. It was very informational, for sure and thank you for all the very cutting-edge information you provided to the audience, we have multiple questions now. Graham is going to read it to you and you can answer it to the audience. So, Graham you can ask certainly.

[00:46:22]

Graham Taylor, PhD

Sure! So, yeah, Dr. Sahay, thank you very much. We had a few questions, for instance from Stephanie Khan, she was asking about some of the sax data that was shared in regards to the interior structures of the LP and how we might review, how we can identify different components of the LNP using sax analysis?

[00:46:44]

Dr. Gaurav Sahay

Yeah, so I think, you know, that is a great question and again this was done with collaboration with Moderna at that time and I think, you know, they had some very similar professionals to do it. But from my vantage point what you could do is look at just the internal structure and there is a paper by extrazynic as well where they are, you know, looking at the internal structure of these particles and initially we thought that there might be a change in the internal structure. What we found was, you know, no differences. Again there are previous methods where you could basically use, you know, UHPLC and things like that, just to look at the different components of the lipids. So, there were papers published in 2013 that how you can look at, once you have made the particle how you can characterize, you know, how much lipid content is there. But coming back to the question of SAKS, I think it reveals an internal structure and I think more needs to be done there. What we found was no differences and it took us some time to do that because there was also, you know, the issues of noise and things like that.

So, I think com combining Saks with things like, for us I think because we have like NIH based center of EM, just on our basement with like six seven different cravings, it was much easier to look at that and where once we started figuring out the, you know, that there is an exterior structure that is changing, we moved in that direction. But, I think combining these different technologies might be helpful just to reveal where and how these, Saks might give us, you know, not where exactly the lipids are but at least what the organization of the lipid is internally. We just do not know that, right? Like we know with EMs, you know, morphology is different but how those things are like, you know, is it in an inverted hexagonal sort of morphology inside, what shapes they are taking inside the cell that is the critical element that Saks can give and that might help because, you know, in in terms of delivery how these things are released and in which form that can be something important for us to note.

[00:49:05]

Nima Tamaddoni, PhD

Fantastic!

[00:49:06]

Graham Taylor, PhD

Well, thank you very much Dr. Sahay, and thanks Stephanie for that question. Yeah, so another question, this one is going to be maybe more straightforward from Muhammad Islam, he says great work Dr. Sahay. If we do not deliver LNP via IV or intravenously, does ApoE protein involved in cellular uptake? 

[00:49:33]

Dr. Gaurav Sahay

That is a great question. So, you know, in the eye we do not find that, so when we used an ApoE knockout, you know, model in the eye, we find that the particles are just getting delivered in the RPE as well. So, we just do not know what is binding to the surface of an LNP, in the eye, the same LNP which requires ApoE after an IB administration. I do not know with the muscle, right? So, I have not seen data there but as far as I know for the eye especially, you know, ApoE was not required. So, there might be other proteins that are helping in binding and transduction or transfection with those particles.

[00:50:25]

Graham Taylor, PhD

Okay. Let us see, one question, an additional question was to ask if you could explain a bit more on the endosomal escape ACE that you explained? Maybe this was with the gas, with the protein, the damaged endosomes.

[00:50:44]

Dr. Gaurav Sahay

Yeah, so, I think, so galactin8 is a cytosolic sort of protein, and it was discovered when people were actually looking at mycobacteria. So, in 2012 or something, you know, people looked at, you know, what happens when mycobacterium infects the cells and this cytosolic protein, basically was identified that it binds to the damaged vesicle and takes it to an autophagosome for destruction. In 2013 then Anders Vitro for the first time showed that if you use an LNP with a siRNA, these galactin a sort of also binds to the surface of the endosome and then, you know, in now and like, you know, we showed with mRNA but there are papers with other galectins and some people have shown with polymers as well. So, the story is that this is a cytosolic protein which binds to a damaged endosome which is then engulfed by an autophagosome to be destroyed. So, this basically acts therefore as a sensor, so you take an LNP, you have cells that are stably transfected with galectin, they are cytosolic in nature, you just add those particles and you will start to see those function structures happening. With ELNPs what we found was that it was like a tenfold improvement in sort of having galactin8 buying to an endosome that shows that there is more sort of slight damage happening, and that is helping more in the escape which is then leading to a better transfection that we see with ELNPs.

[00:52:38]

Graham Taylor, PhD

Yeah, fantastic! Really innovative ACE, it was incredible videos to see that happening live in the cells and then it really correlates to transfection, it seems. Let us see. So, this one maybe simple, someone had asked if there is a specific size, target size for particles, an ideal particle size of LNPs for vaccine or gene delivery.

[00:53:08]

Dr. Gaurav Sahay

So, that is a great question and I think there is a recent paper by Moderna, I think the corresponding author where for again this is, you know, you have to be careful with the context because size might matter for eye where the inner limiting membrane is very small but for the immune population after intramuscular injection, they have non-human primate data that it does not really matter there in that case where larger particles were able to give a very similar immune response as compared to a smaller particle as well. So, it really depends on where you are injecting because I think for things to cross after giving an IV injection, the fenestrate are around 200, nanometer in size, so you need a better particle that is below 200, nanometer to hit the liver. The muscle, it seems like it does not really matter. I do not know, you know, if charge matters or not and in the eye definitely you need smaller particles if you have to cross certain barriers. So, it depends on the cell type and, you know, immune cells can gobble up big things and the organ and the barriers it has to cross before you start designing those particles as well.

[00:54:30]

Graham Taylor, PhD

So, yeah, in a way it depends.

[00:54:34]

Dr. Gaurav Sahay

Yeah, it depends. I mean it might not matter for certain, you know, so if you are working on the immune thing, it might not but it might matter if you are giving it to other organs.

[00:54:49]

Graham Taylor, PhD

Sure! Let us see we do have a lot of really good questions coming in. Someone had asked what determines the tissue specificity of an inner particle or can it be engineered to be specific say for the brain? So, yeah, maybe any comments on what to determine specificity or what we know there 

[00:55:09]

Dr. Gaurav Sahay

First of all I think LNPs have been successful in hitting the battle sides because they ended previously, I think, I am listening some… so they, you know, basically take up ApoE in the serum and this also took time, so people think oh you deliver nanoparticles, they go to the liver, yes, but the kuffer cells, it took al-ilam and MIT and UBC like 10 years of, you know, working together sort of to figure out ionizable lipids that were potent enough that can hit the liver hepatocytes and that was because they had the capability of binding to ApoE. Then there are other particles that are being discovered like the sword and others which, you know, might require other serum proteins that can take them to a certain specific organ. But, you know, the serum proteins can also be lower in concentration. So the question of can you target them is also interesting, if you look at, you know, the body of thinking, like we have done this before where we have put targeting ligands on.

Once you put a targeting ligand on a particle, the issue is that there is also a lot of optionalization, so serum proteins are binding too and there has been difficulty in delivery, you know, after targeting, having said that that does not mean that this would not happen. I think it is a still a valuable idea and like people like Dan Pierre are working on putting targeting ligands on it and with the advent of newer technologies and faster ways to evolve these particles, we might be able to target them as well, it has been challenging not easy. The best has been a particle which has just taken up a serum protein and gets delivered because of the nature of the particle but I think targeting capability, we cannot undervalue it and again, the field has worked, so if you talk to people in the field, they will be like, you know, we have done this before, has had incredible challenges. But, I think, as I said before with newer technologies coming in for screening and things like that, it is possible that, you know, we will be able to target them as well. So, it is possible to do both, it is just like, I think we need to like do some good science to resolve some of those challenges. So, it could be, again coming back to your targeting question, it could be something that the particle picks up in the serum and that has been very successful till now. Or I think it could be targeting approaches too, they have been less successful but there is a possibility that they will start working as well after some time, if that answers it…

[00:58:15]

Nima Tamaddoni, PhD

Okay, very good, very good. Okay, go ahead. 

[00:58:24]

Graham Taylor, PhD

Okay. Fantastic! Sorry for the echo there. So, let us see, this is a question from Yamin Zhao, considering the lipid exchange and interactions with lipoproteins and plasma, the original LNP formulation before injection may not be the same formulation that is internalized by cells. Is there any way to measure or control the LNP structure that finally hits the cell?

[00:58:51]

Dr. Gaurav Sahay

I think that is a great question. I think that is something which we, you know, like as a long-term goal of my lab is to understand trafficking in vivo, right? Like the steps I showed you, again this has been harder because the escape is so low, that you cannot really visualize anything, with these newer ACEs like galactin and you can do that. But the challenge is how do you do this in vivo and once you start doing this in a faster way in Vivo, can you then start looking into the structure as well in vivo setting. So, I agree with Yemen that there is an exchange happening and then once things are internalized, inside they might be in a completely different way. So, for example MC3 based particles, in vitro they are okay, right? And there is again the question of in vitro to in vivo correlation which is very limited in our field but having said that if you look at MC3 in vitro, it works okay, but in vivo it is just fantastic, right? So, there is something happening there where the particle becomes even more potent because its structure is such that it can interact with endosomal vesicles, so that is a more fundamental question. I think definitely we need to like figure out ways to answer that. We are not there yet though on that area and that has to be actively researched. 

[01:00:26]

Nima Tamaddoni, PhD

Go ahead.

[01:00:27]

Graham Taylor, PhD

So, yeah, I think that kind of goes to a question about, you know, when in formulation development and testing, is it best to go straight to individual testing and start there to go to in vivo testing and pass, you know, any in vitro work or both and maybe does it change based on the application? So, maybe you could talk a little bit about how you approach this, if you have a preference to go straight to animal models or maybe it changes.

[01:00:57]

Dr. Gaurav Sahay

So, I think, you know, there is the… in vitro to in vivo correlation in the field is pretty bad and so if you have the capability of going directly in vivo, I think that is the best strategy, having said that I think a lot of fundamental studies are limited that you cannot carry them out at this juncture. We will have to develop ways to do them in vivo, having said that some of the particles which, you know, sort of like were not working at all, once we fundamentally started to understand them. For example if you add a particle that is ApoE dependent it would not transfect cells and as soon as you start adding ApoE, the in vitro to improve your correlation with those particles started to increase. So, I think once you have a fundamental understanding then that correlation becomes better. But, when it is a newer field, basically then going directly in vivo makes sense. Sometimes it is not possible just because of resources or it is also if you have primary cell types, they might be much more representative of the in vivo setting. But, you know, I think if there is a way of like testing for you that is easier and like which makes you step away from in vitro I would go directly in vivo as well. But there are certain considerations, especially if there are primary cells, primary human cells that you can use then that makes sort of sense. At the end of the day you will have to test this in vivo and unfortunately the correlation is pretty bad. So, if you could skip certain steps to reach directly in vivo, I think that is good enough but again it depends on the type of studies you are trying to do as well. That it might be limited resource to go after it.

[01:02:54]

Graham Taylor, PhD

Sure! Okay, let us see, I think we have time for a few more questions. Susan Kiati has asked to see, we will see if this rings a bell. Can you comment on FDA draft guidance from December 2017 on nanomaterial's? Are there any specific challenges coming from this guidance?

[01:03:18]

Dr. Gaurav Sahay

I will have to say I have no idea there. I have not looked at the guidance, you know, not, you know, I should, basically I think good idea, especially I think now with the vaccine, I think one of the things would be crucial is like how fast can FDA approve, if it is the same LNP, and like similar RNA. So, all those things are very, you know, interesting to know and I will be looking forward to that but unfortunately I do not know about the 2017 guidance.

[01:03:54]

Graham Taylor, PhD

Yeah, I think it is good guidance from 2017, so, it would have come pre-COVID technically but it was certainly being drafted with these kinds of therapeutics and vaccines in mind, you know, biological products that contain nanomaterial's which itself is kind of broad but can be applied so.

[01:04:14]

Nima Tamaddoni, PhD

And this question can be asked for many of our speakers on Friday.

[01:04:20]

Graham Taylor, PhD

This is true. So, yeah, we will have a chance to talk a little bit more another Q/A sessions, and other panels on some more kind of industry targeted topics. So, let begin, let begin. So, this is something you may be able to speak to, another question from Susan with respect to skin delivery, solid nanoparticle versus soft nanoparticle which is better delivery? So, look maybe what do we know there?

[01:04:50]

Dr. Gaurav Sahay

Yeah, I think, you know, skin again is one of another barrier and I think people, you know, might try to use like some micro needles to study them. So, you know, we did some work with like where we were putting like, you know, particles in a hydrogel, just did not pan out that well. So, I think that is, you know, something where the solid particle might be much better. It depends on what kind of formulation you are making is it in a hydrogel or is it in a mirror or a patch. So, those are basically formulation considerations that could be iteratively checked. I was listening to someone talking about solid sort of particles for skin delivery yesterday. So, I think, again I have not personal experience with that but it is more of a iterative formulation things that one can look after and the dosage form design one can look after to really figure out if one thing works or the other, you know, whether it is a lipid like we sometimes, you know, are underestimating the power of polymers because, you know, of course, I work on lipids but before that like I did my PhD, you know, it was on polymer based system, so I think those also have a huge merit, so if you go after those for local delivery, there might be a way to pursue them as well. So, it is newer materials but also dosage form and it is an iterative process which I think, you know, people are working on, and hopefully we will know more in the future about.

[01:06:37]

Graham Taylor, PhD

Fantastic! Let us see, we will ask one more question and then another panelist Hardik, I think he has a question to share. One question is there any comment on structural relationship difference between LNP with modified or unmodified mRNA?

[01:06:57]

Dr. Gaurav Sahay

Yeah, okay. So, again this is based on papers that have come and not, you know personal experience but basically I think, you know, modifications sort of are essential if you are eliciting some, you know different responses. But, I think people are still working on making a very pure formal, you know, RNA formulations as well and that can actually be really well that there were papers were modified and unmodified after giving IV were not showing much of a difference but for maybe like what it shows with vaccines. And, you know, these clinical trials that modifications have really helped giving some sort of a good immune response after it. So, I think it is still a work in progress even though the clinical trials have shown that the modified versions are better but for other delivery or other target delivery and the purity of these mRNA. More, you know, can come out of it from them, so there is not a definitive answer but I think it is more work in progress still.

[01:08:14]

Graham Taylor, PhD

Okay. Well, let us see. One question, Muhammad Islam asks he has another important question on mRNA-LNP stabilization and can we measure water content inside LNP or is there any analytics you did or can suggest, can we reduce or eliminate water content inside LNP?

[01:08:40]

Dr. Gaurav Sahay

Yeah, I do not know, I, you know, it is a great question. Again like so the structure of LNP is still as I said up for debate, you know, there are papers from Esta Zeneca where the mRNAs and like, you know, basically in water and then the ionizability is forming some sort of these tubes. So, I do not know if removing all the mRNA water content is a good idea or not because it is again like solubilized in that sort of tunnel that has a water inside it, and has RNA in. So, you know, how much of that is essential for efficient delivery, I do not know. Again internal structure is something which we are still figuring out, right? Like we now know that the peg lipid is in the on the surface, followed by maybe DSPC and then there is ionizer blood and cholesterol. So, where the water pockets are and are they crucial whether removing them is a good idea or not is, you know, it is unclear but people are working and doing like computational studies to work that out as well. So, again I think those are all great questions and I am not sure about an analysis of like how can we… that is a good question, I will have to actually look into it, like how can you look at the water content inside the LNP at this point.

[01:10:13]

Graham Taylor, PhD

And then I think 

[01:10:15]

Nima Tamaddoni, PhD

Oh, Hardik is coming live, so now you can ask him.

[01:10:18]

Graham Taylor, PhD

Okay, yeah, Hardik.

[01:10:20]

Nima Tamaddoni, PhD

Yeah, Hardik is one of our panelists joining from university of Montana; he has some questions, thanks.

[01:10:26]

Dr. Gaurav Sahay

Hi, Hardik!

[01:10:27]

Dr. Hardik  

Thank you. Oh, thank you Nima Tamaddoni, PhD. My question is about the temperature stable mRNA LNP development, what do you think in terms of timeline it will take or any kind of papers that you have come across that looks promising?

[01:10:44]

Dr. Gaurav Sahay

Yeah, I think, you know, papers by Yuzu Dong, they were using, they are like different, you know, like sucrose and things like that which are already in LNP formulations. I think this is an active again area of research especially since this got so much attention that I am pretty sure, you know, BioNTech, Pfizer, Moderna, their formulation scientists are working day and night and perhaps they have already figured it out, right? Like when this started and people were saying, oh, is this minus 80, I said like, with so much interest and this being a pharmaceutics based problem you will start seeing solution and then it became hey it is -20 and at that time like these LNPs were not well characterized for like, you know, for Pfizer because they were getting it from like I think Acutas and BioNTech to really know what the stability characteristics were. So, to de-risk it, minus -80 was a good idea but I think as they get more stability data, I think they might reach it to like minus 20. But making it room temperatures stable that I think would have a higher impact. I think they can reach very quickly at 4 degrees with some of these changes that they are adding different additives there. But I think making vaccines to be room temperature that has been a long-standing goal of gates foundation. I know Bob Langers and Anna Jank, like she is working on making, you know, other protein kind of vaccines that are room temperature stable

So, I think this, the timeline I think is much faster because of the pandemic, it is hard to predict science. I do not want to, you know, be a scientist or humble enough to, you know, not to say this will happen by this time because science can give you, you know, can make you humble at a certain time, but I think with so many people working on it, just especially to four degrees, I think they will reach pretty much quickly and I think a lot of people are working on it but I think we also have to understand why the stability is an issue, right? Is it the lipid nanoparticle, right? Like is it the LNP that is unstable enough? Is it the mRNA? Is it interactions with the environment? So, it is a lot of physical chemistry sort of in my mind that needs to be well understood to resolve this problem and I think it is much more of a simpler problem than escaping the endosome which has so much biological barrier. So, this problem, you know, is a main fundamental pharmaceutics problem like pharmaceutical industry does this for a living. So, I am pretty sure they will come up with ways, I think one way is lyophilizing, right? Like lyophilization of these particles, they might be trying that as well.

So, in a powder form and then you re, you know, and then we put that in our review as well. So, there are other methods to make things stable like lyophilization and things like that that also could make it much faster. So, I think it is going to be faster than we think. But I think to bring them to room temperature like that is something which will really have a big impact. So, if you could come with formulations that are room temperatures stable, especially for the whole world where then you do not have to maintain the supply chain would be just transformative, and for therapies as well then. Again whatever happens for the vaccines and making them better potency wise or for making them more stable helps therapeutics that would come in the future as well.

[01:14:40]

Dr. Hardik  

Thank you, Dr. Sahay.

[01:14:43]

Graham Taylor, PhD

Yeah, thank you very much. So, there are quite a few questions and we are picking out some of the questions that Dr. Sahay might be best suited to answer, and there are a lot of other good questions. If you have a question that does not get asked or does not get answered, know that we have it and we hope that we can follow up and address it at one of the later Q/A discussion sessions on maybe some technical aspects of sample preparation or what is the best way to prepare samples things of that sort. One, let us see, so on point, Yemen Zhao had mentioned lyophilization formulation of LNP which you touched on, so that is another possible avenue to try to help with stability to stabilize vaccines or other drugs. Mosain Ramazan Poor has a good question, is the molecular mechanism at the LNP endosomal membrane fusion and gene room? So what do we know about molecular mechanisms of LNP endosomal fusion or release and do you know anything about the local lipid phases that assist with that gene release, so…

[01:15:50]

Dr. Gaurav Sahay

Again, a great question! I think, you know, we are getting like some clues of how this is happening, still, you know, I think it is much harder, especially what happens in vivo. But I think, you know, we had a review where we thought there are different processes, right? Like the proton sponge was there, like fusion was there but I think it is becoming more and clearer that there is some sort of an endosomal fusion that is happening. There are papers even recently where the concept that there is an inverted hexagonal phase that happens once the ionizable lipid interacts with the negatively charged endosomal membrane and that causes this inverted hexagonal phase of an arrangement that leads to a better escape. So, if you can get from that lamellar phase to an inverted hexagonal phase, once the particle lands on an endosomal membrane in the luminal side that arrangement sorts of helps in the escape process, having said that I think, you know, those are all good hypotheses to have and have some, you know, already some biophysical merits and people have studied, like Peter Cullis, recently Dan Siegert had a paper where based on that hypothesis, he made a lot of different lipids that can help in better gene transfection, possibly through an endosomal escape. So, I think those are fewer methods, initially people thought oh back fusion from an endosomal membrane that could be something. So, I think there is fusion that is for sure. Inverted hexagonal phase may be one of the ways of happening but what are the molecular regulators that are doing it, right? You are having this much of galectin8, binding on an endosomal membrane.

So, I think with things like ELNPs, now we can pinpoint what are the other molecular regulators that are helping in that fusion process, it is just the inverted hexagonal phase or, you know, I consider like ionizable lipids as an active ingredient because, you know, once… so, for encapsulation for example the particle is just encapsulating it, it is just an excipient but once it starts to interact with an endosomal membrane, and then changes that membrane permeability through fusion, and causes things like galectin to go up, it becomes sort of in my mind an active sort of an ingredient engaging with the biological membrane and again coming back to your question, the other regulators, therefore there are other regulators in the story galactin8 is one that might help us understand how this transport is happening, is there like a molecular transporter that becomes active and helps in this fusion process.

So, I think there is a lot to know there as well but coming back to your question about what are the particle characterization, it is I think it is fusion and an inverted hexagonal phase, what are the biological milieu, what is changing there, I think that if we start figuring that out that can give us a better understanding of how this is happening from the biological perspective and how fusion is occurring because like a lot of these viruses have different sort of peptides that interact with the biological membrane in a certain way and my hypothesis is that LNPs might have similar characteristics as well and that can lead to a better fusion too. Again long-winded answer but I am an endocytosis guy for a long time. So, if you ask me an endocytosis question, get a long-winded answer, if you will ask me regulatory I will be there.

[01:19:46]

Graham Taylor, PhD

Yeah, we will stay away from the FDA guidance although. I think we have tackled quite a few of these excellent questions. Thanks, a good pouring in, Dr. Sahay, we cannot say thanks enough, we really appreciate you taking the time to share with all of us here, and so…

[01:20:08]

Nima Tamaddoni, PhD

Yeah, well, we are truly humble. Thank you so much Dr. Sahay for your time and I am sure 400 attendees enjoyed the talk and ask a lot of good great questions. We are going to go to a social session, it is going to be open to all, and it is kind of a break basically, we are going to come back at one o'clock with Daryush Muhammadyani from Johnson & Johnson, he has worked in Moderna previously and now he is working at Johnson & Johnson, he has also played a key role in computational approach of these vaccines and therapeutics, the RNA, LNP. So, thank you all attendees as well, we are really grateful to have you all, and this is a start of a platform, educational platform that we hope to keep you all within this community. So, thanks.

[01:21:14]

Dr. Gaurav Sahay

Thank you, this was my pleasure. Thank you so much. Bye.

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