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Speaker Doctor Syed Reza, the scientific and sales consultant in NOF Corporation, so Syed served as a drug delivery consultant to the company since 2017, and in this role, he has been responsible for managing industry partnerships for the company's Code Sum technology and voices on strategic partnership and licencing.
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Previously, Syed was head of U.S. business development for OctoPlus and managed industry alliance and partnership for the company's protein nanoparticle technology platform, and prior to that, Syed served as director of business development for Hoffman Peptide Technology Group in Colorado.
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He is a graduate from of the New Jersey Medical School with an MD and PhD degree in molecular biology.
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And today, without further ado, he's going to talk about the biodegradable nanoparticle, lipid nanoparticle for mRNA therapies and vaccines.
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The floor is yours.
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Thank you.
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I'll just use this mic.
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Thank you everyone.
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I don't think I need to use a mic so much.
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I'm guess I'm loud enough so to get started.
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Thank you for that great introduction.
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So let's see.
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So I just want to give you a quick background on NOF, NOF is a company, we're based in Japan.
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We've been in business for more than 80 years and our specialty is lipid technology and PEG polymer technology.
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We are a diversified company within that technology group and our products and are used in the whole range of applications ranging from automotive, industrial chemicals all the way up into pharmaceutical and life science products as well.
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And then earlier this year, we also announced a collaboration with Phosphorex where we have developed a relationship to provide a one stop shop for LNP development to enable the next generation of mRNA medicines.
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So since this is a little bit more of a general audience, I'll explain a little bit about LNP technology.
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So lipid nanoparticles are now widely emerging as a well-used modality for delivery of nucleic acid medicines, for vaccines, for gene therapy, for protein replacement, for protein expression in vivo like enzyme replacement therapies.
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And LNPs are generally of two types.
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You can make them with lipids that are relatively passive.
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So we have a structure lipid and ionizable lipid.
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In our case we have the SS lipid.
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We also have other helper lipids to modify the function that can be phospholipid or other types of fatty acids and some PEG lipid to provide a hydrophilic layer on the outside.
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And then we can have active targeted LNPs which can include some molecules to attach a ligand and functionalise the LNP on the surface.
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And both of these are complementary modalities or approaches that can be used to allow the LNP to go to specific tissues or cell types in the body.
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So in terms of the nature of this formulation is actually very complex because not only are we dealing, we're trying to control the physical properties of the nanoparticle size and size distribution that has a role in by distribution and safety.
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But there's other components.
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First of all, the mRNA itself or the DNA as a nucleic acid is relatively sensitive.
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So there's a lot of, there's some specifications and control and design around that.
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Then we have to also think about the inner relationship between other lipid components, and they'll affect both the kinetics, the drug products stability, the tolerability aspect of the LNP as well as the tissue targeting.
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And so if you change one, you'll end up sort of affecting all of these other buckets of performance as well.
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So the development of the LNP formulation is a fairly complex activity, but one of the things that we can look forward to in spite of the complexity and the reason why our company and many others in the space are pursuing this modality is that it has a promise really enable plug and play therapeutics in the future.
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So one of the things we're finding out is that if you have a single LNP formulation and you swap the nucleic acid inside the formulation itself behaves the same.
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So it's stability as a drug product is the same, manufacturing process is almost the same.
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The analytics because they're mostly you're analysing the lipids are still the same and then the by distribution in vivo targeting is also the same, of course, you know, in terms of where the lipid goes.
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So once we have identified a lipid, for example an LNP that goes to the liver, we can now use the same formulation.
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And once we prove that out in human trials, we can use that to plug in a range of other nucleic acids and then hit a further range of targets within the liver.
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And that shortens the development and risk associated with new therapeutics.
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Similarly for CNS, we have a new LNP that delivers to the CNS as well.
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So we can enable a whole range of different applications to be addressed within that particular approach.
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At NOF, our approach is really to start with a large lipid library.
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So we focus on synthesis of diverse lipid library.
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Then we take those lipids through formulation assessment, in vitro reporter assays to understand the activity of the different lipids.
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1 is able to express more or less amount of mRNA and then additionally reverify the in vitro to in vivo correlation with rodents.
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And we also get some safety readouts to make sure that none of the lipids that we're selecting for further development in our library are toxic.
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But the key feature of our lipids is that these are highly biodegradable.
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So we found that the biodegradability is very well correlated with both the activity of RNA expression as well as the tolerability.
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And so to a certain degree, the lipids have tuneable immunogenicity in that we can select different lipids that are non-immunogenic versus ones that are more immunogenic.
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And that allows us to create LNP's with different for different applications.
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And then even though these are biodegradable, we've been able to create stable drug products both as a lyophilised LNP with mRNA and for a frozen formulation at -20°.
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So a little bit about the chemistry of our lipids.
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As the name implies, these ionizable lipids are called SS lipids because they all have a disulfide in the head group.
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And then we have an ionizable heterocyclic amine here that serves to provide an ionizable group that allows for endosomal disruption in fusion.
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And then there's also an Ester bond that can also be cleaved in vivo in the cell after deliveries accomplished.
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So what happens with these lipids is that once they are internalised into the cell is a highly reductive environment that allows the disulfide to be cleaved and that then allows the Ester bond to have access to water.
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And that water further catalyses the hydrolysis of the Ester bond as well, resulting in four different degradation products to be made from a single lipid molecule.
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And that is one of the that is the key reason why these lipids are highly biodegradable.
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So this just shows the overall scheme of how these lipids function.
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There are many.
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You can think of them as like little molecular machines because they have so many moving parts to them in a sense.
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But once we have a LNP that's circulating in the blood compartment or let's say if it's locally injected in the extracellular fluid that has a pH of around 7.5, 7.4.
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So the ionizable group is not really charged at all.
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That's here.
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Sorry, we're starting over here.
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That's not charged.
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And then the disulfides are connected together.
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But once you have internalisation, then these LNPs will end up in the endosomal compartment that undergoes acidification.
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Then the LNP picks up a charge on the surface because of the ionizable group and that allows endosomal fusion.
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And then the next step that happens is that once the LNP is released into the cytoplasm, the disulfides get reduced and that allows the Ester bond to be cleaved.
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And then I'll further wrap allows the lipid to dissipate relatively quickly and that frees up the RNA.
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The RNA can be translated, and everything is good.
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So the two main types of lipids that we that I want to discuss in this presentation are is a type of lipid called COATSOME SS-CC, which has toco feral, which has immunogenic properties.
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So it induces certain cytokines and activates macrophages and T cells.
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And then we have COATSOME SS-OP, which is non immunogenic.
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It's very well tolerated, does not induce any inflammation.
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And so as you can imagine, we can use them in different settings.
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Here we just want to show a little bit about the benefit of a biodegradable lipid.
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And this is an idea that's I think is also gaining more traction with other innovators as well.
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But if you look at the example of a LNP with, for example here, DNA that's used to transect cells, what you can see is if we make the LNP with the lipid that just as a carbon-carbon bond, not a disulfide bond, that lipid LNP just sticks around in the cytoplasm.
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It does not dissipate.
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But when we have a biodegradable reducible lipid here called SS-M, you don't see the lipid signal.
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You see just the DNA, the cargo, meaning that the lipid has degraded and has been eliminated from the cell.
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And similarly, we see a correlation with expression that when we use a biodegradable lipid versus an analogue that's slightly less biodegradable.
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So just a difference in the degree of biodegradability, the more biodegradable lipid has high levels of expression, whereas the less biodegradable lipid has lower levels of expression in vitro as well.
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So that again correlates with what our hypothesis is about the mechanism of biodegradability and its impact on or any expression.
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Similarly, in vivo, if you look at the tolerability, you can again see that in the case of SS-OP single dose into mice, it's pretty well tolerated.
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There's not a lot of hepatic tox that's observed.
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But in the case of MC3, which is another lipid, another ionizable lipid that's been used in the clinic and it's part of an approved product that has higher toxicity because we know from our own experiments that MC3 is less biodegradable than SS-OP.
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So using these types of lipids, we're now also using them to create LNPs that have a specific pharmacokinetic, pharmacodynamic profile.
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So we have a LNP that goes to the liver for hepatic targeting.
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We have another LNP that delivers to the CNS again via a non-invasive route.
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And then we have another LNP that goes to the spleen from the IV route, another one for transforming primary T cells and cell culture and then two different kinds of vaccine formulations as well.
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So I'll focus on just a few select ones to give you an idea of what is possible with the LNP technology these days.
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So common LNP applications delivery to the liver and usually when you make any kind of initial LNP, you'll find that a lot of these LNPs by default will just end up going to the liver anyway.
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So here we can see that LNPs made with SS-OP are no different with the luciferase cargo.
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We can see very good liver expression.
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But one of the interesting things is that the kinetics of this expression really fast.
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So if you use for example, the EPO mRNA to track the kinetics, you'll see that after injection just within one hour we can see the protein expression and then it peaks in three hours and starts to decline by 12 hours.
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So the kinetics are really very fast.
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These LNPs are taken up in the target organ very quickly, quickly depackaged and the RNA is expressed.
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Recently, after the advent of the COVID mRNA vaccines, we also went on to do a second comparison of our LNPs with the two vaccines that were approved for as an mRNA LNP.
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And we basically put luciferase mRNA inside those LNPs as well as compared to with our hepatic LNP.
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And you can see that the activity with the NOF hepatic LNP is higher.
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And again that's correlate with the fact that these lipids are in a sense highly biodegradable.
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So that seems to have some benefit for increased expression compared to some of these other ionizable lipids.
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And again, it's just about the degree of biodegradability.
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I don't mean to imply that other lipids are not biodegradable, but it what we have also observed is that it's has to do with the speed of biodegradability, the faster it degrades, the better it is.
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So the case of CNS delivery, we again performed a screening where we looked at different ionizable lipids and some of the other components in our formulation.
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And by varying the formulation ratios and the identity of the lipids, we're able to test some new LNPs.
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So in our assay we basically take mice, we tell them to lie back down on their back and stay still.
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And then we put a little couple of drops of the LNP solution in the nose.
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And what happens is that the mice nasopharynx is highly innervated with a lot of olfactory nerves and trigeminal nerves.
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So it is thought that the LNPs get absorbed and they'll get trafficked into the CNS. And so, but you can see that in the case of LNPs that are labelled just for the dye, so it's a lipid dye that's in the LNP lipid layer.
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You can see that we can start to see some tracking of the LNPs inside the brain itself.
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So this is a non-optimised formulation that our starting point was our hepatic formulation delivered by the nose, not a great effect, but when we improve and optimise the formulation, we can actually see a good amount of delivery.
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And the expression in the brain of a luciferase mRNA is also higher compared to the liver compared to the hepatic formulation.
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And we've also performed additional studies with directly injected LNPs as well where we can show that we can see expression both in neurons and astrocytes.
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So that's one of the benefits of mRNA that all you have to do is get the mRNA to the cytoplasm.
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If the cell is not dividing like a neuron, you can still get the protein to be expressed.
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So we feel that with some of the work that we're doing to show the safety and tolerability of these lipids that we have the potential to create perhaps new CNS targeting applications that are non-invasive, that are more patient friendly, that are non-toxic, so that we can give relatively repeat doses and be able to address a wide range of indications.
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Finally, we'll go with some of the vaccine data real quickly.
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So for vaccine applications, we've actually been trying to profile the cytokine expression induced by different lipids.
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And one of the things that we find is that very immunogenic lipids tend to cause a lot of hepatic injury as well.
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And so the toxicity of some of these lipids goes hand in hand with their inflammatory properties.
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And usually the lipids that are highly toxic are the ones that tend to induce a large range of cytokines.
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So some of the cytokines that we focus on are what are called pyrogenic cytokines like TNF Alpha, IL-6, that if you get high levels of it, you're going to feel, you know, they introduce myalgia and they have other very broad inflammatory effects that are that lead to adverse complications.
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But for vaccines generally, if we can just get interferon beta, then that's really the ideal cytokine for cellular immunity.
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So we went out and did a programme to screen LNPs for different cytokines.
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And I'll just jump to this slide over here where we screen some of these lipids in vivo and mice as well.
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And what we find is that some of our lipids do induce interferon beta, but they tend to also minimise the secretion of pyrogenic cytokines like IL-6 and IL-1 beta.
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And interestingly for this lipid, it tends to do this only when it has a cargo inside.
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So if it's the empty LNP, we don't get the cytokine expression.
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And so we think that what's happening based on what's known about how mRNA and DNA induce immunogenicity and how lipids can also induce immunogenicity is that there are two parallel pathways.
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The lipids when they're degraded can induce some stress related pathways that synergize downstream of the toll like receptor that is response for immunogenicity from nucleic acids.
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And so the two molecules in the same formulation are actually performing sort of crosstalk that gives us a synergistic response and tailored immune responses.
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So using that know how there was a lot of other work that I've had to skip over, but we've been able to optimise an LNP for inducing T cell responses, for cytotoxic T cell responses for things like tumour immunotherapy and a second one for inducing antibodies preferentially.
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And so we have a spectrum of activity, and we can actually tune these formulations to favour one or the other.
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But looking at the extremes of the formulation, you can just see that with our CTL inducing LNP, we can actually get good T cell killing against an OVA mRNA antigen.
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And similarly, when the same OVA mRNA is placed in our LNP for antibody production, we can get a good amount of antibody produced in mice against OVA as well.
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So it's very interesting that we're able to tune the activity this way and we're exploring what we can do next to really take the immunology applications further.
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And finally, we've also been able to take these biodegradable lipids and really be able to scale up the process while conserving the activity and purity of these lipids and then package them into a drug product that's stable under different conditions.
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So we can make a freeze dried LNP with through lyophilization so that when we rehydrate it by just adding the rehydration buffer, we can recover almost all of the activity that was in the neat formulation that went into the freeze drying step.
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And this drug product is stable at -20 and 4°C for up to one year and at room temperature for three months.
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Sorry, for two, yeah, three months.
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And then we also have a liquid frozen formulation.
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So it doesn't involve lyophilization, but that can be stored at -20 or -80 and that again is stable for up to four weeks, at -20°C and now we're doing long term stability studies on that as well.
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So provides a lot of flexibility if you're looking for an LNP that you can take forward into the clinic and how you would work with it.
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So in terms of NOF, we are really offering end to end LNP expertise starting from our library of lipids.
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So a lot of knowhow around specific formulations and then we also offer manufacturing and a lot of the support to run a lot of the critical assets to get you to the pre IND stage with an LNP programme.
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And we're very glad to do a lot of this work in partnership with Phosphorex.
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If you'd like to learn more, please stop by at our exhibition hall booth.
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Thank you.