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And hello everyone.
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Thank you for taking your time to introduce our technologies.
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My name is Kazuki Ohsakama, Senior Scientist at NOF Corporation.
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NOF Corporation is a chemical manufacturer based in Japan, and we supply many kinds of chemical materials such as surfactants, the speciality chemicals, toiletries and so on.
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I'm from Life Science division and has been developing novel DDS materials and lipid nanoparticle formulation.
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Today I'd like to talk about our biodegradable lipid nanoparticle technology for gene therapy and vaccines.
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That is COATSOME SS series.
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Non-viral RNA delivery is biodegradable ionisable lipid.
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I will start with LNP background. Nucleic acid therapeutics encompass a wide range of applications ranging from vaccines, enzyme replacement, and gene editing.
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Lipid nanoparticle LNPs have been developed for delivery of RNA.
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However, the toxicity of the ionizable lipids limits their clinical use.
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Our molecular design to minimise the toxicity should be pursued to broaden the pharmaceutical and clinical options.
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A key issue for reducing size toxicity and accelerating the intracellular release of cargos is biodegradability.
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For RNA vaccines delivery to antigen presenting cells, antigen expression and T cell activation are also important.
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Here is a component of LNP.
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As you already know, LNPs are typically composed of four lipids.
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ionizable lipid, cholesterol helper lipid and PEG lipid.
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The ionisable lipid is the key component to get LNP delivery efficacy and cytotoxicity.
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We developed our proprietary ionisable lipid COATSOME SS series SS lipid.
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SS lipid can be easily degraded in cytoplasmic condition and enables efficient delivery to delivery of encapsulated nucleic acid such as messenger RNA and plasmid DNA and so on.
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On this slide, I'm showing our product lineup of SS lipid.
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We have developed two SS lipids COATSOME SS-EC and SS-OP.
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They are used in preclinical development with our partners.
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We recommend SS-EC is used for cancer vaccine applications and SS-OP for applications such as hepatic delivery, splenic delivery, infection vaccine and ex vivo transfection.
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I will explain the characteristics of these lipids at next slide.
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SS lipids are unique ionisable lipids are designed to be biodegradable.
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SS lipid has three key units and the first is, as the name suggests, disulfide bond shown in box 1, and this bond can be cleaved in the cytoplasmic environment.
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Then in box 2, tertiary amine head groups with PKA below 6.5.
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Therefore, these lipids are not ionised under physiological conditions but positively charged under acidic condition such as endosomal environment.
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And book three shows hydrophobic lipid tail.
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SS-EC has tocopherol backbone that functions as an adjuvant, so SS-EC is useful for cancer vaccine application.
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SS-OP has a phenyl ester linkers which is highly biodegradable.
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So SS-OP is very effective for RNA delivery.
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To understand how SS lipid works, let's see the mechanism of action.
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This picture shows the process of LNP taking into cell.
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Under physiological conditions, the LNP is not charged.
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But once the LNP is taken up into the cell by endocytosis, the LNP becomes positively charged in the endosomal acidic condition.
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Then the LNP interacts with negatively charged endosomal membrane and leads to membrane fusion and release of the LNP core into the cytosol.
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Glutathione with high concentration is in the cytosol.
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So the disulfide bond of SS lipid is reduced by reduced rapid rapidly by glutathione.
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At that time, the encapsulated nucleic acids are released in the cytosol.
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This is a mechanism of action for SS lipids which is highly biodegradable and very effective for RNA delivery.
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Here we checked the gene expression activity of SS-OP which has higher potential for RNA delivery from self-degradable phenyl Ester linker.
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The thiol group is generated after cleavage of disulfide bond then it can be attacked to this phenyl Ester as therefore SS-OP is self-degradable lipid.
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On this test.
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We compared SS-OP and SS-OB which has no self-degradable benzoyl Ester linker.
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The bar graph shows in vital transaction results using MAF cell line and GFP mRNA, the transfected cells as blue bus were almost the same between SS-OP and SS-OB.
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But in the black bar, GFP fluorescence of SS-OP has higher than SS-OB.
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This result shows the highly degradable SS-OP can enhance gene expression activity.
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We tested systemic toxicity and genotoxicity of SS-OP.
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Here we showed hepatic enzyme levels as an indicator of hepatotoxicity with competitor lipids contained in marketed drugs.
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In the case of IV injection to mice as 230 milligramme ionisable lipid per kilogramme, we can see SS-OP stands out as a very safe lipid.
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On the other hand, competitor lipid C has a higher response. In a single ascending dose study in rats SS-OP indicate safe lipid around 200 milligramme ionisable lipid per kilogramme.
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Also, genotoxicity study of SS-OP was negative, so more biodegradable lipids were shown better tolerated.
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We have developed six types of LNP formulations as shown here, hepatic delivery, ex vivo transfection, splenic delivery, RNA vaccine, active targeting and SS lipid library screening.
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Today I'd like to talk about some representative data.
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At first I will explain hepatic delivery.
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Liver is a common target organ for gene therapy of LNP delivery.
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This slide shows in vivo study with erythropoietin and erythropoietin encoding mRNAs compared between SS-OP and competitor lipids A, B and C, which are used in marketed LNP formulation drugs.
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As shown here, SS-OP had higher mRNA expression activity than competitor lipids.
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For hepatic delivery, we tested lipid dosing study using SS-OP.
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In this study, 5-methoxyuridine or pseudouridine modified EPO mRNA which are chemically modified were used to avoid side effects of RNA derived immune stimulation.
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Injections were repeated every seven days for five weeks.
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As you can see, SS-OP showed consistent gene expression with repeat dosing.
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Our hepatic formulation is also useful for gene editing with the CRISPR-Cas9 system.
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Here I'm showing transthyretin gene targeted gene editing.
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Cas9 mRNA and PTR targeting guide RNA were co-encapsulated in LNP and LNP were IV injected to mice.
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As a result, 55% of TTR encoding genome were edited and serum TTR level were decreased by more than 90%.
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Here is the next application.
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Splenic delivery data.
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We prepared SS lipids LNP containing for splenic delivery formulations and hepatic delivery formulations.
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After injection to mice, we conducted each organ extraction and luminescence measurement.
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The blue bar indicates the activity in liver and orange bar means in the spleen.
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As for hepatic formulation, the accumulation in liver is higher than spleen, but in the case of splenic formulation, both SS-OP and SS-EC showed efficient spring delivery.
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Therefore, SS lipids can be selectively delivered to the desired organs, spleen or liver depending on the formulation design.
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Next, I'm showing the RNA vaccine application.
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Firstly, we evaluated gene expression and CTL activity between SS-OP and SS-EC by SC injection.
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As a result, gene expression activity was higher than SS-OP, but CTL activity was opposite to the result.
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We believe this is because of the adjuvant effect of SS-EC and we think SS-EC would be potentially used for cancer vaccine.
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We investigated humoral and cellular immunity.
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In the photograph.
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We compared humoral immunity of SS-OP and competitor lipid B.
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Mice were immunised at two times via IM injection followed by measurement of IgG production.
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We can see that SS-OP showed much higher IgG production than competitor lipid.
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For cellular immunity study in right graph, we used SS-OP and SS-OP/EC combination formulations to induce CTL activity.
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The mice were immunised by over mRNA encapsulated LNP's.
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Then at day 7 target cells having antigen were injected to mice then measured CTL activity.
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The result showed OP/EC combinations induced a higher level of CTL activity.
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This result supports a high CTL activity of SS-EC, as shown in the previous slide.
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From these examples, SS lipids encapsulating mRNA expressing antigen proteins efficiently induced humoral and cellular immunity.
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We also evaluated the antitumor effect of three types of formulations.
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Here mice were inoculated with over expressing tumour cell lines.
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Then ten days later they were vaccinated with over encapsulated LNP.
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As you can see, SS-OP and SS-EC combination LNP encapsulating mRNA expressing antigen protein showed a significant antitumor effect.
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So we recommend using this OP/EC combination formulation for cancer vaccine application.
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This is recently published data of low inflammatory vaccine of the influenza virus.
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In depth and middle graph.
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The data of IgG assay compared between SS-OP and competitor lipid B.
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Mice were immunised at two times via SC injection using hemagglutinin chain and neuraminidase encoding mRNA followed by measurement of IgG production.
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As shown here, SS-OP had comparable IgG production in comparison with competitor lipid B.
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On the other hand, for inflammatory cytokine production in the right graph, the amount of interleukin 6 of SS-OP had obtained a lower than competitor lipid.
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Other cytokines were produced at a similar tendency.
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So SS-OP LNP can be used for low inflammatory infection vaccine.
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This is freeze thaw LNP stability.
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As you know mRNA LNP has a programme on storage stability.
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To overcome this issue, we have developed freeze thaw LNP.
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Here we showed the preliminary data of EPO mRNA encapsulated freeze thaw LNP and it was stable for at least one month at -80 and -20°C.
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Long term stability tests are ongoing.
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Also, we have developed lyophilized LNP stability, and it was stable for one year at 4°C and three months at room temperature.
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On another note, for extrahepatic delivery, we think active targeting LLP such as ligand modification is needed.
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This channel is an example of active targeting with using SS lipid to lymphatic endothelial cells.
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A quick reaction was used for conjugation with ligand between DSPE, PEG, DBCO and azidated antibody and anti-mouse podoplanin antibody was used for a specific ligand in vivo study.
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The results are shown here that targeted LNP are taken up by LECs with a higher efficiency than non-targeted LNP.
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The uptake was significantly enhanced only by the anti-mouse podoplanin antibody modification and the fluorescence from the targeted LNP was selectively localised in the lymphatic vasculature.
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So SS lipids were applicable for active targeting.
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This modified this ligand modified LNP.
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NOF can supply not only SS lipid but also a wide range of activated PEG lipids for active targeting.
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So if you are interested in our product lineup, please feel free to contact us.
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Finally, I'd like to talk about our SS lipid library.
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This slide indicates the hepatic mRNA expression activity and hepatotoxicity. Bar graph means that this activity in liver.
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In particular, green bar indicates the competitor lipids and blue bar indicates our SS lipid library. As shown here SS lipid library has higher activity than competitor lipids.
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As for hepatotoxicity, orange dot means ASD and red dot means ALT at 24 hours after injection.
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And that this dotted line means the AST/ALT upper limits as normal mice competitor lipid C indicates AST/ALT levels over normal, but meanwhile SS lipids library were under normal level.
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To summarise, SS lipids library showed better activity and lower toxicity than competitor lipids.
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We have succeeded in developing SS lipid library with significantly higher activity and lower toxicity against competitor lipids.
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NOF can offer LNP formulation development service using our proprietary ionisable lipid SS lipid library by utilising NOFs collaborator Phosphorex.
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It's a CDMO company located in Massachusetts State in the United States of America.
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If you'd like to know more information about this service, please visit our booth number 86.
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In conclusion, SS lipids are biodegradable ionisable lipids that are well tolerated in people.
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SS-OP having a self degradable linker enhanced gene expression activity.
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SS lipid induced both humoral and CTA responses with low inflammatory demonstrating efficacy as a cancer vaccine.
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Long term storage of lyophilised LNPs and freeze thaw LNPs containing SS lipids was demonstrated and I know if we can offer LNP formulation development service using our proprietary ionisable lipids.
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I got some accessories for large variety type of application by utilising our collaborator Phosphorex.
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If you have any questions or inquiries about our products, please feel free to contact us.
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Of course, we are waiting your visit to our booth 86 at this conference.
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Thank you very much for your time.
