Nanoparticle-based mucosal delivery of RNA interference therapeutics

so I came up with the title nanoparticle-based mucosal delivery oh this one as well hello okay okay so um yes so I am focusing on a different route of administration I don’t think this has been covered at all during this course yet but that is to use nanotechnology to facilitate delivery of rnai rnai therapeutics across the mucosal surfaces but I also want to try and give you an impression or sun a description should i say of my experiences setting up a a spin-out company in denmark based on the technology which i’m going to focus on which is very simple technology which is based on the poly cation keitus on for delivery across mucosal surface but i think you know the thing i want to stress is that and I’ve got another title simplicity the key to clinical translation and we’ve heard a lot about elaborate designs of nanoparticle systems but the way I see it is the simpler the better in terms of getting a product on the market so you’ll see that this this system is pretty simple but I think you also see that we’ve had some quite good success by using it so as this is a nanomedicine school I should set some educational goals and that is that what I’ll do is I’ll describe systemic barriers and what is the motivation behind alternative routes of administration so why do we need to to deliver materials for example across the mucosal surfaces rather than than injectables I’ll focus on mucosal barriers and delivery solutions that will be half of the talk and the other half of the talk will be utilizing the same technology for the treatment of macrophage base diseases so just a you know to show you where we are who we are what we do so Aras is in the peninsula that reaches out from from Germany it’s on these coasts of Jutland not far from Britain I suppose across the North Sea which we’re based at the University of hose and the nano science center which was spawned out of the physics department at the University in 2002 since those days we’ve now got a purpose-built I nano house as you can see here that was opened in 2012 is it now 2012 and as you can see we’ve got 55 pool full professors associate professor assistant professor 66 postdocs and approximately onion 40 PhD students but just to say that this does look at a quite small nano house but underground and in the basement two-thirds of the area is actually underground so a lot of them that the machines etc are buried beneath the this this this building so you know in years to come will be talking about nano or would it be femto etc I’m not so sure I think that nano may come and go but I think what we do need is interdisciplinary and the former director of I nano in 2002 I think was very forward-thinking because the name I Nano is the interdisciplinary nano science center and I think that interdisciplinary is the key word so in I nano we can draw upon the combined expertise of physics chemistry biology for example all working together on projects and interdisciplinary to me is not a scientist who branches out in a completely different field that’s beyond these expertise interdisciplinary for me is to have possibly a house and nano house where you’ve got experts who are specialized in their particular subjects who can contribute together in combined projects I don’t think scientists who branch out of their expertise or too far out works I think entices interdisciplinary is sticking to your expertise and contributing that expertise to a combined project Luna Luna is our our nanomedicine program it’s sponsored by lung back the Danish pharmaceutical

company and it’s focused towards treatment of musculoskeletal and neurodegenerative diseases and as part of that we work on drug design target validation drug delivery of which I’m prominent and bioimaging cell-based therapies and preclinical models but as you can see we combine all these expertise together in these and combined efforts and just to say that this was established approximately two or three years ago and it was we got funding for about 50 million dollars for this okay so let’s go on to a bit of the background and the motivation why we need delivery solutions for SI RNA now we do know that SI RNA let’s just talk about conventional srnas for the sacred have a poor farmer kinetic profile due to susceptibility to serum nucleases their size allows them to be readily excreted within a couple of minutes you get nonspecific accumulation and their poly ionic and so it’s very difficult for them to cross the biological membrane that is the cell surface in the early days there was a lot of optimism for the use of SI RNA in the clinic some early studies that one my McCaffrey in 2002 and Sue shake in 2004 in the McCaffrey one they used naked SI RNA and showed that by dynamic injection so that’s in ject in quite a lot of volume of the material in the tail vein of a mouse they could downregulate egm luciferase that is in the liver in the sous-chef work they conjugated a cholesterol to the SI RNA and cholesterol is metabolized in the liver and again they could see some therapeutic effects by down-regulating up OB which show then add knock-on effect of showing them down regulation of systemic cholesterol but we’ll come back to this but even in those early days the liver seemed to be the primary target or the target that was most easily approached should I say so naked srna didn’t seem to work and unless you add these artificial situation so we had an old range of nana carrier systems lipid-based or poly cotton based we work on a lot of different systems and elaborate designs as well so we’re part of this trend to to to invent the the spaceship should we say but for this talk I’ll talk about a very simple conceptually simple system that’s based on a kite is on a polysaccharide that self-assembles with SI RNA or nucleic acid should i say to form nanoparticles or polyplexes these can also be called polyelectrolyte complexes and poly poly cotton based systems but we’ll just call them polyplexes but we know there was problems with delivering conventional nucleic acids or should we say naked nucleic acids we thought that having these nano carrier systems would overcome the problems associated with naked delivery but I think in this one slide you can see the complexity of delivering something systemically so we can have a beautifully crafted nanoparticle with the previous speaker should fantastic data of a well-crafted monodisperse system and that’s fine on the bench but what really matters is what happens inside the body inside the bloodstream so if you inject a nanoparticle and this really applies to most none of particles that are not surface modified within a few seconds you can get protein adsorption to the surface which causes aggregation and this is one of the reasons why you know systemic delivery of nanoparticles that are not surface engineered can find accumulation in the lung because you get aggregation so you have this nice nun a scale system that suddenly becomes a macro scale system and then it gets trapped in the first capillary capillary bed which is in the loan so in the early days of polyethylene amine based systems they got aggregation and could actually get silencing in the lung but obviously this is not transferable to the human situation we can’t have aggregation in the lung but one of the main barriers to a systemic delivery system is the mononuclear phagocytes system which is a series of circulatory monocytes and fixed tissue macrophages I know one of the speakers is talking about cut for

cells in the liver that are basically designed to pick up following materials entities floating around in the body of which nanoparticles are seen their phagocytose they’re elaborately designed to destroy this angled material and this is one of the reasons why we’ve had so much success with nanocarriers when we delivered to the old sure si si RNA delivery to the liver because one of the main sites of these phagocytes or the population is in the liver plus it’s highly profuse it’s got a good blood supply but you know it’s no surprise that people are getting delivery to the liver the question is are they getting delivery to the right cell population and I have not really seen definitive evidence from anybody working in the field on the ratio of derm accumulation in say the cup for cells which are the phagocytes and for example the apat asides which is the target cells if we can reduce this so interaction with the MPS we still have to get extra possession across the endothelial barrier and then into the cell so you can see there’s a myriad of barriers when you try and inject something systemically we can surface engineer the surface of particles so pegylation is the common practice for this you can have different graph densities so you can have different and configurations on the surface the most notable configuration is the one in B which shows a brush border a bush configuration which does show reduced interaction with phagocytic cells but then you have the stealth dilemma it’s one thing reducing interaction with a cell type such as a phagocyte but then you’ve got to retarget that particle into a target cell and that is the problem where do you tether the Ligon do you put it on the end of the peg if it’s a peptide it can then be immunogenic or do you bury it deep down in the brush border in which case it’s not accessible to engage with its target so from my point of view using another carrier system that’s pegylated is not the ideal scenario that you want to go for this is not the full shebang of clinical trials but I focused on one of the main players in the RNAi field Alnylam and this is sort of a recent search I did and I think what’s the most striking thing is that in 2014 where we are now that most of the indications which they’re working on are focused on the liver and in fact in their website on their website one of the criteria is that they want a liver targeted a liver target and that’s primarily because accumulation in the liver they’ve had problems directing the nanocarrier to any other sites as we all have and so now they’re basically trying to make the most of where they are and that’s what ever done system they’re using they’ve used lipid base now they’re using sugar conjugates it’s still accumulating in the liver which really reduces the the the the old span of all should I say the range of diseases that she can treat with any RNAi based therapeutic interestingly there was this naked SI RNA without any nanocarrier formulation which was against the respiratory cynosure virus that actually reached phase 2b and this was injected directly into the pulmonary track for delivery to the lung and they seem to get obviously quite nice day too early on but this I think has recently been withdrawn so now they solely focused on liver indications as far as I know technion which is also working on lipid based systems again are working on liver targets I just put this calando which was one of the first polycationic polymer systems to reach clinical trials it’s um it came out of the lab of marquis davis in am Cal Tech and where is used in an elaborate system with a cyclodextrin ways some adamantine with pegylated and transferrin for targeted delivery to tumor cells but as far as I know this has also been withdrawn so where we are now is that the indications that we’re focusing on is in clinical trials is mostly the liver so I put a focus on living indications

predominantly due to physiological consequences after IV injection that can be phagocytic uptake this can be accumulation due to highly perfused and blood supply it can be the disrupted endothelial you find in the liver it can be picking up by hijacking the appo protein II I think that then goes as a matter of consequence to the liver so I would propose that alternative strategies are required and simple strategies so here it go Eli go with this very simple system which is a poly-cotton base nanoparticles for Asano we use a poly-cotton that electrostatically interacts with the srna to form a nanoparticle entropy driven it’s a Niley efficient process but this simple system actually installs many properties into the nana carrier is a self-assembly system so so some degree the SAS RNA is protected and entrapped within the system it’s nanoscale so it’s small enough to extrapolate across certain diseased tissues it normally has a positive a net positive charge and when you make these systems they’re more stable when you have an excess of the poly cation which is got the predominantly amino group so it’s a positive charge entity which interacts very well with the cell surface you can control the functionality which is a form of kinetic cellular targeting up take root and RNA release by the design of the poly cotton so you can do this by synthetic systems but I would say that this has got clinical translation potential because it’s a very simple system we’re not relying on any post modification I won’t go through this slide but just to say there’s an OL array of polymers both synthetic and natural are focused on ones that have been used in vivo that have been used for delivery of a SI RNA when I say si a this could be the conventional srna or this could be precursor of micrornas primary micrornas for example but we’ll just stick to the srna is a general term we can change the polymer architecture especially if the synthetic systems and you can see that you can change the functionality by using different polymer systems I won’t go into detail on this but just to say this versatile and flexible even though it’s conceptually very simple so here we go this is a simple system that I proposed this is the kite assign system which is cosine is a poly-cotton derived from Titan which is found in the skeletons of insects the shells of crustacea by deacetylation you install an amino group and a glucosamine and it’s this amino group that term facilitates this interaction with for example a poly ionic nucleic acid very simple it’s the second most abundant polymer natural polymer shell assay but what people are found even before the days of SI RNAs it’s been used for its mucoid easy properties and that is that it facilitates interactions with glycoproteins that are found in the mucus that lines the mucosal surface on the epithelial cells I’ll go into a bit detail on that it’s also been shown to transiently open so for a short period of time open the tight junctions between the epithelial cells at the mucosal surface the system itself is this poly cotton plus SI RNA into a nanoparticle and just to say although this is a conceptually very simple system it took about two years to optimize the type of Titus on to be used for a system that would work biologically so what you can change is the molecular weight and the degree of deacetylation the amount of amino groups and the polymer backbone so the two main attributes of a chi design system is that it’s mucoadhesive so if we just look at the mucosal surface so the mucosal surface is the line of the respiratory gastrointestinal genital urinary tract and if you look at the the ball the epithelial surface you’ve got tightly packed epithelial cells that vary in morphology dependent on the anatomical site but normally they tightly packed together in the long they display these sillery out pushes which push material away but then overlying these epithelial service you have this gel this physical mucus gel which is primarily composed of mucin glycoproteins but just to say that the mucus layer that overlays these surface is in two layers so you have a more viscous fixed layer at the bottom under underneath borders the epithelial surface then you

have a more watery fluid system at the uppermost surface and it’s in this upper low surface that’s brushed away and replenished depending on the site in the and the mucosal surface it is replenished every minute few minutes or every hours or hours should i say if we zoom in on the mucous itself the Menken the main constituent of mucus is these music like a proteins which are polymers that form a network and this network work forms pores in which material as to penetrate through the pores in able to reach the underlying epithelial cells so when we think of a delivery system we need to consider the this this microstructure that’s composed of these glycoproteins the other main attributes of Titus on is that it’s been shown to enter macrophages more avidly than a lot of materials now we know that particles have a predisposition for phagocytic phagocytic capture this is one of the problems of a delivery system it’s recognized and captured by phagocytes but manners receptor on the surface of the macrophages engaged with the polysaccharide and the kite azam and you get more uptake into these cells and i’ll go go into that just to say that it’s one thing getting uptake of an ana carrier system into a macrophage it’s another matter to actually get for example gene silencing because of the lab network of materials of lysosomes that can destroy him break down ingested material so we started this work let’s say in 2004 for the next two years we gathered data we optimize the system and then i’ll show you some of the data that we published but what happened was the series was that we we came up with this idea of using SI on a with high design now caisson had been used extensively for the every of peptides and also plasmid DNA but we were the first to actually use it for sa RNA and we secured a patent on the SI on a so you can see that although this system had been used extensively for other molecules we were quick on securing IP on this technology and this is important moving forward if you want to establish a spin-out so we didn’t publish at that point we prepared a patent application so we formed a company called nano feirense based on the car design system based at I nano the anticipatory is to inter discipline and a science center and as I said the actual spin-out was derived from the technology surrounding the the chitin system at the University and just to say I don’t know what it’s like in other countries but in Denmark if you do some scientific work in the laboratory the patent is owned by the University so what we have to do is as soon as we thought that we add a novel idea we act to approach our technology transfer office and prepare a patent disclosure and this is a requirement as a university working in Denmark that you have to consult with the tto and offer them the chance have taken on the pattern if they don’t want the pattern then the inventors themselves can take it forward but they did like the patent they did their due diligence and thought that there was some way forward with it so actually the patent is actually controlled by the university but it’s partly owned it third for the the university a third for the inventors and a third for the department so if we make any any money out of this then I nano itself will get something back we launched it in 2010 and this was based on securing seed capital from two Danish companies one Nova seeds which is a derivative of Nova Nordis the big pharmaceutical company and another small investment company seed capital and they invested near on eight hundred thousand dollars into this spin out so this was seed capital but to convince them about this technology so we filed a patent and then the tto actually reached out to potential investors we add clinics where we we met investors so the university itself holds sessions where investors can meet potential in vail inventors for

potential investment and they they were happy that we add IP they don’t want to invest in anything that’s not protected and they were all so happy that we published in good journals for example of this work went in molecular therapy which is quite a good journal in the gene therapy and rnai field so those two plus the fact that they like the enthusiasm of the inventors it’s amazing that you know they put a lot of they place well let’s just say that they they feel that in order to invest they must feel the enthusiasm from the inventors and we were very enthusiastic about this so I I did a lot of this initial discussions with with Nova seeds and seed capital they light the way that we worked and so they they gave us this investment and the the company itself the founders of myself and I was placed in a CEO position young chems a professor who’s an expert in RNAi and Fleming Bay’s mbaka who was the director of vine a know who was actually involved in some of the scientific work and then what we did is we we base our company actually within the labs of the University but what nano ference does is it sponsors University people so the the people working for Nana feirense are actually employed by the university we just sponsor that funding and again what they liked about is the the the investment company we had a strong patent portfolio we had approximately four patents surrounding this technology based on different routes of administration and different treatments and there we go so just to say that you can attract interest by using a very simple system I think one of the important things to consider when you when you utilize an ana carrier system is you have to understand the biological barriers and I showed you the two properties of keitus on and if you know those properties and you know also the limitations you can target that particular delivery system for a different certain application or route of administration and as I said that we feel that it’s mucoadhesive system that can penetrate these mucosal barriers so it was obvious that we would focus on pulmonary delivery delivery to the lung I’ll just describe some of the work we did extensive work with an egfp model system and work we’re continuing in cystic fibrosis so we worked with this transgenic mouse that expressed egfp here we have this simple nanoparticle image with atomic force microscopy and just to say that we administered these particles intranasally was it over a period of five days consecutively into the nose of the mouse and then after a period of five days we sacrifice the mouse and harvest of the tissue and remember we’re trying to float with focusing on delivery to the epithelial layer of the mucosal surface and you can see you in the control tissue we’ve got the egfp expression in the epithelial cells that border the bronchial escape and with the egfp specific SI RNA delivered in our kite is on system administered intranasally you can see that we down regulate the egfp expression and with co stained with Dappy a nucleus stain to show they’re still in tact border so we’re showing that we have got knocked down we then developed this system into an aerosol because we want to reach possibly down into the deep and one of the problems of these biomaterials that mucoadhesive is if we apply them into the nasal cavity that they’re going to stick in that region and they’re not going to reach down below so we nebulized these systems we optimize that procedure and we could I don’t think you can see it here but we are the srna that were presently labeled and we can show extensive distribution in deeper regions for the long if we form an aerosol with this system we also did in collaboration with the Max Planck Institute in Berlin and the lab of tomas maier who’s working on RNAi interference approaches for treatment of influenza an issue we did some in vitro studies show though that we could silence a conserved site in the influenza which was the nuclear protein we then used that si RNA in an animal model that was infected by influenza and we got some degree of knockdown or should I say reduced infectivity in the lung using a this was

an LNA Ln a stabilized assign a system this work is ongoing and actually we’re working now on using targets of the host cells so you know that viruses can hijack host cell processes and pathways and in conjunction with tomas maier we’re now focusing on using srna to interrupt erm some host pathways that are utilized by the influenza virus this is some recent work and we’ve only just really moved into this but this is utilizing this very simple technology for the treatment of cystic fibrosis so cystic fibrosis is caused by a mutation in the cystic fibrosis transmembrane conductance regulator which regulates the the transport of anions and sodium should have say sodium and chloride across the epithelial surface which is is needed to maintain the omis statius of the mucosal fluid if there’s a mutation in this in this gene you find that you get a retention of the mucus in the in the respiratory tract and you get a builder / from you because it’s not watery enough in effect to to be moved from the site and you get a gradual buildup and this is a terrible disease because even now between the age of 20 and 40 is the victim should I say die or they need a lung transplant and there’s no real cure for cystic fibrosis the moment so our reason allows therapeutic strategy was to target this one gene now it’s been shown that this mutated c CF TRG the regulator although it’s misfolded and mutated if it reaches the epithelial surface it can still have some functionality and reduce the cystic fibrosis condition and now how one is actually a mechanism by the cells to remove this mutation so it’s a degradation pathway that actually is there to to to repair the mutation but if we can knock down this degradation pathway we can still utilize this mutated gene which would then still have some functionality and reduce the build-up of the mucus I must say that you know it’s one thing going straight in in vivo but obviously when you do RNA I works you’ve got to screen for example different sequences in vitro so there’s no way that you can go straight in vitro in vivo and do these systems so what we’ve got here is we use different designs of s Lyonnaise designs that would reduce off-target effects or immune stimulation also stabilized SI RNAs targeting the a1 and we used a human CU f1 cells which are pulmonary epithelial cells that express their how one and then we looked for knocked down and you can see that using our very simple system in vitro we can down-regulate this this target gene now moving forward there’s no real good neuron model for cystic fibrosis that mimics the human condition but what they have found is the the pig model does and in collaboration with Paul McRae at the University of Iowa in the USA they’ve got a pig model that as the common mutation which is a delta 508 that basically mimics the human condition so our way forward is to try and get delivery in pigs because we at the end of the line we’ve got a very relevant model to test our system that’s that mimics the human condition but we don’t want to just look at it sells on ourselves on a plate we want to try and replicate the liquid air interface that you find in the pulmonary track as a as a model for delivery so what we’ve done is we take the tricky or Braun key from pigs we isolate the cells and we digest with collagenase we isolate the epithelial cells and then we basically form a liquid air interface model that mimics the the pulmonary track if we do

TM you can see that we’ve got these types of cells but what they also show is that we’ve got goblet cells and goblet cells of the cells in the new kozel cells that produce museum so if we want to replicate the barrier that will combat when we go in in pigs or humans we’ve got to have a cell line that expresses mucin and we can see that we get cob let cells and we do actually seen using produced in these cells this is actually an elaborate in vitro modern itself it’s pretty difficult to silent we have done that we’ve used a housekeeping gene hprt that’s found in in Pig epithelial cells and using different ntp ratios the ratio of the nitrogen to the phosphate in the formulation you can see that we get some degree of knocked down but this is a tough model to show knockdown in but it’s looking promising that we can you isolate this model and then we can show knock down the next step is to show delivery of our our system in a pig model and we’ve got good collaboration with medics at the University of our who’s in the in the cardio unit that use pigs quite extensively and in here where we actually apply an hour delivery system directly into the trachea of the pigs using nebulizers first steps is to use fluorescently labeled and model systems such as latex particles just to see what size will get good deposition into the inter lung and you can see that we can show with this system good deposition just to say that this was imaged with one of these ivis and bio imaging devices so the future work now is we can show that we can get knocked down of the haha one gene we can show that we can silence and get transfer across a liquid air interface model the next thing is to show knockdown of epithelial markers in primary pig airway epithelial the liquid air interface then to show knockdown in our one in the pigs knockdown of epithelial markers in the pickup in this in the pig and then eventually knockdown of our one in CF pigs to see if we can reverse the CF condition and this is where we are at the moment I’ll then now switch on to switch to all delivery using this conceptually simple system now you would imagine that a lectro static interactive system that’s delivered orally would be susceptible to break down we thought that and so we tried some initial studies where we’re focusing our target disease for for the delivery by the oil route is inflammatory bowel disease which is an inflammatory response that’s primarily caused by the production of proinflammatory cytokine tnf-alpha there’s two types of well there’s an old range of inflammatory bowel disease but there’s the Crohn’s disease which is located in the small intestine and then you’ve got the oscillating colitis which is more in the in the colon and just to say and you’ll I’ll show you why but we tried two approaches conventional SI RNA approach as a therapeutic but also locked nucleic acid anti-sense and I’ve come on to that we did some initial studies where we buy all gavage administered the kites and system with a SI are in a model SI ona and then we r visit the tissue at one hour or five hours in different locations in the alimentary canal and as you can see when you compare it with the assignee naked SI on a administered material that we buy northern analysis we can see deposition throughout the all of the intestine is quite surprising that we’ve got stability in the in the in the stomach but you can see that this very simple system can be administered albeit by oil gavage so it’s an artificial approach in a way because you’re flushing down and with a quite a large volume into the into the stomach that’s flushing it through but we are getting preserved integrity of our SI on a when it’s within this very simple we then extended these studies to look if we could get translocation across the intestine to the peripheral tissues such as the kidney spleen and liver we also so we looked with northern analysis and also qpcr and what was surprising is dependent on the ntp ratio so when we

add a high title and content in the empty 120 you could see that we could see the vehicle observe or detect the SI RNA in the kidney and this is amazing really that you can have this very simple unstable you would think system there can be other administered orally but it can reach these peripheral tissues intact we then try to investigate the uptake at the epithelial surface so we use for lesson SI RNA we’re using Citrix we like eight either side of a certain region of the intestine and in jet fluorescent material and what we did see is this this fluorescent term halo around the epithelial surface now the problem of these polyelectrolyte complexes is when they’re in a particulate form you can observe them under a fluorescent microscope but if they disassembled and the srna is inter is naked should I say in liberated is very difficult to pick up the punctate fluorescence so we weren’t sure actually what was going across we know that the material was reaching the kidney for example so the srna does reach that site but is it going as an intact nanoparticle or is it going as a free SI RNA you would think not because the srna shouldn’t cross these borders just to say that we’re working in collaboration with the lab of George Kollias it was a pioneer in tnf-alpha therapeutics and he’s got this transgenic mouse that over expresses tnf-alpha that exhibits symptoms of crohn’s disease and the idea next is that we’re going to use a tnf-alpha SI RNA in our kites on system and see if we can reverse the inflammatory bowel disease condition that song going but we’re intrigued on what’s actually happening at this mucus nanocarrier interface is the nanocarrier going over intact or is the srna moving unassisted we did some simple studies where we add cell lines and we put the particles on top these these polyelectrolyte complexes fluorescently labeled and they could reach the cell surface as you would expect when we add the cells such as goblet type cells that secrete new scenes you could see that well I don’t think you can see it clearly but there’s a disruption of the particle and there’s a definite barriers you would expect so we feel that you know there is definitely a barrier to penetration by these nanoparticles systems use a very simple system where we used a fluorescence-activated reporter assay where by tethering or conjugating fluorescent tags to to determine of the 5 prime end of the SI on a that we found that when they were in close proximity that you had a self quenching effects so when they were when we had the srna in a free form that we had fluorescence when we condensed it down into a polyelectrolyte complex without a self quench in effect we saw a decrease in fluorescence and as you can see here we have the srna that was labeled with a fluorescing type tag and then as we increase the entropy ratio and we form complex you can see we get a reduction in fluorescence and i’ll are going to detail and why we need this this reporter system so the idea is that we wanted to see if the muezzins which is part of the mucus barrier can actually disassemble these polyplex system and way of doing that is to incubate them with different concentrations of musings and then to see if we can then increase the fluorescence which shows that we’ve got disassembly of the particle and lo and behold as we increase this was Porson gastric musings we could see that we get an increase in fluorescence which indicates that the the kites and system is disassembling with it we we saw this also on gels we could see liberation of the chitin system when we add I content of the muezzins we wanted to investigate more thoroughly this interaction of the mutant barrier so in collaboration with Katarina rebec at MIT in Boston she’s got a microfluidic system where she has mutinied rajel and then she flushes materials across it and then you can investigate the interaction at this mucus or mucin interface we use porcine gastric mutants in this case and then we’re looking for diffusion of the particles remember that will detect the SiO a when it’s liberated from the kite sound system so where we are now to

investigate not only the movement of the srna will actually investigate the form is it a particulate or is it disassembling and we investigated two different types of systems so here we’ve got an n to P Phi n 2 p-6 tntp 5 and we’re looking for the movement you can’t really see it clearly air these are depicted by ours we can see the movement of free SI RNA when the particles reach this interface and surprising we’ve got greater movement of the particles that were composed of the higher Chi design content and what we think is happening is the kites and all the excess kite Sun in these systems is interacting more more so with these mutant glycoproteins cyanic acid predominantly interacting and ripping the polyplex apart allowing movement of the particle in there we also tried to the diffusion of naked sra so wanted to see if naked SI innate unassisted could migrate across am using barrier these are just fluorescent beads that are used to measure the diffusion of the system but you can see that over time we get a diffusion into of our fluorescent srna and this is interesting that means that as naked srna can actually cross am using barrier the problem is that naked srna is not very good at cross in cellular membranes it’s Polly ionic it’s quite big and so we have a problem so the proposed model and the new design is that we have srna kanana carriers they interact with the mucin glycoproteins they disassemble and then we back movement to the free srna so what we’re thinking now is we can utilize this process for mucus mediated triggered disassembly for cargo release so only when they come in contact with the mucus barrier though these nanoparticles disassemble and release their cargo and the way forward is to use a different RNAi therapeutic that can enter cells unassisted we know that double-stranded SI rnase conventional 21 MERS do not cross the cellular membrane but anti-sense des so we’ve used locked nucleic anti-sense so these are called gap musze so they’re essentially antisense DNA molecule single-stranded that are flanked by locked nucleic acids that stabilize the structure and these single-stranded materials have been shown to enter cells unassisted without any nanocarrier system and no one really knows how what the mechanism is what is the you know the driving force of these the Estill poly ionic to actually cross maybe their small size you can’t really see it air but we’ve tested the ability of these anti-sense molecules to cross macrophage cell lines caco-2 which is epithelial models but also be taken primary murine epithelial cells in intestinal cells from mice and we can see very good uptake it’s not very clear on this picture but believe me these things go into these cells quite avidly so I’m new system that we’re going to develop is a caisson based system incorporating these types of materials that can once release enter these epithelial cells just to say a number of techniques were utilizing at the nano science center to investigate the interactions with with mucus and new sins so we use an atomic force microscopy obviously to see the morphological changes we’re doing a lot of work by applying different types of biomaterials other than keitus on to these mucus the the musings that interact with the new sins and can disrupt this micro mesh and can maybe open up the pores which allows greater penetration by nanocarriers so doing a lot of work we apply different types of biomaterials looking at the morphology of this museum Network we’re also looking at the bio adhesion of biomaterials as an alternative as alternatives to kite San by using force pulling techniques we’ve also developed a very simple system utilizing a very common piece of equipment the Nano site which is used as a particle tracking analysis technique that’s used to measure particle size it’s it works by shining a laser at particles that move by browning a motion and deflect the laser which is is that deflect the light which is then captured by a camera and it’s routinely used to measure size of nanoparticles an alternative to photon

correlation spectroscopy but we’ve developed this system to look at the movement of particles in Newson hydrogels by looking at the diffusion coefficient and what we can do then is to apply different biomaterials to these mutant hydrogels and then to see if we can alter the movement of the nanoparticles as a prelude to use in these types of systems therapeutically and this is just showing that by using different systems we can alter the pause Network which enables greater uptake we’re also using particle tracking using confocal microscopy we’ve got a well in a well technique where we use a mutant I’d rajel we apply kitem any types of particles onto the surface floor lesson and then we track by confocal microscopy through the museum hydrogen hydrogel the movement of the particles in real time we’re doing a lot of work in in-situ got loops so this is real as i said we we do a laparotomy we open up the animal we isolate a section of the intestine for example that may it be of interest for uptake we inject materials that then allow to to penetrate the mucosa we isolate or dissect that system and then we can isolate the different cell types and by confocal microscopy or float isometry we can we can visualize if these particles enter how am i doing for time is it a keep on okay so i can is something completely different but it’s utilizing the second property of kites and remember mucosal delivery but also phagocytic capture so we want to exploit this predisposition of Qaeda son to be captured by phagocytes so I’ve got it here again the money mononuclear phagocytes system is a series of circulating in fixed tissue macrophages that recognize capture we move nana carriers from the body this is bad the flip side is to exploit this for the treatment of macrophage associated diseases so using a natural stealth approach where we want to basically mimic what happened in with the Trojans when the Greeks entered the city of Troy that they harbored inside a device that was then they were undetected they could enter into the city walls using the same concept we want the phagocytes to pick up how mcafee and pick up our kites and systems and then let the microphase themselves migrate throughout the body we know that these polyelectrolyte complexes are almost useless you inject them intravenously I don’t believe that you can use polyelectrolyte complexes even the one surface modified for systemic drug delivery I think you’ve got to use them for mucosal delivery if you inject them intravenously they’re going to aggregate within minutes and you’re going to get macro size systems the idea is that the macrophage picked them up and they move undetected I’m going to show you some preclinical we’ve worked on two different preclinical models one way we use kites and system for the treatment of radiation-induced fibrosis and I’ll get on to that and one for rheumatoid arthritis both these diseases are predominately caused by the production of the pro-inflammatory tnf-alpha tumor necrosis factor which sets of Cascade inflammatory responses we’ve shown that al Qaeda and system containing an assign a targeting the TNF alpha can down-regulate the TNF alpha levels in macrophages so as we expected when we incubate a fluorescent keitus on system containing srna that’s the red system with a macrophage it gets captured we’ve got a Dappy stay in blue nuclei it’s one thing getting into the cellars i said before can we actually knock down jeans once it’s inside there and we can so we used our kites and system with a the TNF alpha SI RNA we we took peritoneal macrophages so they were primary cells from the potent IAM of mice we stimulated them with lipid polysaccharide incubation so we get these cells kicking out large amounts of tnf-alpha we normalized everything else to those and you can see that when they have a nanoparticle contained in tnf-alpha after 24 hours we get knocked down compared to the consulate system way of a mismatch control at 24 hours and 48 hours so this we didn’t really

think that we could utilize our kites and system for knockdown of of genes in macrophages but this was remarkable to us that we could deliver something into a macrophage that’s there to destroy materials and we could still get the srna to work and that then stimulated us to to to use them for macrophage associated diseases so I’ve got it here Kaiser honest honest system can enter and silenced genes in macrophages this opens up anti-inflammatory therapeutics using this simple system rheumatoid arthritis chronic inflammatory condition 1% of the population mainly mainly women in the 40s it’s manifested as a destructive joint disease although it’s a systemic disease innocence and it’s the main culprit is the inflammatory cytokine tnf-alpha we know that macrophages produce the TNF alpha and they are play play a prominent role but importantly we know that the peritoneal macrophages are recruited during states of inflammation even in the joint so it’s a reservoir of macrophages in the peritoneal cavity that are recruited at heightened immune states and we didn’t really target rheumatoid arthritis but the more reading we did around it and we found that peritoneal macrophages were implicated in the disease and we could not down tnf-alpha in the macrophages then we add a therapeutic strategy that peritoneal macrophages were the subtler target for anti TNF alpha srna intervention therapy in rheumatoid arthritis and the strategy is this we know that these polycationic systems are positively charged if we inject them into the bloodstream they’re going to they’re going to aggregate by same induced aggregation in the peritoneum there’s no serum but there’s lots of macrophages we know that we they get picked up by the macrophages the idea then is that we we inject them into the peritoneal cavity it’s a sale and free environment we don’t get aggregation of our system we exploit this capability of macro pages to to engulf the nanoparticles and we also exploit this added effect of the mannose receptor on macrophages that engages with the polysaccharide and the idea is that they go inside these macrophages the macrophages are then recruited during the inflammatory state and it carries the nanoparticles to those particular sites the Trojan horse effect we could say natural stealth we did some some studies where we injected Virginia and we injected fluorescent si on a kite some particles into the power team of mice after two hours we harvested those peritoneal macrophages and we could see that these fluorescent nanoparticles were collecting in these peritoneal macrophages as expected working with Mark Bell key from integrated DNA technologies in Iowa who are working on dice a substrate SI rnase 27 months we use a 27 27 against tnf-alpha even in unmodified form or with a 2 prime oxy methyl modification which blocks the toll-like receptor engagement of double-stranded sra and reduces the immune stimulation but just to say what we did is we injected our kites an anti TNF alpha SI RNA into the peritoneum we are harvest at the part and the peritoneal macrophages after two hours and then we measured the TNF alpha levels and we got some degree of knockdown compared to the mismatch control with both the non modified and the modified but you must consider that this experiment is very difficult to show good knock down because we inject in and then after two hours we harvest in those macrophages now it’s known that when macrophages engage with a nanoparticle they’re activated and they move away from that site it’s a known phenomenon that macrophages that pick up nanoparticles in the peritoneum then migrate from that region so we injecting these particles and possibly the part of the macrophages a good population of those microphone are moving away so it’s very difficult to get then harvest them and show the knock down one of the considerations was to use a this to prime oxy methyl modification the reason behind this is that we didn’t want to initiate a neat immune response to this 27 such as type 1 interferon and as you can see with the modified form we got a reduction in this response so it seems you know we could use a modification that doesn’t show

this nonspecific effect of target affects your say moving on to the preclinical work so we worked on a model type 2 collagen-induced arthritis in DBA mice and what we did is we you induce this this condition by subcutaneous injection of collagen type 2 i’m collagen type 2 over a period of 28 days so here so what we do after 28 days is we measure the swelling which is is as a measure of the inflammation in those joints day 0 here is actually day 28 so at day 28 we can correlate these the size of the swelling to an arthritic score and you can see that after intraperitoneal injection so we’re not applying this material in the dial directly into the joint which our therapeutic strategy of picking it up by the macrophages moving to the joints you can see that we get some degree of arrested development of the of the swelling with the kite assigned system with the TNF alpha this is just a dexamethasone dexamethasone steroid control and anti-inflammatory that the animals were responsive to treatment but we did get some degree of Arrested Development it was more apparent when we took the pause and did esta lista logical analysis so one of the if we just move to this image here this is sort of the natural state of an arthritic joint so you get disruption of the cartilage barrier you get Heidi of infiltration of immune cells such as macrophages lymphocytes and you can see get a totally discouraged destruction of this here and this is what was seen when we injected our nanoparticles were control SI on a when we have the dexamethasone positive control we saw an intact cartilage layer and no immune activity when we injected the nano particles containing the SI on a against tnf-alpha we got something that was likened to the dexamethasone and this was remarkable because we’re injecting into the peritoneal cavity and we show an effects in the pores I don’t ok I think I can just go over this in a couple of minutes the second model that we’ve tried this this strategy with is radiator a DA shin induce fibrosis so one of the one of the drawbacks of radiotherapy for cancer is that the radio therapist as to limit the amount of radiation that they can administer because a common condition is fibrosis that can manifest itself up to two years after the radiotherapy so to ensure that in two years time the patient doesn’t get this condition they reduce or limit the the radiotherapy so it’s sort of a drawback in in radiotherapy treatment fibrosis is predominantly caused by pro mac pro inflammatory responses orchestrated by tnf-alpha it causes tissue remodeling hardening and deposition of collagen should I say which causes these these types of conditions where you get these strictures where it’s very difficult to to move because you’ve got the fibrotic tissue and there’s actually no treatment for this and it’s quite a common late side effect of radiotherapy what we did is we add a mouse model where we could irradiate the hind leg and induce this radiation induced fibrosis over a period of time in humans it can take up to two years in mice it can take up to mum’s so it was a lie experiment a bit again we used a kite son system containing SI RNA against tnf-alpha exactly the same as the rheumatoid arthritis intraperitoneally and we could see that when we inject so the actual model itself the way that they give it a fibrotic score is when you irradiate and they get this fibrotic condition you get reduced move ability or flexibility of the of the hind leg and this is due to this and this scar tissue this fibrosis and you can correlate this to a score and you can see we get this as a severity of fibrosis and you can see that when we other can control nanoparticles or mismatch control that over a period of months that these animals did in fact show signs of fibrosis remarkably when we gave these systems where the TNF alpha intraperitoneally it was a Flatliner and these were quite high groups 11 animals

in this group and we could see that we recurred them we could stop this this condition because we have ministered them up to nine months because we are to ensure that you know it was a late that we could stop the late onset of fibrosis we had missed these particles up to nine months which was 78 intraperitoneal injection so it gave us an opportunity to look at the toxicity of these systems if you imagine the amount of administration’s we gave and compared to the control mice that which has given no formulations and the nanoparticles I’ve missed over nine month period in all the major organs we saw no it’s illogical evidence of toxicity a final slide we think that our nanoparticles are being put picked up in the peritoneum by the macrophages and their move in from that site the inflammatory site in both these animal models we wanted to prove that so we we did an experiment where we add fluorescently labeled SI RNA in a chiton system and then we injected intraperitoneally neely these systems into the mouse and then we try to visualize the trafficking of these particles after irradiation so we’re using the radiation-induced fibrosis model in the non-irradiated mice you could see that there was no movement of the particles from this region however in the animals that we were ready radiated in the in the footpad you could see over a period of hours that we got movement from this intraperitoneal cavity into the footpad and we do know that the inflammatory state after it in mice the acute phase is is ISM is exhibited at day is it 13 so it was it was remarkable that when we tried to traffic the movement of these particles may be at day 85 even after irradiation we got no movement it was only when they exhibited the inflammatory state at day 13 that we saw this movement that links it with the inflammatory state we then localized that we did astrology on the tissue and we used macrophage markers and we could see there was some accumulation it’s very difficult to see on here but it is logical II there was evidence that there was co-localization of the system the difficulty is actually knowing if the particles move to win the macrophages during this transit period or they moved on assisted and then were picked up by macrophages locally that’s something we haven’t proved okay just to say this is an interdisciplinary a piece of work and you can see in the blue this is I nano so a lot of the works been based in molecular biology and a lot of the work is being in collaboration with young chems who is a 9-hour rnai expert and he’s leader or LED or is leading the pulmonary delivery side we’ve got numerous people working on that but just to say that we’ve been very strong in the physical chemical characterization atomic force microscopy with a physics part of I nano but it’s very important in this is interdisciplinary project to work with medics and all these preclinical model have been in collaboration with different medics on the project I have to thank people IDT and Iowa have worked on the different constructs the sio a the the liquid a air interface modeled the pig model Thomas Maya who’s worked on the influenza all these ongoing collaborations Katrina rebec MIT and we’re in collaboration with different companies Novozymes in in Denmark and also kite design in Belgium that are supplying different biomaterials thank you