Assessing the effect of Liposomal Spherical Nucleic Acid stability on Vaccine Function for TNBC

[00:00:00]hi everyone my name is Lia broker and in this presentation I'll be talking about the research I've been conducting this past year in the Merkin lab which focuses on creating a novel cancer vaccine delivery mechanism and it involves these structures right here called liposome aspherical nucleic acids but since that's a bit of a mouthful we're gonna call them SN A's for short so to explain how these can be exploited for vaccine delivery I will begin by elucidating the SMA structure so spherical nucleic acids are a noble class of nucleic acids that exhibit completely different behavior from their linear analogues and this is because the SNe architecture is defined by these dense highly oriented nucleic acids which are packed into this concentric morphology as shown by the middle bigger the core can be composed of different materials such as gold or other metals but in my research specifically I use liposomal SN Avios vesicles meaning that the core is composed of phospholipid bilayers and I do this because these are an especially attractive scaffold since they're completely biodegradable in addition liposomes are highly modular in their assembly which allows for these constructs to be able to be tuned and lastly the hollow core of liposome assays can encapsulate a variety of chemical cargo and for the purposes of my experiments I encapsulate them with tumor associated antigens and I will explain later on why this is significant in addition as you may have noticed the constructs I create have these small protrusions which are anchored effectively in the outside of the bilayer and these are small fragments of DNA sequence that are designed to behave as adjuvant again the significance of which I will make apparent later on so given this tunable architecture my research centered around improving the structure of these liposomal essays so that they could disintegrate properly and at due time when administered to the patients so that the vaccine could be released effectively and this was centered around the premise that SNA constructs weren't remaining intact upon administration effectively leading to a loco delivery of the antigen and adjuvant to target answer cells I've mentioned antigen and adjuvant quite a bit so why are these relevant to cancer vaccinations you may ask well in general a cancer vaccine is comprised of two main components which is where our adjuvant comes in and this is a molecule that boosts the immune system and the response to recognize an antigen and antigens are tumor associated molecules that have inductive response on the immune system as well so this is a simplified schematic overview that outlines the step of this process but essentially what happens is that when these boxing concoctions are administered they're engulfed by dendritic cells this leads to the maturation of these dendritic cells which is all that means is that the antigens are presented on the cell surface this process is then iterated again and these naive T cells become active T cells which then go out recognize and kill cancer cells and effectively this innate machinery is what we're trying to up regulate and induce so that cancer cells can be killed effectively there's a small caveat however and what happens when there are no known tumor associated antigens well that happens to be the case in triple negative breast cancer and the development of a therapeutic cancer vaccine for T NBC has been hindered because there are no tumor associated antigens that have been identified in fact there's no estrogen progesterone or her2 proteins present hence the name triple negative and also why I picked T NBC because administering lies aids with s na s particularly favorable for this cancer which doesn't have any antigens present in addition as an alternative to common vaccination strategies tumor cells can be extracted from patient disrupted and the protein can be isolated and within this protein isolate there are many lights that can be behaving as tumor antigens which is shown in the previous slide is necessary for an immune response to be induced so ultimately creating these tumor lies aids have incredible benefits over normal vaccines because you have access to the total Cybil antigen pool and you're also personalizing this to a patient's own tumor since these tumor cells are extracted individually for each patient so coalescing these two ideas together if we package tumor lysis and SNA s we could simplify and expedite common vaccination strategies by a massive amount and again this involves isolating tumor cells breaking them down encapsulating these into these vehicles that are then injected subcutaneously back into the patient now to reiterate my research is really focused on this latter part of the approach which is the disintegration of the SMA when it's in the patient so that the antigen and adjuvant can be effectively released so how did I do this well it was a two pronged research investigation firstly I looked at core fluidity and modulating that part and basically my hypothesis was that modulating the stability of the liposomal core of an SMA will impact the antigen delivery and I did this by using four sets of liposomes each composed of a single type of lipid where and and these were prepared from lipids bearing different melting temperatures but the same head group now the reason why I use different melting temperatures is because as you can imagine different melting temperatures affect the fluidity of these liposomal cores and thus the way that they disintegrate in the body now lipid chains with carbon-carbon double bonds which are unsaturated are more fluid than the bits that are saturated with hydrogen's and that's because in the molecular level unsaturated double bonds make it harbored for the lipids to pack together and they put kinks in the hydrocarbon chain as you can see and so these molecules do not stack very well and the intermolecular interactions are weakened second of all I also looked at the DNA anchor and specifically I wanted to see whether using different DNA anchors could affect nanoparticle stability and I did this by using anchors of different hydrophobicity and hydrophilicity is a fancy word for the degree to which a surface repels water and this you can imagine is important since it would affect the dissociation rate in our bodies which are predominantly aqueous physiological environment so combining these two yielded the following results now we looked at SNA stability by looking at hydrophobic hydrophobic anchors and different lipids of different melting temperatures and we measured the rate of decay through fret signaling and this was essentially done by putting fluorophores which are just bright lights onto the anchors so that when these days associated the difference between each fluorophores could be measured and the dissociation rate would be calculated from that aka a loss of fret signal meant greater dissociation so using this data this figure was created and here we can see that the initial rate of decay was calculated for each construct run in triplicate and graphed in decreasing order of hydrophobicity and increasing melting temperatures so the SBC had the highest melting temperature and this is all as a function of initial rate of fret signal decay so what this graph is telling us in a nutshell is that SN ace our dissociating more slowly as higher melting temperature lipids are used and more hydrophobic anchors are used and from these investigations it was found that clearly SN a stability in the presence of serum is governed by both liposome membrane fluidity and anchor hydrophobicity so given this what are the following steps well basically we now know that liposome constituents and DNA anchor moieties affect sna stability so now we can test this in stability on vaccine efficacy by seeing whether each of these constructs the ones that dissociate faster or dissociate more slowly have a different propensity to stimulate d c-- maturation then we could also test the lysate cargo itself which was the stuff that's encapsulated inside the SNA and combine the immunogenic construct from experiment one to the best lysate construct from experiment two to really create an optic optimum vaccine delivery vehicle and ultimately exploiting his parameters could spur the development of an incredibly effective therapeutic counselor vaccine 14 BC which is exciting and to that extent I'd like to acknowledge the Merck in lab and of course the undergraduate research grant for providing me with the funding to do this research thank you for listening to my presentation