T_T Scientific_ Dr. Michael McCaman_ Gene Therapies and Vaccines_ Development to Manufacturing
Dr. Michael McCaman
There we go. Alright, if we progress to the second slide, this is just the usual disclaimer that everybody puts in and now we will go to the next one. Alright, so I would like to thank Nemo and DJ scientific for inviting me to present to you today, a little bit of LNP gene therapy overview, and sort of with my personal experiences involved. The intent that I have with the agenda that you see in front of you was to cover a series of topics that I think are crucial to present the overall picture of what LNPs can do and like with any, you know, valuable product it is clearly very much dependent on the quality of the components coming into it. So, I want to make sure that there is a little bit of time, you know, devoted to that to make sure that we have the overall perspective of what LNP therapy is all about and then I will tell you a little bit at the end about where that story took us.
Alright, so in the next one, the next slide please. Just a quick introduction and as Nemo has already indicated that much of what I am drawing upon is the experience that I had for producing LNPs for therapeutic use and using the crisper cas9 technology as the cargo components and this started back in 2015 when I joined Italia and at that point there were three companies in the gene editing field, so the Editors, Italia and Christopher therapeutics. But only Italia took on the challenge of systemic delivery using LNPs and so my challenge then was to build a CMC manufacturing team, they could assemble the network and the materials that were going to be necessary to put this novel therapy together in a way that was going to work in the clinic and then basically to build a supply chain that did, simply did not have any, you know, parallel or corollary in early 2016.
In order to do this I want to make sure that I have quickly identified for you the components that you need to be aware of that are going to go or play a key role in production of the final LNP and for gene editing that includes a guide RNA, a linearized plasmid that is the basis of making the mRNA, the mRNA itself, the crystal ionic lipid which is the most important part for actually packaging the nucleic acid, and then the production of the LNPs themselves. So, if we go on to the next slide please. I just want to throw out a few questions that I thought are sort of interesting to discuss and to think about here and I will address most of them here quickly. One is the gene editing process itself and understanding how the LNP cargo, you know, plays into that, what are LNPs being used for in this particular situation and Italia, the focus was encapsulating and delivering messenger RNA that encoded the casino nuclease. There are other applications coming for encapsulating plasma DNA, so clearly any kind of nucleic acid, encapsulation here is fair game. The production of them will also be addressed briefly and I think the rest of the symposium will actually bring a beautiful focus to many other technical aspects to the LNPs and I am looking forward to hearing about those and then I will address a couple of the bottlenecks for the production of our LNP products. Alright, and the next one, we will do a quick couple of slide intro to your just of the gene editing just to make sure that there is a little bit of a background here. Again the cas9 nuclease that is crucial, here is part of the Nobel Prize that was awarded in 2020 and I think it is deservedly so given the impact on the medical and science community. But the crucial part here is this enzyme has to pick up an oligonucleotide guide.
So, 100 nucleotides of RNA is the piece that has typically been used here and that complex then is basically the guide RNA is guiding the nucleus to the segment of the gene that is going to be edited, so that there is a hybridization event that is driving this and then the nucleus simply makes a double strain a cut unlike the diagram that shows sort of a single strand being nick that actually makes a double stranded cut and it is the repair mechanism within the cell that then leads to the editing outcome. If you want to see a wonderful video of that I would put a link in here that would be worth anybody's time to do that and I think we will move on to the next slide, if we are good. So, this gives you some repair options when using the LNPs and so if you throw in the cargo with the cas9 messenger RNA which will make cas9 protein once it gets into the cell that protein will then be able to bind to the guide RNA which is also co-formulated in LNP and that complex then will find the gene, it will do the double-stranded cut in in a miss-repair will end up in the gene knockout, that is what you see on the left side of this diagram, and so that will that knockout is a disruption of the open reading frame, you will lose, you know, or gain one or two nucleotides and physically stop, you know, translation and so block production of whatever that protein, so that is called the knock out.
If you at the same time happen to have thrown in a small piece of DNA that could support homologous recombination and repair something in that same region where the guts have been made that could be a very small-scale repair. If you needed to do a gene replacement or an insertion event then you need to deliver an additional cargo besides the cas9 and the guide and at the moment that is either being done with AED but I think the old I will show you there is another option after doing that. So, again it can be a very a multi-step editing process in terms of disrupting the gene that you are trying to knock out or substitute in and then putting coming in with either smaller or larger fragments, depending on the magnitude of the insertion that needs to be done. Alright, if we go to the next one, some obvious examples of LNPs that you either know about or should obviously are one here with the Moderna vaccine, this is a little bit simplified and it is simply obviously, you know, presenting the mRNA that drives the production of the immunogenic captain protein for the vaccination and for those of us who have the Moderna vaccine and very thankful for it. The LNP technology is potentially a lifesaver. Italia is leading the charge as I said for in vivo and I will show you this a little bit more at the end for treating liver disease. Again a lifesaver for those people, and I threw in here an example of a company cellulogen, a new Cambridge startup who has plans to be formulating plasmids into LNPs to achieve larger edits than anything that an AAD is capable of the packaging. So, AAV has that is an effective payload limitation in about 5kb and these plasmids will be able to go as large as one can formulate in an LNP. So, I think those are some interesting options coming down the road. So, I was going to do just a few slides quickly here about the guide RNA itself, since it is a key component of the Chicago in the next one if you would please.
Alright, so the guide RNA as I said is an elegant, there is a synthetic oligonucleotide, it is been used for several years, well it is been around for many years but in 2016 and Nylon was able to advance this as a therapeutic product within inhibitor in it, as an inhibitory RNA so penticiren was approved in 2018 or by product name on Patro and it is comprised of double stranded RNA but these oligos are only 21 nucleotides in length. But they are encapsulated in an LNP deliberate IV and again are excellent for treating the liver disease by suppressing the mRNA that is making the protein that is creating the problem. In the case of cas9 here on the bottom part that I mentioned before that the oligo nucleotide needs to be 100 nucleotides long and while from a manufacturing point of view this is simply more cycles on the synthesizer and seems to be fairly straightforward to make it. The analytical challenges I think were unexpectedly demanding and I will show a little bit of that later. The diagram on the right side is just to give you an idea that the guide RNA actually has several different domains within it which are sort of functional and each of the domains has some function in the interaction either with the cas9 nuclease or with the host genome.
Alright, the next one please, this is a table of some attributes of synthetic guides and I am not going to go through each one of these this is for anybody who is interested sort of a reference perhaps to give you a sense of how things are being done around identity potency and impurity assays. I just, the footnote here is that the purity may surprise you a little bit. That was evident to me that the first time that we had a hundred mirror produced. It was something that was doable but were not in any way routine and so the people the QC groups were using HPLC methods that were designed for a much smaller oligo and they showed me a purity profile and I said great, you know, your oligo is like 98 percent pure and I said that seems a little optimistic and so we pushed it a little bit and pushed it more in and found out that one needs to then adjust the methods, significantly is a function of the size of the oligo. I think on the next line, I have actually sort of more briefly summarized sort of the product testing expectations that I have come across in sort of a stage specific manner. So, depending on if you are, you know, sort of doing in vitro screening versus more visual animal studies, getting into your NHP, you know, talks and then clinical very obviously increasing demands in terms of the testing that needs to be done. But I do not think there is anything that is a surprise there. I think the surprises will really become more around what the purity numbers actually turn out to be. In the next slide I have I have captured the video database that helped establish where for the sake of avoiding the appearances of supporting any particular company I have blacked out the company names but I have left you a little bit of a hint in terms of who they are based on location. Then we have color-coded them to give an indication of which ones are in a position to support GMP production of guides at this time. So, overall there are over 40 companies in the oligonucleotide CMO space. So, I view this as an indication that there is ample capacity in the marketplace but they are not equal capabilities and so this is where you really need to be able to do your homework and understand who is worked with larger oligos, what do they understand about the, either the optimizations of the synthesis but certainly some of the specifics around analytics that will get you through an ING in the clinical study.
I have summarized this on the next slide in terms of some sort of required inputs and challenges and clearly this is sort of a boilerplate template in terms of what you want in life with a CDMO. In this case you want one with a good quality system, you know, obviously robust equipment and process knowledge, going into the guide itself I mean you really need a well-screened sequence, but that is where your bioinformatics group or organizations going to support you, giving you the right material to make. From a supply chain point of view there were a few interesting surprises for us along the way, you certainly need to be able to have access to all of the amadites which are the building blocks of the oligo and there are natural versions in modified methylated versions. Obviously, you need solvents because it is an organic synthesis. What I was, a little unhappy with the outcomes in our experience was that the solid phase that was used for producing the polymer went through a bit of a shortage.
There was an acquisition of one of the big makers that took a little while to get sorted out and the ionic exchange resin that is used for the downstream purification turns out that there was a global shortage for reasons I still do not understand back in time and 2017. So there are some things that you need to anticipate or as they say expect the unexpected, just to be sure that you are not, you know, caught short at a crucial time. Alright, on the next slide, I was going to say a few things about plasmid, the linear plasmid in particular, as this is the template from what you are going to be making mRNA. So, the plasmid is basically there is an upstream and a downstream aspect of it and again these are typically done at CMOs and just to be aware that obviously there is a plasmid construction, there can be cell banking for the E-coli, that is your typical host strain, you are going to go through some high density fermentations in order to generate self-paced that is easily done, like that show obviously going from shake flats where you might start up to, you know, computer-controlled bioreactors, and give you an idea that the plasmid range, you know, in terms of yield should, it should be between, you know, 0.5 to a couple grams of plasmin per liter culture. The downstream purification is this fairly well established I do not think there are too many surprises but it does need to be executed though and with some success and inconsistency should not be too much of a problem. I will flag something on the next slide that is unpredictable. Actually I think it is going to be the one after this but this one, so this slide again is just sort of a summarization of the typical tests and testing your analytics package that one would expect for the Q-seeing of the plasma itself. Again, I am not going to spend much time there but the big thing that it is, it is got the elements that you want. I think what is useful also is again there is a here on the slide you are seeing down 18, there is there is a growing list of companies who produce plasmid, most of them are offering this is a GMP service. So, again I think there is ample capacity for making the plasmid. There are some limitations in terms of the number of companies who do the linearization and provide you that as a GMP quality material.
So, if you go to the next slide, I have a sort of a summary here. Then clearly you are going to go in with a very well established sequence but there are some things that you need to be looking out for depending on what you are asking the classmate to do. In the case of a mRNA template, the two things that can be most troublesome, it is the poly (A) tail that if you are encoding the poly (A) tail of the message as a string of T’s within plasmid, E-coli tend not to be happy with the repetitive sequences of any great length and so there can be some genetic instability that you have to be on the lookout for and the way that you deal with that and just to back up for a second then there. Another concern that we had bumped into was the inverted terminal repeat sequences that are typical for viral genomes and if you put that in a plasmid and those tend to encourage sort of a hybridizing loop and structure that can actually be eliminated. That again you will have plasma instability, that is quite annoying, but the answer to that is engineering the host brains, and a little bit of modification of the back home plasmid sequences.
So, once again you just have to do your homework, you have to be on the lookout for anything like that to make sure that those things do not slow you down and then obviously finding a CD who is either experienced them and figured out how to fix them or understands the nature of the problem and again gives you these sorts of same standard attributes I think is important. Interestingly, I have heard recently that there was some issues in terms of a supply chain, the need for the ionic exchange resin for some of the downstream purification, seem to have gone through a bit of a short supply and some panic buying too. So, I think in this this still slightly postcoded world, your supply chain is very much, their mission has to be staying a step ahead of whatever the next shortage is and again expect the unexpected. Alright, so let us get into the mRNA production, on the next slide, next one please. Alright, so the in vitro transcription is the production of mRNA using the linear plasmid DNA as the template. So, the components that need to go in, obviously is you are going to be providing the template itself, you are going to need an enzyme like T7 polymerase that is able to read that template and then off that provide or synthesize and the RNA polymer and obviously the nucleotides which are the building blocks of the mRNA, I need to go in. There are some other goodies that can go into the mix of viral phosphatase to deal with the initial hydrolysis products the phosphate, typically or oftentimes people pull in an RNAs inhibitor in hopes of preventing any degradation of the mRNA during the portions of its lifetime. That it is around that RNAs inhibitor and there are some capping materials that will go in and we will describe that a little bit later. So that there are two ways of putting sort of a block as it were on the five prime end of the message to stabilize it and to drive its update. So, the ideal reaction temperature for these IVTs is typically around body temperature 37 degrees, plus or minus. People can optimize the buffer conditions as they want, it is a function of the template, there are some natural and engineered versions of the polymerases that are available that will have slightly different optima. So, there is all sorts of room for enzymology to play a nice game here in terms of, you know, maximizing the yield that you are going to get.
Typical reaction time is relatively short, a couple of hours at most and you typically stop the reaction by adding a DNA that effectively chews up the plasmid substrate, so there is nothing more for the T7 polymerase to be guided by. Then you start the purification of the message and depending on the purity there are some techniques that are available to you. In the next slide I want to do a little bit more of a dive into the mRNA domains. So, there is sort of color gutter here and again this is a single stranded RNA that we are looking, at the box on the left side is the cap structure and again this is important both for guiding the translation for recruiting the materials that you need to do the protein production off of this.
But this is also important for blocking exonuclease degradation from that end of the message and so it is critical for projecting as it were the green sequences that follow, so that the first little green sequence will call that an untranslated region but it is got regulatory and non-coding sequences, it can modulate bindings of the polymerase, it can control the rate of the individual transcription reaction and so it is crucial for, I am sorry for the translation, so it is crucial for expression levels and then you will find people who are substituting different UTRs, five prime UTRs into to get the level of protein production off of this message that they seek. The red section in the center is the thing that people think about most which is the open reading frame itself that encodes the particular target protein. There is up there is room there for actual codon optimization to make sure that you are not using photons of the host cell, will not be able to present with sufficient frequency and speed to keep up with the synthesis needs. Then there is a three prime untranslated region which again can play some roles in terms of regulatory and modulation of translations. Again, also add some stability by providing sequences and then there is the poly (A) tail which again is one that is primarily perceived as being there for protection from three prime exonuclease digestions and typically those can range from, you know, 100 up to thousands in certain natural states. So, again one can play around a little bit there. So, the hard work here for around mRNA, the hard work really is the initial design of the sequence structure and the optimization of it, which then is translated back into the plasmid sequence that you had to produce. If we go to the next slide, we will just touch briefly on the downstream, so that the processing, this particular diagram is for what we call a post-transcriptional capping which means that you will isolate the mRNA with that free fiber phosphate, and you will do an enzymatic capping. So, in this case you see that there is the linearized plasmid in the process flow diagram feeds into the IBT or the assembly reaction, then you will stop the reaction with the DNA, you can go through a buffer exchange with a potential flow filtration. Often there can be an affinity purification step or iron exchange and then out of that you will then put that into a second reaction which we show here on the right side with the enzymatic capping that installs that sort of reverse nucleotide structure on that and go through the purification that you need to there.
This is the approach that Modernist has used for their COVID-19 mRNA vaccine. The other option involves the use, instead of an enzyme is that you do what is called co-transcriptional capping. So that you can provide something at the time that the IVT is set up, in that you put in a sort of a blocking or a partially blocked nucleotide which is in the initiator of the mRNA and so it can be built, they can be extended upon and then each extension from that molecule gives you a nicely capped mRNA. So, the molecules that you might have heard of are called either ARCA or clean cap, are the two that are the most popular out there. So that eliminates during enzymatic capping. But for either one of those, you end up with mRNA, and then this testing table that we have here on slide 24. Again, as I gives you an idea of the… my experiences has been what are sort of the stage appropriate essays and you can see as with the other elements that we have talked about that there is the focus on the quality of the mRNA itself.
The concerns about, you know, any residuals from the reaction and functionality assay and bio potency are things that are coming along as is more talked about but a little bit more challenging figure out how to make those quantitative and robust assays. They could turn into release assays but all together this is sort of the package of information that you are looking for to have qualified your material. On the next slide is a small table of companies who are doing mRNA manufacturing. Again, I have sort of locked out the names to protect the innocent and again give you some hints about where they are located. But this is a growing capacity because clearly the world has seen the value of mRNAs as therapeutics. But again not all of them are equivalent, some of them have much more experience, obviously those are directly involved in the COVID programs, have sort of had that crash course experience, and others are trying to follow on their in their footsteps but I think there is, again some homework to be done in terms of who has the experience and the process knowledge to give you what you need. In the next slide I have summarized a few observation points; the mRNA field has grown so quickly that it is just remarkable to me that in 2017, there were maybe two CMO choices that one had. That list is that we just saw is rapidly, it is up to 10, I am sure it is going to double to 20 within the next year or two. So, again you are going to, it will be important for the or in coming up on you as the customer to make sure that you are finding the right kind of CDMO service that you want there with both experience capability quality systems. There is a couple of supply chain thoughts just to throw in there that if the affinity resonant the oligodet resin that is being used instead of a one-step purification is as popular and continues to be. Then you can imagine that for the vaccine manufacturers alone that their consumption of that material could be enormous.
So, once again you think I have to think about the affinity of resin supply. There are only two makers of that material at the moment. You might consider initiating some kind of a reuse or recycling program, not single-use resins and I would not take my eye off the enzyme and especially nucleotide supply sources either because not quite sure how long it is going to take for them to develop the capability to support an ever-growing market. So, again stay vigilant and make sure that, you know, who some of your backup supplier options might be. In the next slide, we will move on here to the ionic lipid, sort of the cornerstone, let us do the next one and so here I have shared a picture from a maternal publication of that happens to summarize a number of the different key ionic lipids that are in play now, including the ones that Moderna has patented, there is one from a couple from acuities arcturus. To give you an idea of some diversity of the structures that is built. The issue for this lipid is not really the chemistry. I am delighted at how the innovative the chemist has been for scaling things up.
It is the shortage of CMOs to do that work, so with a very limited number of CMOs, able to provide quantities of GMP lipid that one needs for the current mRNA vaccine market and those of us pursuing other LNP sources, clearly you are going to have to figure out how and where you are going to get your material made and I am hearing stories from some of the small startups that there is quite a backup in terms of availability. So, if you go to the next one and here unfortunately, I do not even have enough CMOs to justify a table of who they are and where they are. I am surprised to say that they led less than a handful and so from that point of view custom lipids are really the things that are sort of the bottleneck, as I see it right now for LNP therapies. I am aware of CMOs at least one in Europe, in the US, India and china but unfortunately I do not really have words of wisdom to share right now how to get to the head of the line regarding supply contracts, given the demands from the other vaccine makers and stuff. I think this is a very much get in there quickly and somehow make enough noise, get yourself established and good luck. But assuming success, let us move on to the LNP itself. So, if we go to the next one, here is a sort of nice little sort of historical summary and an evolution of the increasing complexity of synthetic nanoparticles that I found on the precision Nano systems. Website, starting from the polymers, nanoparticles from the bottom, then there were lipids for a while that were the molecule, and now we have progressed to an even more complicated structure in the nucleic acid, encapsulating lipid nanoparticles. If you go to the next slide, this gives you a beautiful sort of artistic rendition of what we think this might look like. But the advantages here are very high and efficient nucleic acid encapsulation process which makes a potent as a transduction tool and then based on the surface coating of the LNP, you will be able to modulate and maximize your penetration into tissues, into tissue distribution in fact and that lipid structure is also going to be key part in understanding the solder to the cytotoxicity and any immunogenicity.
But, overall I think that these molecular characteristics, the benefits of the encapsulation process make these lipids, these ionic lipids are just absolutely fantastic as key reagents that enable these nanoparticles. To be candidates force, so first just a delightful variety of medical applications and again I mentioned before the first one with the small inhibitory RNA cargo is an approved product. So, LNPs are actually a commercial success and so I think we are just going to see an ongoing string of these as we find more opportunities. In the next slide I was just going to talk briefly about the… from my point of view sort of an overview of the process technologies that go into play. I suspect again that there will be other speakers in this symposium that will help address other more technical aspects of this one. So, one of the two ways that I know of making on these is this impingement jet mixing, sort of a high energy forcing of streams as you can see. In the middle diagram so that is the hardware, so a T-mixer, so the lipid stream dissolved in organic like ethanol and the mRNA and dissolved in an aqueous solution are forced together with a under relatively, well in intermediate sized pressures and high throughput, so you get a lot of turbulent mixing and out of that come the nascent the nanoparticles.
They go through some size adjustments but bottom, at the end of the day it is based on the plumbing and the configuration and then sort of the physics and engineering within that T-mixture and then you allow for a certain thermodynamic. I guess preferred, you know, structure to emerge out of that and that is the stream of lipid Nano particles coming out the end and then with some tweaking in terms of inputs and, you know, actual conditions there one can optimize a little bit for the particle size and certainly you can look forward to what we have seen as a very nice and consistent particle size distribution, and an excellent production efficiency. On the next slide, there is a different technology that is performed for using microfluidics system and a much lower energy mixing. These have involved or have evolved, I should say to a sort of a cassette system, it is, there is a GMP device as pictured on the right and this is this involves a convoluted fluid fat, where the liquids are mixing but not with the same kind of turbulence this is more of a subtle, sort of an edit mixing, that again allows the same sort of interaction between the organic and aqueous phases, and the same thermodynamics come into play and it seems to work quite well on these small scale channels and so the microfluidics has been scaled, not scaled up so much; it is scaled out, so putting multiple of these cassettes with these little fluid pads in them, as a way of producing enough material to be of a therapeutic interest. Alright, if we go to the next one, I found a publication that I thought was comforting and reassuring in terms of talking about the quantitative encapsulation opportunities that LNPs represent. There is some pictures that are probably have to see the journal article to get full value from them but what I found quite interesting was the description of these unusual structures that are more prevalent on the mRNA side, in the diagrams that I am looking at.
But they look a little bit like an acorn, so on the lower left you see a sort of a bleb structure or a two ring structure and my experience has been that we first saw that as we were doing that with mRNA and could not really make sense of why there would be that kind of anomaly and whether or not that was a good thing or a bad thing but I think this paper from the coleslab has done a nice job of validating that it is a real thing and it appears to be a rather lipid dependent distortion of the structure. But I do not think it has not gotten in the way of the therapeutic efficacy but it represents an example of the ever-growing need for more analytical characterization in understanding and certainly being sure that these attributes are things that you see as you either scale your process up or out, so that there is process consistency. On the next page, we will just run through a couple points here the key points then for LNPs are the random molecular size and understanding if what the size variation would be. So, there is the difference there, the first one being sort of the average size, the second one is the diversity or the polydispersity crucial to the economics of this situation is how much of the RNA or nucleic acid, the payload was actually encapsulated since you are paying a lot of money to produce nucleic acid, you certainly want to maximize the amount that is actually encapsulated and then you have to go through the fair amount of sort of biochemistry work due to then sort of verify the correct content of these by a deformulation step, where you are basically breaking down or effectively destroying the LNPs in order to analyze the components to verify that the materials that you put in, assembled in the way that you had anticipated.
Obviously, you know, checking for the integrity of the nucleic acid is a straightforward crucial step and then the usual sort of safety test would do and I have summarized that a little bit on the next table. So, again it is sort of a stage appropriate manner that as you are progressing your LNPs through different levels of testing, if you have you will see a growing list of assays that need to be brought to bear. I would point out that the potency assay, the one up from the bottom. It is the one that gets the most interesting conversations with regulatory authorities because as the LNP is effectively the drug product that you are building your case around for an ID. It is also challenging to ask how I will demonstrate its potency and what does that really entail. Agencies are understanding of that but it is not a trivial undertaking but something you need to be thinking about from the beginning, if there is a way, there an individual assay that one might come up with that at least demonstrates that all the components that are there are functioning, in the way they are supposed to. Alright, and then the next slide, I found a reference, next slide. Please. Thank you. I found a reference that I thought would be helpful for describing this and translated a little bit of that information here, and to give you an idea in terms of what the actual capacity of these LNPs, and typically is. So, from something that is in the 15 to 16 nanometer range, there is some calculations that you can do in terms of estimates and sort of based on charge by ratios from, can bring the nucleic acid to the cationic or ionic lipid that you will come up with some numbers that will give you a sense that the content can be significant.
But at the same time small that is as an example here I have highlighted that you might imagine 200 SIRNA molecules. Remember we said those are 21 nucleotides, double stranded. But if you are getting up to something sizeable in the way of an mRNA, you may be down to single digits in terms of the number of molecules within a particular LNP. So, one has to be cognizant of the fact that this is in a sense, then the therapy becomes dependent on how effective, how many cells you will be able to deliver to, and how many molecules have to be delivered with the probability that one of them at least will be able to get to its end goal and achieve the editing of therapeutic benefit. If you go up in terms of sizes there, as I was saying there at the end for plasm but the numbers can go down even more, so at some point if the nucleic acid gets much larger, it is you are going to be down to a single molecule for LNP or you are going to need to make a bigger LNP.
Alright, so in the next one then, we will get into GM, I think I have slided to for the clinical success and so for this one I wanted to point out that this is what happens after we have gone through everything that we have just talked to us through in terms of collecting and assembling all the GMP grade components, found a way to put them together into an LNP, you know, developed all the analytics, convince the FDA and ourselves that were prepared to introduce this into humans and so in the case here, this was the press release for a successful entry into a phase one study that the Delia had announced last year, and as of June, and the bottom statement there, as of June there had been a delightful set of positive results coming out of this and I have even seen a vision testimonial, it certainly makes me believe that we have done everything that we thought we were going to do and that their people are deriving their benefit from this LNP therapeutic and I wanted to throw in one last slide here, this is an acknowledgment that this is, there you go that this is the army of people that in the team that I built that were instrumental in developing all of these, both the components and the final product and that without this team that we would not have had the success, so kudos to them and a great appreciation. So, that will be the last slide, and I will end with… thank you to you for listening and happy to address questions either now or through email and appreciate the opportunity to address you.
Nima Tamaddoni, PhD
Thank you so much Dr. McCaman, we do have several questions, maybe we do not get to all of them today but we save it and pass it to you and we pass it to the audience after what Graham wanted to get to a couple of them, I think.
Dr. Michael McCaman
Graham Taylor, Ph.D.
So, yeah, we have a few questions here coming in for instance Muhammad had asked, does it make any difference between modified and unmodified mRNA with crisper cas9? So, modified and unmodified mRNA with crisper cas9 and did you use conventional or the modified margin?
Dr. Michael McCaman
Typically the modified means incorporations of methylated nucleotides and the incorporation of the methyl groups provides additional stability, both chemical and resistance to enzymatic degradation, it also actually converts a certain rigidity to the RNA, and in the case of the guides it is quite interesting that there are segments that you can put them from the methyl modified nucleotides in, and it is very good and there is some sections where you clearly need some secondary structure, and these methylated ones can interfere with that. So, it is not uniform and you have the luxury that I mean the optimization of modifications is something that this sort of case-by-case. In the case of cas9, we have sort of mapped out where those regions are that impact the interaction but it can be very dramatic. So, it is one that there are benefits to having modified nuclei, modified nucleotides in it definitely.
Graham Taylor, Ph.D.
What about, from Brett Cauldron, Can be the 100 nucleotide guide RNA abates using plasma DNA and vitro transcription like the long mRNA?
Dr. Michael McCaman
And so this actually dies back to the previous question. The answer is yes that one can imagine doing either in vitro or even in vivo, if you want to do the purification. But yes that if you do this with a, like a PCR-based reaction, the problem there is you do not have the ability to control positional insertion of modified nucleotides, right? So, if you are doing an enzymatic assembly and you put a methylated cytosine, in every cytosine or randomly, I mean either everyone is going to be modified or that is not good. Or even if you try to put in a 50-50 mix of methylated and non-methylated C, it will randomly distribute, so you do not really have control over it. So, if you want positional control, the organic, the synthesizer provides that. If for some reason you did not need methylation then you are right the enzymatic assembly would certainly be a viable alternative.
Graham Taylor, Ph.D.
So, I think we might take one more here, and Leanne Menaul asked a good question, Michael. So with the lack of correlation between in vitro and vivo potency demonstrated before nanoparticle drug delivery exclusions, how should, how do you demonstrate potency of the FDA for clinical batches in a cost and time effective manner? Thoughts on that!
Dr. Michael McCaman
I think, the thing that you are trying to address from a potency point of view of a product would be is it capable of doing in general and capable of doing the thing that I said. So, as an example, if I build an LNP with a crisper cas9 on, you know, toolbox as I have described to you, if I applied those to cells and in some, you know, passive transfection mode or electroporation or something. However, I got that material into cells in a culture dish. What my measurement for potency will be is do I see an editing event, right? Now in a cell that is really the only thing you can do, if the cells that you are growing this on happen to be liver cells that is better than if they are fibroblasts but if you can show that, you know, the mRNA made case-9 and you can do that, you know, in a number of different ways if you want to show that cas9 is active by combining with guide, you can digest a plasmid, you can all sorts of ways that incrementally, you can show that each of the components were active but at the end of the day if you are trying to show the LNP is, you do not have to prove that it is therapeutic, you just have to prove that it is capable of entering a mammalian cell and achieving a genetic edit intent with the specificity that it is supposed to. So, I think that is where you build your model system that serves as the surrogate of potency without actually being the disease model per se.
Graham Taylor, Ph.D.
Okay. Great answer! So, we have a few more and we are going to start the talking in just a moment but Michael one more question. Would you recommend or do we need to do toxicity studies for all of the components in these LNPs?
Dr. Michael McCaman
So, as far as I can tell, and again I am not the toxicologist but the bulk of the concern and the effects actually are from the lipids and in particular the lipid that is the least natural that is the ionic one. I think the other lipids that tend to go in to these LNPs are things like cholesterol. Well, everybody has cholesterol in their system. The DSPC I just do not think that those are pose the challenge that the ionic ones do because they tend to be very large molecules with complicated structures and some, you know, potential for degradation products that are not particularly well tolerated in the liver and I think some of the early history around the MC3 lipid would verify that. So, I think that once we found a couple of lipids that are shown to be, you know, quite nicely tolerated. I think the toxicity concern is pretty much going to vanish.
Graham Taylor, Ph.D.
One last question, Michael… So, this question is about the FDA approval for the ionizable lipid and other formulation components. So the question is do we need to get a separate FDA approval for the ionizable lipid and other formulation components? Or rather if the FDA approves a specific formulation for emergency use, does it also give FDA approval by default to all of those formulation components?
Dr. Michael McCaman
I think the last question is easily answered is no there are no freebies. The FDA will always want to see, you know, the context of the delivery, the ratios of the materials, the composition could be a little bit different from one user to another. So, I think each of those deserves its own safety evaluation. I do think that once, you know, a couple of these lipids have gone through the process and in the case of the Moderna been used in, you know, 100 million people, you have sort of proven human safety in principle, right? So again that does not mean that it is guaranteed but it is a huge de-risking of the LNP technology, by saying that I have, you know, lipids that have been used extensively. So, I think, again it does not take it to a zero risk but I think it drops it, you know, a couple of orders of magnitude from what it might have been viewed as, you know, a couple years ago. So, again the focus should be on not just the lipid itself, it really needs to be in the context of the LNP with some kind of a cargo to make sure that you are truly mimicking the, you know, that you have got the right sized particle, that you have got the right surface composition, and that if there are impacts on cells in the body, that are, you know, part of a toxic response, that you are presenting it with this close a mimic to the product as you can, you know, if you are going to set up preclinical studies.
Nima Tamaddoni, PhD
Michael, we greatly appreciate your time. I know we all do and thanks for the questions from the attendees. We are looking forward to the rest of the talks today and we are going to switch over shortly with Valerio and I think, yeah, Michael we cannot say thanks enough, thank you one more time.
Dr. Michael McCaman
Nima Tamaddoni, PhD
Thanks everyone for your patience this morning, we will see you in the next hope.
Dr. Michael McCaman
Nima Tamaddoni, PhD
Thank you, Michael.
Graham Taylor, Ph.D.