Dr. Hardik Amin is an experienced drug delivery and formulation development scientist who worked at the National Cancer Institute (NCI) of NIH. He is currently a Postdoctoral Scientist at the Center for Translational Medicine, University of Montana. Hardik's current research focuses on COVID, Opioid, and Flu vaccine formulations for clinical development, taking molecules from drug discovery to the development phase, working with academic collaborators and a start-up, Inimmune Inc. Hardik's past research focus has been on cancer targeting, breast cancer metastasis, tumor microenvironment, delivery to the brain, bio-therapeutics delivery, and immunotherapy.
Dr. Hardik Amin
University of Montana
Aug 4, 2021 Presentation at the Global Conference for Lipid Nanoparticles & Other Non-viral Nanocarriers
Redefining the Dogma of mRNA Formulations
T&T Scientific, Dr. Hardik Amin: Redefining the Dogma of mRNA Formulations
Nima Tamaddoni, PhD
Dr. Hardik Amin, he is an experienced drug delivery and formulation development scientist, who worked at the national cancer institute at the NIH, he is currently postdoctoral scientist at the center of translational medicine at the university of Montana. Hardik's current research focuses on COVID opioid and flu vaccine formulations, for clinical development, taking molecules from the drug discovery to development phase, working with academic collaborator, and startups. So, I will also want to share a personal view of a great friend, I found in a conference and NIH, he, I got to know Hardik two years ago at NIH, and we have met a couple times in different conferences but one of the uniquely good characters I know and I really appreciate for all the help you have done for this conference and hopefully we get to work together at soon. I hand it to you.
Dr. Hardik Amin
Thanks a lot, Nima Tamaddoni, PhD. Thank you for your kind introduction. Thank you Alex as well. Let us see if I can share my screen. Okay. So, let us change the mode. This looks fine?
Nima Tamaddoni, PhD
It does look very well. We do see a screen. We do see you and we do see the slide, so, yeah very well.
Dr. Hardik Amin
Perfect! So, the title of my presentation is redefining the dogma of mRNA formulation. I want to mention first that I work on the vaccine development side, we specifically have our own proprietary TLR cellular adjuvants, and my today's presentation on this mRNA formulation is going to be from the vaccine side and not from other purposes like therapeutic protein, replacement or something else or maybe even cancer vaccine. So, this is specifically mRNA formulation for infectious vaccine development. When I say dogma, what I mean is that principle or statement of ideas, especially when considered to be authority or accepted uncritically and what I felt researching on these mRNA formulations, specifically with mRNA and Hypnano particles, I realized that there were some sort of dogma or I would say like particular thought process that were like fixed, people thought oh this is how you should make the formulation and this is how it should be and I went much deeper into literature and realized that it might not be the case always, so I want to discuss some of this dogma with you today.
One of this is from the mRNA side which is unmodified versus modified mRNA. Second is about the LNPs that whether you should use ionizable lipids or cationic lipids and whether the absolute PKA of lipid matters or the total PKA of the whole lipid nanoparticle matters. The third one is on mRNA plus LNP side and this is about the encapsulation of mRNA within the LNP, do you really need that or you can just absorb the mRNA on LNP and still can have a very good formulation and the last one is from my domain about Adjuvant that do we need to add any Adjuvant ones into the mRNA vaccines for infectious disease or we do not need that. So, we will go through this one by one. The first one is about the mRNA unmodified versus modified and you know that there are lots of modifications of mRNA that can be done. This is a very good paper that describes in this one figure different modifications. If we have to put it into different category, I would say that we can do base modification, sequence modification and we can do shape modification.
When we say base modification, I think professor Kariko and Drew Wisman and others have done tremendous amount of work on base modification that what sort of different modification can be done to make mRNA less immunogenic. It can be pseudo-uridine or one methyl pseudo-uridine, 5methyl-cytosine or something else. There are lots of options available here in terms of base modification. For sequence modification, you can increase the GC content of the mRNA sequence or you can add long-lived UTR’s or UTR’s and for un-translated regions and viral replicase ORFs, so viral replicas is being used for the self-amplifying mRNA that you add those sequences and it will help in more and more translation of that mRNA. Or some others anti reverse cap at five 5’ end which helps increasing the translation and poly (A) tail and there can be some others as well. So, forgive me if I am missing something else. In terms of shape modification, there is this new thing which is called circular RNA where instead of making linear RNA you actually join the five prime, three prime ends, make it circular and that has its own advantage. If you think about the modification and its primary purpose, this is how you can decide that oh, so if you want to do these modifications to prevent immune stimulation, high GC content, again to prevent immune stimulation whereas long-lived UTR’s or viral replicase, a poly (A) Tail or at 5’ end capping can be used for increase the translation of mRNA, whereas circular RNA unmodified and modified can be used to increase the stability of RNA and of course this will have other impacts as well, for example if you are increasing the stability of RNA and at the same time it is modified it can have this translation as well. For my purpose, I have a collaborator at Houston Methodist research institute, and we are using circular unmodified mRNA from them, and I want to mention here that this circular RNA, sorry circular mRNA, they are very stable, they are lyophilized form, you can easily lyophilized them and they are stable for at least few months at room temperature. So, it can have very fast applications in the real life situation.
This is my personal opinion about how these different modifications can impact these four different parameters like reactogenicity or immune simulation, or temperature stability, physical chemical stability of mRNA and increased translation and the dark blue color is bad it means like it is high for that parameter and the light or white color is that it is really good and it can be good at that parameter. For example circular RNA if they are modified, I can see that it can have very good, very less reactogenicity but at the same time they can have very high translation capacity and physical, chemical and temperature stability and of course, if you and this is just one slide of the discussion where this is just mRNA but if you combine some of this mRNA, modified mRNA with a proper formulation you can still achieve all of these parameters and make a very good formulation out of it. In terms of unmodified mRNA, I want to clear some doubts here. First of all that it is definitely easy to manufacture unmodified mRNA but it is not that much relatively easy. One advantage or disadvantage with TLR 7/8 activation with unmodified mRNA is that sometime it can be good and sometime it can be bad and in most of the cases it is bad. TLR 7/8 activation by the unmodified mRNA will lead to a reduction in the translation and you may not see much better vaccine response.
Also, this unmodified mRNAs, mRNA, they are not stable, and they get degraded by RNases more easily, to back the claim that why unmodified mRNA are really good I want to draw your attention to this paper and in this paper they check, they compare actually unmodified and modified mRNA, and they also, so this is the paper which says present 2016 and they also compare their data with earlier data from other papers. What is important to note down here is that between on unmodified and modified mRNA, they found very equal efficacy from both unmodified and modified mRNA. At the same time immunogenicity was equal at high and low doses. So, based on this paper, we can say that unmodified mRNA can be quite useful, they can have very good efficacy and low immunogenicity as well but it will depends how you are formulating them into lipid nanoparticles or some other delivery vectors. One criticism if I have to make from this is that they ran 30 plus cytokine panels, in this which had interferon gamma but not interferon alpha or beta and mRNA are supposed to activate interferon alpha and beta more than gamma. However, there can be some cell line rated C or something which might be the case where why they went for interferon gamma panel over interferon alpha panel. But they required good study with 30 plus cytokines and they did not find any higher immunogenicity with unmodified mRNA. This is one more data from them where this was efficacy of unmodified versus modified mRNA, it is almost equal for all the case, different doses. I want to tell you one thing about the unmodified mRNA that lots of people have idea that CureVac’s COVID vaccine was unmodified mRNA which is not the case. CureVac’s mRNA was actually one of the most modified mRNA if you compare that with Pfizer and BioNTech vaccine.
The reason is that they did not do any pseudo uridine modification but at the same time they did other modification like 3’-UTR of Homo Sapiens alpha-2 globin, Poly (A) tail, Poly (C), sequence, Histone-stem loop, all of these modifications can help with increasing the translation and to certain extent eastern standard can help with the stability as well. So, there is some confusion that CureVac’s vaccine did not work much because it was unmodified, that is not the case, and it was modified but not optimized modified. So, before we move to the next section I want to give you one piece of advice that you have to consider unmodified and modified with your formulation and not just alone when you want to develop some sort of formulation, you can still get away with very good unmodified mRNA formulation but modified seems far better option as of now. The second is about ionizable lipids, so in terms of lipid nanoparticles we have few different options, ionizable lipids versus cationic and we will discuss this here. There is this really good paper where they use different kind of cationic lipids and compared them with ionizable lipids, and they selected two cationic lipid nanoparticles and tested them against one ionic lipid nanoparticles and what they found was that the efficacy was actually same at 1.5 microgram dose of self-amplifying mRNA for both cationic lipids and ionizable lipid containing nanoparticles.
However, if you see this data here for 0.15 microgram dose, the ionizable lipid nanoparticles were far better than their counterparts cationic LNPs. So, this shows that your formulation can be also dose dependent, for example for self-amplifying mRNA here you do not want to give too much of mRNA because they are already self-amplifying, they will translate, no matter 10 times dose difference but they are translating very well. So, it can be mRNA dependent if it is self-amplifying or not and same time you can still get some sort of good response with cationic LNPs. One more thing I want to discuss is about the COVID vaccines which are in the market and under trials now. So, Moderna-NIH and BioNTech-Pfizer, they are using ionizable lipid containing nanoparticles and on the other side HDT Bio which is a Seattle based company startup, they are using actually a cationic hybrid lipid nanoparticle, lipid inorganic nanoparticles and they also got some good response and they are under clinical trial now for the cold vaccine. So think about this that it is not that only with ionizable lipid nanoparticles, you can have a based formulation, and you can probably have the based formulation even with some liquid hybrid nanoparticles or with the cationic liquid. So, in HDT bio’s vaccine they have no tape, as cationic lipid they have some other components surfactant span 60-80. They have SPIO2 which is, SPIO which is iron oxide nanoparticles within the nanoparticles and the orange color is scaling actually which I will discuss into last section about that one that why they are adding squalene. I want to move to the second part of this dogma with the LNP that whether we should use a parent versus absolute PKA of ionizable lipid. So, there is a trend in the industry that lots of people are making different kind of ionizable lipids and then they will check the PKA, absolute PKA of the ionizable lipid and try to tell it the overall lipid nanoparticle formulation capacity to release the cargo into a late endosome from just absolute PK but that should not matter much because the moment you mix your ionizable lipid with other lipids cholesterol and your cargo, mRNA here, it will change the overall PKA of the LNP.
So, this is kind of now accepted that instead of just checking the absolute PKA we should actually check the PKA of apparent PK of the whole LNP by using two different methods. We have Potentiometric titration method where you start from acid and start adding a base and check the change in the PH and you can check the PKA for your LNPs or you can add some fluorescent tag and get the data out of your plate reader and decide what is the PKA of your LNPs. So, based on this, there is again two more strategy to make a formulation with ionizable lipid. One is where you just have one ionizable lipid and after mixing with different components it has a proper PKA of between 6.2 to 6.5 which is considered as like good PKA to have to have late endosome release but another strategy is where you have like two different ionizable lipids, you mix them into a different proportion and figure out the optimum amount of each to have the PK between 6.2 to 6.5. So, there is lots of patent issues right now with all of these companies developing their own ionizable lipid and a good way to get away from this sort of patent war is to have some sort of ionizable liquids which are just not within the range but at the same time when you mix them into proper proportion they give you a proper PKA of LNP.
So, the third part is about mRNA plus LNP, here I want to discuss about why absorption of mRNA can be also okay instead of encapsulation of mRNA into the LNPs. So, we know that encapsulation of mRNA is done within the LNP to prevent the degradation of RNAs, prevent the hydrolysis of RNA, and release of the mRNA from endosome to cytosol. However, same can be achieved with adsorption as well. So, first before we go into that I want to talk about stability. Stability, when we think about the mRNA stability, we think there are different parameters that can affect the stability of mRNA physical or chemical aggregation precipitation whereas for chemical it is hydrolysis occultation are in by RNAs. But in these parameters hydrolysis is one of the most important parameter, I think that is the main reason for the degradation of mRNA and hydrolysis actually happens by three different ways either by itself, by the mRNA or by the Lowry-Bronsted Acid Base presence or by the water and to prevent that you can do base stacking where you select a sequence of mRNA by using particular algorithm, so that it has maximum double standard regions. So, you have to focus on the secondary and tertiary structure of mRNA and make sure there is more stacking so that it can prevent the hydrolysis and if you think about the LNP which has, ionizable LNP, which has mRNA with in the LNPs, there is this paper that describes that even within the LNP’s the mRNA is surrounded by water channels. So the hydrolysis can still happen within the LNPs. So, again I want to mention here about HDT Bios delivery system where they have this rotate cationic mediated lipid inorganic nanoparticles, inorganic is because they have this iron oxide nanoparticles within the nanoparticles and they showed very good TH1 response with their spike protein vaccine. The mRNA vaccine and even the single dose that we are saying to the non-human primate cause very good antibody response, neutralizing antibodies response against SARS-CoV-2 protein and in this one, the mRNA is stabilized on the surface of cationic ally charged nanoparticle.
So, mRNA is actually just stabilized by the charge-based infection and it is not within the LNPs. I mean the theory is that that as long as it is stabilized by charged based interaction or something it can prevent the hydrolysis. This is another paper published few years ago for the ZIKV virus vaccine by the same group when they were at E3 Seattle, it is in fact, this is research institute and this, the same group they came up with the ZIKV virus mRNA based vaccine, at that time and that was one of the best vaccine that you can find for the ZIKV virus at that time. The neutralizing antibodies are really good and in this one they used, they did not use any iron oxide nanoparticles but instead they use Dynasan 114 which is a triglyceride. So, there are some options that you can use, basically the reason for using some sort of triglyceride or iron oxide nanoparticles, it is just to provide stability to the whole structure. So, the takeaway and this is one more data from that ZIKV virus challenge that all of these NLC’s which is the cationic nanoparticles that they developed, they against the challenge they were really good there was no depth, as you can see here by the orange lines.
So what we can learn from this is that you can still develop a nanoparticle system where mRNA is just on the surface as long as it is stabilized by the charge with this interaction, you can have a good stability. The last part is about the Adjuvant and whether to add or not and this is in the context of infectious disease vaccine, please keep this in mind, this looks like a busy slide but from the figure what I want to show you here is that when mRNA is given, if it is unmodified it can really trigger your TLR 7/8 and TLR 3 receptors which can result into interferon one, interferon alpha-beta release, cytokine release and that can actually in the long run, it can prevent the translation of mRNA. However, if it is let us say modified mRNA and it does not like trigger this TLR receptors, it just get translated into a protein, this protein will be presented by MHC class one or two peptides, if it is by MHC class one or the cytotoxic site will get activated and they will do their job. Whereas if it is MHC class 2 the t helper cell response will happen it will lead to like missile maturation affinity maturation and memory cell response. So, when we think about adding Adjuvant, we have to think a few things together. For example mRNA modification itself can lead to Adjuvantation and then what is its effect on the translation. So, let us say we have mRNA and it will get translated into the cell into the protein and then our T-cells are responding, our cells, our ABCs are responding in terms of interference response. So, if our cytokine response is after translation of mRNA then we are good, we will have beneficial image stimulation which is really good. However, after giving mRNA if the cytokine response happens before the translation of the protein, then it can reduce the translation not only that because you have lots of cytokine in the body, it can lead to some adverse effects.
So, timing is the key when you want to add Adjuvant or when you want to add any Adjuvant with the mRNA and of course on modified mRNA, they have high adjuvant capacity to add to induce tier 78 week one, these are three kind of responses stimulation, and thus they can have like low translation whereas for modified mRNA it is high translation versus low adjuvantation. Sorry, I am running out of time but I will show you something quick. One thing about the CureVac is that they have their own mRNA formulations where they use two component approach where they have Adjuvant as a… which can trigger TLR 7/8 whereas at the same time they also add some RNA that can translate to flu and region and they have shown very good data with the influenza vaccine that by adding Adjuvant, you can have very good DNP cell meditative responses.
Again, with the HDT bio, they have squalene and this is what I want to mention is that squalene is an adjuvant. So, actually HDT Bios coping vaccine is the actually the vaccine which has actually Adjuvant inside and squalene can do immune cell recruitment and the more mechanisms are not yet known but any formulation which has squalene has shown in stimulation. So, yeah, this is the first vaccine which has adjuvant inside, like intentionally added. They also have some other papers with the ZIKV virus as I mentioned earlier which also has squalene. So, there is a literature precedent for addition of Adjuvant to mRNA vaccine for the infectious disease but it is very important to figure out that what kind of stimulation you want, do you want TH1 or TH2 or TH17 response, do you want lymphocyte response, so do you want cytotoxic T-cell response and then you have to choose your adjuvant, so timing is key and with these I want to end my presentation and I am open to any questions, thank you.
Thank you so much. We do have a few questions here. The first what do you evaluate the perspective of circular mRNA with the point that circular RNA could invade total-like receptors 3.7 & 8?
Dr. Hardik Amin
Alex I did not get the question, let me see if I can read it.
Nima Tamaddoni, PhD
Yes, it is in the Q/A
Dr. Hardik Amin
Okay. What do you evaluate the perspective circular mRNA with the points to like receptor 378? I think it is under testing at my place. So, I do not have the data that how much that three seven and eight activation that circular mRNA can does. One thing I know for sure is that my circular mRNA is actually unmodified. So, it can probably do activation of seven eight. However, if it is on if it is modified and it is also circular then probably, we would not see any image stimulation and in those cases we may have to think about addition of some other adjuvant ones and sorry I do not answer this question.
Nima Tamaddoni, PhD
It is what it is but we do have another one. Do you think ionizable or slash cationic lipids can cause chemical degradation of mRNA and affect its integrity? For example interaction with these lipids can induce hydrolysis.
Dr. Hardik Amin
I do not think so and I have not come across any kind of literature which mentioned that cationic lipid itself can produce degradation of mRNA based on hydrolysis. So, I would say no to that.
Nima Tamaddoni, PhD
And then we have another one. Do we really need modification of circular RNA or is RNA or circular RNA inherently can minimize the innate immune response?
Dr. Hardik Amin
I think we may need modification of circular mRNA in case of some indication, for example think about therapeutic protein replacement therapies, where you want to give mRNA based therapy for long term more than few doses and in those cases you want to make sure that that you have the least amount of immune stimulation because the more and more doses you give it can increase the immune stimulation and it can like reduce the protein translation. So, it will depend on what indication.
Nima Tamaddoni, PhD
Very good! Alright, thank you again so much. We are going to end it here. If anyone else has any other questions, please stick around, or email her directly, we are going to go hop over to the other session now.
Dr. Hardik Amin
Thank you so much.