Development of mRNA for Therapy

Summary

The development of mRNA technology has been a significant milestone in therapeutic applications, particularly highlighted by the rapid creation and deployment of COVID-19 vaccines. This advancement is attributed to decades of research and innovation by scientists like Dr. Katalin Karikó. She has been pivotal in overcoming challenges related to messenger RNA's stability and immunogenicity, which are crucial for its medical application. The talk at UC Davis emphasized the journey from fundamental discoveries in the 1960s to present-day applications, including ongoing clinical trials for various therapies. This exploration promises future breakthroughs in both infectious disease treatments and chronic illnesses, underscoring the vital role of multi-disciplinary collaborations in scientific progress.

Highlights

• Dr. Katalin Karikó's research has revolutionized mRNA therapy, leading to COVID-19 vaccine breakthroughs. 🎉
• The story of mRNA: from a 1961 discovery to a pillar of modern medicine. 🚀
• UC Davis is at the forefront with innovative collaborations, enhancing therapeutic developments. 🌟
• mRNA is transient, making it ideal for therapies that don't require permanent genetic alterations. 🧬
• Nucleoside modifications in mRNA are crucial for reducing immunogenicity and enhancing stability. 🔬

Key Takeaways

• mRNA technology, once unstable, propels life-saving vaccines like Pfizer's COVID-19 shot! 💉
• Collaborations during pandemics lead to groundbreaking innovations in therapies. 🧑‍🔬
• mRNA offers transient solutions, ideal for some therapeutic applications. 🕒
• Challenges like immunogenicity have been addressed through nucleoside modifications. 🔍
• The future of mRNA: healing therapies, reduced immunogenicity, and even possible hair regrowth! 🌿

Overview

mRNA technology has come a long way since its discovery in 1961, primarily driven by relentless research and innovation. Dr. Katalin Karikó's work at BioNTech has been instrumental in developing techniques that stabilize this once-unstable molecule, paving the way for modern vaccines and therapeutic applications. Her team's research contributed to significant breakthroughs, especially in creating vaccines like the Pfizer COVID-19 shot, which rely heavily on mRNA's ability to produce rapid immune responses.

The COVID-19 pandemic underscored the need for rapid and effective therapeutic solutions, bringing mRNA technology to the forefront. At UC Davis, interdisciplinary collaborations between the School of Medicine and the Office of Research have flourished, leading to major advancements and substantial funding for further exploration. The efforts in asymptomatic COVID-19 testing and vaccination trials exemplify how academic partnerships can accelerate scientific discovery and public health advancements.

Looking forward, the potential of mRNA extends beyond vaccines. Its transient nature offers promising solutions for temporary therapeutic needs, ranging from chronic disease management to potentially novel applications like aesthetic treatments. However, challenges such as targeting specific cells and managing immune responses remain, requiring continued innovation in delivery systems and nucleoside technology. The journey of mRNA is a testament to scientific perseverance and the promise of even more life-saving applications.

Development of mRNA for Therapy Transcription

• 00:00 - 00:30 okay good morning everyone my name is prasant mahapatra i am the vice chancellor for research at uc davis it is my pleasure to welcome everyone to our exciting program this morning as a part of the lung center carol c cross visiting professor and distinguished speaker series in research and innovation today's event is a part of the robust collaboration between the uc davis office of research and the school of medicine
• 00:30 - 01:00 that in many ways flourished in response to the kovit 19 pandemic bringing together research teams and experts from across the disciplines and schools at uc davis to find innovative ways to unite in our battle against the pandemic one prominent example of this is uc davis novels deployment of asymptotic covid19 testing and our community partnership with the healthy davis together
• 01:00 - 01:30 which has kept our students and communities safe and has put uc davis in the national spotlight for our innovation and collaboration this period of collaboration has yielded tremendous results with nearly 36 million dollars in covid19 research funding at uc davis since the beginning of pandemic across the school of medicine school of veterinary medicine engineering and many other programs this speaker
• 01:30 - 02:00 series is a part of uc davis office of research and school of medicine's desire to feature innovators and groundbreaking scientists who are changing the world we are pleased that you can join us today in this exciting program now it is my distinct pleasure to introduce my colleague the dean of uc davis school of medicine alice in russia thank you so much vice chancellor
• 02:00 - 02:30 mahaptah we are just delighted with the success of this series as you noted the level of collaboration and dedication and innovation at the school of medicine at all of our partners across uc davis has truly been inspiring and resulting in amazing achievements during the pandemic not only did uc davis treat the first known case of community acquired covet 19 in the nation last february but we've been at the forefront of clinical trials for
• 02:30 - 03:00 covent treatments and vaccines including the pfizer mrna vaccine as a neurologist and researcher i'm a strong believer in the importance of academic medical institutions and multi-disciplinary research in discovering life-saving treatments and health care innovations as one of the country's leading medical schools ranking seventh in the nation for training primary care the heart of our mission is to provide
• 03:00 - 03:30 everyone with the best patient-centered care and overall health our ground-breaking research is vital to making that possible and now it's my pleasure to introduce dr angela haaksu the uc davis school of medicine associate dean for translational research and director of the lung biology center and dr satya danikar professor of microbiology and chair of the department of medical microbiology and immunology at uc davis school of medicine dr haksu will introduce dr carrico
• 03:30 - 04:00 thank you dean brashear and thank you chancellor mohapatra for your opening remarks it is my absolute privilege to introduce our distinguished guest dr catalin karikou little did i know when she and i were colleagues at upan that katie would do what all clinical researchers and scientists only dream of doing making discoveries that would save lives
• 04:00 - 04:30 by the million dr karikou is senior vice president at biontech rna pharmaceuticals and associate professor at the pearlman school of medicine at the university of pennsylvania where she served for 24 years for the past four decades her research was focused on developing mrna and mrna mediated mechanisms with the ultimate goal of making therapy
• 04:30 - 05:00 replacement protein and vaccine development dr karikou is the co-inventor of mrna related patterns for application of non-immunogenic nucleoside modified rna she also co-founded and from 2006 and to 2013 served as the ceo of rnax a company dedicated to developing nucleoside modified
• 05:00 - 05:30 mrna for therapies she's a founding member of the international mrna health conference an annual non-profit meeting for advancements of mrna technology inaugurated in 2013. her patent co-invented with drew weissman on nucleoside modified uridines in mrna is used to create the anti-source covey to mrna vaccines utilized both by the moderna and the pfizer vaccines
• 05:30 - 06:00 it is my greatest honor to welcome our extraordinary guest dr catalin kariko katie you are still muted
• 06:00 - 06:30 i haven't seen a word yet i try to set up my computer here to share the screen so excellent thank you thank you andrew for this uh intro nice introduction i remember uh fondly those days when we did together messenger rna therapy to the lung and i am very happy to be here and uh um uc davis we just heard was participating in the clinical trial of the pfizer vaccine
• 06:30 - 07:00 and um i am also very grateful all of the volunteers who participated in this program so i am here today to talk to you about development of the development of mrna for for therapy and um okay so first of all messenger rna was discovered in 1961.
• 07:00 - 07:30 in 1961 it was a very exciting time because actually on that year also uh genetic code for two first amino acid was solved that was the phenylalanine and proline and 1961 the space phrase and space age started because gagarin was went around once in the but in our field this was a very exciting time what
• 07:30 - 08:00 happened is that people um scientists realized that um how could it be that uh cell would know what kind of protein to make when all of the information is in in the chromosome under under the dna oops and um and the translation actually happening in in the cytoplasm and they found in a ribosome that there is an rna and at the beginning they thought that
• 08:00 - 08:30 maybe this is the rna but turn out that it was ribosomal rna and it was for every cell it was the same and it was the size of the same so it couldn't be so just like for the mandolin system they suspected there has to be one rna something which some information is coming out and this is one actually discovered that there is a messenger rna is produced from the chromosome and it comes out from the nuclei
• 08:30 - 09:00 and in the ribosome it translated to protein and this publication came out in actually 60 years ago 1961 and the both paper stated that this was a very unstable molecule the rna which is coming and carrying the information to the ribosome what kind of protein has to be made so many times you know this unstable
• 09:00 - 09:30 characteristic was something that even later years and years later you know made it difficult to introduce to the therapy but it was 1961 and when we could first time make mrna in vitro it was 1984. so what happened during these 20 plus years first of all uh the technology had to be the molecular biology had to be advanced so plasmid was introduced
• 09:30 - 10:00 and many other features what uh but for this one for our mrna was most important that the scientists couldn't find rna polymerase which they could generate in vitro rna so they tried bacterial and eukaryotic rna polymerases but none of them worked up until scientists douglas melton and paul krieg published this finding phage rna
• 10:00 - 10:30 polymerase sp6 rna polymerase which could translate uh transcribe very well and from the plasmid and the sp6 promoter here is you know just a 17 base pair sequence and so it's very small and very uh feasible so the first uh rna actually what they made it was um coded for interferon beta and uh bernard most gave
• 10:30 - 11:00 them the captain enzyme who worked in nih and worked on the vox senior project so they had a lot of help and then they made a kept uh rna which are polyatl and five primes three priming tr in addition to the coding sequence to find out that whether the rna what they made is functional what they did they inject it to frog oversight in 1984 that was the way to introduce an mrna inside the cell and the frog side is large so that with the
• 11:00 - 11:30 needle it could be injected and then they injected like 20 uh such or site and incubated and they collected the medium and tested out whether the medium contains functional uh human interferon and then they found yes it was it was functional so this is a 1984 when we can start and saying that yes messenger rna
• 11:30 - 12:00 therapy can start but of course you know this is not feasible micro injecting two cells because for that one you need huge cells so uh for me uh the next milestone is really 1989 when mrna delivery by lipofectin was solved so in that time t7 rna polymerase was the favorite phage
• 12:00 - 12:30 polymerase which people used it was introduced one year after the sp6 polymerase was used and and uh also instead of enzyme this was that time it was not available capping the enzyme it was the cap honolulu was used it was also um important uh because this uh lipofectin because um people before lipofectin and i worked with rna in hungary but those were not messenger rna short rna
• 12:30 - 13:00 then those could be only introduced to the cell with the osmotic shock or calcium phosphate or precipitation something which is uh medically is not feasible only on cultural cells we could subject such a severe treatment but lipofectin was very mild lipofectin actually is a positively cartionically personal positively charged and you have to know that the rna is negatively charged and the
• 13:00 - 13:30 surface membrane is also negatively charged so if we put the rna to the cell it's not getting in so so lipofectin uh was introduced actually uh with phil fagner introduced in 1987. and uh it was also very important that um in in that second part of the 80s you can see there's so many things happening because uh here for example the trisol was introduced and those people who struggle with isolating rna
• 13:30 - 14:00 you know this champion the championship introduced this uh method it's very simplified how we could make rna otherwise it was very tedious and very many times ended up with degraded rna so for this uh second part of the 80s also so characteristic that some scientists at the bench discover something and immediately was commercially available so that like the trisory agent or the lipofacting or
• 14:00 - 14:30 the rna polymerase because in the same year when it was published by douglas douglas that it was already companies already were selling it so it was very easy it was also very important that um the second part of the eighties already tech dna polymerase was available and uh was the molecule of the year in 1989 and we could buy
• 14:30 - 15:00 pcr machines so all of the sudden if you had a cell which expressed the interest of protein you just could sitting at the bench you know at the laboratory and then you could make the rna you could generate and analyze in different cells it was like so empowering that uh if if you live in that area you just can fight like you are a woman you can do anything and so it was um that's how it happened that uh it was
• 15:00 - 15:30 this time 1989 i went to penn and um my colleague was interested in eurokina's uh receptor and so um eliot barnatan uh and together we decided to make messenger rna therapy what was interesting is that um the euro kinase receptor was a highly post-translational modified protein needed a gpio anchor and highly
• 15:30 - 16:00 glycosylated and to our biggest surprise when we put this mrna this eurocoin is mrna to the cell with hyperfecting we found that the functional receptor was made and we together tried to use for different application and from cardiology i moved on to neurosurgery and there again using different mrna coding for uh
• 16:00 - 16:30 nitric oxide synthase for example where you know not the protein but the generated product was functional and tried to use for fighting the vasoconstriction so all of these experiments let that we can do any kind of mrna and we can use it for treatment and so very exciting time but uh all of the sudden in 1998 i learned that the rna this in vitro transcribe rna is immunogenic
• 16:30 - 17:00 and this has happened because um true weissmann arrives 10 from fauce's lab and i met him and he was interested to develop hiv vaccine and i told him that i can make rna and he was interested to test out because he was using plasmids and it didn't work very well on human dendritic cells and when he tested this rna i gave him
• 17:00 - 17:30 he was very happy that this is the perfect uh vaccine because not only coding for the antigen but it is also like a work as an urge one because it induced immune response and how happy he was i was so disappointed and so sad because for 10 years almost i was working to make a therapeutic protein mrna coding for a carotid protein and all of the sudden turns out
• 17:30 - 18:00 that it is immunogenic and i have to tell you that we are here in 2000 and at 2000 none of the um none of the rna sensors were identified the first one was store-like receptor 3 and it was discovered in 2001. so we didn't know at all that is why the messenger rna we are making would be immunogenic and we know that in our body we have a
• 18:00 - 18:30 messenger rna and they are not immunogenic but of course they are coming out from the nuclei and and what we are doing we are delivering the rna from outside of the cells and so concluded that most likely these um cells recognizing as a foreign material and maybe this is why generating such an immune response and so i don't have the whole afternoon to tell you that how many things we tried but then we come to the conclusion that
• 18:30 - 19:00 maybe we should test out whether the rna which is inside the cell and we can isolate back and put it again to the immune cells whether this is immunogenic so we isolated rna and on monocyte derived human disease we tested out and measured inflammation and what we found is that rna here in a purple what was in vitro transcribe
• 19:00 - 19:30 generated a lot of tnf alpha and it was all rna related because when we when we digested with rnase it all disappeared so the rna was immunogenic but we found that those other rna which were isolated back it was not as immunogenic and surprisingly the trna was not immunogenic at all and so we were wondering why trna is not immunogenic and if we look at there we know that trna is
• 19:30 - 20:00 highly uh modified it contains a lot of modified nucleus all of them which is pink and red here in this picture and so he gave the idea that maybe this modification maybe these are responsible for lack of immunogenicity of the trna so when we reploted this same uh graph here we could see clearly that those which had no modification this was the
• 20:00 - 20:30 invitations five rna it um uh generated uh it a lot of tnf alpha while those which had the most uh like the trna was not immunogenic so we were wondering sorry we were wondering about that how we could make this in vitro transcribe rna to be non-immunogenic and introduce modification and so for this one we looked at that how what we know about
• 20:30 - 21:00 modification and so you have to know that all of the rna whether it is messenger rit rna or whatever small nuclear rna all of them is made from the basic four nucleotides but to push transcriptionally modification are introduced and right now we know more than 100 different type of modification and these are part of the rna maturation process there are more modification because oxidative stress can generate and modify the nuclear size for
• 21:00 - 21:30 different kind of articulating agent but we are talking about just those which is natural process and some of them is generated by isomerization or ventilation and at that time we are here in about between 2000 and 2004 the enzyme or how these modifications occurred not only the enzymes were not available it was not known and so the modification
• 21:30 - 22:00 as you can see on the left can happen in the in the the base and on the sugar rule methylation this is showing for the pyrimidine and the other is the puri so this is a lot of different modification and very complex and how could we introduce this to our in vitro transcribe rna so uh we wonder about and we decided that since the enzymes are not known that probably we have to purchase something
• 22:00 - 22:30 and try to see that whether the t7 rna polymerase would incorporate and we finally we purchased which was available these naturally occurring modified nucleoside it was a triphosphate and these were ten different uh triphosphate which was available i insisted that that time that we will do a lot of unnaturally occurring modified nucleoside is also available in triphosphate form but i remember in 1993
• 22:30 - 23:00 reading about the theoloridine trial which was introduced this modified nucleoside for hepatitis b and to treat that and the the 15 volunteer uh five of them died and they at that point you know we are in 2004 it was not known the reason why why this modified nucleoside is so toxic whereas when they tested out thousand times
• 23:00 - 23:30 higher dose in rats and even in monkeys that they couldn't find any any kind of toxicity so actually in 2006 when finally the answer came and turned out that nucleoside transporters and only the human nuclear site equilibrated nuclear site transporter in the middle of the coding sequence has a mitochondrial targeting signal and what happens is that in the case of human all of this modified nucleoside pump
• 23:30 - 24:00 into the mitochondria and then the patient lost you know the kidney and and died so i insisted that when we do mrna we will never ever use any kind of unnatural because the unnatural nucleus side because our body only knows the natural one how to get rid of and will not re-enter with a different kind of transporter i might just mention here that in addition to equilibrium there are concentrated transporter and all of them have several alternative
• 24:00 - 24:30 variants so what we did we tried to incorporate these nucleotide triphosphate and generate the rna not how nature generated but how we could generate and from the tan five of them did incorporated by the t7 polymerase in this experiment we uh changed the corresponding uh nucleotide transfer with the natural with the modified one and so
• 24:30 - 25:00 in all of these uh rna all of the labeled nucleotides were modified and then we test it out to see that whether or not these are will be immunogenic or not so in this experiment first because my by 2004 it was known uh that tlr3 was activated by double standard rna so we generated stable transform hack 293 cells and what
• 25:00 - 25:30 we found is that um when we uh generated two like receptor seven and two like receptor eight over expressing hex cells we found that the unmodified rna induced high level of il8 this is another rna what we generated and when we did introduce these different modifications we get a much lower iolate induction so subsequently we did trans tested out whether it is true for
• 25:30 - 26:00 uh primary human dendritic cells so again the same rna we tested out in primary cells and what we found is that only those which had a uridine was modified those uh did not induce any tnf alpha so now that the other modification did not influence the immunogenicity so why why it is uh why it is uranium what is so special about why nature selected
• 26:00 - 26:30 to recognize foreignness in an rna by uh uridine so this was a question and and so indeed we could see that um when we had pseudo-uridine fiber material in thyroid you know they we could get no immune response i wonder about that why people could not see it why scientists did not realize that uridine is such a foreign so i like to go back and read
• 26:30 - 27:00 all the papers and so i went back all the way to 1963 and i realized that actually scientists knew that that time she could because what happened is that um isaac who actually in 1957 he discovered in deferent he did an experiment and he isolated the rna from mammalian cell and he put on cells and realized that there is no um interferon was induced but when he treated that rna with nitrous acid he found that
• 27:00 - 27:30 the rna became foreign how he said that and it was now induced high level of interferon and what this nitrous acid is doing it converts the ura the cytosine to uracil so when he had an rna for example you see showing a sequence the treatment resulted that all of the sick would be converted to you because domination occurring and so uh he also mentioned that
• 27:30 - 28:00 how much percentage of cytosine had to be changed to uh uracil so actually scientists in 63ish already suspected but they didn't know so when we did this recognition that uh uridine is uh so critical for activating immune immune sensors uh later in the crystal structure of two like receptor seven and two like receptor eight they identified that urine containing
• 28:00 - 28:30 rna as well as uridine the nucleotide by itself can dimerize two like receptor eight and only uridine fits right to the to this um crystal structure interacting with the different amino acid and most likely when it is modified is not fitting there so of course now we are happy that we have a urethane modified uridine containing rna is not
• 28:30 - 29:00 immunogenic but we have to see that whether it is translating because after all we want to make therapeutic protein mrna coding for therapeutic protein when we test it out we were surprised to find that when we tested in 293 cells for example uh pseudo-you're reading containing rna translated so much better than the unmodified rna some which was modified didn't translate it at all while um
• 29:00 - 29:30 also we found that in murine dendritic cells we found that this pseudo-uridine containing rna translates so well here i just show you that the uridine and pseudo-uridine when people are saying that it is chemically modified it is really not chemically modified because uridine and pseudo-uridine both of them has the same base uracil it is just linked differently to the to the sugar so and the weight is the same and that was the reason maybe that when
• 29:30 - 30:00 we are here in 2008 this was not known at all that uh messenger rna actually has a pseudo-uridine now we today we already know and know the enzymes that can incorporate that so we also were interested whether it is translated or in vivo and again the formulation was critical as i mentioned the lipofacting was important for uh delivering mrna to the culture cells now that transit was
• 30:00 - 30:30 important and other complexing agent to deliver in vivo to mice and as you can see when we injected the recombinant protein ipob to the animal it is uh it's a half-life is about two hours so the injected protein quickly disappears when we had the blue shows the euro uridine containing rna then we get we get
• 30:30 - 31:00 translation is shorter and when we had the uh pseudo uridine containing uh mrna coding for educator poetine and here is a very small amount was injected to the animal and up to four days translation was detected hippo could be detected in the blood and most importantly the animal did not generated any interferon in response to
• 31:00 - 31:30 the injected mrna whereas the u containing rna-induced interferon so it is very important now that we have a pseudoiridium containing rna which translate very well and which is not immunogenic it is like the dream come true and so the next after 2012 uh yeah so that i so
• 31:30 - 32:00 an important part was actually to show that uh sorry i should learn handle more okay so the important part was that the ipo was also functional we could see that um hematocrit increased even after injecting once the mrna and repeated injection we could see a weekly injection of ippo mrna maintained hematocrit high level
• 32:00 - 32:30 uh what happened after this after 2012 we further optimized the mrna it is very important to mention that in the last 20 years you know it will became available that we can just order and the gene can be synthesized so let's say the covey the pandemic is happened 20 years ago all of those who tried to develop
• 32:30 - 33:00 vaccine had to be maintained and and obtain some material which contains the virus but today it was sufficient just to get the information and the gene could be synthesized it could be incorporated whether into the plasmid or whether it just used as a pcr template transcription can be made and many improvements were performed like a cap structure were introduced now is a cap one which is uh
• 33:00 - 33:30 improved greatly the translation and uh coding sequence optimization and so here for example the y type rna with codon optimization could be increase the translation and for this one i would show you that what kind of way we can optimize the coding sequence and how it can change the immunogenicity of the rna so when we introduce a pseudo-already
• 33:30 - 34:00 modified rna we can eliminate all of the immunogenicity if if we um let's say make the rna ugc rich then um some of the code we can change it and the uridine to c or g but actually not all of the amino acid can be coded by a code that has no uridine and the right
• 34:00 - 34:30 i am showing you here that which are those amino acid and what is their frequency invertebrate that requires uridine in the present to the code so what it means that um when somebody is trying to reduce immunogenicity of the mrna by making it gc rich it will be greatly depend on the amino acid composition of the target protein so it might be that
• 34:30 - 35:00 if it is loosing rich then it will be difficult to make non-immunogenic that rna in addition the codon optimization the other sequence other a part of the rna the utrs can be optimized and of course it was a great enhancement of translation if the rna is purified i don't want to talk about it but again
• 35:00 - 35:30 like we spent like five years trying to figure out how to uh purify the rna to make it now that finally remove all of the double standard rna and so eventually the rna will not activate 2 like receptor 3. so here we know that in 2021 and we can make a optimal mrna with the optimal cap and
• 35:30 - 36:00 already clinical trials are ongoing so those who were not engaged in the messenger rna field they thought that maybe this first mrna which went to human trial was the vaccine but actually there are already phase two trial is ongoing to use a vgfa mrna in a heart failure patient in addition cancer treatment with intra-tumor injection of cytokine
• 36:00 - 36:30 rna again these are protein waves replacement therapies because the mrna code for a therapeutic protein and in addition for passive immunization when messenger rna coding for monoclonal antibodies uh targeting for viral protein so these are already clinical trials which are ongoing with the nucleoside modified rna in a protein replacement field but and and and we as and by on tech
• 36:30 - 37:00 also contributed to this and our program generating by specific antibodies which one part is recognizing the immune cells the other is the cancer cells already uh moving to clinical phase we generated these messenger rna and test it out in in mice and showing that ensuring that
• 37:00 - 37:30 the tumor could be reduced greatly in animals that were treated with this by specific antibody encoding mrna so many protein replacement therapy is incl is in a phase of pre-clinical stage those are mrna encoding for secreted proteins
• 37:30 - 38:00 and those are kind of easier because any kind of cells may be able to make the protein of interest so the mrna can be translated to functional protein and these are for regeneration and healing of a bone or or injury of the skin or treating the anemia with epo i just show you and the other part is when mrna encoding intracellular
• 38:00 - 38:30 protein those are much more challenging because in that case the mrna had to enter to the to the cell which requires the missing protein or protein which has a lower level and so again we will see in the future that the most important part would be again the formulation i told you first was the lipofacting which made it possible to work in cultural
• 38:30 - 39:00 cells then it was the transit which we have which helped us to investigate the mrna translation in the inner vivo in animals and know that we are needing a kind of formulation which can target specific cell types so that all of the intracellular protein can be introduced of course you know all of my primary goal was during these years is
• 39:00 - 39:30 to develop mrna for therapy therapy where mrna code for a protein that is beneficial and not an antigen but how we get there i have to show you here my colleague norbert party at upan who came there to 2011 and we started to work together and later he kept working uh further with uh drew weissman
• 39:30 - 40:00 and what he did is that he generated an mrna coding for a pre-membrane envelope glycoprotein and this he introduced this lmp formulation and this experiment was done in 2017 and reported that nucleoside modified mrna formulated with lmp can protect even monkeys in very small
• 40:00 - 40:30 dose against the zika viral challenge and the importance was that know that even for the vaccine nuclear site modified rna was working better and subsequent work he demonstrated and compared with the eu containing and pseudo-reading containing rna differences in vaccination identify the different kind of molecule the follicular helpers as a role
• 40:30 - 41:00 uh in that process and so that was the first time that in 2017 that messenger rna nucleoside modified messenger rna were formulated with lipid non-particle and used as a vaccine successfully in not just in mice but larger animals so here you can see that very very small those even 50 microgram was sufficient i have to emphasize again that when a
• 41:00 - 41:30 dna was used primarily that for a vaccination it had to be scaled up for animal so larger animal needed higher dose but it turned out that is not case for the rna i might mention here also that the at present pfizer vaccine has bioenterpriser vaccine has 30 microgram mrna and the same those were needed for mice to protect them so
• 41:30 - 42:00 for me is uh uh these are the major timelines you know what i can say here uh to you as a summary here uh that we disc the rna messenger array was discovered in 61. 1984 was the first time that we could synthesize mrna in vitro 89 was that lipo affecting finally helped us to proceed and deliver mrna
• 42:00 - 42:30 and doing many experiments and in 2000 we realized that mrna in vitro transcribe rna immunogenic 2005 that the nucleoside modified rna is not immunogenic and 2017 as i showed you lmp which was uh containing modified rna was used as a vaccine and now we are last year 2020 we get the
• 42:30 - 43:00 emergency use authorization approval and uh and now that the vaccine entered to the uh i am not talking here about the vaccine because our ceo biotech ceo uhu zaheen just gave two lectures uh very recently and he is the best person who would talk about the development of the different kind of vaccine including the biotech pfizer vaccine so please
• 43:00 - 43:30 listen to his presentation and learn from it i just would like to finally acknowledge my colleagues at the university of pennsylvania who um you know at cardiology that eliot barnatan who believed me and then who we together started to perform mrna therapy at the beginning at the neurosurgery david langer who managed to save my program
• 43:30 - 44:00 by convincing the chairman neurosurgery chairman to open a lab in neurosurgery for for a molecular biology and this is the first time that i had my own lab and a salary and and of course you know that true basement and the norbert party whom i work together and even today and develop further the modified rna program and
• 44:00 - 44:30 for the bi-specific antibodies program christianist toddler and and hayat were instrumental and of course i am very grateful to uber zaheen and islam turesi working in bayern tech and hiring me there and making providing an opportunity for me to to do my best and try to bring the messenger rna to the clinic and um
• 44:30 - 45:00 and from aquitas you know the tom marin who uh ceo of aquitas that formulated the rna for the vaccines and thank you very much for your attention thank you very much katie this was really really fascinating to listen to and thank you so much for reminding all of us to read old papers not only the new west
• 45:00 - 45:30 ones all the time so i'm going to give it over to my colleague sacha here and she will open up the q a session uh thank you angela i am delighted to join our distinguished guests and panelists for this it's going to be a wonderful discussion we'll begin our session with questions from our panelists as well as questions from our audience
• 45:30 - 46:00 due to lots of questions on on the question and answers we will try to group uh recurring question themes to address as many as possible so our first question is from vice chancellor mahapatra thanks dr kirikou it was such a pleasure listening to the detailed presentation you had so the question that i
• 46:00 - 46:30 have is how do you see the messenger rna platform shift future research and are there any specific collaborations that you recommend engaging in order to advance the impact of this approach so messenger rna is a therapeutic use you know those people who were not engaged in this field would think that you know it is just emerged and only these companies like
• 46:30 - 47:00 biontech and modern is doing but i have to tell you that there are many messenger rna companies all over the world from japan and korea and and and smaller companies and here in the u.s also that is specializing for certain application field and some of them was emerged as a formulation companies and then they needed rna and then when we came together on the mrna meeting you
• 47:00 - 47:30 know everybody who is looking for help whether it is regulatory because they want to enter to the clinic or whether they need a better rna or better formulation or different one so that's the meeting when the people are coming together and helping each other and i have to tell you an interesting thing about this field because there is a lot of competition there are qrak which was the first mrna company uh biontech and
• 47:30 - 48:00 moderna but we are also rooting for each other because any failure would be detrimental for all of the mrna programs so we make sure that everybody is all of these companies are successful so it is a very unique situation but um the platform so at present you know the modified rna is used by uh by uh biotech and and moderna
• 48:00 - 48:30 and other companies like translate bio and curved they are using unmodified rna for different applications not just for vaccine but also for protein replacement therapies thank you katie and the second question is from dean brazier thank you so much um uh dr carrico as a woman scientist and leader there um i'm sure been many
• 48:30 - 49:00 challenges that you've faced and have indeed overcome um really uh what advice would you give for women in science uh particularly stem researchers um and what perhaps is the biggest challenge that you had to overcome yeah so so i i as a book and you know in here in the us i'm more felt because my heaven have accent and then you know i start to learn only in a university in english so sometimes
• 49:00 - 49:30 there was ask a question you know that who is my supervisor because when i presented the modified rna program some assumption was that probably somebody smarter than me has to know all of these things but um you know the the thing is that you know what i can suggest that don't bother by comments or something that focus focus on the science as much as you can and um and do what uh
• 49:30 - 50:00 you know what you can do and um i also mentioned before when we chat here that um many times it is very discouraging for people seeing that around them that you know they people who work less and earn more and they advance and somehow if you focus on your own stuff and then try to not to bother by these things you know that sooner or later you know your advancing
• 50:00 - 50:30 science will pay off and then eventually it will be turned out fine and uh of course it is difficult for a woman because they have to give birth they had to you know what if they want to and so so that the help from a husband is good because i also have get a lot of help from my husband who you know didn't want me to be to be in the kitchen and cook and he he agreed that if i go
• 50:30 - 51:00 at the weekend you know and even coming back with a pcr machine that didn't close the lid and he had to fix it he didn't complain but he have so everybody's help is needed but yes so i don't know that yeah so so i i don't know that why you know girls would less interested maybe in the science but i i think that they are
• 51:00 - 51:30 everybody is interested everybody likes flowers birds and and in some level everybody would like to know something like you know when the birds are migrating where they are going how they find it you know and any different level if the teachers would keep the interests of the children then you know would be more scientists and we need more scientists because now you can see that the mrna will be entering in many many different areas of therapy and we need the next generation of
• 51:30 - 52:00 scientists to come and also the women look at you know differently land the women can be multitasking can do so many things so it would be great in the workforce if women and men would be you know working together on different projects okay the actual next question is from me so when there are new ideas and new concepts that are ahead of their times and they
• 52:00 - 52:30 are not readily appreciated or accepted by peers reviewers nih reviewers and funding agencies and you are well experienced with these challenges what are your suggestions or advice to those researchers who are navigating the same situation you you know that when we didn't i couldn't receive the funding with eliot bernadette and we
• 52:30 - 53:00 went out for venture capitalized we presented to them you know i don't name them because now that maybe you know they kicking that but that they didn't help us and it was 1993 and very small amount actually it was seventy thousand they promised but never delivered so that um you know we tried that and i realized one thing that um you know our education did not include all of these different
• 53:00 - 53:30 skills so for example as to be a salesman you know i i just wonder that uh uh stefan banzer you know he was a salesman and he was so good and he did a great thing for the mrna research because he could uh invite a lot of money you know uh ostrazeneca get 240 million that was the biggest mrna you know kind of gap and so we need other skills and and i i didn't had that probably when i presented i
• 53:30 - 54:00 as a scientist you know when tried to get money and or explaining to others it was not articulated well once i remember when on the ip office said at 10 you know when finally they asked what is good for i i told the guy there who didn't have hair that probably mrna is good to grow hair and then he was immediately excited it was the only time i was you know i was a little bit the salesperson but um
• 54:00 - 54:30 so that uh and the language you know that i i don't blame the people that didn't give the money probably you know it was too risky and the kind of the funding is going to science which is kind of half established or well established and not something that and maybe 99 percent is not fundable really crazy idea and or maybe we have to decide that uh fund all of the hundreds because
• 54:30 - 55:00 maybe one will be the great one right i don't know because you can see that i tried many things i tried to establish the company but uh you know i couldn't get the pet my our patent couldn't get with a drew wiseman we couldn't get for our company so you know try to ask other people try to ask the government and others so so that's difficult yeah thank you katie this is um
• 55:00 - 55:30 this is very encouraging and also actually it answers some dean larmor's question regarding persistence and how how you overcome how you overcame these odds and and what you would suggest for up-and-coming um scientists in regards to persistence how they could persist but i think it's it's been um very helpful um anything else if if you wanted to
• 55:30 - 56:00 add in regards to how a person how a scientist can persist um in facing failures all the time yeah so such as they say that you don't get until you get a failure because the successful will we get that you can go get over on that and uh so for me i was um you know everybody's saying no no no but not everybody because as long as i had one person who who was for it you know and could
• 56:00 - 56:30 get enough money i mean my salary was meager and uh was like the technician get more but if you focus and you do not interest about that then you can stay on course and i have to tell you also that it is important to see progress so i don't say that you just keep doing something and without not seeing some kind of positive outcome because then it would be you know it is just a waste of your time so it
• 56:30 - 57:00 was important for us that we could see that okay now we can get more translation and we could see um progress that no we could see on on on animal and we could see the function and then we could imagine more and more application actually it was always there that oh it will be good for something at the beginning when we could do only ex vivo we focused on you know treating a blood vessel for you know
• 57:00 - 57:30 bypass surgeries because that's what my colleague uh eliot was a cardiologist and they were doing so that i could have a blood vessel and human blasto in my hands and then we flush through the rna it is enough that one minute is going through the liquid already the rna is picked up and then you know 10 minutes you already can see the protein so it is instant and so we just have to figure out which kind of um protein that mrna had to code which
• 57:30 - 58:00 would make this vessels more patent and would stay you know and survive longer and we also proposed and visited other colleagues at campus with drew weissman we tried to increase for example the bone marrow half-life by extending the tip of the chromosome meaning that you can apply less bone marrow for one person and it would be more people could get it or older people
• 58:00 - 58:30 could give a bone marrow because now that we are extending the tip of the chromosome and then it will be so we try to constantly it is kind of you have the button and try to find the code because you you you have the rna and now that you try to find what is good for of course you know when it is the therapeutic window is very narrow i wouldn't suggest that but in a 24 years at university pennsylvania i attended twice a week for a lecture excellent
• 58:30 - 59:00 lecture and i also went prepare and then at the end i always if i could feel that it is good for mrna this kind of uh treatment i i made the list and there so i arrived at the biotech with with the 30 proposal that's 30 different and at present we are not doing this because we had to do other things you know for two years i was spending there try to see that what we could do at the bench how we could
• 59:00 - 59:30 upscale because then we had to find other technology another so it went and then we had to develop programs which we could get funding for again so even in the company but maybe now that things change and we maybe go back and then see that all of these including you know getting hair growth okay good message write 30 proposals and don't don't get disheartened so i
• 59:30 - 60:00 love it thank you and now um i have a few um science related questions that i i'm trying to learn together um so one question actually the very first question was um similar to to what i wanted to ask um it's kind of um interesting that still in younger children on mrna um vaccine is
• 60:00 - 60:30 not not well um researched and and i was wondering if there is any age related difference and if you noticed um anything um between um young and old on subjects how they would respond to repeated um mrna uh therapy or also it is um it is a similar question to what i got from my colleague who is actually calling in from beirut
• 60:30 - 61:00 lebanon irani um she was wondering um how you could optimize on mrna related therapy to avoid non-specific immunity but still evoke the kind of immunity that that you want so you are not talking about vaccine now you are talking about therapeutic because i mean one for therapeutic many of the diseases rare diseases
• 61:00 - 61:30 you need to apply for very early age you know the newborn had to be treated right away but you know that you cannot use any kind of mrna or any other drug before you have to try out on a dog and then you have to scale back and you look at the younger and younger before you apply uh for the newborn let's say for several rare diseases and so this is a big challenge and also a big challenge that
• 61:30 - 62:00 some of the rare disease are ultra rare or you you know it is hard to find a patient and but we we are also have program and we are working on rare diseases and actually again when i went to germany at the last time i took a deep breath and then wrote one more time in my life a grant because totally i received only once as a company crime and that was for uh dystrophic epidermolysis bullosa
• 62:00 - 62:30 i felt so passionate about those and again i did not get the grant but we you know i am still want to use this messenger rna because i find it is very applicable and could you know the children could benefit from that so so that um for the clinical vaccination related uh you know the uh visor already uh testing between
• 62:30 - 63:00 age 12 and 15 already that was close to this uh trial already and and now it is recruiting for the between i think it was five to 11 or something so it will be come so it is you know when the people are saying that uh it was pregnant woman who was not it is natural that any kind of drug when they are testing out you know they are never the first line to test out the pregnant woman you know that would be usually the last
• 63:00 - 63:30 one so it is obvious that first you know and here had to first try the trial also went on you know the elderly because they were the most vulnerable and and so those were those will come it will just take more time i understand there were a couple of um questions related to to the chemical modifications and one of the first one was asking
• 63:30 - 64:00 the fire luridin um where was this this toxic version uh found originally um was it found in nature or it's a synthetic material and the the other one was related to the the thiol atoms and in some of the nucleosides and how would those be affected by in vitro modifications manipulations such as some use of dtt
• 64:00 - 64:30 okay so actually i work at the camper university and my supervisor professor suladonic wrote the book on the textbook on modify nuclear sites so and if i say that my phd thesis i i use modify nucleosides those were cordyceps those are naturally occurring but it was occurring in fungus and uh you know and those were
• 64:30 - 65:00 you know three prime dioxin and so there are many naturally occurring but occurring in bacteria or fungus and for the theory best minorities was not naturally occurring it was you know the iodinated and the ribo was fluorinated and nature doesn't have the only sugar modification in nature is the methylation two primal methylation is the only one so it is not natural
• 65:00 - 65:30 it is just um uh what was um already known that all of these unnatural modified nucleoside is is a antiviral component in civil and even some of the natural which is present in bacteria uh co-formicin and others they are using against uh treating of cancer treating of viral infection you know that because they are incorporating to the
• 65:30 - 66:00 growing chain and then it chain terminators and inhibiting cell proliferation so so those different kind of whether it is present in naturally in some kind of small other organism that mammalian so it is important also for us to know that pseudo-uridine which is our fifth most abundant modified nucleoside in our body we we know our body knows how to handle and
• 66:00 - 66:30 we cannot break the carb you know the glycolic linkage to the carbon so that actually we pee out pseudo-uridine and but it is not coming back it cannot come back because the nucleoside transporters make sure that it is just one way way out and so that's why it is critical and this understanding is actually just ongoing and you know when when somebody is um using
• 66:30 - 67:00 like mrna for therapy we had to figure out the translation what is required for translation what we know now we are taking from outside the mrna we have to know that all of this what we are potential sensors not just in the endosome but even in the cytoplasma is there then we have to know that how the rna what will be the fate how it degrades how it will dispose so um and you know all of these nucleoside transporters so many many uh other things we have to know to make
• 67:00 - 67:30 sure that everything will be safe and so that's what i at the beginning i i insisted because more modified nucleoside triphosphate was available in the chat about rna viruses
• 67:30 - 68:00 they do induce strong inflammatory response and and many of them dampen the immune response and any comments on that how the rna gets modified or how that is used in these situations so actually the modification nuclear site modification the heydays were in the 70s when 1975 the cap was discovered and they said found that
• 68:00 - 68:30 the m7g is there they they thought that oh there's these there are modifications here and and then they looked at there and they found also that for example m6a present in the messenger rna another place so when they looked at there people realized you know the scientists that actually very specific position and it was also when they discovered in the ribosomal rna very specific position was modification and they thought that if it
• 68:30 - 69:00 is so specific must be important so they changed it and when it changed nothing happened there were no phenotype so you could see that the seventh is you know so many publication and modification nuclear site modification and then after nothing because there were no phenotype so i looked at the back again you know like to see that why they didn't see it so one of the interesting thing was when uh for example m6a or an other modification was present in the
• 69:00 - 69:30 and then the scientists changed the sequence so that it's still coded for you know the virus everything but now that the modification couldn't be incorporated they checked out on the on the cultured cells if they would have looked at animal then they would see that now that there are there is a difference because the modified rna would be you know uh less immunogenic and it would be more virulent in the nmr but they they they couldn't see because they were tested out on so i tried to see just like i mentioned
• 69:30 - 70:00 just for the sake of it you know that looking back is why they didn't see that did they see that and then it was the same when i looked at their scientists that johns hopkins already changed but they did not inject it back to the anamar the virus and that's why they couldn't see so the virus is using modification the corona has nuclear site modification that's that's a trick to hide you know that is a fascinating point you have
• 70:00 - 70:30 brought about the in-vitro cell culture-based testing and then in vivo because in the context of immune system and how response takes place and corrections take place which you can't see in cell culture that's a great point uh another question is which is i had but there's also one in the chat uh so right now it's a one candidate vaccine mrna that's been
• 70:30 - 71:00 placed for delivery to the cells but maybe you could enhance immunogenicity or enhance the immune response by combining it with a delivery with an immune modulating mrna or some other ones so how do you see future and what may be the hurdles for increasing the payload of the that delivery system and uh include more mrnas
• 71:00 - 71:30 and deliver and enhance the whole process is this uh possible and what would be the challenges i mean if you say 30 microgram rna is protecting a human being that's kind of very potent already but um you know what happened is one and i didn't elaborate on this one the messenger rna is very immunogenic
• 71:30 - 72:00 and why we have to reduce the immunogenicity and health finally in the vaccine also is is the lmp actually which is activating the immune system because the messenger rna inducing interferon and this interferon is interfering actually with the immune response from the molecular keyhole process so the antibody response will be much lower if we are in using interference
• 72:00 - 72:30 and that's what the unmodified mrna is doing but if you try to make g series like qrack is doing you can reduce the uridine content and you might get less interferon induction interferon is good against the virus but not good for inducing immune response and when you you are using the when the lmp is activating and the work as an ultra one is not in using interferon it in using other kind of cytokines which is critical
• 72:30 - 73:00 to get good immune response uh just to add on to that uh uh can you comment on the advantages of self amplifying rna in compare in comparison to uh regular mrna that you are talking about this is a question in the chat yes so this is a my own private opinion because i work with a self-amplifying rna here at penn and then it was
• 73:00 - 73:30 it was very good it is very potent but um you know one one problem is that you are not just delivering mrna coding for the critical let's say antigen coding sequence but it is also contains 260 kilodalton of replicas which are you know that you are applying and so it is a huge rna it's challenging because it at least 7000 or longer nucleotide
• 73:30 - 74:00 long has to be because it it replicative self-replicative rna codes for the replicase and so this is one disadvantage the other thing is that um it's not working well in the larger animals that's what is the problem i see we know already that first it was no artist was pursuing and then it was
• 74:00 - 74:30 jsk was doing the clinical we have to see the clinical trial i haven't seen clinical trial data from and it was already started last year in imperial college of london the robin shattuck team started a replicative rna for covet 19 vaccine and the data are not out so we don't know the data that why it is not advanced why it is not published but maybe it will be out and we will see
• 74:30 - 75:00 that it is also good i have some but again this is not shared by biontech opinion we also tested actually in phase one clinical trial we had self-replicative rna ureading containing rna and pseudo-iridium one methyl pseudo-reading containing rna and we proceeded with the modified artery i have some questions uh related to to toxicity and also
• 75:00 - 75:30 uh several um people in the audience were wondering about the 30 microgram dose and and how is it possible that it works the same way in a mouse which is a thousand times smaller than um um than human and um um also um um questions are related to the blood brain barrier whether mrna is capable of of bypassing it and get expressed
• 75:30 - 76:00 in the brain and then an addition to this is totally unrelated but still related we have a plant geneticist um little micromore in the audience and he would like to know if mrna therapy could be used in plants too sorry for lamping all these together yes so last one about plant and rna actually
• 76:00 - 76:30 right now double-stranded rna is used in plants and those are very specific so like the colorado bug for example which is eating the leaves and so when they spray double-stranded rna and those rna actually contains some complementary material which would interfere with the digestive system of
• 76:30 - 77:00 the bug so they are eating and then they cannot grow those kind of bugs like the uh insects so that's actually rna and that's x s x as si rna okay so the the sequences contains complementary sequences and and interfering with the digestive system of the insect and because we are not eating the potato leaves so it is
• 77:00 - 77:30 this is why they are using for the those plants and actually in different countries already spraying the double-sided rna to to the crop which yeah so that's rna is already there but um you know injecting rna so there is a more challenge there because the plant has uh very thick walls so uh it is a cell cellulose and then it is uh you have to penetrate through on that
• 77:30 - 78:00 and so the same way like the bacteria is also you put an rna it won't get inside so but in a in protoplast when you remove the wall probably you can deliver rna in plants um right now i cannot remember that somebody was doing that but they demonstrated that actually extracellular rna is circulating in the plant inside the plant so naturally but not
• 78:00 - 78:30 what uh therapeutic but uh could be used uh what was the beside yes the the other was blood brain barrier and also the how come that the 30 microgram yes so the the blood-brain barrier these are not going through the but recently somebody created some just yesterday i was reading that created a tiny particle which finally and modified it in a way surface had a
• 78:30 - 79:00 nucleic acid there that could they demonstrate it could go through blood vein barrier but these others one is not not going through it has had to be applied through the skull and that's actually we did a lot in neurosurgery we delivered it intraventricularly and interactively and and that time you know what we could use is only secreted protein which was therapeutic because the rna even if the particle is there it
• 79:00 - 79:30 won't go through several layers of cells it is just the surface so if it is an epithelia lining up the and the with the csf is circulating the secreted material can reach certain part of the brain but delivering the rna it won't go through layers of cells it is also when you deliver you know we did together to the to the lung you know it in a mucus it won't go through you know there is no no way that the
• 79:30 - 80:00 formulation it gets through and the 30 microgram of course you know that um improving the rna and making it highly translatable and so it is um making that sufficient i mean we did a clinical trial and we did higher those and lower dose and this was selected maybe less is enough you know we just and and why uh you know in the mice actually with the
• 80:00 - 80:30 norbert party showed 30 micrograms he showed that one one injection generated the high dose so that you know if you would go higher those maybe one injection would be enough so but for this development of the vaccine the primary goal was as quickly as possible so that you know and that was the reason maybe the it was the minus 70 celsius because we had the most experience with it and and then it needed to generate data
• 80:30 - 81:00 to show what happened in -20 and i would emphasize that when you keep a lmp mrna in minus 70 then celsius then it can be kept there for years and when you are minus 20 it is just six months and it meant that you know you have to throw away uh your inventory if the expires so minus 70 still has a big advantage thank you uh
• 81:00 - 81:30 that is one question from uh dr bill murphy and uh this is more regarding the mechanism maybe is the is this the stability of antigen expression that is what is giving better immune response and boost to immune response using in vitro transcribed rna as compared to modified one or having to use an adjuvant so is there a faster clearance
• 81:30 - 82:00 an impaired expression so if you could comment on that i did not understand exactly what is the question then uh so does the immunogenicity of in vitro transcribed rna actually help boost immune response so i mentioned that the rna is immunogenic it seems that is in using interferon and if we have the uridine in it because
• 82:00 - 82:30 it activates a 7n primary tool like receptor 8 and it will induce interferon and so and we have demonstrated and others also that if the interferon was in use then it uh interfered with a good uh high affinity antibody response so so that it you we have to select what you want which is not in using in their film and that is what is in the lmp is doing okay
• 82:30 - 83:00 another question this is more related to delivery is that what are the challenges to deliver mrna to specific types of cells or the parts of the body and how do you see that is it possible to deliver this mrna specifically to some virally infected cells for example yeah that's a good question so so one
• 83:00 - 83:30 one part is of course when you are delivering locally so that if you are try to you know have a hair growth then you are delivering where you need the hair growth or you know intramuscularly that you are injecting in the muscle of course when it is injecting iv and you want specific whether to go to the lung or the spleen changing the charge on the particles the formulating particles we already demonstrated that it can
• 83:30 - 84:00 greatly enhance whether it will go to the spleen or whether it will go to the long wear mostly epithelial cells actually picking up the other is um when um scientists like dan peer and colleagues like they are adding the antibodies to the surface of the particles and then targeting for specific cell types so then the lmp rna were picked up by those cell types
• 84:00 - 84:30 additionally scientists because you know there is no tissue specific promoter so it's just the rna so that if they want to avoid the translation in certain type of cells if it is known those cells which they try to avoid what kind of micro rna is over expressed then incorporating to the three prime utr target sequence for those micro rna and then the rna degrade on those type of cells so this is how you can reach specificity
• 84:30 - 85:00 and but you know formulation also that my colleague drew wiseman and team you know they are doing also targeting like a bone marrow the specific targeting they can do and everything is not the rna but the form formulation is and that's why you know the formulation will be so critical because that was uh not just for the targeting but also when we want to induce immunogenicity the vaccine
• 85:00 - 85:30 or whether when we want to induce tolerance of course when we want to induce tolerance the lnp has to be different it cannot activate because repeatedly and you know biontech team demonstrated in a multiple sclerosis animal model that repeated a presentation without activation could induce tolerance in autoimmune disease so the mrna encoding the auto antigen then tolerance can be reached but then again the lnp has to be
• 85:30 - 86:00 different the lipid cannot activate so that will be the next big chapter in in our science is that the development of better and better formulation absolutely that's the importance of the whole delivery system and gene therapy folks always faced with that so there is one nice question from uh priyasha uh and this is more to do with the challenges
• 86:00 - 86:30 uh faced with scaling up uh so she's asking about uh the aspects of the scale that you worked on when you first arrived at bio in tech and can you reflect on some of the more challenging aspects of getting this technology to scale yes so it was uh from the microgram to go to milligram and then like in the laboratory
• 86:30 - 87:00 we usually just precipitated the rna but you know in the industry there is no precipitation because the precipitate won't be this tiny something you will have like a tennis ball size of precipitate and you cannot put it back to solutions so this precipitation was had to be excluded it was also you know the uh purification which we have done on on a hbse and it was uh there is you cannot get a bigger column and so it is
• 87:00 - 87:30 you had to change the technology you know they're just like you know they used to say that the light bulb is not coming to improving the candle you know you can put a bigger bigger candle but you know finally you just cannot do and that is so the technolo technology had to be completely different now you cannot precipitate no you cannot do those different uh purification how we did and we had to come up with a new way and so that's uh that was a little bit
• 87:30 - 88:00 frustrating uh frustration for me you know that uh we knew what to do and the the we could do it but now that we have to figure out how we can scale up and this is again when when the vaccine is produced that rna is not as much difficult to scale up it is more like a formulation or component of the lipids synthesis and others and then you know for for people just um i
• 88:00 - 88:30 mentioned to cooking for two people you know it is easy you steer it and ready and then tomorrow is 1000 the guest is coming and even if you have the same stove or bigger stove and bigger pop maybe you cannot steer anymore and the bottom is burning and up is cold and and you have to come up with absolutely different not this way and then it is when you are doing something it is very difficult okay forget it and we have to do somehow differently so
• 88:30 - 89:00 it is you know i have so great respect for all of the people working in the industry that and and figuring out in advance and doing all of these things it is just um you know fascinating and for this vaccine development all of them my fellow scientists and people at biontec at the wiser and all of the technical personnel that figuring out in the early on that we have to put more doors in one we need you know
• 89:00 - 89:30 figuring out how we would ship we need a new aeroplane we need permission to more co2 can be shipped on airplane somebody look at that oh there we don't have permission we have to go to the aviation and everybody thinking so much ahead not mentioning all of the clinical trials that how they could work with the hospital you institution like with your institution as well so it is a specialist also so so much respect i have for them
• 89:30 - 90:00 so you brought really new way of uh i mean you answered about scale up it's not just simple scale up you go from two to four or two to twenty you totally have to put a new thinking gap on how to amplify angela yes uh thank you this this was really uh eye opening and actually we have a lot of questions regarding that now you put mrna
• 90:00 - 90:30 um on the map so both for for vaccine which is a very uh different question scientifically but also for replacement therapies and some of the questions are related to the advantages and disadvantages of mrna versus dna related gene therapies that's for general gene replacement would you um
• 90:30 - 91:00 be able to just briefly um summarize what would be the advantages and disadvantages one versus the other and whether mrna would be more the future rather than a dna in terms of therapy so you know the rna was always criticized that it is transient which i always said that this is good right now we could say that you know who who wants to make spike
• 91:00 - 91:30 road and rest of their life and of course you know that permanent changes can be done also with the mrna and all of this uh general editing which is ongoing they are using uh word cas9 and no other can certain another they are using uh editing enzyme which is delivered as an mrna so they want to very transiently present there and change the genome so that
• 91:30 - 92:00 even the rna is we just wrote a preview one that rna is fulfilling the promise of the gene therapy because actually permanent changes with together with the genome editing we could make permanent and of course it is very good which is many times when they want to use something even gene therapy they check out whether the rn with rna because the rna will be gone
• 92:00 - 92:30 and the effect is gone and then you can see that whether something is good or not i mean the rna is more like a conventional medicine in many and many people more likely to have acute disease and need transiently some kind of over-expressing thing and it is also important because if you use this way you don't have to worry about like immunogenicity because if somebody is missing the protein or had a mutated one you know it they might generate immune
• 92:30 - 93:00 response against the one that you introduce and secrete it of course if it is not secreted it's less danger but if you are introducing but of course for dna is everything is permanent and uh and uh when when my first grant i wrote about the cystic fibrosis i was reading about that the epithelial cell is turnover is high so i thought that oh if you would permanently introduce dna into the epithelia and the cells survive is
• 93:00 - 93:30 two weeks and gone then it is also not a not a permanent you know changes you know because the cell is gone then you have to re-apply even the dna but um i am sure that um there is place for for both of them and of course the aav which is mostly used viral therapy and but because i focused on so many times on the arguing against them so all of the
• 93:30 - 94:00 argument is coming just why the rna would be better but probably there are many good arguments for them also but i didn't practice that yeah i understand you're a bit biased so yeah i totally agree with that so but okay so let me be the devious advocate here and and um several other questions are related to the adverse um outcomes of of the necessity of repeated
• 94:00 - 94:30 administration um and also the possibility that even though you carefully modify the rna it might still evoke residual innate immune responses and thirdly the rna always would need to be packaged into something to be uh delivered so would you care to um to comment on that
• 94:30 - 95:00 yes dystrophic epidermolysis bulowser which i mentioned to uh apply i've found it's very important that the repeated application like for example to a blister is it won't be so frequent because the uh if it is causing by collagen seven uh mutation the collagen seven half-life is uh half a year so you know months and months you know that and that that protein had to be there on
• 95:00 - 95:30 that layer of cell type so then can be delivered there and then it will be not so frequent repeated application i i understand then i agree that you know if weekly or bi-weekly has to be applied that it has a challenge but um yes so if the gene can be altered so with editing then it would be more feasible and better somebody asked
• 95:30 - 96:00 is phenocatanuria um being treated with mrna yet if you are yeah i i am not aware of uh so you know that moderna had several more uh program and they terminated some of them uh so it it is challenging i understand and what is the future of um of mrna uh
• 96:00 - 96:30 packaging carriers how how you're gonna move forward with those challenges so as i mentioned on the mrna this in vitro transcribe mrna therapy meeting you know that the biggest section is is formulation so that people are coming up coming up with new ideas and presenting and testing out trying and so um you know i mentioned the
• 96:30 - 97:00 targeting is a major issue and different kind of uh ligons are linked to the formulation and try to use this way to in vivo deliver specific cell types so it is a challenge but you know everything is a challenge yes you you did show that um um if you can dream it it's gonna be
• 97:00 - 97:30 possible when there is a there is a there is a wheel there is a way and actually our last year this pandemic uh witness this possibility so um uh reinforced our beliefs in uh in science and and i hope that the standing on scientists in society increased as a result and i know we had many many more questions but we are out of time and um
• 97:30 - 98:00 this should conclude um this q a session and thank you so very much for your time and your effort and we all are very appreciative this was a wonderful and um really eye-opening session thank you katie very grateful thank you thank you yes yeah thanks again and you know i
• 98:00 - 98:30 i can just imagine how busy your schedule would be um once again thank you very much for being with us and sharing your expertise um so much in detail and congratulations on all your remarkable achievements and contributions to the society i also take this opportunity to thank dean bressiers dr haksu and dr dan dicker and to all the audience out there for joining
• 98:30 - 99:00 us today enjoy the rest of the day