68 – Harnessing Messenger RNA metabolism for the development of precision gene therapy – SYNGAP1

Jeff Coller, PhD

These are our Introductory Comments

Our presentation today is ‘Harnessing Messenger RNA Metabolism for the Development of Precision Gene Therapy.’

Dr. Coller is a Bloomberg Distinguished Professor of RNA Biology and Therapeutics at Johns Hopkins University. His lab has made seminal discoveries in the area of messenger RNA stability and translation.  He received his PhD in cellular and Molecular Biology from the University of Wisconsin and was a postdoctoral fellow in the Howard Hughes Medical Institute at the University of Arizona.  Prior to moving to Johns Hopkins, Dr. Coller served as Director of the RNA Center at Case Western Reserve University where he held the Henry Wilson Payne Distinguished Professorship.

He studies the very essence of life: translation of the genetic code. His work has led to fundamental shifts in the understanding of gene expression by demonstrating that the genetic code is a major determinant of mRNA fate. He is the Co-founder of Tevard Biosciences and Wyve RNA Therapeutics. In 2018, Tevard Biosciences was awarded Pfizer’s Golden Ticket award for promising neuroscience startups. His publications have been cited over 6 thousand times and he currently holds numerous patents for RNA-based therapeutic applications.

THIS IS A TRANSCRIPT ONLY:

0:07
welcome to today’s webinar my name is Lauren Perry I’m operations manager at
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srf and a parent to a 12 year old was sitting up one um our presentation today is harnessing
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messenger RNA metabolism for the development of precision gene therapy and I have the pleasure to introduce
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today’s speaker Dr Jeff collar um Dr caller is a Bloomberg distinguished professional professor of
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RNA biology and Therapeutics at John Hopkins University he
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um his lab has made several excuse me has made seminal discoveries in the area of messenger RNA and
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stability and translation he received his PhD in cellular and molecular biology from the University of Wisconsin
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and was a postdoctoral fellow in the Howard Hughes Medical Institute at the
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University of Arizona prior to moving to Johns Hopkins Dr Coller served as a director of the RNA
1:04
Center at Case Western Reserve University where he held the Henry Wilson pain distinguished professorship
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he studies the very essence of life translation of the genetic code his work has led to fundamental shifts
1:18
in the understanding of gene expression by demonstrating that the genetic code is a major determinant of mRNA bait
1:25
he’s the co-founder of tevered biosciences and why of RNA Therapeutics
1:30
in 2018 tuber biosciences was awarded Pfizer’s golden ticket award for
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promising Neuroscience startups his Publications have been cited over 6 000 times and he currently holds numerous
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patents for rna-based therapeutic applications a recorded version of this
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webinar will be available on srf’s website under webinars on the family menu and you and at the end of Dr caller’s
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presentation you’ll have the opportunity to ask questions please write your questions in the Q a
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below and for those of you just joining us our speaker today is Dr Jeff caller
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it’s now my pleasure to turn things over to him great thank you Lauren you can hear me
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okay yep okay great let me share my sides thank you Lauren and Mike for the
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invitation to talk to you guys today uh let me make sure that we can share and you can see everything
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everything’s okay that’s good good okay great uh again
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thank you to um the syngap research fund for the opportunity to give this webinar today
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um I’d like to tell you about some of the work that we’ve done in the RNA therapeutic space uh that um
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is being done both in collaboration with tevert but then independent work that’s being done at my lab at Johns Hopkins
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and I’ll and that’s actually the work that I’ll talk about where uh relates specifically to cingo
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and just as a brief uh conflict of interest statement as Lauren said I’m
3:01
the co-founder one of the co-founders of covered biosciences and a member of their Sab
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and so you know this group needs no introduction to this but what I have
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become interested in over the last few years are genetic disorders called
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hapler sufficiency disorders where um single mutation within one of the two
3:24
copies leads to half the amount of protein that’s needed for appropriate uh
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wild type function and the um uh really fascinating aspect about this
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is that the mutation is not necessarily a gain of function it’s simply that it’s
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reducing the amount of protein and you need a 100 of the protein expressed in
3:50
order to have a normal uh individual and so this seems like it should be a
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relatively simple fix from a genetic standpoint where if there’s a mutation in one of the two alleles that all we
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need to do is raise the amount of protein two-fold and while that seems simple it’s actually very difficult to
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do and what I’m going to tell you about are three technologies that my lab has
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been involved in developing where what we’re going after is in fact the MRNA
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and the uh in the attempt to either Express the mutant copy
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of the restore expression of the mutant copy of the RNA and this is specifically
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through very specific types of mutations called stop codon mutations
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or uh two technologies where what we’re doing is in fact stabilizing and
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improving the translation of the wild type copy of the MRNA and this is the particular technology that I’ll spend
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the most amount of time on today because it’s very powerful for genetic disorders like syngap because it’s
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mutation agnostic and I’ll explain what that means in a few minutes
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and so if we think about an mRNA an mRNA which Everybody by this point uh in the
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world has heard about which makes my job a lot easier mrnas are of course the
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communicator between genetic information from DNA to protein they’re the intermediate that carries that message
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that information to translate the genetic code into protein and if we look
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at features of an mRNA that are important for its stability and its
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translation everything that I’ve shown here in red is a feature that we know contributes to the overall amount of
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protein that comes from that mRNA and how stable that mRNA is within the human
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body and that includes the structure called the cap it includes What’s called
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the three prime untranslated region and then codons themselves which my lab has
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demonstrated a great deal of evidence about and stop codons and then this
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thing called the poly a tail and we’ll talk about that at the very end of my talk
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and so in my lab what we’ve tried to do is leverage what we know about mRNA
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metabolism to improve the overall expression of the MRNA and we’ve focused
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really on these three features of the MRNA the codons the stop codons and the polyun tail and so I’ll tell you three
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separate stories about each one of these and the first part of my talk will be
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work that I did in that case Western Reserve University in collaboration
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warmings have art and now and then in collaboration with tavard which is use these unique RNA molecules that are
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called trnas in order to improve the expression of an mRNA
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and then at the end of the talk I’m going to tell you about work that we’re doing independent of Tabor which is
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through my lab at Johns Hopkins on poly a tails and this is really the work that
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we focused on the syngap but all of these approaches have the potential to
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be used on syngap deficiency and also show why that’s true in a few seconds
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and so just as a brief introduction to tevard biosciences this is a biotech
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company that um I’m a scientific co-founder with Dr Harvey Lotus of MIT
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and uh Daniel Fisher and Warren Lambert are our Founders and both Daniel and
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Warren have children who have drave syndrome and this group all knows what
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drab A syndrome is um and tevard in fact is drave written
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backwards and the idea here being that tevart’s mission is to turn these
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recalcitrant disorders like drave and Rett syndrome and syngap to turn them
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around some fashion so this company was started in 2018 in
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my lab and then um spun out to Cambridge Mass at Lab
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Central and now has grown quite a bit over the last two years and just entered into a unique partnership with vertex to
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look at other disorders including duchesne’s muscular dystrophy and so the two technologies that we
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started tevart on were the usage of trnas to leverage mRNA metabolism so
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what is a TRNA a TRNA is a small RNA in the body about 75 nucleotides in size
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very small and what trnas do is they communicate the genetic code
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it’s from nucleic acid sequence to protein sequence so an mRNA carries the
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information and that information is read by a ribosome and the TRNA and that TRNA
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there’s a TRNA for each of the 61 codons within the genetic cone more or less
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there’s some redundancy in that but that’s all that’s really important to know for today
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and the two technologies that we have are what we call suppressor trnas and enhancer trnas and I’ll talk about
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enhancer trnas first now why do I use trnas we’ve heard a lot
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uh with covid about mRNA Therapeutics uh mRNA Therapeutics are um given have been
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given a lot of interest in the biomedical space as of late
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um but uh outside of vaccines we haven’t shown uh that they have
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um efficacy at this point but it’s um it’s likely that they will um but trnas are unique in that they can
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address the root cause of a disease especially a haplo insufficiency disorder because they can restore and in
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the case of the suppressor can restore the exact amount of protein that needs
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to be expressed or at the very least they won’t go over their target I’ll explain why that’s true in a minute
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the other thing is that gene therapy is through aav everything that we’re going to talk
10:14
about at tepard is going to be a based gene therapy is delivered by AV are limited by this 4.7 KV Target size you
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can’t get a gene into an AV Vector that’s larger than this and many human
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genes are much larger than this 4.7 KD limitation trnas are incredibly small
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they’re only 75 nucleotides in size and so that means you could you could
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literally put hundreds of trnas into a AV vector
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in order to reach a therapeutic margin um in addition one of the things that is
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the most attractive aspect of trna-based therapies especially a suppressor therapy is that it’s a one-size-fits-all
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that one particular formulation could be used to treat multiple rare disorders in
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such as drive a rat syngap and others could use the exact same therapeutic and
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so we shouldn’t think about this type of approach as we’re going after drave syndrome or we’re going after ret or
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we’re going after syngap we’re actually going after most of these things all at once
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using any of the vectors of course is a one-time delivery mechanism
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and as I mentioned that um in the case of suppressors this
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actually can restore the normal expression of the Gene and for some disorders like ret syndrome it’s known
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that over expression going over that normal 100 amount of protein is in fact
11:47
just as bad as having the Apple insufficiency and there are other reasons why
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um TRNA suppressors are in fact useful so
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TRNA suppressors and TRNA enhancers can be used to treat a variety of mutations
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of a variety of indications as I said in the case of suppressors they treat
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nonsense mutations and even though disorders like syngap are the result of
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spontaneous and random mutation many of those mutations that occur in these
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haplo insufficiencies and all genetic disorders are in fact nonsense mutations it’s about 15 to 50 percent of all
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genetic diseases are caused by a nonsense mutation so having a suppressor
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that can read a nonsense mutation allows you to go after a large variety of genetic mutation genetic diseases
12:45
Capital insufficiency is of course there’s at least 300 it’s predicted up to 1000 in the human population and our
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technology can be used for um most of those We Believe
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and it really our sweet spot is somewhere in between here in diseases that are not amenable to gene therapy
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and these these figures actually just show many of what I just said in the previous slide in the Y tyrannos are
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available okay so trnas are a critical intermediary in the translation of the
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genetic code as I mentioned RNA mRNA messenger RNA has transcribed from your
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DNA and this mRNA encodes a single Gene in a human in the human body and those
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nucleotide sequences are then read by the TRNA the TRNA basically determines
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essentially it reads these individual what are called codons and parlays that
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information into protein identity so that at the end you get the exact protein that was dictated by the DNA
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sequence and so this intermediary is essentially what we’re focusing on to improve mRNA biology improve mRNA
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expression and I’ll go through why that works in right now so the first approach I’m going to talk
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about is enhancer trnas and it’s really based on work that was conducted in my laboratory
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um from the year 2015 to 2000 2010 to 2015. and for the last
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30 years my lab has been interested in the process of mRNA stability how fast
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an mRNA is um destroyed by the cell so when DNA
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makes Mr and air when mRNA is made from DNA well DNA is very stable the MRNA
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that is made is unstable it goes away over time it only sticks around for the
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most part for a few hours and the interesting observation that we’ve known
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about since the 1970s is that all mrnas are degraded at a different rate this is
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what we call a northern blot and all it’s showing you is an mRNA and just need to understand that it disappears
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and this mRNA is disappearing very quickly what three minutes this mRNA is
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very stable has a half-life of about a half hour and then this mRNA is somewhere in between with a half-life of
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about 15 minutes and if we look at all 26 000 or so mrnas that we know about in
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the human body they all Decay at a different rate and that rate is variable meaning that it’s not quantized like
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this where it’s you know short medium and long it’s every range in between
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and so my lab has been interested in how is that regulated so why do we care about that we care
15:46
about mRNA destruction because if you were to calculate how much of an mRNA is
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present within the human body its function of this equation which
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basically is that the rate of synthesis of an mRNA impacts the overall abundance
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of that mRNA and that is equal to how much mRNA decay occurs on that
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transcript and so an eight-fold change in synthesis of the MRNA we’ll have the
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exact same effect as an eight-fold rate of destruction of that mRNA in terms of the overall abundance and now you just
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have to think about this in terms of hablo and sufficiency disorders where we’re trying to change abundance and so
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if we can modulate mRNA transcription rate of course we could change abundance but another way to do that which is
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equally as power powerful is to modulate the K rate and that’s what we’re doing
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and so what my lab had discovered in 2015 was that the major mechanism for
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how these transcripts elicit different Decay rates is
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the genetic code itself that codons can broadly influence mRNA stability any
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everywhere from yeast to humans and these codons these individual 61
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words within the genetic code and it’s actually 64 but three of these are not good
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61 words are what dictate the identity of a gene but it’s the constitution of
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these codons within an mRNA that dictates how fast it destroys and I’ll
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just briefly show you that and basically how this works is every mRNA has codons
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within it and those mrnas are read by a machinery called the ribosome and how
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fast of ribosome reads these codons is what dictates their stability
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a ribosome is moving along the transcript the MRNA of the body
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deciphering these codons by use of the TRNA and if that ribosome is moving fast
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that mRNA is very stable but sometimes these codons are difficult to read by
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the ribosome and the reason why they’re difficult to read is because the TRNA and how much the TRNA is present within
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the cell is limiting it’s low in concentration and so the ribosome finds
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this codon for example and has trouble finding the right TRNA there’s a cloud
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of trnas around the ribosome and it has to sample these in order to find the cognate or the right one and if it’s low
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in concentration it’s going to hesitate there and those hesitations are cumulative across the body of the
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transcript and read by the Machinery that destroys the MRNA and so if the
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ribosome is hesitating this Machinery binds to the ribosome and then destroys it
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and so this is what’s called codon optimality this term was actually coined back in the 1960s as a theoretical
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possibility and my lab essentially showed that you can classify codons into either optimal or
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non-opt based on pools of trnanges and that results in either faster or slower
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the coating of the genetic code and faster or slower rate of mrnado Decay or
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opposite actually and so if we quantitate TRNA levels in
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the cell this is actually from another group’s Steve breitman’s lab and this
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quantitation of the trnas breaks those into optimal codons or non-optimal
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codons non-optable meaning the ones that hesitate optimal being the ones where the ribosome moves fast
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and if we take how individual codons influence mRNA stability and we overlay
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that with TRNA levels it’s a perfect match such that the TRN the codons that
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stabilize an mRNA are all optimal meaning that they have large or high concentrations of trnas and the codons
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that destabilized and mRNA are all non-optimal their trnas are relatively low in abundance
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and the way this looks from a gene by Gene basis is it’s a ratio of all of
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these optimal to non-optimal codons that dictates their stability and this is really kind of the critical
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experiment for the therapeutic that I’m going to talk about which is that we can then modulate the
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stability of an mRNA without changing its polypeptide sequence by simply affecting the optimality of the codons
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and here’s an experiment where we’re gradually replacing codons for
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synonymous codon pairs that are optimal and as you can see that in the course of
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the experiment we haven’t changed the polypeptide C once but I’m just showing this experiment twice
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that you get a dramatic dramatic stabilization of the MRNA just by
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changing the optimality of those now in the human body we can’t change the optimality of the codon Itself by
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changing the codon but we can change its perception by altering the TRNA
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concentration and that’s what we do in tevart I’ll talk about and so the idea
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here is that if we can identify slow codons within a gene like syngap or
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scn1a which is related to drive a syndrome if we can find codons that super slow down the ribosome
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and we now Express their cognate TRNA to speed up the ribosome and essentially
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what that’s going to do is stabilize the MRNA this in fact works the experiments
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that I’ll show you are really with scm1a that’s the only test case that we’ve done sn1a has five groups of very
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non-optimal codons and by expressing their cognate TRNA we can then improve
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the expression of scn1a in a message specific fashion and that’s just showing over here that these cocktails and trnas
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it turns out to be three unique trnas all shown here in green will lead to a
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dramatic increase in scn1a levels and this is extraordinarily specific because
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these three trnas are only sorry these three codons triplets are really only enriched in
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scn-1a and not at the same proportion in any other Gene in the genome and so this
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is a mutation agnostic technology because it’s not working on the mutant
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copy it’s working on the wild type cotton of this particular Gene and so it allows us to improve its expression
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now the other technologies that I’ll talk about are much simpler to understand and the one that has the most uh promise
22:44
to get into the clinic the earliest is in fact suppressors urinas and this this technology has been Advanced by Tavern
22:51
considerably in the last few years and just to introduce this TRNA suppressors
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are a platform that as I mentioned provides a one-size-fit-all cure for a
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broad range of diseases that are caused by nonsense mutation and this includes
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syngap for patients that have a nonsense mutation what are nonsense mutations I’ve already
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shown you this is the genetic code that was elucidated in the 60s and we know
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that three of the codons encode what we call stop codons this tells the cell these stop codons
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tell the cell when to stop making the polypeptide and there are three of them UAA UAG and
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UGA and the stop codon mutations often occur in human genetic disorders
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and there’s particular reasons for that that I could always answer another time
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but what that ends up doing is that the stop codon causes the probably polypeptide to prematurely terminate and
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therefore a loss of function of that protein in one of the two copies in the case of a apple institutions
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and so what the game is that we play here is we now take a TI so just to back
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up there is no naturally occurring so trnas decode all
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trnas decode all of these codons in the genetic code but there is no TRNA or
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these stop codons in the human body they are in fact fed by a protein not a TRNA and so the game we play in this
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type of therapeutic approach is to alter to chemically alter a naturally
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occurring TRNA so that it can now read those three style codons
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and put back in the right amino acid that was missing from the patient
24:47
in their protein and so that’s what we call a suppressor TRNA
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it turns out if you look at this is clinvar date if you look at all genetic mutations that have been associated with
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some sort of Clinic clinical pathological outcome
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um most nonsense mutations within the human
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genome or human population are the result of an arginine
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codon going from a CGA to a UGA this would
25:23
actually be used and tgit is human RNA and so this stop codon is the most common stop codon that you find in all
25:32
human disorders and that’s going to be true and these others of course are also
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present at um high levels but if we can develop
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suppressors for just a handful of these then we can cover almost 90 percent of
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all known stop codon mutations within the human population and so it does become a
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one-size-fits all for a number of genetic abnormalities and this is really
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what we would call Precision medicine where we’re identifying a therapeutic
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based on the patient’s mutation now just uh for the aficionados that
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might be in the audience they might add I do suppressors not read through normal stop codons
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and the fact is they don’t um there is a mechanism that’s poorly
26:28
understood but does exist which discriminates the normal termination codon from a premature stop codon or
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premature nonsense mutation and in actuality if you look at
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ribosomes and their movement across an mRNA you can see that the ribosomes will
26:48
pile up at the normal stop codon but if you express if you express various
26:54
suppressor trnas you’ll never see that ribosome move Beyond that normal style codon but it will read
27:02
through a premature style codon and so it becomes an Exquisite therapeutic that
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can be used to Target only the diseased allele with minimal opportunity for
27:14
off-target effects and because it’s only working on the
27:20
mutant mRNA and it it only act on the actual amount
27:27
of mutant mRNA that exists it will never over Express the protein it will only
27:33
restore the protein at most to Wild type levels and so here’s an example of this
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in practice where this is a cell line that’s expressing scn1a this is the
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normal amount of expression and this has a mutation so this is a actually haplord
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this is a normal amount of expression over here and we have a mutation in arginine at position 1407 that’s taking
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this CGA and turn it into a UGA style codon
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and so without a TRNA suppressor you get no expression of the protein but if we
28:09
add back the TRNA suppressor arginine is restored at that stop codon and now you
28:15
get appropriate expression of the protein and this is comparing 10 of Arts TRNA
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suppressor technology to small molecule drugs that have been touted as
28:28
translational read through drugs including these aminoglycosides and
28:33
adaluron and you can see that at the levels that we have presented here and we see very little
28:39
um read through at all um in this case not only is it expressing the um wild
28:47
type amount of the protein that amount of protein is actually or the cell is now functional and so scn1a is a sodium
28:54
Channel Gene and using a patch gland asset you can see that when you have a mutant scn1a protein you get no opening
29:03
and closing of that sodium Channel but when you restore the Arginine at that
29:09
stop codon mutation you now get the appropriate wild type opening and closing of the sodium Channel and so in
29:16
fact it restores protein activity and protein abundance and activity
29:21
one of the beauties of suppressors is that as I said you can use this for all types of
29:29
disorders as long as it’s the same stop codon and so here’s an example
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using scn2a and these are various mutations all Arginine stop mutations
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Arginine CGA going to UGA at different positions within that Gene and
29:47
expressing the exact same suppressor TRNA and that’s important every one of those
29:53
is being restored to a wild type function and so not only can you do this within a cohort of patients that have
30:00
the same mutation but at different positions you could also use the same suppressor across indications and so
30:07
that was dravay syndrome and now this is Rett syndrome that’s linked to the mecp2
30:13
gene and in the case of mecp2 this assay is essentially looking at
30:20
expression of macp2 which is a nuclear localized protein and in the absence of
30:27
a suppressor you get very little expression of MSC mecp2 but when you express the exact same suppressor that I
30:34
just showed in the sn1a and the sn2a we’re getting the same restoration of
30:39
mcb2 function and so it’s a drug that can be used across indications including in syngap patients who would
30:47
have a UGA mutation and just to point out sevart is working on all
30:52
three stop codon mutations and we’ll have suppressors soon for most of those
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for the next year okay so just to wrap up um this is the work that has been done
31:07
by tevard in collaboration with my lab back at Case Western and it’s a TRNA
31:12
based Therapeutics that have the promise to ameliorate many haplo insufficiencies
31:18
and other genetic disorders based either on improving the wild type wild type
31:23
mrnas expression or helping to restore the expression of alleles that have
31:29
nonsense mutations and so now I’d like to move away from the work that we’ve done with tevart and
31:36
talk about new technology that my lab has developed in the last six months at
31:41
Johns Hopkins and this is really work that we’ve done in collaboration with
31:47
the syngap research fund through the funding that they have so generously provided us and this is what we call an
31:54
mRNA booster therapeutic and so Not only was this supported by the srf also I’d
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like to give thanks to Carl Hall and Mrs Nancy Hall for a generous charitable
32:06
donation as well as the shoddy translational fund here at Hopkins Johns
32:12
Hopkins University so what are mRNA boosters so in the last
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few years I’ve thought a lot about genetic therapies linked to aav vectors and there’s a lot of Promise of aav
32:26
based approaches it’s a one and done um but it may not be
32:32
always the right choice depending on the indication and I’ve wanted to develop
32:39
novel Therapeutics that could be delivered not by aav but by a
32:44
nanoparticle or an LNP a therapeutic that could be transient um a therapeutic that could be used to
32:53
um um you know improve expression of an mRNA for a brief period of time or be
33:00
manipulated to express an mRNA over a long period of time but that we had much
33:07
more control over the chemistry of the RNA and so returning back to
33:13
the identity of what an mRNA is as I mentioned mrnas have caps and they have
33:19
codons and they have poly a tails and what we focused on for this technology is in fact the poly a tail
33:27
what is a polyatil well every human mRNA with the exception of a handful and
33:32
we’re going to talk about what those are um almost every mRNA has a poly a tail
33:39
and the function of that poly a tail is to drive the translation of the MRNA
33:46
the polyetale is variable in size it usually is about anywhere between 50 to
33:52
300 nucleotides and the length of just poly a and as long as that mRNA has a
33:59
poly a tail it will bind to ribosomes and those ribosomes will translate making polypeptide and you’ll get
34:06
protein expressed from that fully functional mRNA but as soon as that mRNA loses that poly
34:13
a tail so that is now what we call de-adenylated no protein is made anywhere and that
34:19
mRNA has one of two shapes either it’s translationally inactive and stored or
34:25
most of the time it’s destroyed and the function of a polyatail is in
34:31
fact to act as a slow burning fuse that dictates how long an mRNA is going to
34:38
express a protein there are enzymes that are constantly degrading the polyetail
34:44
as soon as the MRNA reaches the cytoplasm these are called diadentalases or poly shortening enzymes and this is a
34:51
clock the rate at this removal is a clock tells the cell how much protein should I
34:56
make and as long as that tail is there the MRNA makes protein and as soon as the
35:02
tail is gone it stops making protein and so the goal here is to trick the cell
35:08
into thinking that this polyetale remains on an mRNA and continues to
35:14
translate and so that’s what we call an mRNA booster and what we have done is
35:19
we’ve taken synthetic rnas where we have a short guide sequence this is about
35:25
anywhere between 30 and 50 nucleotides in size and it specifically binds to the
35:31
three prime untranslated region of an mRNA and when it binds what it’s doing is supplying a polyetail mimetic and we
35:40
can modify this transcript to maintain its overall stability within the cell
35:45
and improve the gene expression of that mRNA and so what this booster RNA transcript does is
35:53
it fools the cell into thinking that this mRNA still has a poly a tail and
35:59
should still make protein longer than what it normally would from its normal endogenous polyethyl
36:05
and the benefit of this is that this is really small this is only about between
36:11
70 to 100 nucleotides in size it’s very analogous to what you might think of as
36:18
a antisense polygonucleotide but it’s not the same this is in fact an RNA
36:23
that’s improving translation of an mRNA out in the cytoplasm
36:29
so this technology Works here’s one of our initial experiments that we did in vitro and we’re doing this again on the
36:37
mecp2 gene me cp2 linked to rep syndrome and this was just a test of our
36:43
technology and various ways in which we organize the polyetale relative to the
36:49
guide and different modifications we put on it and essentially what you can see is that this particular booster is the
36:56
winner where if we have a modification present on the Spy Prime n and we have
37:01
the polyatil memetic going this way followed by the guide we get a dramatic increase in the expression of that
37:08
protein within this cell line and that’s like a 10-fold increase it’s very
37:14
dramatic the same exact booster now we don’t see
37:19
the sort of dramatic effect in Vivo which is important um that same exact booster can be
37:25
injected into a mouse and in this case what we’ve done is we’ve injected into the tail of a mouse and we have three
37:33
mice that we’ve injected controls and two four mice and Jack one booster or
37:39
normalize another booster and if we look at the amount of mRNA expression that we
37:44
see with this booster too that I highlighted in the previous slide we get an increase in the MRNA stability
37:53
and if we look at the actual protein that comes from mecp2 from that mRNA msc2 mRNA in the
38:00
liver this is specifically in the liver then you can see an increase in protein
38:06
amount you assuming that this is just darker and so in a mouse we can improve the expression of mecb2 expression in
38:15
the liver this technology is portable across indications and what I mean by that is
38:22
if I go back to my slide here all I have to do is change this guide sequence to a
38:27
new Gene and so in the previous example I showed you this would be complementary to the mecb2 gene but I can change this
38:34
to any Gene that I want any mRNA sequence that I want and redirect the booster to that transcript
38:41
that mRNA and so here’s two additional cases these are genes also linked to how
38:48
fluent sufficiency the this is the gene for beta-catina CT and nb1 and this is a
38:54
gene linked to a very ultra rare Apple insufficiency called Pure Alpha and in
39:00
both cases we can develop boosters that will enhance the expression of
39:05
those specific genes in a specific map so what about syngot so we’ve done a lot
39:12
of work on syngap with this particular technology um I don’t need to introduce and GAP
39:18
deficiency to this Crown but syngap is like many of these Apple insufficiencies that it’s a debilitating monogenetic
39:26
encephalopathy um it does have some similarities to other disorders that I’ve talked about
39:33
um and if without the right expression of syndrop there we we see an increase
39:39
in the excitability of synapses which makes it difficult um for neurons to communicate and this
39:45
is what often leads to many of the issues that we see in syngap patients
39:52
um the phenotypes obviously everyone knows this phenotypes of syngap deficiency are shown here this includes
39:58
moderate to severe intellectual disability Tremors and epilepsy as well as
40:06
autism-like behavioral issues and one of the things that is really uh
40:15
difficult with many of these disorders like syngap and drabe and ret syndrome
40:23
Etc is that they tend to result from sporadic germline mutations they’re not
40:28
necessarily inherited it can be but they’re not necessarily inherited it’s a sporadic mutation and this causes a
40:36
problem when you try to develop Gene therapies because there are patients
40:41
that have all sorts of mutations they have stop codon mutations they have Miss sense mutations frame shift mutations
40:47
spliceight changes and so how do you develop a gene therapy to deal with this
40:53
and the trick is to really go after the wild type RNA or the wild type Gene and
40:58
that’s essentially what the booster technology allows us to do in addition like many genetics like many
41:06
genes in the human genome there’s one gene but it actually produces multiple isoforms and so there
41:13
are 19 exons in the syngap Gene and
41:18
those are stitched together to make the full length isoform of syngap1
41:25
but then there are other minor species that also uh exist that are the result of alternative splicing but one of the
41:32
most important things about so what this means and there’s some evidence for this in the syngap community is that the
41:37
expression of all four of these isoforms is in fact important for wild type
41:42
function and so how do we improve the expression of all four and what’s
41:48
important here from our standpoint is that they all share the exact same three prime untranslated region and this is
41:55
the region that we target with our mRNA booster
42:01
so we’ve developed a mRNA booster that specifically recognizes the syngap
42:07
mRNA and that’s shown here where we have this two sb1 and sb2 some Gap booster
42:14
one some Gap booster two and in the case of mRNA levels this is actually done in vitro these are in these shsy5 cells
42:22
which are neuronal line we can get a dramatic Improvement in the mrnas expression in the cell culture system
42:31
and so now what we’re doing with this technology is we have to go through a lot of pieces a lot of things to do to
42:36
make sure that it’s going to work and that it’s going to work in a clinical situation so we use in Vivo it’s already
42:43
in vitro systems like these cell systems that I just showed you we use patient
42:49
derived IPS zones that and we’ve gotten some of these from the srf
42:54
um and thank you to the donors who have given us those we will take those IPS
43:00
cells and turn them into neurons and then test our our uh our technology and once we
43:10
develop a technology we have to de-risk it to lead candidate optimization and then what importance we have to test
43:15
this in Mouse models and so one of the aspects that we’ve gone through in testing and doing lead candidate
43:22
optimization is to find boosters and I you know that give us the biggest amount
43:29
of mRNA expression uh in the syngap-1 MRNA and so in this case we’ve done a
43:35
limited scanning of the three prime ETR where we have five boosters and some controls and you can see that this
43:42
particular booster here is giving us the biggest increase in overall expression
43:47
now we’ve actually gone ahead and taken one of these we actually started with
43:52
sb1 we haven’t gone on to these next guys yet but we started with sb1 and
43:58
developed that further in Mouse model systems so sv1 is nice because well the syngap
44:08
MRNA is nice because it is 82 percent identical in the three prime utr between
44:15
human and mice and this becomes really important for us because that means that we can do experiments in the mouse
44:22
and take that information and translate it right back to the human so this
44:28
particular booster sd1 binds with perfect complementarity to this region
44:33
in the human three prime ETR or syngap1 and that’s identical to the same region
44:38
in the mouse three prime utr and so it allows us to test this clinical
44:44
candidate on inner Mouse and so what we’ve done in this first set
44:49
of experiments there’s a lot of things we can do with this technology we can make lnps and nanoparticles and try
44:56
different routes of administration but in our first set of experiments we wanted to do a direct deep brain
45:01
profusion of our booster into the hippocampus and this has been done in
45:06
collaboration with our colleagues here at Hopkins
45:11
Mao over in the institute for nanobiotechnology on the Homewood campus
45:17
at Hopkins intentions experiments what he has is a really unique system where he can using
45:25
artificial intelligence and pretend to understand how this works but you can develop a development device which has a
45:34
stiff too and a super elastic to encoded inside that tube
45:40
and then what you do is you insert that stiff tube into the brain a single point of penetration
45:46
and then insert this super elastic tube and it’s here where the artificial
45:52
intelligence has plotted out the path it needs to go in order to move through the
45:59
hippocampus and so this tube has been engineered in a way that as it’s pushed
46:04
in it moves in this pattern and this has been shown as a curve for Simplicity but
46:10
then what happens is that when you retract that tube it leaves behind this super elastic nanotube that then
46:19
can be perfused with small molecules or in our case a small RNA that has been
46:25
encapsulated in a neuronal specific LNP and the neural specific OMP was
46:31
developed by heshwan Mao over at the Homewood campus and so we’ve done this deep brain
46:37
diffusion experiment with our booster and you can see sort of two results in
46:43
this figure that we injected into the hippocampus and then dissected the hippocampus the
46:50
cerebral cortex midbrain and the cerebellum from these mice and this is the MRNA quantification but
46:58
really what’s important is to look at the protein quantification and I’m only showing a western for the full length of
47:04
syngap one protein and cameras a little bit in the way but what
47:10
you can see here is in the hippocampus you get a nice increase in the expression of syngap1
47:16
that Telegraph the the boosters telegraphing into cerebral Gore-Tex and
47:23
the midbrain but by the time it’s just the cerebellum is way too far away and we’re not having much of an effect there
47:30
so there’s actually not much singap expressed in cerebellum at least in in our experiments and so this gives us a
47:36
lot of hope that this technology will work and we can further develop it and
47:42
modify the RNA to increase not only its efficacy but its durability to maybe we
47:48
need to make sure that you know this can last a long time in a mouse um you know
47:54
will the mouse Express and Gap at this level for days weeks months what we
48:00
haven’t gotten to that point um but those are our next steps is to improve the overall durability of the
48:07
small molecule and then also to do risk it in terms of making sure that it doesn’t have off-target effects we’ll do
48:14
both the rna-seq on this as well as proteomics to ensure that we’re only having this effect on syngap expression
48:21
um and then from there we’ll take it into model systems this is these experiments were all done in Wild type
48:27
mice perfectly happy mice um we’ll take that into Mouse models
48:34
where they have a syngap deficiency we didn’t want to do that right now because those mice are very precious and they’re
48:40
delicate and we really want to do a lot of our um tinkering with the booster and uh the
48:48
best case scenario which is really a wild type Mouse okay so I have 10 minutes left for
48:54
questions let me just summarize um by saying that this new technology that my lab has developed we call an
49:01
mRNA booster it is a novel gene therapy that enhances gene expression it really
49:06
is a first in class technology we’re not aware of any other technology that does
49:11
this and it leverages the power of RNA Therapeutics in order to
49:17
in order to do what it does it’s We Believe it’ll be disease modifying
49:23
because it restores the Apple insufficiency
49:28
um it also is mutation agnostic as it works on wild type mrnas and it really
49:33
is a platform technology we’ve we’ve chosen to develop this for syngap but
49:38
this could be used in theory for any one of these Apple insufficiencies that I’ve
49:44
talked about today or the 300 other that I haven’t and there are also other
49:49
applications for this technology that go beyond the treatment of Apple insufficiencies
49:56
okay with that I’ll just thank the people who’ve done this work this is all done at Johns Hopkins uh the booster
50:02
technology was all developed at Johns Hopkins everybody here is past or present member of my lab the present
50:07
members are violated asterisks and these are the people who give us the money to
50:13
do what we do and especially the syngap research fund with whom out whom we
50:19
never would have gone down this road and tried to develop technologies that
50:24
really can help this particular Disorder so thanks to you for all of your efforts
50:30
your your precious donations and um the time and reagents that um given
50:36
to us so thank you awesome can you guys see me hear me I
50:44
gotta stop share yeah okay uh thank you so much Professor
50:50
Kohler that was incredible and um I did not prompt or pray Professor caller to
50:56
um talk about the value of reagents or the importance of us being able to
51:02
support work like this when it comes to our attention but they’re valuable and see previous discussion on making cell
51:08
lines so people have jumped in on the Q a and um I imagine the parents are a
51:14
little overwhelmed at this point um if they’re seeing this for the first time but I want to encourage the parents to
51:20
ask questions as we have seen today Professor Kohler is is adept
51:25
at making the incredibly complicated accessible and I and I urge you not to hold back but um
51:33
let’s let’s go through the Q a while people are collecting their thoughts um
51:38
and before we do that I just wanna because you because you covered three Technologies
51:44
I think some people might walk away from this and say okay only if my patient has
51:50
a nonsense which is an art our stopper a cue stop is this applicable to me and
51:56
can we just clarify at the highest level um the the the different things in the
52:01
poly a which ones work which ones are mutation specific and which one’s a mutation agnostic
52:07
the only one that’s you can still hear me right the only one that the only one that’s mutation
52:13
specific is the nonsense suppressor that’s only for patients that have
52:19
stopped codon mutations but in some indications that represents a lot of patience yes and
52:26
um one of the beauties of that of course is that once it’s approved and been shown to work in drive a the same exact
52:33
formulation could be moved to other indications and so you can do all the de-risking of
52:41
the technology the talks everything on one indication and then move it through everything else you know wrap send out
52:48
you know go down the list of and neuronal encephalopathies that could
52:54
benefit from that and so by having one therapeutic maybe it doesn’t help as many patients Within syngap but
53:01
collectively it’s helping lots of lots of kids that’s the beauty of that technology
53:06
right but the poly a would be mutation agnostic so anyone with a frame shift or that’s exactly right and the differences
53:13
between those Technologies the enhancer and the booster the enhancer is a aav
53:20
based approach and the booster is LNP nanoparticle
53:25
approach and so what it really does is gives you multiple shots on goal of
53:30
what’s going to work right you know if in my career what I’ve decided to do is
53:36
not stop with one you know I’m not one and done that I’m going to go after a particular
53:43
technology and I think the model that works better for researchers like me develop a technology then give it to
53:50
biopharma to to de-risk and do all their development that’s what tevar’s doing with the enhancers and suppressors the
53:58
booster technology that you know we’ll talk about that at another day where that will go um but that’s really the point is
54:05
you know and we’re not the only game in town you know Stoke has Asos and other groups are
54:11
developing Asos um and hopefully you know one of these things works and you know the more
54:18
opportunities we have the better chance we have to get success I mean look
54:23
making a gene therapy is not easy it’s tough yeah it’s tough
54:29
yeah lots of mice have been cured yeah no and another time we should talk about Zen
54:36
plenty um it was another srf grantee where we’ve talked about delivery and as I think about sticking your Tech in his
54:42
delivery it could be pretty exciting well let’s stay on topic so Ashish uh tp53 has you’ve really woken him up
54:51
and I have no idea who this is but he has a lot of questions um and then but before we go to him there’s
54:58
the anonymous questions about missence can you briefly address why or why not this might work for missense mutations
55:03
same same questioner um yeah
55:09
I’m not you know I don’t know exactly what technology they’re talking about but I’ll just answer
55:14
that so the suppressor will not work for missense mutations that it can’t missense mutation is a mutation that
55:21
made the wrong amino acid turned it into something else right but the enhancer in
55:28
the MRNA booster would work for patients with mid-sense mutation because again it’s helping improve the
55:35
wild type copy as long as that distance mutation is not leading to what we call a dominant negative
55:41
which for a lot of these we don’t know all the time whether that’s true but we
55:46
suspect that most of the time these innocence mutations are simply a loss of function meaning that the protein no
55:52
longer works um but to be honest I’ve talked to some very smart people recently
55:58
and they even think that with some of the missense mutations that sort of a
56:04
partially functional protein and maybe improving its expression would give us a
56:09
little bit of a therapeutic window um so you know I think and that’s in
56:15
fact we’re going to test that with our RNA booster on uh that very idea whether
56:21
improving some of these missense mutations will give us um a therapeutic margin so hopefully
56:28
that answers your question well there’s people in the audience so I’m curious if you just triggered a question but um so
56:33
ashish’s questions she has a lot of questions but I I like his last question best because he’s asking you which of
56:40
your children is prettiest so picking one of the three booster enhancer
56:46
suppressor who’s going to make it to clinical trials first it’s a hard question well I have three daughters and
56:51
they’re all beautiful and I have one boy and he’s beautiful too but um
56:57
I mean My Pet Project right now I I don’t know I think they’re all wonderful Technologies
57:04
um who’s gonna reach the clinical stage first that’s actually easier to say because it’s going to be the suppressors
57:10
that’s already advanced I mean we started to have art in late 2017. you
57:16
know it’s been what what is that six years of development
57:21
um that will get the clinic way before anything you know the booster technology is still within my lab it’s not even
57:27
ready for um there’s a lot we have to do before biopharma picks that up
57:33
um suppressors are you know most of this work on enhancers and suppressors since it’s not done by my lab anymore that was
57:39
developed back in 2018 in my lab and it’s moved on to cover they do their their stuff with it
57:44
so and is the poly a stuff delivery agnostic
57:51
mode of delivery agnostic like could you put it in multiple theoretically you can put it in an aav
57:57
in theory we have actually shown I didn’t show these experiments here but we’ve packaged it into a nanoparticle
58:04
that our colleague made we packaged it into an LNP that our colleagues made and it works in both cases
58:12
um you know and we’ve shown that it works in the liver which only now works in the brain
58:18
um two different genes um we could put it in aav I see no reason
58:23
why you couldn’t um but we haven’t done that yet got it
58:30
um do you want to go through the shisha’s questions or do you want me to read them it’s a lot I can answer some of them so
58:40
let me start from the back how do you choose among the various booster guides do we need to perform wet lab experience
58:46
to find the most optimal guy yes you always have to form wet lab experiments to make sure
58:53
um can use computational approaches and if scene-based learning to play a role to arrive at optimal boost guides faster
58:59
potentially um but you know I’m an old school molecular biologist I don’t think
59:05
you you have to do the experiments because you are as smart as we think we
59:11
are and as smart as we think AI is biology smarter so
59:17
um you always have to do wet lab and this is very important because you never know when you’re going to get off Target
59:24
and talks uh associated with a particular trans particular small RNA or molecule
59:31
um so wet lab experiments need to be done okay so that
59:37
okay um is Tethered your lab trying to apply the TRNA suppressor enhancer approaches
59:43
to other rare diseases yes um it is um they have to talk about their
59:50
candidate um they have to talk about what they’ve announced already I mean what we can
59:55
what I can tell you is that brave is their number one uh indication of choice and
1:00:00
um duchesne’s muscular density now with our collaboration with vertex they are
1:00:05
working on other rare diseases um but those programs haven’t been announced
1:00:12
um how would you rank enhancer TRNA
1:00:17
suppressor technology in terms of the following factors time to develop a drug cost to develop a therapeutic drug epic
1:00:24
see adverse risk and side effects I think I answered that question in some some sending the the suppressor
1:00:30
technology is the one that is going to result in the drug fastest um
1:00:36
okay and then the first question would be fair to summarize as a Layman the enhancer technology has increasing
1:00:43
protein levels by modulating the functional wild-type copy of the gene in question yes that’s exactly what it does
1:00:49
it’s it’s essentially improving the stability of the MRNA because the MRNA
1:00:54
stability goes up protein level goes on
1:01:00
super um everyone else has just been wowed into submission I’m I’m I’m I’m
1:01:06
fascinated um I’m very grateful Professor caller for doing this this is I this thing this
1:01:12
is a webinar people will go back to I thought your isoform slide was really accessible too I don’t think people always get that
1:01:19
um and so I guess on the isoform slide my
1:01:25
given that this is so specific would would the different isoforms still
1:01:32
be expressed I mean would it yeah I can I don’t have the data to show
1:01:39
you because it’s red hot off the presses um and you know I always want to see things a couple times before I but yeah
1:01:46
when we’ve looked at the other isoforms we’re seeing that they go up as well and it’s just what syngap is
1:01:53
um you know as you know those some of those ice forms are really close together in
1:01:59
size and so it’s difficult to resolve like when I show that ban on the gel that’s probably actually the two isoforms two
1:02:06
biggest ISO forms um I’m not using the antibodies that I know that there are researchers that
1:02:12
have developed antibodies that are for the specific isoforms I don’t have access to those antibodies
1:02:18
um but it looks to me like off at least the visible isoforms that I can see which I
1:02:24
think at least two are both going up yeah so I
1:02:30
I wasn’t even being that specific I I just framed the question poorly what I was trying to say at is given that this
1:02:37
is affecting the way the proteins are made for there
1:02:43
there are other therapies out there that are just delivering alpha one right one specific ice form and then people are saying oh no what if we leave out the
1:02:49
other isoforms whereas with these approaches am I am I safe to assume that we don’t
1:02:54
have to worry about that because the body will still Express the isoforms that are needed that’s right okay that’s the theory
1:03:02
because it’s binding to the three prime utr and the three parameter are shared between the four
1:03:08
um they also go up and so yes it’s it should be more
1:03:15
um amenable to disorders and genes that have multiple ice forms but sheer common
1:03:21
three prime meteors superb thank you very much this is your
1:03:27
last chance people no further questions we’re grateful for your time Professor Kohler I’m happy to be here and you know people
1:03:34
can always ask me questions by email you just Google my name you’ll find my email address
1:03:39
great thank you so much for your time thank you Mike

1:03:24
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Lunch & Learn: Gene Therapy 101