55 – Evaluation of a Stem Cell Gene Therapy Approach for SYNGAP1

Here are our introductory comments: 

Our presentation today is ‘Evaluating Stem Cell Gene Therapy Approach for Syngap1’

I have the pleasure to introduce today’s speaker Dr. Joe Anderson at the University of California, Davis.  

Dr. Anderson is the Associate Professor of Internal Medicine in the Division of Infectious Diseases at UC Davis.  He has been in the field of stem cell gene therapy for the past 20 years and has developed numerous therapeutic lentiviral vectors for HIV, Tay-Sachs/Sandhoff Disease and Angelman Syndrome and has evaluated their safety and efficacy both in vitro and in vivo in adult and pluripotent stem cells.  He performed the in vivo safety/efficacy data for the current pre-selective anti-HIV lentiviral vector being used in a Phase 1 clinical trial for HIV-lymphoma patients.  Dr. Anderson has also developed stem cell gene therapies for both Tay-Sachs/Sandhoff Disease and Angelman Syndrome that are currently in the IND-enabling experiment stage.  Dr. Anderson has successfully written, submitted and received approval of NIH RAC, pre-IND, and IND applications for his HIV stem cell gene therapy work.

THIS IS A TRANSCRIPT ONLY:

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hello everyone and welcome to today’s webinar my name is Olga Bothe and i’m a syngap parent and part of the team at SynGAP research fund our presentation today is evaluating stem cell
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gene therapy approach for SYNGAP1 and i have the pleasure to introduce today’s speaker Dr
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joe anderson at the university of california davis dr anderson is the associate professor
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of internal medicine in the division of infectious diseases at uc davis he has been in the field of stem cell gene therapy for the past 20 years and has developed numerous
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therapeutic lentiviral vectors for HIV tay sachs sandoff disease and angelman syndrome he’s
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evaluated their safety and efficacy both in vitro and in vivo in adult and pluripotent stem cells
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he performed the in vivo safety efficacy data for the current pre-selective anti-hiv lentil
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viral vector being used in a phase one clinical trial for HIV lymphoma
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patients excuse me dr anderson has also developed stem cell gene therapies for both tay sac standoff
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and Angelman syndrome that are currently in the investigational new drug enabling experimental
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stage dr anderson has successfully written submitted and received approval of nihrac
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pre-investigational new drug and investment negotiational new drug applications for his hiv stem cell gene therapy work a recorded version of this webinar will be available on
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the SRF website under webinars in the family menu by the end of this presentation you will
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have the opportunity to get to answer your questions we’d love to hear from you so please write those questions in the q and a below for those of you just joining us welcome and
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again our speaker is dr joe anderson and his presentation is on Evaluating stem cell gene
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therapy approach for SYNGAP1 it’s now my pleasure to turn things over to dr anderson thank you
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thank you olga and thank you everyone from SRF for inviting me to give this webinar
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on our research so Olga mentioned my name is joe anderson i am an associate professor at the
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university of california davis my research focuses on stem cell gene therapy for multiple diseases
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from infectious diseases to lysosomal storage diseases and now we’ve moved those strategies and approaches into some neurodevelopmental disorders to
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evaluate whether this approach will work for those disorders as well so today i’m going to
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talk to you about our stem cell gene therapy approach as a potential treatment for syngap
Cross correction
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the way that this approach would work is through a mechanism called cross correction so instead
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of directly gene modifying neurons we would actually be using microglia cells that are in
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the central nervous system to transfer wild-type and functional syn gap to the affected neurons so
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this has been successfully used for some lysosomal storage diseases and leukodystrophies that are
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currently in clinical trials we’ve shown that this works well for tay sachs disease which is a
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lysosomal storage disease and then also for angelman syndrome which is a neurodevelopment
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disorder the way that this would work is the microglia cell which is a progeny from the
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hematopoietic stem cell which is the blood forming stem cell that would be the cell that would be
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genetically modified with the functional and wild type form of syngap these cells
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home into the central nervous system where they’re used as accessory cells and they’ll
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be able to express SynGAP and then transfer it to affected neurons so that’s the overall
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strategy scientifically and in our hypothesis on how this would work so we wouldn’t be
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directly reprogramming or genetically modifying the neurons but we’d be using these microglia
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cells as protein factories to then transfer the functional protein to the neurons
insertional gene therapy
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type of gene therapy that we use is called insertional gene therapy so we’re actually
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permanently integrating the wild type and functional copy of syngap
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into these blood stem cells so it will be in there for the life of the patient and every cell that’s
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derived from that stem cell will have that copy of that gene as well and so we’ll insert this
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copy of syngap using a lentiviral vector so this vector system has been engineered to
selfinactivating vectors
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allow permanent integration it’s derived from the HIV system where HIV permanently integrates into
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the genome however these lentiviral vectors are stripped of all the pathogenic genes that HIV has
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the modifications made to the vector system also don’t allow the vector to replicate once in the cells so they’re called
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self-inactivating vectors and so they are still able to integrate their genetic information
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into the genome of the target cell but they’re not able to replicate so it’s more like a one-way
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street we’re able to get the syngap gene in with its respective promoter to drive expression but
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then that’s all the vector will do it will drive expression of the syngap gene the cells will make
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the protein and that’s all that the vector will do it won’t be able to replicate it doesn’t have any
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of the components that could cause hiv this vector system has been used in numerous clinical trials
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for other diseases so it’s it’s a safe method of inserting a gene that you want into a target cell
microglia
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the reason why we wanted to use the hematopoietic stem cell is that the blood system and the blood
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system cells are found throughout the body even in the cscns so as i mentioned the
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the main cells that will be doing the work for this approach are the microglia which are found
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in our brains they are accessory cells just like the other glial cells and so they
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they know to home into the brain and then do the work that they will do and then
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in what we want to do for syngap is to program them to express
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the functional copy of syngap transfer it to the neurons and then the neurons will be able
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to use this protein for normal function and hopefully correct clinical phenotypes
hematopoietic stem cell
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this is a classic tree of hematopoiesis it shows the pluripotent stem cell at the very top
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and this is the hematopoietic stem cell this stem cell resides in our bone marrow
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there are a very small percentage of the cells that circulate throughout the blood but then
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eventually they go back into the bone marrow that’s their home that’s where they reside and
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they constantly make our red blood cells and our white blood cells which are immune system cells
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for the life of us babies 80 year olds all of us have these cells in our body because
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we constantly have cell turnover and so our immune system has to constantly make new immune
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system cells including red blood cells and so if we’re able to target this hematopoietic stem cell
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with our gene therapy there’s the potential of it being just a one-time treatment if we’re able to
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get enough of these hematopoietic stem cells gene modified with our vector and our syngap protein
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then the body would make all of these progeny and daughter cells that express the syngap protein for
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the life of the patient including the microglia in the CNS that will transfer it to the neurons
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and that’s the reason for targeting the stem cell we could target these other daughter cells and
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the microglia themselves possibly transplant them into the brain but those cells do have a half-life
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they do turn over and so we would have to repeat that procedure potentially
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a number of times but if we target the stem cell then potentially there’s just a one-time treatment
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because those cells constantly make our immune system cells but also make more of themselves
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so there there could be an arrow up top at the very top by the stem cell
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kind of pointing back at itself because it’s not only makes more of these immune system
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cells but it also makes more of itself so that you have them for the life of the individual
blood stem cells
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so this is just a basic schematic of what we would do we have these blood stem cells they have this
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marker on the surface called cd34 so we’re able to isolate them we have our syngap expressing
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lentiviral vectors and we have a number of them that we’re going to be trying some of them
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are wild type like some of them have different modifications the challenge of this approach for
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a neurodevelopmental disorder like syngap is that SynGAP is normally found inside of
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the cell so we have to sort of manipulate the protein to get it to be transferred from the
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microglia to the neurons so there are numerous modifications that we can make and so we have
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a number of these syngap expressing lentiviral vectors that we’re going to test first for
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functionality make sure that they function correctly and then also for their expression
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and so these syngap lentiviral vectors will gene modify these blood stem cells
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and then we will have these syngap expressing immune cells which will
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hopefully be the the therapeutic approach to transfers and get to the neurons
mouse model
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once we evaluate these syngap expressing lentiviral vectors in our immune system
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cells once we find out which ones function the best express the most and all of those details
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then we’re going to evaluate whether they can correct some of these phenotypes in a syngap
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mouse model the way we’ve done our previous experiments with other diseases is we’ve
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first made a humanized immunodeficient mouse model that’s a disease specific
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so there are SynGAP deficient mice available at JAX and they’re currently being cryover covered
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so they don’t they don’t have these mice readily available they’re frozen down as embryos and
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on their website it takes about 12 to 14 weeks for them to cry or recover them we just
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got the funding for this grant from SRF March 1st the grant started so that
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we ordered the mice right away on day one because we knew how long they would take to cryo recover
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so three months later hopefully we will get them so in june at some point hopefully
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and then what we want to do is cross them with an immunodeficient mouse strain so this il2 rg
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deficient mouse this il2 gamma receptor is used on t cells and b cells to activate them so they
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can attack foreign material pathogens but if you don’t have this il2 gamma receptor you’re actually
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immunodeficient and you don’t have a functional immune system and so if we are able to use a
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mouse model that has this immunodeficiency cross it with the syngap deficient mice we can then
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transplant these mice with human hematopoietic stem cells or human cd34 positive cells the mice
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don’t have an immune system so they can’t reject these quote-unquote foreign cells which are human
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technically a immune competent mouse that has a normal il2 receptor gamma gene it has a normal
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immune system if we put human cells into that mouse they will reject them they will see them as
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foreign and we won’t get good engraftment and we won’t be able to evaluate our therapy and for
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the FDA for other funding organizations like cern they like us to use as close to the therapeutic
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candidate that we would use in our pre-clinical experiments and so being able to use human cells
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that have our therapy our gene therapy in them is a lot more advantageous than doing everything
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in mouse cells gene modifying the mouse cells and then putting them into the mice
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because eventually we’re going to go into humans and human cells so if we can skip that step by
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making this immunodeficient mouse model then we can use human cells in these mice and we’ve
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we’ve done this before for our tay Sachs work and then our angelman work which is published and it works great the human cells and graft in
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so once we get these mice from JAX we’ll cross them with these il2 rg deficient mice
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and then we’ll genotype them make sure that they have this immunodeficiency and then we’ll able
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we’ll be able to start evaluating our gene modified human cells in these mice so we
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would take cord blood cd34 positive cells we can isolate them for cd34 positive so we get over a 90
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pure population we’re then going to evaluate the therapy in adult syngap deficient mice
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and then in newborns we know that the newborn mice that are deficient they die within
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a week a week and a half but we’re able to transplant these newborn homozygous pups at day
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two so we can transplant them with our gene modified cells and then we can see if we get
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an extension of lifespan in the newborns the adults are different the heterozygous adults
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will live longer they do have some behavioral phenotypes such as a motor locomotion and
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on this rotor rod assay where they don’t have a good enough claw grasping and so
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we’ll condition the mice transplant them with the gene modified cells and then we’ll give them
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eight six to eight weeks for the cells to engraft and then we’ll start doing some behavioral assays
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on them including the the rotor rod to look at grasping and then also a number of indications
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in the open field including center time that’s more of are they scared of the environment that
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they’re in we’ll look at locomotion like how far are they moving around we’ll look at vertical
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rearings can they get up on their hind limbs and then we’ll look at just total total locomotion
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as well and we’ll be evaluating wild-type mice the syngap hats or the homozygous mice as
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newborns pretty much just as an extension of lifespan if we’re able to extend the lives
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past a week week and a half that’s great and then eventually we could do the behavioral assays but if we’re able to get the newborns to live past the week then that’s
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that’s significant by itself we have an empty vector does not have the syngap gene at all it’s
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just a control to show that the transduction with the lentiviral vector with an empty lentiviral
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vector doesn’t do anything and then we’ll have our syngap vectors that we’re going to use
therapeutic timeline
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so currently we’re at the beginning of this therapeutic timeline we are currently
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developing the vectors testing the vectors and then generating the mice so we’ve made
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all of the syngap expressing lentiviral vectors that we want to evaluate and we’re currently
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tightering the last one tightering is just how much vector is there per volume so that we know
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how much vector we need to add to gene modify our cells we currently have a syngap b cell line that
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has a point mutation in exon 17 that causes a stop codon instead of an arginine and so
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there’s no express well there’s not complete expression of the protein it’s truncated
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and so we’re initially going to evaluate the function and expression of our vectors in this
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syngap b cell line and then we’ll look at expression of these vectors and then we’ll
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also look at functionality in these ras and gtpas assays just to see if the modifications
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that we made to some of our vectors if syngap is still functional in its normal activity
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and so it is at the beginning of this therapeutic timeline but once we have the mice generated
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and all of these experiments the functionality and expression and all that will be done prior
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to when these mice will be made it just takes a long time because they had them cryo recovered
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and so it’s three months just to get those mice out of the freezer and then we’ll start
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on the efficacy experiments and then if we see correction of some of these clinical phenotypes
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then we’ll move on to the safety experiments one of the big potential risks with what we do is that
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we’re over expressing these proteins in cells and so will it cause some detrimental effects
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on engraftment of these hematopoietic stem cells on generation of the t cells b cells microglia
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that are made from the stem cells and then will those cells function because it’s one thing to
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express these therapeutic proteins but if we’re causing detriment to the immune system
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then there will be other problems
bone marrow transplant
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the way that this was work for a clinical trial it would be like a normal bone marrow transplant
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so there are mobilization drugs most of our hematopoietic stem cells live in the
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bone marrow as i mentioned before there’s a small percentage that circulate in the blood less than
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one percent and so we wouldn’t be able to get enough stem cells out so what the patients get
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is a mobilization regimen which actually pushes the stem cells out of the bone marrow into the
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blood and then the patients just get a blood draw just like if you’re going to donate blood
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they sit on a machine and then cells are taken out of the blood into a lugo pack and then what
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we do the arrow pointing down is that we will then isolate those stem cells to greater than
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90 purity and then we genetically modify them those stem cells with our syngap vector x vivo so
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outside of the body and then a patient gets a conditioning regimen so that they can make
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a room in the bone marrow for the stem cells that we then want to transplant and then the
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patient just gets an intravenous injection of the gene modified syngap expressing stem cells these
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hematopoietic stem cells know where to home we can put them in the blood but they know to go back to
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the bone marrow and so we don’t actually have to do anything to the bone marrow itself as far as
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like sticking a needle in the bone marrow taking bone marrow out that way or putting the cells back in everything can be done ivy
Angelman syndrome
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and so the reason why we wanted to test this approach for other neurodevelopmental disorders
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is because of what we saw with our work with angelman syndrome this work was published
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in collaboration with jill silverman’s lab last year where we showed that we could both
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prevent and rescue clinical phenotypes in an immunodeficient humanized mouse model
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of Angelman syndrome in both adults and newborns the adults typically don’t show
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clinical phenotypes till about six to eight weeks and so what we did was we transplanted these mice
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both as newborns and as adults once they started showing phenotypes and then we looked at open
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field locomotion beam walking gait correction which are all sort of motor and behavioral
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assays but then we also looked at novel object recognition was more like a memory and learning
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and then we also looked at a decrease in an eeg delta wave spike so in angelman patients they have
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a large jump in their delta wave when you do an EEG and what we saw we didn’t see a correction
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all the way down to wild type but the spike was cut in half and that that was significant to
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actually show that we could take this delta wave down and so after seeing all of this
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for angelman syndrome we are now looking at other neurodevelopmental disorders to see if this
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approach could work similar to syngap UBE3A which is the protein indicated in in angelman syndrome
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it’s an intracellular protein it’s a ubiquitin ligase so it doesn’t normally get secreted
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out of the cell and taken up by other cells unlike hexa and hex b for tay sachs or some
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of the other proteins for the lysosomal storage diseases or leukodystrophy so we had to make some
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modifications to UBE3A to allow it to get out of our gene modified cells and some of those similar
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modifications we’re testing in syngap but this is the reason this publication if anyone wants it
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i can email it out it’s freely available online anyone can access it and look at it
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but that’s the the main reason for our endeavor into into syngap is because of how well it
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worked for angelman syndrome we wanted to look at it for other neurodevelopmental disorders
Angelman data
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so i’ll show i’ll just go through this quick it’s just showing a little bit of the data from the angelman syndrome work you know same setup as far as the mouse model we made a
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immunodeficient humanized mouse model took a cord blood gene modified with our ub e3a vectors
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you solve them for adults or radiation for the newborn mice and then eight weeks later post transplant we did behavioral assays
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and then i’ll show some data for that this is an open field essay the blue line so the mice
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go in this box and then they just walk around and they explore and you can it has
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beam breaks so you can tell how far they’ve traveled how many vertical rearings on their hind
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limbs how much center time it actually calculates how much time they’re in the center or how much
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time they’re on the perimeter and then we can look at total activity so the blue line is our gene
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modified cell transplanted mice this top line is wild type and then these bottom two lines
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are the ube3a deficient mice and we saw some correction of this total activity
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oh yeah this the video shows the mouse going around so this is what we’ll do for the syngap mice as well
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this is a beam walking assay the increased number of rod number actually decreases the diameter so
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it’s harder for the affected mice to cross rod three because it’s a thinner rod so they
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they take more time the blue again is our ub e3a vector transplanted mice and we can see
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that these mice on rod 3 acted similar to our wild type mice where these two
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higher bars are our ube3a deficient mice
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this is the novel object recognition there’s a familiar familiarization phase where there’s a
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similar object and then when you throw this novel object in there the normal mice will actually
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go and sniff and hang out around that novel object a lot more than the
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disease affected mice and we can see that in this blue bar here with our ube3a vector transduced
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cell transplanted mice they actually spend more time around that novel object similar to the wild
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type compared to the ube3a deficient mice and then this shows the delta wave spike so in humans
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an a.s child has this red spike in the delta wave where a non-angelman
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child has this normal delta spike and then in our mouse model this lower line is our wild type mice
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the blue line is our vector transduced cell transplanted mice so you can see that it was
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almost cut in half from the spike of our ube3a deficient mice so seeing
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this was pretty significant that we were able to take that delta wave down
Project update
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so where we are for this project we’re currently doing the IND enabling studies the FDA for an
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adult trial for angelman’s they wanted us to do one more toxicity and expression study
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so we’re currently doing that but then for a pediatric trial they wanted us to do three more efficacy experiments a sleep disruption seizure threshold
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and one more and so in order to show direct patient benefit for a pediatric trial we have to
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complete these three additional efficacy studies and then for the adult trial which might start
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sooner than the pediatric trial they might just be separate trials just depending on how the experiments go we’re just doing one additional toxicity in an expression study
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which is going to be similar to what we would have to do for syngap if these vectors work in the syngap mouse model so i’d like to thank the people in
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my lab julie and camila and then everyone in the vivarium which is the where the mice are kept for
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all of their work and then of course everyone in the silverman lab that worked with us on the angelman syndrome study and then i’d also like to thank the SynGAP Research Fund for
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funding our current research for the syngap therapeutic approach
Questions
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and thank you i’ll take any questions awesome joe thank you very much
30:33
i’ve got a couple of things off the top i want to say for the families who are watching because
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great presentation it feels a little sci-fi to some people and i just want to emphasize to people that you have made good progress in angelman and
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people who follow our work at SRF know that i follow angelman very closely and
30:58
on the one hand this work if successful would require our kids to go through a bone transplant
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a bone marrow IV infusion which sounds a little scary to people
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but is it could you speak to the relative theoretical efficacy of this versus say an ASO or
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an AAV in terms of percent of brain that would be affected and i realize that this is a theoretical conversation but it is an interesting feature of this approach do you know where i’m going
31:34
yes so the microglia these accessory cells are found throughout the brain and so by
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transplanting the blood stem cells that have this gene modification we potentially could
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reach every part of the brain that these microglia are in they’re also going to be
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i guess you can call them protein factories where they’re just going to be expressing this protein
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and secreting it out of their cells whether into space or in direct contact we know that
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in neuronal damage there is direct contact where these accessory cells like astrocytes and glials
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glial glial cells are in direct contact with these neurons so they could actually just be
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sending it across the cell membranes and into the neurons but the fact that these microglia and accessory cells are found throughout the brain there’s i think a greater potential to
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send these proteins to more neurons compared to an AAV or an ASO where
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it might be injected in multiple parts of the brain but is it going to get to
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a majority of the affected neurons i i don’t know that but just knowing that the microglia are
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found throughout i think this approach has the potential of
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affecting more of the neurons and then plus like i said before we’re not directly
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gene modifying the neurons themselves yeah yeah so then there’s a couple of there’s a few
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science questions pouring in and before i i defer to them i want to i want to make another point
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so we are funding the early work on this and as you pointed out so frustrating we have to uncryo
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these mice and it takes so long but the sooner we start the sooner we get somewhere right and
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then if this works there’s a lot more work to be done and the 130,000 for this initial experiment
33:49
really is just the tip of the iceberg so one of the first conversations you and i had was who’s going to keep paying for this and hopefully you know there’s enough good evidence that we we want
33:58
to keep supporting it at whatever level we can but can you talk a little bit to potential other
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funders as as this work gets gets its sea legs like california stem cell what are they called
34:14
yeah so the california institute for regenerative medicine they have a huge
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neurodevelopmental pipeline that they want to develop and if we’re able to show
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efficacy in the mice we should be able to get funding from whether it’s NIH whether it’s serm
34:36
if yeah if we’re able to show it in the mice then i don’t really see a problem of getting funding
34:44
especially with with something that would work just well
The Point
34:51
yeah exactly so i think i think the point i’m trying to make for for the families is
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this will take a lot a lot more time and a lot more money but yes hopefully our money will be catalytic and could could support the creation of more money for this work
35:08
point one and point two even though it is far off and you know we we have now two companies publicly
35:14
working on ASOs for SYNGAP1 just yesterday praxis announced they were working on this
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i think the the the potential to have a one and done treatment that would as
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you just said create a protein factory in the body is just too tantalizing not to go after
35:34
in my opinion right yes and that’s the point i want the families to land on there’s a lot
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of questions here there’s a lot of what about this and what about that but the punchline is if we can pull this off there’s thousands of syngapians out there we got to help them
35:47
so with that i put Hans up on the panel Hans do you want to ask your question and then
35:52
oh i want to i want to honor one more question then give it to Hans to handle the science stuff um pat brelin said if all goes well how many years are we away from dosing a human in a trial
If All Goes Well
36:07
a big if but it will probably take
36:14
8 to 12 months to evaluate this in the mice
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we then have to do you know if we get funding right away for the toxicity
36:30
then that would take another year or so
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i don’t know it’s really hard to say if everything goes well i would say
36:43
three four years depending on funding
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but you know everything has to go perfectly the big thing right now is the mice getting these mice
36:56
up we still have to cross them with the il-2 mice that takes generations of mice to make so that’s
37:02
months because once you cross a wild type il2 with a homozygous il2 deletion you just get heteros
37:11
and then you have to cross those heteros to get your homozygous mice and then you have to grow up those homozygous mice to then get enough to do the experiments with and so getting this mouse model
37:23
up and running is the first thing we may actually do some experiments that i didn’t put in the initial grant where we use the mouse cells and go into the mice while the
37:35
humanized mice are being made we’ll have these we’ll have these immune competent syngap
37:44
hetero mice that we could transplant so we could get some initial indications those experiments are
37:52
quick and easy to do while these immunodeficient mice are being made
38:00
so yeah but you know it’s just it’s just hard to say what’s the FDA going to come back are our
38:08
open field and roto-rod assays enough to do an adult trial for our tay sachs our standoff
38:17
that was enough we just had to do a toxicity and expression study which we’re doing now
38:22
for tay sachs and then we’re able to put in our ind for an adult trial for tay sachs so
38:29
it might it might be enough and and that’s a big if as well we thought we did enough for
38:35
angelman’s with all of that efficacy to be able to do a pediatric trial but they want
38:40
us to do three more it was enough for an adult trial but not for pediatric trials so
38:47
it’s hard to say um no it’s i mean it’s a hard question and i and i i’m not asking it to
38:54
to push you to say give us an answer we’re going to be happy about but just to highlight for the audience that science stuff takes time and it’s unclear but again as soon as we start soon we get
39:05
somewhere okay hans you want to do the science questions well joe it’s good to speak again
malignancy risks
39:13
thank you to some degree i’ve asked these questions before but um you know we just have
39:19
to be so careful can can we talk further about the malignancy risks in the sense that the the
39:25
mice are treated with buscell to condition them for space in their bone marrow for the engraftment
39:31
is that right correct and are there you know is it similar in the human then in terms of
39:39
of conditioning regimen and there are their malignancy risks secondary to the conditioning
39:49
um we are actually moving into a new conditioning regimen it’s actually a cd45 antibody that method
40:00
that is safer less toxic and it actually clears more of the bone marrow out so they wouldn’t get
40:09
puck and there’s already trials that have used it um we’re actually adding it into our hiv trial for
40:18
our next trial and so there are safer ways as far as the conditioning regimen causing the malignancy
40:26
there’s always that potential especially with chemo conditioning regimens but with this
40:34
45 antibody it’s different and it acts in a different way
40:40
so i can’t really speak as far as potential malignancy for that because it’s more new the
40:48
FDA is always concerned about integration status of a lentiviral vector where is it
40:56
integrating this and that the fortunate thing is that HIV which the vector is based off of
41:04
there’s been no report of it causing a primary malignancy obviously it causes other ones because
41:11
you decrease the immune system response and you get all these other cancers but as far as where lentiviral vectors integrate compared to retroviral vectors
41:20
they have a different integration pattern we do those experiments that’s part of the toxicity in
41:27
the mice we also do these immortalization assays in vitro where we look at transducing the cells
41:34
and then passaging them for a long period of time and look for outgrowth of these cells just based
41:41
off integration pattern we also do integration analysis on where these vectors integrate and
41:49
we take a sample of different cells that have been transduced gene modified and we do an integration
41:56
analysis and look for any hot spots we’ve done that for others and we don’t we don’t see it
42:05
but yeah it’s always something that we’re looking for and is in the back of our heads because the
42:12
vector integrates and it’s permanently in there and that harkens back to the skid trials right
bluebird bio mds
42:19
i mean the kids who were getting gene therapy for severe combined immune deficiency there was
42:24
some malignancy signal there but the more recent you know bluebird bio aml mds it sounds like the
42:34
the the treatment itself has sort of been exonerated yeah and so for the the skid
42:42
those are using retroviral vectors so they integrated near a gene called lmo2 which is an
42:48
oncogene and with these retrovectors some of their ltrs are active and so they integrate near a gene
42:57
ltr is the normal mouse retrovirus promoter it can up regulate nearby genes so the lmo2
43:05
gene was upregulated and that’s what caused the cancer but now people have moved into lentivectors with the bluebird yeah that was just hard to say whether it was
43:18
the promoter they were using the gene they were expressing whether some of those kids were predisposed to any of that what kind of conditioning regimen i haven’t heard
43:31
anything yet as to what actually caused the mds i know the kids were treated for the mds and they
43:38
recovered but it was a small percentage i think they had done at least 60 or 70 kids or 80 kids
43:48
and it was in the low single digits i don’t know are those numbers correct is that what you’ve
43:55
heard like three or four of the kids had mds i i thought it was an in fact lower even maybe
genome sequencing
44:01
just one or two and then there was an aml that was written up in the new england journal in january
44:09
okay so yeah one thing that would be good so what we can do is we can actually sequence a person’s
44:17
genome and actually look for any of these predispositions and i was talking with our
44:22
bone marrow transplant physician because we use the similar promoter that they suspected was
44:28
causing this in our taste sax vector and our angelman vector because it’s such a good promoter on expressing proteins and they wanted me to change it out and so we’ve
44:41
been changing out this promoter for other high expressors just because there’s that potential
44:47
and then i was talking to him and was like well maybe we should you know sequence some of these
44:55
patients prior to enrolling them see if they have any predisposition to any of these diseases
45:01
sometimes that’s done with these lymphoma patients that we have in our hiv trial we actually
45:07
sequence them and see if their lymphoma is caused by a specific mutation that they had and so we’re
45:14
able to look at predisposition of some of these diseases based on sequence and so if
45:19
it is something about these conditioning regimens or something like that there there’s potentially
45:27
things we can do pre-enrollment into these trials to see if there is any you know potential for
45:35
malignancies well i have to admit i mean to hear that there’s you know an improved conditioning
improved conditioning regimen
45:42
regimen you know an anti-cd45 is so much more appealing than some you know caustic chemotherapy
45:49
agent yeah and the fact that it clears more out so we’re able to get a higher level of
45:55
engraftment for some of these monogenic diseases we may not need the large level of engraftment of
46:03
gene modified cells as we might for our hiv stuff so sickle cell i think they only need around 15
46:12
percent gene modification to actually get correction of phenotypes from sickle cell
46:18
because then you get enough of those red blood cells that are corrected so you’re able to
46:24
function normally know it’s probably lower hanging fruit in terms of correcting that phenotype
Science questions
46:33
but i hear you oh it’s fantastic can we get to the some of the science questions there
46:39
was one in the chat from shin chen to what extent can microglia graft in the cns is that
46:48
um we are kind of looking at that we’re doing immunohistochemistry
46:56
on the brains of our transplanted and engrafted mice and that’s actually part
47:02
of the our syn gap proposal we have that in there so we can we can look and see how many
47:10
of the human microglia engraft into the brain we’re currently doing that with our angelman
47:16
um with a conditioning regimen and a strong one i feel like we can get a large number of them
47:25
but we we will be looking at that we’ll be doing co-localization of human microglia and
47:33
mouse syn gap in our studies so we’ll be able to look at those and quantify them
47:46
great thanks and then ray holden asked a question i’ll let hans parse there’s a number of other questions from jj in here but i’m gonna
47:55
just throw rays at you i don’t i’m not quite sure what he’s saying be honest but i don’t
48:01
want to talk anyone out of a job or research but would parent-to-child stem cell donors create corrective glial cells or the modified gill cells imperative to the success of this method
48:12
isn’t the answer that you need to modify them to have them make the syn gap or am i making that up
48:20
can you repeat the part about parent to child i can’t find that question in here you can’t
48:25
see it if you scroll down the open it’s about halfway down if you scroll down on the q a it’s in the q a and i i think it’s you know naturally a dad oh in the q a okay a dad is interested in
Dads cells
48:38
giving his cells you know and not putting his child through the effort but then you’ve got a degree of mismatch oh yeah yeah and yeah i don’t think that that would that would work that
48:49
would be an allogeneic and there’s actually more toxicity in that graft versus host disease all of
48:54
that compared to just using autologous cells and a patient’s own cells so just as a as a sort of
49:05
maybe a little bit of relief um you know bluebirds done this in children with their cerebral
49:12
adrenal leukodystrophy and don cohn at ucla has done this with the ada skid
49:17
kids so this is done all the time in kids and juveniles and the protocols are there
49:27
so i would be confident and i would feel comfortable if i had a child because it’s been
49:36
done so many times and the protocols have been there and to the point you
49:41
know it’s going to be a few years until we’re doing this to humans assuming all goes well and in those years we would expect the protocols to get even more refined much like the
49:49
the clearing regimen you were talking about becoming a better choice right so yeah
49:56
the only thing we can be sure of is that science continues to happen at lightning speed so the fact that we’re a few years out in that sense is good news because things should get more refined hans
50:06
you want you want to manage the rest of these well so one of them is is how are you going to control
SynGAP1 integration
50:12
where where the syngap1 lentivirus integrates and it’s not something under your control but
50:18
the lentivirus integration sort of profile is seemingly benign
50:26
correct yeah and we’ve done these integration analyses on our patient samples for our hiv trial and there’s actually over 13 to 15 000 integration hits they can be in introns exons
50:42
close to cpg regions they’re just all over the place but where they like to integrate compared to
50:51
where the retros like to integrate is completely different and plus the vectors are inactive the
50:57
the ends of the vectors that actually do the integration they have no promoter activity
51:04
there’s a poly a tail in the three prime ltr so it stops any transcription from going through
51:11
the vector so the only thing that’s in there that’s active is our promoter that’s driving the
51:16
expression of syn gap so these self-inactivating lengthy vectors have been designed to not
51:24
cause much damage once they integrate in but yeah we cannot control where
51:31
our specific vector integrates and then there’s a question of whether there’s toxicity observed
Toxicity
51:38
in the in the pre-clinical work whether it’s for angelman’s or or another another disease
51:45
we have not seen anything yet in our angel mice we just got our adult uh toxicity data back and
51:53
there was nothing that we saw that was toxic as far as this one we just don’t know yet we’ll have
52:00
to do those experiments we’ll get an initial idea when we actually transplant the syngap heteromice
52:11
you know if something happens whether it’s weight loss or some other thing that we observe
52:17
then that could show some potential toxicity because those could technically be called our
52:24
you know our clinical trial patients because they have the syngap deficiency
52:30
so we’ll be able to get some idea once we start transplanting the mice and um so have you done a second species as reported as a part of the safety studies for
Second Species
52:41
for angelman treatment no i think because we use the human cells and we use these immunodeficient
52:48
mouse models that we haven’t had to go to non-human primates we haven’t we weren’t asked
52:55
for hiv we weren’t asked for our taste acts and we weren’t asked for our angelmans in our pre-ind
53:00
meetings with the fda so i think using these human isomice where we’re actually using the human cells
53:07
and not just mouse into mouse that actually helps that’s fantastic thank you for your patience
How to get SYNGAP1 protein into neuronal cells
53:17
mike yeah this is this is great um
53:26
i just wanted the the this one question up that i just want to cover i think i think we talked about it but how are you going to get syngap1 protein into neuronal cells
53:36
so if we go back to the sort of bears repeating if that’s if people are unclear right yeah so if we go back to
53:45
the second slide the way the hypothesis works is that the microglia that are transplanted and
53:55
then engrafted into the cns will act as protein factories so in the upper left corner that’s our
54:03
gene modified microglia and then in the bottom right that’s our affected neuron and
54:10
just based on modifications that we’re making to the protein we should be able to transfer
54:15
it to the neurons whether in the extracellular space or the microglia express it and then the
54:23
neurons take it up or with a direct contact so with neurodegeneration or neuron injury sometimes
54:31
there’s direct contact with these accessory cells like the astrocytes and the glial cells
54:36
and so it could just get transferred in between the cell with close proximity or
54:43
cell membrane contact we don’t know the exact mechanism and i don’t know if we’d be able to
54:50
identify that just with some immunohistochemistry and looking at expression one cell takes it up one
54:58
cell expresses it we see that co-localization but how it’s actually getting there um we just
55:07
don’t know that exactly but knowing how the cells work and interact and what we’re doing to modify the proteins there’s a couple hypotheses one might
55:20
work one other one might be right but they both might be right but yeah
55:26
got it and just for the people on the call who don’t think about neuroscience all day long
55:34
the people like me who’ve been trying to figure it out for the past three years and they’re still struggling broadly speaking there’s at least two kinds there’s neurons and there’s glial cells in
55:43
the brain right and so what you’re talking about is affecting the brome marrow to create the glial
55:50
cells which would produce what we need to go from the glial cells into the neurons and the neurons
55:57
is where we find synga like at that at that sort of cartoonish level is that accurate
56:02
yes and then what we are hoping is that once syngeb gets into the neuron the neuron will know
56:09
where to put it so it would be in the synapses so you know normally sin gap is made the rna is
56:16
made in the nucleus just like any normal cell and then the protein is made and then it gets transported you know whether it’s a cell membrane protein it has cell localization signals in it
56:27
so that the cell knows where to transport it so we are hoping that once syngap gets into the neurons
56:35
the neuron will then put it where it needs to go at that point we have no control because
56:41
it’s in the neuron and the neurons gonna do what it wants to do but it should put it in the right
56:47
spot it should have all the all the correct signals we don’t modify any of the major uh
56:56
like reactive regions the enzyme active regions any of any major structural regions
57:04
and the proteins we try and stay away from all those active regions or binding binding
57:09
domains things like that and we just make these modifications in in other regions maybe in some
57:16
like other folds that don’t seem to be necessary just based off of current research and what we
57:22
know about the proteins right i mean so i’m just going to editorialize for a hot minute
57:29
you know i went to the angel in meeting where you presented this work and you’ve been driving this and i talked to a lot of people and there are some people who are like well there’s still some things
57:38
we don’t understand and um there are other people who are like yeah that’s kind of how science works
57:48
but that they’re seeing things in the mice and if this does work the thing that struck me and
57:54
where i really decided we needed to fund this i talked to one very smart angelman parent i said
58:00
it was off the record conversation i said what do you think of this like are you give me tell me the and and she said i would be the i would i would want my child to be patient one because
58:12
it’s one and done and she said if this works and it’s a huge if but she was very enthusiastic and
58:17
i’m not saying that you’re going to create a silver bullet here but i am saying that um
58:24
i think the purpose of the syngap research fund is not to fully fund the creation of entire therapies
58:31
that is just a stupid amount of money the purpose of the singap research fund is to make sure we leave no zone unturned and when we find something like this that could be a winner
58:42
and we find someone like you who’s courageous enough to push the ball up the hill it’s it’s i think it’s i think we owe it to our kids to support you until you can get enough data
58:52
to get the nih and the california institute for regenerative medicine and others to keep funding
58:58
the work so one of the parents asked how do we support this work the answer is simple you help us you raise money for srf and you help us support joe and other people who um
59:11
have the courage to go after this stuff so thanks joe keep it up i know it’s um it’s tough to get up here and be like what about this
59:21
but we are grateful and uh we look forward to working with you yeah thank you and thank you for
59:27
funding the research because it could be this it could be something else it could be a combination
59:32
we just don’t know when there’s nothing currently out there you know we’re grateful and appreciate
59:39
the opportunity to you know test our our theories and our hypotheses on what could happen
59:48
it seems like it would work just based on what we know of the hematopoietic system and you know you
59:54
have a cell that doesn’t have a protein that works so we need to figure out how to get that protein
1:00:00
into the cell once we get that protein in there hopefully it does what it needs to do and
1:00:07
we can correct these neurons but yeah okay thank you very much joe good luck thank you thanks joe
1:00:28
you