78 – Therapeutic Strategies for Autism: Targeting Three Levels of the Central Dogma of Molecular Biology – Presented to the SYNGAP1 Community

Prof. Lilia Iakoucheva, Ph.D. & Mr. Derek Hong, M.S.

Speaker Bios

Prof. Lilia Iakoucheva, Ph.D.

Dr. Lilia Iakoucheva is a Professor of Psychiatry at the University of California San Diego. She obtained her B.S. in genetics from Kiev State University in Ukraine, and her Ph.D. from the Institute of Immunology in Russia. After completing postdoctoral training in protein biochemistry at the Pacific Northwest National Laboratory (PNNL), she joined Washington State University to study Intrinsically Disordered Proteins. During that time, she made a series of fundamental discoveries about disordered proteins, including their role in cell signaling and cancer. Several of her papers on disordered proteins are highly cited, and accumulated over a 1500 citations each. Lilia then joined the Rockefeller University studying protein interaction networks and pathways underlying human diseases. She joined Psychiatry Department of UCSD in 2010, and her current research focuses on understanding of the molecular basis of neurodevelopmental diseases, with the focus on autism spectrum disorders. Dr. Iakoucheva has been the Principal Investigator on numerous research grants from NSF, NCI, NICHD, NIGRI, NIEHS and NIMH. She is a Member of the UCSD Bioinformatics and Systems Biology Graduate Program, Biomedical Sciences graduate Program, and an UCSD Institute for Genomic Medicine. She serves as an Associate Editor of PLOS Computational Biology Journal, and has received multiple awards, such as student-nominated Outstanding Faculty Mentor Award, and Outstanding Dedication to Undergraduates award for mentoring high school and undergraduate students.

Mr. Derek Hong, M.S.

Derek Hong is a research associate working in Dr. Iakoucheva’s lab at the University of California San Diego (UCSD). He received his B.S. in Biochemistry and Cell Biology, and M.S. in Biology at UCSD. His M.S. thesis was devoted to reviewing current approaches used in the development of autism therapeutics, and their future in clinical translation, which is the topic of his today’s presentation. Currently, he is working on utilizing a CRISPR toolkit with the aim of restoring the expression of autism-linked haploinsufficient genes. He serves as a mentor to several UCSD undergraduate students working on diverse projects that are related to developing therapeutics for autism.

THIS IS A TRANSCRIPT ONLY:

hello everyone and welcome to today’s webinar my name is Olga Bode and I’m part of the team here at syngot research
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fund our presentation today is therapeutic strategies for autism targeting three levels of the central
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dogma of molecular biology I have the pleasure to introduce today’s speakers Professor Lilia yakuchova and
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Mr Derek Hong both from the University of California in San Diego
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is a professor of Psychiatry at the University of California San Diego she obtained her BS in genetics from Kiev
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State University in Ukraine and her PhD from The Institute of Immunology in
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Russia after completing postdoctoral training in protein biochemistry at the Pacific Northwest National Laboratory
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she joined Washington State University to study intrinsically disordered proteins during that time she made a series of
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fundamental discoveries about disorder proteins including their role in cell signaling and cancer
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several of our papers on disorder proteins are highly cited and have accumulated over 1500 citations each
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uh Lilia then joined the Rockefeller University studying protein interaction networks and Pathways underlying human
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diseases she joined the Psychiatry Department of UCSD in 2010 and her current research
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focuses on understanding of the molecular basis of neurodevelopmental diseases
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with the focus on autism spectrum disorders Dr yakotva has been the
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Principal investigator on numerous research grants she’s a member of the UCSD bioinformatics and system biology
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graduate program biomedical Sciences graduate program and a UCSD Institute for genomic medicine
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she serves as an associate editor of plos computational biology journal and
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has received multiple Awards such as student nominated outstanding faculty
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Mentor award and outstanding dedication to undergraduates award for mentoring high school and undergraduate students
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uh Mr Derek Hong is a research associate in Dr yukonis lab he received his BS in
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Biochemistry and cell biology and Ms in biology at UCSD is anesthesis was
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devoted to reviewing current approaches used in the development of autism Therapeutics and their future in
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clinical translation which is the topic of today’s presentation currently is working on utilizing a
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crispr tool kit with the aim of restoring the expression of autism length how the insufficient genes
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he serves as a mentor to several UCSD undergraduate students working on diverse projects that are related to
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developing Therapeutics for autism a recorded version of this webinar will be available on the srf website under
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webinars on the family menu by the end of this presentation you will have the opportunity to get the answers
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to your questions and we’d love to hear from you so please put those questions in the Q a below
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for those of you just joining us welcome again our speakers today are professor
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yakuchova and Mr Derek Hong from the University of California at San Diego to
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present on therapeutic strategies for autism targeting three levels of the central dogma of molecular biology and
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it’s now my pleasure to turn things over to Professor yokico okay hello everybody
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and first of all I would like to thank Mike for inviting us to share the
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research from my lab resume and you probably don’t get uh two
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speakers at the same time usually um but um the reason for this is that um I
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wanted to give Derek a chance to present his work that he did in my lab as a
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master’s student so the story was that Derek started in my lab
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um earlier before pandemic and then pandemic hit and we had to kind of stop
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some of our experiments that we were doing and then we got together with Derek and
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we decided that um the best you know use of his time would be to review the literature on the
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therapies that uh that exist for autism and there weren’t very many Publications
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specifically addressing this topic so direct did an excellent job reviewing
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the literature um the recent literature on how you know we approach the treatment strategies for
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autism and he is going to present his basically his Master season that was unpublished
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as a research paper or scientific paper review uh and I decided to also
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um you know show you a couple slides um to just introduce um the research that has been done
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um in my lab um it’s not uh directly related to to
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Derek’s talk but um of course we are always thinking about
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um you know therapies for audism and we study um several autism genes nuts and GAP but
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everything that I’m going to tell you today is relevant to syngap because we
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study other genes that are you know might act through the same mechanism as
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syngap so let me share my screen
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so just just to begin um I wanted to introduce a little bit
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ogism genetics to you um so autism is neurodevelopmental disease
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that affects um about one in 36 people
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um in the world and the recent CDC report States it’s prevalent as 2.8
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percent as and as many of you might know the prevalence of ogism is
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um increasing over the uh with each year so autism has four to one boys to girls
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ratio so boys are affected four times um more often than girls and it’s
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typically diagnosed before age of three so autism is characterized by Three core
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symptoms deficits in social interaction language deficits and
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restrictive and repetitive Behavior children with autism have other
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comorbidities such as medical comorbidities immune deficit
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gastrointestinal problems and and other comorbidities as well as cognitive
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deficit and anxiety and ADHD
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unfortunately despite a hard work of scientific community
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there is still no reliable diagnostic biomarkers to diagnose autism and even
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more upset and of course is that there is not not an FDA approved drugs that
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targets the score you know social deficits or in autism
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oops
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somehow my slides are not advancing
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hmm
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am I a end presenter oh here you go
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so um what sorry so as any
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um uh you know multifactorial disease autism is caused
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by the interplay of the nature of the genes and the nurture or the environment
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we still don’t know a lot of sins about autism but one sin
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um that we do know is that um autism is has a very strong genetic basis
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so the recurrence of autism that was measured in
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monozygotic twins that share hundred percent of their genetic materials
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compared to disagoitic Twins and siblings that share about 50 of their
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genetic materials and general population as you can see on this graph the
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prevalence in MZ twins is very high meaning if one twin has ogism
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in 70 to 90 percent of cases also will have ogs whereas the prevalence for
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Jizzy Twins and sibilance is about 10 to 20 percent and general population this is an older figure was one percent but
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now as I mentioned is more like 2.8 percent so what what’s the status showed is that
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the proportion of autisms that can be explained by genes is estimated at 70 to
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90 percent so if autism is genetic then why you
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might wonder why does it happen that you know two healthy parents might have a
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child with autism why does it not you know run in families like other
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monogenic diseases well the reason for this being that
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autism is frequently caused by so-called de novo mutation this mutation is
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usually not inherited from parents but arises uh after conception so not after
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conception that it arises either in sperm or the Egg of the parents and then
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um you know during fetal development it will be present in all the cells of the
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fetus so in us and this genome mutations you
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can imagine them it’s pretty much like a dart thrown into genome so it can kill
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any Gene in the genome with almost equal possibility
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so then over mutation could explain the
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heterogeneity of audism meaning that there are many different genes that can be impacted by those
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Genova mutations in other words to just you know explain the genetics of autism
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I will quote Steven Shore who is audism Advocate and also a person with autism
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who said if you met one person with autism you’ve met one person with autism
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so this pretty much explains uh genetic of Buddhism
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so if OG is a genetic what genes are involved
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so recent whole genome and whole exome sequencing studies implicate that very strongly more than
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100 genes with so-called Regional mutations in autism
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and what this if it’s 72 genes being a very strongly implicated
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so what what this slide shows that as you can see more than 90 percent of
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these genes carry the so-called GN or Genova mutations and there is very small
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proportion of inherited mutations and as you can see by this pink color the
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majority of the mutations in these genes are actually protein truncate invariants
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and I just wanted to point out to you since Gap is one of the strongly
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implicated genes as you can see that carries a lot of Broad and truncation
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mutations and these other graphs again just shows that that the majority of the
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mutations in syngap are protein shrinkage and although there are also
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some missions mutations but they are less frequent in in this Gene
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so then just to recap what what other genes
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could be implicated in autism as I told users genes that were detected by
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sequencing axons of genomes of the trios mom dad and the and the child affected
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child identified so-called genes with their Genova mutations and implicated
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them strongly in autism and as a set of genes so-called common
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variants that were detected using genome-wide Association studies were
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also implicated and now we have about from 5 to 12
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um strongly implicated common variants and those those variants could be inherited from parents
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there are also several syndromes such as fragile Acts or
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um other syndromes that that can be caused or red syndrome when we know which Gene
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actually causes the autism in in these children and another class of genes so-called
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copy number variants those are a large duplications or deletions of several
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genes sometimes even up to 100 genes that can cause autism
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and another class of you know Gene that I’m not mentioning here or mutations
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that I’m not mentioning are actually located in the non-coordinary regions of the genome
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um that could also be poisoned origin spectral disorders so now that we have
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all this you know autism candidate genes ultimately what we want to do to
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understand the disease we want to know how these genes connect to each other
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and what Pathways could be disrupted by this genetic mutations and another
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question that we want to know that at what times of brain development these
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genes cause the disease so just by looking at the you know
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Pathways that this genes could affect it’s clear that there are a lot of genes
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that participate in the synaptic processes and again you can see syngap
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it belongs it’s a synaptic genosynaptic protein that that could cause autism
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another set of genes uh chromatin genes
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such as ched and said D5 and other ones that can uh that implicated in autism and again
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yet another set of genes involved in post-transcriptional
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regulations and they can act through the RNA messenger RNA
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so now is that we more or less know what Pathways those Gene effects we would
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also want to know um at what time points of brand
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development those genes uh disrupt brain function
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so to understand this um we and others use a data set so it’s
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called brain span so this data set is a gene expression data set of the
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developing human brain starting from the fetal stages and up to the doubts and
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what we can do with this data set we can build so-called special temporal brain
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transcriptome when we combine specific developmental periods of the brain with
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specific regions and we can build so-called spatial temporal networks and
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what do we do with these networks what we can do we can map the genes that
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carry those autism-associated mutations computational to those networks and by
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doing so we can see where these genes are functioning in the
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brain and when and how we can infer using machine learning approaches how
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these mutations can dysregulate brand development and we and others did a lot
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of this you know computational and bioinformatics work and what we
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discovered is that majority of uh genes that are implicated
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with autism actually functioning during late mid-fetal cortical development so
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it means that these mutations that we are finding probably disrupt fetal brain
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development even before the child is born so now that we discovered this
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computational is that fetal brain could be involved um we will we want to confirm this
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experimental right so basically what we need then is
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uh fetal brain with autism diagnosis and to compare it to the control fetal brain
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right to discover those Pathways and ideally we would also need patients uh
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Branch from the patients with specific mutation in in specific Gene and compare
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it to another you know healthy controls uh fetal brain without mutation in that
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Gene so basically can we get if you talk with
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more than brain with ogism diagnosis um probably not because as we know
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autism is diagnosed after child’s birth
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could we get a fetal postmortem brain with specific gene mutation well this in
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principle is possible however we would need to sequence a lot of fetal brains
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to find you know just one one person with with the specific Gen X mutation
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unfortunately this is also not feasible so what do we do how do we actually
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investigate fetal brain that’s probably the you know the tissue that we need to
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understand better how autism is developing
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fortunately we can use some alternatives to the you know fetal brain tissues for
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example with the development of crispr cas9 genome antigen technology what we
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can do we can insert the mutations that we identified in the patient into the
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mouse rat models and then we can extract the you know fetal developing brain and
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study what what goes wrong in those you know mice and red models this we are
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pursuing this line of research but unfortunately you know mice and rats are
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not human of course and they have a different Developmental trajectories and
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different time points in the brain development so another alternative is
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so-called patient-derived mini brains and of course um they don’t look like this
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they look like like this there are little blobs that we call Brain
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organoids so this uh this is a human derived model
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and there they could be derived from the tissues of patients with autism and
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they’re basically genetically identical right to the to the patients they also
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recapitulate human fetal brain development as opposed to the animal
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right and this way we can also stratify the patient and find several patients
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with a specific mutation that will give us a greater power to to study this
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developmental processes that go wrong in the fetal brain
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and my lab uses as I mentioned was the animal strategies and and the Brain
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organoid um strategies
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so how do we make those brain organoids so brain organoids can be made from the
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patient’s skin biopsy and also from the blood cells as well so if you start from
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the patient’s skin biopsy we can culture the fibroblasts from the skin and then
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add transcription factor and convert those fibroblasts into the induced
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chloripotent stem cells then we can generate differentiate those
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stem cells basically into any cell of the body in in our
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um in our case it will be neuronal cells and then we can produce cortical
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organoids from from the skin the whole process takes about a month and then
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what is really great those organoids could be maintained for a long time even
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for nine months of up to a year and we can you know grow them and study what’s
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how mutations cause the disease so how do you might wonder how do we
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know that this organ oil that we are producing can actually recapitulate what’s going on in the
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fetal brain well the usually we perform the sequencing of
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these organoids and then using a machine learning again framework we can compare
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this so-called transcriptomes from the organoids that we produced from the
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patients with uh fetal brain development or human brain development using again
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this brain span data sets and what this figure below shows that
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when you see the red color it’s more similar that means that organoids are
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more similar to the human brain and the blue color means they’re less stimulant
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so you can see that when we start with this induced pluripotent stem cells
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they’re very similar to the embryonic and early fetal brain development but then when we keep those organizing
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culture and grow them to one month and then to three months you can see that
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their maturity is Shifting and at one month organoids are recapitulating the
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global transcriptional profiles of the early to late mid-fetal human brain and
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then when we keep them even longer for three months it’s even moving into late fetal brain and neonatal or early
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infancy this will tell us that we are producing you know uh this cells or
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brain organoids that that are similar to the developing or human fetal brain
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we also look at the specific cells from this organoids and we see that there are
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a whole diversity of cells from from those organoids that also could be found
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in the fetal brain such as progenitors all different types of neurons so this
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gives again a higher confidence to us that organoids recapitulate human fetal
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brain and even more fascinating this picture is from the paper by our
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collaborator Allison Morty from UCSD but the fasting agency and about organoids
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is that you can also record electrical activity from this organ oil so they
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have synapses they have all the sins that that they’re active and if we
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record those electrical activity from organoids from different time points you
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can certainly appreciate that while organ I know it’s matured in this electrical activity becomes more and
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more complex those spikes that that organoids produce but what even more
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fascinating that Allison showed that when they compared this electrical activity of organoids at the later time
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points of development to the premature baby eegs it’s actually pretty similar
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have some similarities with the pre-term babies EEG
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so that means that this organoids serve as a really good model to study autism
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spectrum disorders so and I’m almost done lastly we in collaboration with Allison
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Morty we performed one study on a different you know genetic mutation in
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autism called 16p 11 2 code number variants where we use cortical organ oil
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so what we did we collected ipscs from this patients with this copy number
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variant and we produce cortical organoids from these patients We performed a lot of different you know
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um molecular studies a lot of sequencing and proteins and a lot of a lot of
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cellular studies to identify specific Pathways that implicated in in the in
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this disease and we did identify one of the pathways that can cause you know the
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phenotypes present in the patients but even more interesting we also were able
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to rescue some of the phenotypes using a small molecule inhibitor
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that correct some of the defects of those organoids
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so just to summarize I hope I convinced you that organoids are really good model
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and they can recapitulate the development of human fetal brain and we
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can identify potential mechanisms that can be dysregulated by those
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mutations and autism we were able to even identify the candidate drug drugs
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that we are currently testing in Mouse models and again in organoids but most
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importantly organoids can definitely be used to search for this convergence of
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different genes and with with OG’s limitation potentially on the same pathway and they can also be used for
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the drug screening more easily and faster I should say than animal models
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um and um yeah I I hope uh you you guys have some questions for me but I’m just
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gonna that stop sharing my screen and
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and Derek is going to share his screen but if anybody wants to ask questions
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meanwhile go ahead and if not we can just you know take questions at the end
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thank you for your attention so yeah I don’t see any questions yet but
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I’ll let you know for sure sounds good yeah
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okay so I’d like to thank um Lilia for providing an introduction with the
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genetic basis of autism and I want to reiterate once again that
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the past two decades have been incredibly successful in identifying these ASD Rich genes
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and a lot of the current progress in therapeutic development has also been
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successful in animal models as well as in vitro or within cell and organoid
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models like Lilia mentioned and in order to really understand the
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core mechanism behind how the Therapeutics work we really do
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need to understand how these ASD genes are interrupted or
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um lose their function and so this is the idea of haplo
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insufficiency and haplo insufficiency is when there is
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a loss of function mutation in one allele and
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when this happens and the remaining functional allele is not able to produce
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enough product to compensate this could ultimately lead to the loss
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of normal physiological functions and this is important because many ASD
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mutations cause haplo insufficiency so they’ll be very promising targets for
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the Therapeutics that we’ll be discussing today and so in order to restore this normal
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physiological function I do want to explain a poor fundamental
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Concept in molecular biology and this is something called the central dogma
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and the central dogma is the process in which all of the information that’s
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coded in our DNA ultimately ends up as a protein
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and so to begin um the genetic information is coded in
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our DNA in the form of a double-stranded DNA and so this information
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is copied over to a single stranded RNA in a process
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known as transcription and the RNA kind of functions as a
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messenger or an intermediary and once the RNA comes into contact with
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a molecular machine known known as the ribosome the ribosome will read the code on the
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RNA and assemble amino acids as per the instructions of the RNA
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and so once these amino acids are assembled we have our protein and the
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protein is basically the Workhorse of our bodies and provides structure as
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well as all the necessary functions for ourselves and so
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as a presentation overview I want to begin by discussing Therapeutics at the
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DNA level such as the crispr mediated modifications
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second I will be delving into trans Gene delivery as a therapeutic
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and as we move on to the RNA level I’ll be discussing antisense oligonucleotides
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also known as Asos and finally the Therapeutics will be at
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the protein level and this will include small molecule drugs
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and in order to wrap everything up I’ll have a brief discussion about the
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delivery of these Therapeutics and two begin with crispr
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I want to explain that crispr is the system that consists of the cas9 enzyme
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as well as a guide RNA that will be complementary to a DNA
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Target and so once this guide RNA travels to the DNA Target
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it’ll guide the cas9 enzyme over and cas9 will function as this giant pair of
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biological scissors and it’ll cut or induce a double stranded break at the
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location indicated by the guide RNA and so when this double-stranded break
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is created um it’s your cells are in emergency mode and they’re really going to prioritize
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fixing the double-stranded breaks as soon as possible and there are two mechanisms in which
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the cell does this the first of which is something called non-homologous end joining
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and this process is incredibly error prone because it’s
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one of the makeshift mechanisms to fix the double-stranded breaks as soon as
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possible and so you can get things like deletions or insertions in the DNA as a result of
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this the second mechanism however is something called homology directed
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repair or HDR and HDR utilizes the homologous
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chromosome as this template or blueprint for repair and it’s way more precise and less error
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prone than non-homologous engine and we can actually take advantage of
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this HDR mechanism and couple it with our cas9 and guide
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RNA as well as a donor template so for example let’s say there’s a
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mutation in our DNA as indicated by the Thunderbolt we can introduce the cas9 the guide RNA
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to direct the cast to this mutation and so
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in addition to this we also have a donor template of DNA that provides the
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blueprints um of DNA that doesn’t contain that mutation so when homology directed
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repair does its thing we can have DNA that no longer has the mutation
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and so to kind of Zone in on a therapeutic
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Target I want to explain the idea of a natural anti-sense transcript otherwise
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known as a net and so some ASD Associated genes have
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Nats and Nats are these mRNA transcripts that are complementary to the Gene’s
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mRNA the target Gene okay and Nest can enhance
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or inhibit the translation of its Target genes and this is going to vary on a
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gene to Gene basis so not every Gene will have the same interactions with their respective Nets
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and so as a syndrome that we can potentially Target
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Angelman syndrome is in normal physiological
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physiological conditions um not only with angeleness like everybody will have
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a paternal copy and a maternal copy of ub3a but the maternal copy of this Gene is
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actually silenced in neurons for everybody and within Angel men’s syndrome
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um there’s also a loss of function in the maternal ub3 allele
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so in addition to this um as we’ve explained before we have
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gnats that are inhibitory and in the case of the paternal ub3a
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allele there is a net that acts as a suppressor for the paternal ubi3a allele
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so what we can do is we can Target the gene that codes for this inhibitory net
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of the paternal ub3 allele at the DNA level using our cas9 enzyme
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and so a cas9 enzyme will come and basically decrease the function of the
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net at this DNA level and this is exactly what needs to be done in order to
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partially restore ub3a expression through this paternal copy and
38:18
one study actually found that using this exact mechanism within Angelman syndrome
38:25
model of mice that they were able to partially rescue the protein expression
38:30
of ub3a as well as partially rescue motor coordination in these mice
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and so this is in animal models but um there is an example of using crispr in
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clinic and um this crispr cast 9 was used for A
38:51
congenital um blindness and it’s in a drug called edit 101.
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and with edit 101 they delivered a crispr cas9 construct into the retina
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and it’s a good example or it is the example of um the first use of a genome
39:12
editor within the central nervous system and this therapeutic is currently in
39:18
phase one and two clinical trials
39:23
and moving on to the next mechanism we have transgene delivery
39:30
and trans Gene delivery is the administration of genetic material to
39:36
affect it cells in order to treat a disease or a condition
39:41
and this genetic material is actually packaged into a carrier vector or
39:48
delivery vehicle that does not produce an immune response in patients
39:57
and so one potential targetable syndrome with transgene delivery is Rex syndrome
40:04
or rtt and Rett syndrome is a neurodevelopmental disorder or ndd that
40:12
lies under the ASD classification and it’s characterized by a smaller head
40:20
size or microcephaly seizures cognitive impairment as well as a decrease in hand
40:28
function and at the genetic level this is
40:33
directly caused by a loss of function of the mecp2 gene
40:40
so in one study
40:45
scientists injected the human mecp2 gene into the
40:53
brains of mice that were lacking at mecp2 and what they found was that there was
41:01
an increase in survival of the mice that were treated with this mecb2 transgene
41:08
and there’s also a decrease in phenotypic severity so measures of that
41:15
include motor deficits and nuclear volume
41:21
and again this was an animal model so um to provide a clinical
41:28
example of transgene delivery um oh sorry one important consideration
41:35
um in the development of the transgene delivery as the method is that we need
41:43
to make sure we have the correct dose because at normal physiological levels
41:48
we know mecp2 is expressed correctly in Rett syndrome we know that there’s a
41:56
decrease in expression of mecp2 but on the other end of the spectrum we
42:02
can also have something called mecp2 duplication syndrome where too much mecb2 is being produced and this
42:10
condition is characterized by developmental delay hypotonia and
42:15
moderate to severe intellectual disability and so
42:21
for transgene delivery um dose dependency of macp2 is very
42:27
important because we don’t want too little expression but we also don’t want
42:32
too much expression and recently there was also an FDA
42:41
approved drug named Lux Turner that’s an example of trans Sheen delivery and it
42:48
was used to treat a rare inherited retinal disease that resulted in Vision impairment as well as blindness and what
42:55
they essentially did was they delivered this rpe 65 gene into patient and Within
43:02
These patients they saw an improvement in visual Acuity as well as functional
43:08
vision okay and so at this next level we have
43:16
anti-sense oligonucleotides at the RNA level
43:21
and basically anti-sense oligonucleotides are these synthetic single-stranded DNA
43:29
sequences that are designed to Target mRNA transcripts so we’re basically
43:34
regulating the expression of genes at this mRNA level
43:40
and we have two main functional categories for Asos the first of which
43:46
um are Asos that increase the gene expression through direct interactions
43:52
with the target genes mRNA transcript
43:57
for the other functional category we have Asos that increase the gene
44:03
expression by inhibiting the natural anti-sense transcript of that Target
44:09
Gene but for the purposes of time I’m really
44:15
just going to focus on the first mechanism or the first functional category and that’s that direct
44:21
interaction between Asos and the target Gene mRNA
44:26
and one important mechanism from this functional category is alternative
44:32
splicing and we can think about all alternative
44:37
splicing as a way to rearrange or process how the final mRNA looks
44:45
but first I would like to direct your attention to the little blocks that are
44:52
labeled exons and these exons within the MRNA are
44:58
basically just building blocks that can be rearranged in different ways to
45:04
ultimately produce variants of a protein and in addition to this
45:10
um I have in red here something labeled ISS targeting Exon 7.
45:17
and what this basically is is a red flag that tells the processing machinery
45:24
to remove that Exon 7 building block from the final mature mRNA product so as
45:33
you can see here Exon 7 is not in the mature mRNA
45:38
and so what what we can do is we can actually design an ASO that targets this
45:46
red ISS targeting Exon 7 region to effectively remove that red flag and
45:54
tell the processing Machinery to actually include Exon 7 in the final
46:00
product and one incredibly successful and widely
46:07
used example of this would be spinraza and spinraza is the first FDA approved
46:16
drug based therapy for spinal muscular atrophy known as SMA
46:23
and in SMA people lack a working copy of the smn1 gene
46:30
and for everybody including including those with SMA
46:35
um there we have a different version of The SMN Gene known as smn2
46:43
but if we recall to the previous mechanism that was described smn2
46:48
actually has this red flag that tells the process processing Machinery to
46:55
splice or cut out Exon 7 from the MRNA product so because of this smn2 normally does
47:03
not produce a full-length SMN protein so what spinraza does
47:09
is it is an ASO that goes in and binds to this red flag region and it allows
47:18
for the inclusion of Exon 7. which ultimately ends up translating
47:24
into a full-length SMN protein and so for patients who were treated
47:30
with spinraza um it was found that there was an
47:35
increase in survival probability for these individuals compared to those that
47:40
were not treated with in Raza and as a side note
47:46
we know that the development of these Therapeutics are multifaceted and are at
47:51
every level of the central dogma and currently in phase three clinical trials
47:57
um there’s actually a gene therapy to replace the smn1 Gene
48:05
and it’s it’s certainly in phase three clinical trials but we can see at the
48:11
MRNA level at the DNA level this development of Therapeutics is very multifaceted
48:19
and so in addition to this we have a foundation or there exists a foundation
48:26
called nlorum that looks into these incredibly rare Nano rare genetic
48:34
diseases and they develop Asos specifically for these
48:39
and this is also coupled with um a an ASO
48:46
for Angelman syndrome um recall our previous discussion about the natural anti-sense transcript that
48:54
suppresses the expression of the paternal ub3 allele
49:00
um all of this is very strong evidence as to how Asos are becoming a very
49:06
promising therapeutic technique in not only mdds and autism spectrum disorders
49:12
but also for genetic diseases as a whole
49:18
so moving on to the protein level we have small molecule drugs
49:24
and more molecule drugs are small molecules that have low molecular weight
49:32
and examples of these can include insulin aspirin and antibiotics
49:39
and many small molecule drugs are actually able to be taken orally
49:45
and once they are taken they will bind to the Target proteins
49:52
to produce a response from that protein’s pathway
49:58
and one example that Lilia briefly mentioned earlier is a target called row
50:04
a and row a is involved in the formation of the cell cytoskeleton structure as
50:13
well as cell motility and it’s been found to be upregulated in
50:19
some ASB models while being down regulated in others and a haplo insufficiency of another
50:27
autism risk Gene colon 3 or coal 3 has been found to result in an up regulation
50:34
of row a expression and as a result of this it was found
50:40
that within neurons there is a decrease in dendritic growth as well as network
50:46
activity and within our own lab what we found was
50:53
treating neurons that were derived from Mouse with this colon 3 haploin
51:00
sufficiency with a rose thin or I’m sorry with a row
51:05
a inhibitor um we were able to successfully uh restore the dendritic links as well as
51:14
rescue the neuronal network activity
51:19
and recall our previous conversation about Rett syndrome as well
51:26
um at this small molecule level very very recently actually there’s a
51:33
drug called trophenotide that was FDA approved and it’s a small molecule drug
51:40
that targets the igf-1 receptor and it
51:45
resulted in improvements of clinical scores which include communication
51:50
ambulation and use seizures attentiveness eye
51:57
contact and breathing and although this doesn’t Target
52:02
mecp2 directly it provides kind of an alleviation of the downstream symptoms
52:10
yeah and so I really want to wrap up with a discussion about the how we
52:16
actually deliver all of these Therapeutics and
52:21
um all of the Therapeutics we’ve discussed today have been commonly delivered through viruses
52:28
and these viruses include lentiviruses adenoviruses and adeno-associated
52:35
viruses for aads and all of these have their advantages and disadvantages based
52:43
on differences in inflammation risk packaging size or risk of genome
52:50
integration but in addition to this we have an obstacle that we need to
52:58
overcome and this is the Obstacle of tissue specificity
53:03
because many different or many differentially expressed genes that are implicated in ndds are located in brain
53:12
tissue and so we really do need to be able to penetrate the blood-brain barrier or um
53:19
are in order to have a safe clinical translation of our Therapeutics
53:25
because some current methods include invasive direct brain injections or the
53:32
neurotoxic disruption of the blood-brain barrier so some very promising and interesting
53:41
Innovations to tackle this issue include a different stereotype of aav9
53:48
and this av-9 was actually the vector that was used in the FMA phase 3 trial
53:56
that I mentioned earlier where the trials that delivered the sma-1
54:02
transgene intravenously so right into the circulatory system
54:07
and they found that they were able to rescue motor function in patients using
54:13
aav9 stereotype intravenously another promising Vector is this aad
54:23
cpp16 variant and this variant as an engineered capsid
54:29
to allow for better blood-brain barrier Crossing and it was found that there was greater
54:36
transduction efficiency of the cells compared to just a regular aad
54:42
and in addition to this this is really cool there’s High neuron specificity as
54:49
opposed to other cells and finally we have these
54:54
neurotransmitter derived organoids I’m sorry lipidoids and um they’re basically
55:00
a lipid or fatty barrier around these Therapeutics and
55:06
um Studies have done uh to show that um the neurotransmitter derived lipidoids
55:13
were successfully able to deliver Asos small molecule drugs as well as Fusion
55:20
proteins into the mouse intravenously and so to conclude
55:28
um this identification based Gene research for ASD really did set a very
55:35
strong foundation for the translation into clinical Therapeutics
55:41
there’s also a lot of promise with current studies making progress at all
55:47
three different levels of the central dogma and these gaps in clinical translation
55:54
research are also being filled at an incredible rate because these scientists
56:00
are developing these Innovative therapeutic Delivery Systems to tackle these obstacles associated with ASB and
56:08
ndds and finally these molecular Innovations are only
56:14
beneficial to these developmental disorders as a whole but they contribute
56:20
to um human disease research because we can apply them to cancers or
56:28
neurodegenerative disorders thank you
56:35
well that was great thank you so much of course yeah I wonder if anybody has any
56:42
questions I don’t see any on Facebook but please put them in the Q a if anybody does let’s see Hans has got his
56:50
hand rings I’m going to promote him he’s one of our parents that is also a
56:55
doctor um let’s see
57:01
On Come On In
57:07
there he is hi Helen hi thank you yeah it just rejoined as a panelist
57:13
um that was fantastic thank you very much I feel like we got two awesome
57:18
webinars in in a single hour one on you know the range of Therapeutics and one
57:23
on basic science translational science of autism and this question is for Dr Aya
57:30
kachiva has there been an effort to correlate the organoid findings to let’s
57:38
say the post-mortem findings the the brain structural findings it’s it’s challenging and you have to plan right
57:46
but we have about 10 years now since the New England Journal paper on postmortem
57:51
autism brain demonstrating disorganized disorganized cortical
57:57
Foci so are you guys starting to tease
58:03
out what you see in an organoid to what happens in the brain structurally down
58:09
the road yeah this is an excellent question
58:15
so um organoids that we produce do not have
58:22
actually a specific layers you know that clearly defined layers
58:29
you know complementary to the layers of the human brain
58:35
however having said that we definitely could see
58:41
not not at the structural level but at least at the maturation level that there
58:49
are some you know um defects in the neurogenesis we could
58:55
see this from organoids so for example we could see that
59:02
um in in one of our studies we saw accelerated neurogenesis where we see that organoids with a specific mutation
59:09
are producing more neurons than control organoids and this is due to you know
59:17
increased proliferation or migration specifically so so we can basically we
59:24
don’t see the structural disorganization but we see the disorganization of
59:31
neuronal migration is a neurons more neurons are produced of certain types
59:37
that are not supposed to be produced or they migrate or not migrate there is
59:44
slowed neuronal migration for example in some of mutations that we see so it’s
59:51
it’s it’s not really structural defects but it’s a g Factor neurogenesis that we
59:56
think you know recapitulate the neurogenesis defects in the human brain
1:00:03
so so you see different characteristics in the organoids that reflect
1:00:10
pathology let’s say in humans yeah absolutely we can kind of correlate that
1:00:17
but but not as a structural level and and that makes sense that that
1:00:23
really does I mean these are primitive brains right they don’t they don’t have the fully developed layers as
1:00:30
you just explained um you know a lot of these proteins particularly syngap has multiple
1:00:37
functions multiple isoforms so are you guys
1:00:43
making sort of single changes and seeing the effect on an organoid sort of doing
1:00:49
you know that deductive work because this is facing you know
1:00:58
Us in trying to tease out some of the basic science of syngap and then the
1:01:03
protein itself with multiple isoforms of pdz binding domain at the at the seat
1:01:09
terminal so is this something you’re doing in the the 16
1:01:16
dot yes so actually we are not introducing those
1:01:22
mutations artificially well we do that too but what we are doing we are
1:01:28
actually extracting right the human skin so basically it is organoids should contain
1:01:36
exactly the same isoforms it’s a patient derived tissue right they are not
1:01:41
artificially introducing mutations this is exactly mutation that are in the
1:01:46
patient so if in your patient you know there is a mutation that the fact one
1:01:52
out of three isoforms and by producing organoids from the genetic material of
1:01:58
the patient we also you know recapitulation that that this mutation
1:02:04
effects on the one out of three isoforms so basically it’s you know it’s it’s
1:02:11
exactly what is supposed to go in the patient is also going on in biological
1:02:18
in in organoids that we are producing because they are produced from the from
1:02:23
the patients is a fibroblast or patient cells right of course there there is
1:02:28
also possibility to introduce the mutations or to correct the mutations even better from the patient so if if
1:02:36
you know if you imagine that you know we got a tissue from the patient and produce organoids we can also use crispr
1:02:43
to remove the mutation right and use the control control organoid as our control
1:02:51
to get deeper into those pathways yeah and that’s in fact you can introduce
1:02:57
mutation we can take you know my skin and introduce mutations because it’s isogenic models
1:03:04
um or introduce different mutations but it would be a little further from the patient because it will have different
1:03:10
genetic background because genetically what we know it is genetic background of the patient actually matters a lot so so
1:03:18
the advantage of those patient-derived organoids is that that they recapitulate
1:03:24
boss the exact mutations with patient carries as well as all the Lord of other
1:03:30
mutations all the genetic background of the patient so this is the advantages of
1:03:36
this patient-derived models yeah and and in fact you know some of
1:03:41
the organoid work done in syngap did use isagenic controls you know crispr corrected from the patient uh as a
1:03:48
control you know and be able to demonstrate then a defect in radial uh I
1:03:54
think it was glial cells that you know are different so yeah it’s it’s a fascinating Frontier
1:04:00
for these organoids to tease out the biology thank you so much yes
1:04:10
cool um I think oh unless anybody wants to
1:04:15
put anything in the Q a um I think everybody a little bit dropped
1:04:22
off so it was great we didn’t need any questions it gets covered everything
1:04:27
so it was good all right Olga did you um no this was wonderful
1:04:34
um very informative um I think we probably just went a little
1:04:40
bit over the time so we did lose a few people but that’s that’s okay it was all very important information so um thank
1:04:46
you both so much for taking the time to present and
1:04:51
um we look forward to following your work thank you
1:04:56
well thank you so much for inviting us and um thank you for for listening to