Modeling Neuroepigenetic Disorders In 2D And 3D Neurons
March 30, 2023Information
- ID
- 9778
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Transcript
- 00:00It's my pleasure to
- 00:02introduce Doctor Yang,
- 00:05who is received this MD from Shanghai
- 00:09Medical College of Punan University and
- 00:12his PhD at Baylor College of Medicine.
- 00:15And he completed his pediatric residency
- 00:18and Clinical Genetics Fellowship at
- 00:20Texas Children's Hospital at Baylor,
- 00:22at Baylor and then joined the Yale
- 00:25Department of Genetics in 2019.
- 00:27His research focuses are the discovery of
- 00:30rare diseases and modeling neurogenetics
- 00:33and neuro epigenetic disorders using
- 00:35human derived ipscs and mutant mice.
- 00:48Thank you. All right.
- 00:57Well, thank you for and hyphen
- 00:59for organize this event.
- 01:01Also for the invitation,
- 01:02I shoot the clothes.
- 01:04I'm not stem cell biologist,
- 01:06I'm not biomedical engineer either.
- 01:09So while I'm here,
- 01:13sorry, how do I convince?
- 01:17So how do we get interesting for the
- 01:20bring organize IPSC and 2DS3 neuron?
- 01:24So I'm kind of called genetics. I see.
- 01:26The patient in the clinical. Sorry
- 01:30for real genetic disorder
- 01:31and then back to the lab.
- 01:33Wow, that didn't work. Sorry.
- 01:40Go one more time.
- 01:45Oh, OK. Thank you. That means we
- 01:47need every step together, right? So.
- 01:52Next lab, we can try modeling the
- 01:55genetic new epigenetic primary aspect
- 01:57of brain development in the lab,
- 01:59primarily using the Moss model
- 02:01or other animal model,
- 02:03understanding the function of gene,
- 02:05understanding the disease mechanism,
- 02:07passive Physiology as a physician,
- 02:09definitely interesting develop
- 02:10a treatment back to the family.
- 02:13However, as probably I would say
- 02:17almost all the success we learn
- 02:19primary from the brain from the mice.
- 02:22Did not translate well come to the
- 02:25human we fail miserably for many,
- 02:27many of the successful exciting
- 02:30story for mice.
- 02:31So that's what we're asking could
- 02:34we kind of study for the IPSC,
- 02:37derive patient, derive 2DS3 neuron,
- 02:39feel this gap, but just this challenge.
- 02:42So I'm going to give you two
- 02:44example in in my lab,
- 02:45probably focus on the
- 02:47first one the time I say.
- 02:49So I hope you feel these two disorder
- 02:52are very interesting in general.
- 02:53So this is Endrum syndrome which is
- 02:56oftentimes many syndrome named by physician,
- 02:58the first recognized name endrum,
- 03:02it's a very classical severe end of
- 03:04the new developmental disorder IQ,
- 03:06it's very low IQ like 20,
- 03:08they don't speech at all,
- 03:09don't have any speech.
- 03:11More challenges and they have
- 03:13very severe epilepsy and almost
- 03:161/3 is medically intractable.
- 03:18It's very devastating to the family.
- 03:21It's very interesting molecular basis,
- 03:24a primary genetic defect IS15Q11Q13
- 03:28and matured patient have a 15Q11Q13
- 03:31deletion cross this region,
- 03:34but interestingly because
- 03:35it's imprinting related.
- 03:37So the paternal delition delition
- 03:39come from paternal chromosome
- 03:41caused a complete separate syndrome
- 03:43called the pero alloy and with
- 03:45the delition come from mother
- 03:47maternal alloy cause the end German.
- 03:50So over the time we know the gene
- 03:53response for this larger delition
- 03:55it's ubiquitin protein like this 3A.
- 03:58As more interesting this region is,
- 04:00we also know maternal duplication
- 04:03only maternal duplication from mother.
- 04:06Costs about 1 to 2% in the Ed Pass
- 04:09autism but not paternal so you can
- 04:11see it's very very interesting.
- 04:13If a duplication for father
- 04:15relatively normal
- 04:18so. So with with over the time we
- 04:22learned this is more complex sort of
- 04:24a genetic epigenetic defect majority
- 04:26of the logic deletion we have a poor
- 04:28mutation in the maternal chromosome you
- 04:30put in like H3A we have another two
- 04:33class of a where kind of uniprantal
- 04:35dysomy 2 comes come from same parents
- 04:37and of imprinting defect that's also
- 04:40small number for case color you B3
- 04:43gainer function contributor for autism.
- 04:47So now you put in like a G is an open.
- 04:51H3A some of you very familiarly percolation
- 04:53pathway and the most interesting to
- 04:55us is this is epigenetic phenomenon
- 04:57kind of imprinting the expression
- 04:59for the gene in the next generation.
- 05:01It's depend where this come from.
- 05:03So for the ENDROOM gene actually it's
- 05:06very interesting which is first kind
- 05:09of new specific imprinting gene in non
- 05:11neuro both Gene Express both earlier express.
- 05:14In the neuron in the brain only
- 05:17maternal allele expressed.
- 05:19So that's the how interesting this
- 05:21phenomenon is and over the time we
- 05:23and many other understanding for the
- 05:26mechanism how could this they cell
- 05:28type specific as a less specific
- 05:31infinite phenomena happen.
- 05:32It's actually due to a very very long,
- 05:34almost mega based long non coding a RNA.
- 05:38Are part of what we called also antisense
- 05:40for the UB3 gene only expressed from
- 05:43paternal chromosome then silence for
- 05:46the sense on the paternal console.
- 05:48So that's the mechanism and we also
- 05:51generate a many Moss model over
- 05:53the time to study this mechanism.
- 05:55Overall Moss model provide many many
- 05:58valuable insight however that's.
- 06:02Also the capitulate a lot of the human
- 06:05phenotype reasonable well especially
- 06:07we're interesting for the epilepsy
- 06:10or abnormal EEG.
- 06:11However then we take this one on try
- 06:14understanding it because as you know
- 06:15one set of patient have no control for
- 06:18the seizure or lifetime which is very,
- 06:20very challenging.
- 06:21So we try understanding,
- 06:22use the muscle model industry
- 06:24understanding why this epilepsy so
- 06:27common just highlight one phenomenon
- 06:29we use this is very specific.
- 06:31To Physiology phenomena measure the
- 06:34action potential with particular
- 06:36folks on fast component after
- 06:39hyperpolar polarization.
- 06:40We realized in this particular engine
- 06:42mouse model in the brain and neuron
- 06:45this FHP is increased and we have done
- 06:47a lot of work using the biochemical
- 06:50molecular and linked it to the Ek channel.
- 06:53It's enhanced function for BK
- 06:56channel contribute this phenomena.
- 06:58Then we also show this link to indeed
- 07:00in link to the epilepsy in the mice,
- 07:02which is you can use the antagonist
- 07:05Hassel and can reduce the amplitude
- 07:08and the frequency eventually suppress
- 07:11the seizure in the mice.
- 07:13So that's all good.
- 07:14The question come to whether this
- 07:16is translated between the human
- 07:18so that come to what we got into
- 07:21the IPSC 2D and three you are.
- 07:24That's one time I moved to the
- 07:26Yale and then I realized the world
- 07:28class of the stem cell center.
- 07:30So I talked to Hifi and India and the child
- 07:33home and say hey why we just do this
- 07:36create a repository for the Andrew IPIC.
- 07:40So luckily we got a very generous support
- 07:43for the fast foundation for Andrew
- 07:45and therapeutic they give very general
- 07:48support and right before the COVID.
- 07:50So over the last three years
- 07:51even during the COVID,
- 07:52we were able to generate.
- 07:55One day cell line IPS IPSC cell line from
- 07:59different genotype including the control.
- 08:01This is free to everyone here.
- 08:02If you're interested you can just e-mail me
- 08:05are free to distribute it to each of you.
- 08:07So that allow us to really ask the
- 08:10question whether phenomenon we study from
- 08:12mice is the translator from the mice.
- 08:15So I'll just give you a few slide and
- 08:17the summers are published already.
- 08:18And then to shows the phenomena,
- 08:21same phenomena we observed from
- 08:23the mice which you can see the FHHP
- 08:25which indeed in IPSC the wife the
- 08:282D new one in the cortical new one
- 08:30in this is enhanced the two.
- 08:32So that's increased the frequency
- 08:35suggestion the hyperexcitable new
- 08:36one and then we all can can use
- 08:39in the sort of rescue indeed it
- 08:41shows that deficiency UPC is a
- 08:43responsibly electrophysiology.
- 08:44Phenomena.
- 08:44Now we got to choose also biochemical assay.
- 08:49Indeed it's correlated for the BK
- 08:52channel function in the 2D neuron.
- 08:55Now you can show also the
- 08:57paxilance same as in vivo,
- 08:58in the mice can suppress
- 09:00this hyperexatibility,
- 09:01but in the 2D neuron,
- 09:05now we move on together.
- 09:13That's we cannot advance. So that's
- 09:21right. All right. Maybe I did
- 09:23something which I should not do.
- 09:28So then when you organize 3D neurons, it
- 09:30shows very similar discovery of funding to.
- 09:33Yeah, same thing, the increase the.
- 09:39Frequency then the pass and
- 09:41suppress the hyperexcitability.
- 09:43Of course the question will come
- 09:45to whether you can recapitulate
- 09:46epileptic form in the brain organelle.
- 09:49That's question we still have not
- 09:51get into that very very very detail.
- 09:53So that's all looks good because we
- 09:55can study the mice to translate the
- 09:57human allow us to the confidence
- 09:59maybe indeed we can allow these
- 10:01two system to testing additional.
- 10:04Particular for treatment strategy.
- 10:06So one of the thing we are working on
- 10:09right now is to try to using this strategy.
- 10:12As I told you,
- 10:13I could not get into the very detail.
- 10:15So the mechanism regulate this
- 10:17imprinting is due to the antisense
- 10:20Bay long megabase continuous
- 10:22antisense long line coding RNA.
- 10:24So the one strategy is if we can
- 10:27disrupt this long line coding RNA
- 10:29and then you can reactivate the gene.
- 10:31On matpat and chromosome is supposed
- 10:33as like a gene therapy can approach.
- 10:36So the ASO has proved it's effective and
- 10:40it's also in the phase one trial right now.
- 10:42So we think about it with whether
- 10:44we should do a more permanent fix.
- 10:46The ASO we need every month sort of
- 10:48the eye spinal injection and now we
- 10:51work on this with CRISPR additive.
- 10:55So working with collaborative with the.
- 10:58People from the biomedical engineer as
- 11:01deliver crisp to brain is a challenge.
- 11:03We actually got this piece of very
- 11:05exciting data by our AIS in the lab
- 11:08shall now shows you we use this
- 11:11chemical modified I MP conjugated
- 11:13CAS 9 protein and and gala and
- 11:16they together deliver IT intracego
- 11:18injection which you can see.
- 11:20Amazingly this deliver the editing in
- 11:22the cortical neuro and cerebellum.
- 11:25Which you can see this a new M cell,
- 11:27then this green cell,
- 11:29it's a reactivation after editing
- 11:32the anti sense.
- 11:33It's almost like 70% efficiency for
- 11:36this coach similar to the cortico
- 11:39that is pretty amazing.
- 11:41So we feel like this will be the next step.
- 11:43We are watching actually active working
- 11:45on the 2D and the 3D neuron right now,
- 11:47see if for the same delivery it's
- 11:50effective if entry before we
- 11:52go to FDI&D and to the human.
- 11:55So I'm gonna switch the GAIL
- 11:58for second disorder,
- 12:00which I hope that you will
- 12:01find also very interesting.
- 12:03In the same scene I could
- 12:05genetically related disorder.
- 12:06So this is a patient I saw about four
- 12:08or five years ago in the clinic.
- 12:10It's a very similar to engerman but
- 12:12that's definitely severity it's 11
- 12:14mile and moderated compare engerman
- 12:15and to me it's a severe and and this
- 12:18is a moderate and with autism as
- 12:20a predominant feature intellectual
- 12:22disability interesting they have
- 12:24macrosuppony which the big brain is
- 12:26the bigger they have a low percentage
- 12:28of low frequent preference of epilepsy
- 12:30too and so so this is the the boy
- 12:34and what he was nine years old and then.
- 12:37Later on then was to find interesting
- 12:40another end of a phenotype.
- 12:42This is the
- 12:44sort of a longitudinal sort of picture
- 12:48from the infant to when he was thirty.
- 12:52I hope you probably say okay,
- 12:53that's probably not 30 years old face,
- 12:57it's probably more old than that.
- 12:58So that's a premature Asian phenotype
- 13:00that's we think it's also the other end.
- 13:02It's very interesting.
- 13:03That's a delay early new
- 13:06development somehow later on.
- 13:07Is actually accelerated aging process
- 13:11so that's clear a puzzle and and then
- 13:13we're looking for the genetic we we
- 13:15did a whole accident in the clinic we
- 13:17identified the first mutation is patient
- 13:19other colleague from UK also similar
- 13:23time 2017 2018 report a few other
- 13:26case and eventually last five years
- 13:28will accumulate almost 100 case now.
- 13:31So what you notice quickly matured
- 13:33for mutation in the C terminal domain.
- 13:37And majority for them it's
- 13:39A-frame shift mutation.
- 13:40What's more interesting when you
- 13:42do the computational prediction,
- 13:44the open reading frame,
- 13:45actually it's quite interesting,
- 13:47you can see here if you do the open
- 13:51reading from open reading from prediction,
- 13:54regardless where the mutation
- 13:57location near all and very very same
- 14:00tail about for the amino acid at N.
- 14:04So that's to me is a little unusual.
- 14:07I'm a geneticist,
- 14:08I see a lot of patient database.
- 14:10I have not seen this kind of
- 14:12phenomenon very often.
- 14:13If it happen one of you,
- 14:14you have some case like this talking
- 14:16to me and we kind of working
- 14:18together to figure out this puzzle.
- 14:20Interestingly we also generate and
- 14:23antibody specifically against this tail.
- 14:26The antibody actually very easy
- 14:28to generate because this tail if
- 14:30you against the genome or podium
- 14:32actually pretty unique.
- 14:33So allow you to very quickly
- 14:35generate this antibody.
- 14:36Now you're testing the patient IP.
- 14:38At the same time we generate about a
- 14:41IPSC cell line from this patient too.
- 14:45So you can see this IPSC cell
- 14:47line and this abnormal tail,
- 14:50it's indeed it's stable.
- 14:52So that raised the question
- 14:54whether this abnormal tail.
- 14:56It's actually gain of function or
- 14:58dominant net function because when
- 14:59you now call this gene in the mice,
- 15:01they have no significant phenotype.
- 15:04But in the habazygs in the human,
- 15:06it's very definitely a very severe phenotype.
- 15:09So that we interesting also this
- 15:11is same mutation or same mutation
- 15:13in the mouse gene or mouse gene,
- 15:16you won't be able to create the same tail.
- 15:19So it's only to the human coating.
- 15:22So that create a little challenge
- 15:24to to manipulate in the mice but
- 15:26of course we can't do it.
- 15:28We made in the humanized mouse model
- 15:30by engineering the entire human gene
- 15:32in the marketing of the car knocking
- 15:35and replace the anti mouse genome.
- 15:37So that's ongoing.
- 15:39We're just talking to people outside
- 15:42like it's a mouse have azyg have
- 15:44mild phenol type homozygic actually
- 15:46end the post Natal early so.
- 15:48So,
- 15:48but in any way so we will say OK
- 15:50that would be good to kind of look a
- 15:52little more this is a very definitely
- 15:54because my title is a new epigenetic.
- 15:56So it's a, it's a H1 link protein
- 15:59as you know probably have about
- 16:0011 H one link some of the somatic
- 16:03form is one of five somatic form.
- 16:06So this H1 link and the function
- 16:08for H1 link in largely we still
- 16:10don't know because over the last
- 16:1325 years lot of people study cold.
- 16:15For histone, very, very detailed,
- 16:17but this is almost like a forgotten
- 16:19histone for over the last decade.
- 16:21But now you can come to interesting for
- 16:24many people because the human disease link.
- 16:26So again it's a link histone with
- 16:29the link DNA and it's core histone.
- 16:31Presumably function is making the
- 16:34chromity more compact but it's a
- 16:36very basic component of the histone,
- 16:39but the link to very selectively
- 16:41human neuro behaviour and neuro
- 16:44developmental phenotype.
- 16:45So, so that's what we would generate
- 16:47a panel of an IPSL again,
- 16:49again a child home and in yon.
- 16:51And for this effort,
- 16:53sometimes people ask and say
- 16:55why you didn't do it at Duke.
- 16:57And I would say, well,
- 16:58yeah, we want to do at Duke,
- 17:00but we don't have a facility like here.
- 17:03I find I left two years ago
- 17:04before I joined the Duke.
- 17:06But that's what we lost high
- 17:07final deal and now we don't have
- 17:09the stem cell facility there.
- 17:10So that's what's asking you should have
- 17:13taken advantage of for we have indeed
- 17:15excellent facility environment here.
- 17:17So again remind you this phenotype
- 17:20patient have a macrocell,
- 17:21a big cell,
- 17:22a big brain.
- 17:23So you look at the cell proliferation
- 17:26indeed somehow suggesting that
- 17:28maybe correlate the human phenotype
- 17:30they are proliferated faster.
- 17:32Then both in IPSA and MPSC new one early
- 17:37precursor and we also did it for the I,
- 17:39I and they seek and looking for.
- 17:42Whether it's a chromatin structure
- 17:44affect the downstream transcription
- 17:46indeed is when you have this mutation
- 17:49chromatin sounds like more relaxing.
- 17:51So more gene up regulated and
- 17:53surprising to us it's you actually
- 17:56see the set of a gene regulated,
- 17:58it's actually still in the
- 18:00chromatin related gene.
- 18:01Many ask actually chromatin related gene.
- 18:03So we are very,
- 18:05very intriguing by this founding.
- 18:07We also look at because this is a
- 18:09chromatin structural later protein,
- 18:10we asking whether that's actually
- 18:13indeed affect the chromatin nuclear
- 18:15morphology or chromatin structure.
- 18:17So we did a EM for this IPIC and
- 18:20and the neuron we look in the
- 18:22morphology of the nucleolo and
- 18:24the nucleus indeed that's alter
- 18:25the morphology from the nucleus.
- 18:28So indeed because the unique
- 18:30this protein alteration which
- 18:32are not mimicking in the mice.
- 18:35So that I PRC derived 2D and 3D new
- 18:38one actually indeed it's the one way
- 18:41to to do more sort of investigation.
- 18:43I do have time to show you the
- 18:45organized other data on different time
- 18:47and almost close perfect time there.
- 18:49So I hope that by using the two
- 18:52example illustrated 21 is that
- 18:54you don't have to be the stem
- 18:56cell biologist to study stem cell work.
- 18:59Second is I hope you also.
- 19:02Data from if you study mice
- 19:03actually indeed it would be good to
- 19:06translate what your mouse discovery
- 19:07of funding from mutant mice to one
- 19:09more step between the human device,
- 19:12this neuron before moving to the
- 19:15we call it FDI&D study because
- 19:18we are fail many many occasion,
- 19:22many example everything is
- 19:23beautiful in the mice,
- 19:25it's a fail in the human stage.
- 19:28So large of the people in the lab
- 19:31primarily I think the people Kung
- 19:33Yun is the primary thing is now here
- 19:36working on the andrewman IPSC and
- 19:39IPSC organelle and then so nice data
- 19:42from the mouse for the IP and then
- 19:45a few other kind of also work in the
- 19:47similar sort of standing project.
- 19:49And then of course without your stem
- 19:53cell center support this kind of large
- 19:55scale production will not be the feasible.
- 19:58And all the support I've received and
- 20:00also the collaboration with Duke and
- 20:03the EOS for the engine work together.
- 20:05Thank you.