63 – Targeting alternative splicing of SYNGAP1 using antisense oligonucleotides

Here are our introductory comments: 

Dr. Benjamin Prosser is an Associate Professor of Physiology at the University of Pennsylvania Perelman School of Medicine.  He earned his Ph.D. in Molecular Medicine from the University of Maryland School of Medicine in 2009 and started his own lab at Penn Medicine in 2014, with an initial focus on developing novel therapeutics for heart failure.  In recognition of the lab’s cardiac work, Dr. Prosser was named the Outstanding Early Career Investigator by the American Heart Association in 2017 and received the Outstanding Investigator Award by the International Society of Heart Research in 2022.  In 2018, Dr. Prosser’s daughter Lucy was born and diagnosed with STXBP1 encephalopathy, a rare, genetic neurodevelopmental disorder.  Dr. Prosser started a separate research arm in his lab focused on developing new therapies for STXBP1 and related disorders. With Dr. Beverly Davidson at Children’s Hospital of Philadelphia he has organized the ENDD (Epilepsy and Neurodevelopmental Disorders) therapeutics team, an interdisciplinary group of clinicians and scientists primarily focused on developing RNA-based therapies for STXBP1 and SYNGAP1 related disorders. 


Welcome and introduction to the work of Professor Ben Prosser

0:07hello everyone and welcome to today’s webinar my name is Olga Bodie and I’m a syngap parent

0:14uh and uh work with syngap research fund our talk today is targeting alternative

0:21splicing of syngap1 using Asos and I have the pleasure to introduce today’s

0:27speaker Dr Benjamin Prosser Dr Benjamin Professor is an associate

0:32professor of physiology at the University of Pennsylvania Pearlman School of Medicine he earned

0:38his PhD in molecular medicine from the University of Maryland school of medicine in 2009 and started his own

0:45Lab at Penn Medicine in 2014. with an initial focus on developing novel Therapeutics for heart failure and

0:53in recognition of the lab’s cardiac work Dr Prosser was named the outstanding early career investigator by the

1:00American Heart Association in 2017 and also received the outstanding investigator award by the international

1:06Society of heart research in 2022 in 2018 Dr Prosser’s daughter Lucy was

1:14born and diagnosed with STX bp1 encephalopathy a rare

1:21genetic neurodevelopmental disorder excuse me Dr Prosser started um a

1:27separate research arm in his lab focusing on developing new therapies for stxbp1 and Related Disorders and with Dr

1:35Beverly Davidson a Children’s Hospital of Philadelphia he has organized the end yeah that’s the epilepsy in

1:41neurodevelopmental disorders Therapeutics team an interdisciplinary group of clinicians and scientists

1:48primarily focused on developing rna-based therapies or sdx bp1 and

1:54syngap-1 Related Disorders um recorded version of this webinar will

2:01be available on the srf website under webinars on the family menu by the end of this presentation you will have the

2:07opportunity to get the answer to your questions and we’d love to hear from you please write your questions in the Q a below

2:15for those of you just joining us welcome and again our speaker is Dr Ben Prosser and his talk is excuse me

2:25uh targeting alternative splicing of syngap one using Asos

2:31it’s now my pleasure to turn things over to Dr presser hello can you guys all hear me and seeTargeting alternative splicing of SYNGAP1 using antisense oligonucleotides

2:38my slide and my laser pointer okay we are good to go all right well good morning and good

2:45afternoon good evening wherever you are everyone um it’s a pleasure to be with you guys today to talk a little bit about our

2:50efforts trying to treat syngak disorders using antisense oligonucleotides or Asos

2:57so first it’s a bit of a talk outline I’m going to start with some background on on why I study neurodevelopmental

3:04disorders I’m going to talk a bit conceptually just about how we think about treating

3:10um syngap one loss of function or other Related Disorders we’ll do a little bit of overview on RNA

3:16splicing as this is what some of our therapies are targeting and then the bulk of today’s talk will

3:24um uh be discussing some recent results uh part of which have been uh deposited

3:29on by archive as a preprint around how Asos uh that Target alternative splicing

3:36of syngap could be used to increase its levels and and sort of full disclosure

3:41this is a rather dense rather technical paper even for senior scientists I’ve

3:46designed this presentation today to be a geared towards an informed

3:53patient audience but we will indeed get into the uh into the data a bit and

4:00occasionally I may throw in some technical details for any scientists on the call as well and happy to go into any level of detail in the Q a

4:07afterwards and at the end I’ll chat just a little bit more about some of the specifics I think for for the parents

4:13and families to keep in mind thinking about what an ASO therapy might look likeWhy I study neurodevelopmental disorders

4:19all right so I like to start with this because these these sort of how it started how it’s going beams um are on

4:25social media or at least they were like two years ago maybe I’m not sure if they’re still cool or not but this is mine um I started my lab in Penn in in

4:322014 and for the first five years as Olga referred to we studied really the mechanics and molecular biology of the

4:40heart um and this was going well and then um uh as I guess they often do

4:47um my child decided to shake things up and and these days about half of my lab is studying the circuits of the brain

4:53and genetic therapies for neurodevelopmental disorders which is very different than the part of course

4:59and and this is not a good career move and if if you can avoid it

5:05um for me I couldn’t because in May of 2018 um my amazing daughter Lucy was born she

5:12started having seizures a few days later Dan was pretty quickly diagnosed with STX bp1 encephalopathy a

5:19neurodevelopmental disorder that presents somewhat similarly to singap it was a very tough first year for Lou

5:25eventually we got her seizures under control and now she is uh she is a

5:30largely a happy kid as hopefully you can appreciate here from these photos and of

5:36course her happiness is sort of Priority One but we want more for our kids and they deserve more and so they started

5:42working on her disorder in my own lab and and these are actually Lucy’s neurons firing on the right hand side

5:48here I remember watching this experiment oddly there’s these neurons that are

5:54harboring my daughter’s own genetic code lighting up under a microscope in my lab and I was a bit surreal I remember

6:01thinking that that college Ben really didn’t see that coming I suppose

6:06um but this is where we are now and and I want to leverage my somewhat unique position as a parent scientist to try to

6:13do some good in this community very early on in our efforts we also met

6:21Keeley who was a singampian who was being seen by our neurologists at

6:26Children’s Hospital of Philadelphia Dr Ingo how big and Kaylee’s mom Becky and I we really

6:31instantly connected on our desire to try to help these kids and how we wanted to

6:37go about doing that and since then we’ve been pursuing parallel approaches to develop treatments for both stxbp1 and

6:44syngap disorders and over these last couple of years I’ve really had nothing but great interactions with many in the srf

6:51community including families scientists affected kids and I really do feel like it’s a privilege to be able to work in

6:59this space okay so our goal of course is to cureHow could we treat SYNGAP1 loss-of-function?

7:04these genetic disorders and to do that we first have to understand the root cause and so all of us wild types would

7:12like to refer to us on the call have two good copies of the syngap gene each copy

7:18of DNA can be transcribed into RNA which is then translated into Protein that’s

7:25what we refer to as central dogma of Cell Biology and it’s the syngap-1 protein that

7:30actually does its job in the cell now in our affected kids they have a

7:36mutation in one of the two copies of this Gene and depending on where that mutation lies it can disrupt how that

7:42DNA is transcribed into RNA how the RNA is made into protein the net result is

7:47you’re not getting enough protein in the cell to do its job and this is what we refer to as syngap-1 loss of function

7:56so how do we try to treat this well you can imagine at least three different sort of genetically oriented strategies

8:03to address this first you could try to edit the mutant allele you could use

8:09crispr or its ever evolving toolbox to try to fix um this this root cause you could try to

8:17add extra copies of the syngap-1 DNA into the neuron although this is a

8:23challenge given that syngaporn is a very large Gene or you can also try to get the normal

8:29allele to make more protein by bolstering either transcription or

8:34translation and this boosting of the normal remaining good copy of the gene is what I’m going to focus on a bit

8:40today so the team that we’ve put together at

8:46Penn and at shop which are sort of side by side in Philadelphia is pursuing several of these different approaches

8:53um even though I’m just going to talk really about ASOS A bit today and we’re called the end team for epilepsy and

9:00neurodevelopmental disorders but I’m really thankful um that I get to work with this the

9:07brilliant and and caring group of of scientists and clinicians and and I’m quite proud of the team that we’ve

9:12assembled as outside of Dr Helbig here I don’t think any of us were really working on syngap or stxbp1 three years

9:18ago and now this group is really dedicated and engaged and I think that that also

9:25sort of reflects the broader acceleration in research and therapeutic development into these disorders

9:31um that we’re seeing across the board that has me um uh very optimisticOverview of RNA splicing

9:38okay so let’s talk about Asos and RNA based Therapeutics this requires a

9:45slightly more detailed look into the life of an RNA molecule so we start with double stranded DNA

9:51chromosomal DNA which can be then transcribed into a single stranded RNA

9:57but RNA is not transcribed in its fully mature form it starts as this pre-rna

10:04that then has to be spliced together so these regions called exons shown in

10:10green which contain the actual code for making proteins they get spliced together and these leftover genomic

10:18regions called introns shown in brown they get spliced out together this makes

10:23a mature messenger RNA and it’s that mature mRNA code that actually gets read

10:30out and translated into protein but not all mRNA is created equally

10:37commonly the same pre-rna can be spliced into multiple different mrnas that can

10:44contain different components and this is the process that we call alternative splicing

10:50and often these different rnas they can code for slightly different but still very functional proteins and this is

10:57true for singap one right it’s pre-rna can be spliced to multiple different

11:02forms to make slightly different proteins with slightly unique functions in different neurons

11:08so this alternative splicing it’s not necessarily negative it’s a useful it’s an evolutionarily conserved process that

11:15helps create necessary neuronal diversity but sometimes certain genomic elements

11:22that should have been spliced out are not such as this retained intron here or

11:29what’s called a poison Exon here and when the MRNA contains these

11:34particular elements the cellular Machinery can recognize these defects and degrade that RNA

11:40instead of using it productively to make protein so you can imagine that if we could

11:48redirect this splicing if we could send more RNA down this productive pathway we

11:54might be able to get more protein out of the same gene

12:00okay so how do we Target and manipulate RNA and this is through anti-sensological nucleotides or Asos

12:06and so an ASO is this small chemically synthesized piece of RNA that’s designed

12:13to bind to a Target sequence on RNA molecules through classic Watson and

12:19Crick based pairing to manipulate their biology in some useful way now one advantage of this technology is

12:26very crudely shown here and that’s that an ASO is Tiny it’s about 20 nucleotides

12:32in length often it can vary a bit but that’s about 200 times smaller than the

12:37copy of syngap-1 DNA or for example the crispr tool that we would need to then

12:44package into a virus to try to deliver into neurons so unlike these other genetic therapies

12:51this this particular genetic material of the ASO can be packaged into a drug that

12:57can get into neurons without the help of a virus and this is one potential advantage that could speed its ability

13:04to translate to the clinic which we’ll return to laterHow do ASOs work?

13:09right so what do Asos do how do they work well again they manipulate RNA in various ways and I’m going to talk about

13:15just two sort of high level ways that they can do that today and so the first is what’s called a

13:22silencing ASO so if for example our therapeutic goal

13:28is to reduce the expression of a certain protein you can design an ASO with a

13:34certain chemistry so that it will bind to an mRNA and cause it to be cut up and

13:40degraded and so this type of ASO is is actually currently in clinical trials for the

13:46treatment for example of angelman’s syndrome which is another genetic form of a neurodevelopmental disorder it’s

13:53also in pre-clinical work for targeting certain rnas that could negatively

13:58regulate syngap and some of you may be familiar with the syngak antisense transcript

14:04now second type of ASO is what I’ll call a blocking ASO and this is what we’re

14:10going to use to modulate splicing so to modulate splicing we can use a

14:16different ASO chemistry and if we put that that that ASO

14:22right on the spot around say this element here that we don’t want we can

14:28redirect that splicing so that now you have less of this non-productive RNA and

14:36more of the productive mRNA that you want and so this is what we’ll call a

14:41splice switching ASO and and these have been shown to be clinically effective

14:46and have been FDA approved for example for the treatment of spinal muscle atrophy and they’re in clinical trials

14:52right now also for the treatment of Gervais syndrome another severe pediatric epilepsy

14:58so that’s where we want to get to um in the clinical Pipeline and I’m going to focus on this type of ASO today

15:07all right so so now I’m going to move into sort of the new data slides but just before I do that I want to introduce this this sort of data key and

15:14so in these these upcoming experiments we’re going to use seven several different research models and each has

15:21their own strengths each has their own limitations um we’ll use human cells that are not neurons for example because they’re very

15:28easy to work with with Asos we will use neurons that are generated from patients

15:34using stem sex stem cell Technologies we’ll use mice for some in Vivo studies

15:39and a lot of our work will be done in human cortical brain tissue that we’re

15:45very fortunate to have ready access to so as it can get a bit confusing on each data slide I’ll include one of these

15:51icons and sort of the bottom left hand corner so you know what type of material you’re working with and you can

15:56contextualize any results sort of accordingly okay so let’s start in in human brainIs SYNGAP1 alternatively spliced in human brain?

16:03and really the first question that we asked is is singap one alternatively spliced in

16:09human brain is there something there a signature that we could manipulate within ASO and so this is one way that

16:15we look at splicing of a gene this is called a Sashimi plot and this is generated from RNA sequencing from adult

16:23breed this is technically a bromine area 9 which is part of your frontal cortex so it’s RNA and the neurons right around

16:29here and I’m showing the displacing pattern for stxbp1 first just because it’s it’s

16:36simpler and easier to interpret so these little blue hashes down here

16:42vertical lines they Mark the exons and these are the RNA that’s being read at those exons these connecting regions are

16:49where the introns would have been and these lines that connect show where the splicing is occurring

16:56it it this is where you’re reading from one mature RNA uh from this Exon to the

17:04mature RNA in this Axon and this all looks correct right this Exon splice to this one this one to this

17:11one all the way through this chain nothing too complicated until we get to the end where you see the one bit of

17:18evidence of alternative splicing where here you can splice from this Exon to

17:24here or alternatively you could splice to what’s called this cassette Exon down

17:30here okay so that’s kind of how we look at alternative splicing now let’s look at single app single

17:37and hopefully you can sort of immediately appreciate that this appears more complex

17:43we have alternate transcription start sites towards the beginning of this Gene this creates what are called the

17:49n-terminal variants of syngap we have alternative splicing near the end of this Gene this creates what are called

17:55the c-terminal variance there’s also several introns that aren’t always getting properly spliced out which leads

18:01to some chatter around here this is a poison Exon back here but this little event right here is what

18:08really caught our eye there’s a lot of complex and interesting alternative splicing of syngap I’m going to focus in

18:14now on this particular region which we think may have some therapeutic tractionAlternative splicing of SYNGAP1 Exon 11

18:20so here we’re just zooming in on these exons 9 through 12 of syngap1 and each

18:28of these Peaks here that corresponds to the MRNA that we’re detecting in in human brains

18:34and again this looks good we see lots of mRNA where it should be at Exon 9 its places to 10 to 11 and to 12. what we

18:42also see is this little shoulder uh down here and that is an alternative splicing

18:48event on Exon 11. so what this tells us is that some

18:54percentage of the time instead of splicing Exon 10 to Exon 11

18:59together here there’s this new earlier splice site

19:05now this isn’t for every syngap one mRNA molecule this is happening at a

19:11frequency that sort of crudely proportional to the height of this little Peak relative to this peak so in

19:18other words this is in normal adults if in you and me for every 10 syngap mRNA

19:24molecules that we might have in our neurons maybe one or two has this alternative splicing on Exon 11.

19:33okay so what does this alternative splicing do well in this case it essentially renders this RNA useless in

19:40making protein and that’s because inside that portion of RNA right here marked in red is what

19:46we call a premature stop codon so when the Machinery that is reading

19:52through and turning that RNA into protein called the ribosome it’s reading along this RNA and it gets to this early

19:58stop that will stop making protein now this this is actually also what

20:04happens to sync up patients that have to some syngap patients that have a nonsense mutation or premature stop

20:10mutation they have a variant somewhere in the same Gap Gene not necessarily around here but that causes a premature

20:16stop to be included and this is why they don’t make enough syngap protein

20:22okay but again not to be confused this is not a patient brain sample this is looking at how the normal syngap allele

20:29is typically spliced and so the question then is in a patient population

20:35can we coax the splicing Machinery more down this productive path

20:40less down this futile path to get more syngap mRNA and protein in that context

20:46where we need it so then how do we tune this splicing

20:51well splicing is often tuned by RNA binding proteins or rbps that bind to

20:58that pre-rna right around the region where the splicing is going to occur and

21:04through through some computational analysis we noted that there was a lot of binding sites for one particular RPP

21:11right around this alternative splicing event so we hypothesized that that splicing

21:17Factor might be driving this alternative splicingPTBP regulates alternative splicing of SYNGAP1

21:22and that RNA binding protein is called ptbp or poly pyrimidine tract binding

21:28protein which is obviously a mouthful so I will refer to it as ptbp and this is

21:34just a protein that can bind RNA at specific sequences and modulate its

21:39splicing so we can test whether ptbp is

21:45controlling this alternative splicing of syngap by getting rid of ptbp in these

21:50neurons and asking does that change the splicing pattern so what we did is we treated human

21:56neurons in this case with one of those silencing Asos that I was talking about

22:01earlier to knock down KD or reduce the levels of ptbp and that’s what you see

22:08Quantified here in white and gray in almost all of our plots will always be negative controls and in red is where

22:14we’re measuring the protein levels of pdbp after treating with this ASO to reduce its levels

22:22so we can get rid of most of the ptbp protein in human neurons and the question is what does that do to singap

22:27splicing and so for this we design what’s called a PCR assay to look specifically at this

22:34splicing event around Exon 11. where we can detect bands that will correspond to

22:42either the uh I’ll call it non-productively spliced

22:47version of the RNA that won’t make protein which is what corresponds to this top band or the productively

22:53spliced version that will make more protein what we see is that when we reduce the

22:59levels of ptbp we reduce the levels of this non-productive

23:04band and we improve the splicing to the productive band that’s Quantified down here below and

23:10I’ll present this data as a percent of syngap that is productively spliced productive meaning that mRNA should be

23:18able to make full lengths in gap protein and so again in red you can see that when we reduce ptvp we have more of the

23:26productively spliced syngak isoform now critically corresponding to that

23:32correction of splicing we get more syngap mRNA and most importantly we get

23:37more simiac protein in these human neurons so this is what we want therapeutically

23:44right we want more syngap protein and by silencing PTP we’re able to increase the

23:49levels of syngap but we can’t just silence ptbp as an

23:55actual therapeutic because it doesn’t just regulate syngap it regulates many many different genes in fact this this

24:01ptbp is really an essential protein it’s required for proper neuronal splicing and proper neuronal development

24:09so we measured using a technique called RNA sequencing every Gene whose

24:14expression was altered we’re using some splicing analysis every Gene who’s splicing was altered when we manipulate

24:21these levels of ptbp and this is just showing on the left hand side

24:27or just the synaptic genes genes involved in synaptic function of the neuron that are altered when we

24:34manipulate PTP you can see if you look closely in here this is syngap one this is my favorite Gene is the xpp1 favorite

24:41is maybe the wrong word um but these are both altered as well as

24:46many others when you affect PTP levels and we actually looked a little bit closer at this at just genes that are

24:52implicated in causing rare diseases or associated with rare diseases and a good number of genes are both alternatively

25:00spliced and changed in their expression pattern when we manipulate ptbp these

25:05are genes that we think could be considered for trying to design therapies to to to redirect this

25:12splicing perhaps in a beneficial way and again you’ll see syngap buried in here next to perhaps some other genes some of

25:17you may know like brand one and mvpt MBD okay but the point is pwp controls many

25:24different synaptic chains and fine-tunes their expression to affect neuronal development

25:29so therapeutically we don’t want to just bluntly Target ptbp due to this

25:34predicted collateral damage we just want to disrupt its interaction specifically with syngap

25:40and so to do this we need to know right where it’s binding on syndap and for

25:46this we use a technique that’s called clip sequencing clipseek which is a technique to map where a particular

25:52protein like a ptbp is binding to RNA molecules and we look at this across all

26:00RNA molecules we map where ptpp is binding on every RNA molecule not just

26:05syngap and we mapped this again in both human neurons and in human brain samples

26:12and this little cartoon here just generally shows us across all of those different RNA molecules where ptbp is

26:19typically binding so it mostly binds to introns shown here to regulate the splicing of

26:26nearby exons but the question we care about here is is does it bind near our event of

26:32interest on syngap Exon 11. so let’s zoom in on that region and this is an

26:38example at the bottom here of what clip data can look like so you see these great Peaks the higher

26:44these Peaks that is indicative of more ptbp binding in that particular genomic

26:51region so and sure enough there’s some pretty big Peaks right around that

26:57um syngap Exon 11. and we can use some computational tools to really predict

27:03our highest confidence areas where ptbp is binding and so we kind of grouped

27:09these into two distinct sites of ptbp binding right around this alternative

27:15splicing event which I’ll call site 1 and site 2 and I’ll show them green and in blue respectively from here on out

27:23okay so so now we know where that ptbp is binding on syngap and our goal is to

27:30kick it off and see if that will correct this alternative splicing and that we can try to do using these blocking Asos

27:39okay so here is a zoom in on site one and it shows where PTP is a binding in

27:47blue and then it shows the region in green where we put a bunch of blocking Asos all along that region and we ask

27:55which one can kick off ptbp and increase the productive splicing of syngap which

28:01is what’s Quantified um here on the y-axis so again these are our negative controls

28:07and then these are Asos that don’t seem to be having any effect on improving syngap splicing but then as you move

28:13forward you see several that are able to increase syndex splicing all in this region and then it sort of trails off

28:21and we get a a no effect with Asos that are further located from that site

28:27so from this and other experiments we can pick this this ASO here for example 19 as a lead ASO that’s targeting one

28:36site one to move uh forward into further testing and we do the same sort of iterative ASO

28:43walk around site two we walk Asos all around this particular region and here

28:49we found that this particular ASO number 85 was particularly effective at

28:55increasing syngap splicing so we move forward with that candidate so now we have our site one and site 2Lead ASOs increase SYNGAP1 mRNA and protein

29:02lead potential candidate Asos and we test them more rigorously and first we

29:08do what’s called a dose response study where here we’re showing as we increase

29:14the dosage of each of those Asos in cells and we look at the effect on

29:20syngap splicing on mRNA and protein levels so hopefully what you can see is that

29:25with increasing concentrations which are these numbers down here of either the site one and green or site blue oligo

29:32that we get increased productive splicing of singap and we have increased

29:38RNA levels and hopefully you can also appreciate that this this site to targeting oligop

29:43number 85 this one’s kind of a beast this is increasing syngant mRNA Levels by about 10 fold in these particular

29:50cells and finally again most importantly these Asos are increasing syngap protein

29:56levels this is what’s shown by the Western block here which hopefully you can see this aso85 on the right is

30:03leading to a very dark syngap band which means it’s robustly increasing syngap protein expression

30:10okay so these results had us really excited um but these are cells in a dish and so

30:17Kennedy’s Asos work in the brain and in Vivo and so for this we did a proof ofCan we increase SYNGAP1 in vivo?

30:23concept experiment in mice where we take our site one and site two lead Asos and we inject them directly

30:31into the brain it’s called an ICB injection of young mice and then we harvest the brain tissue about five days

30:38later and ask whether we’ve increased sync app levels so these are actually slightly different

30:44Asos than were used in the past experiments because we have to match them to the mouse syngap sequence which

30:51is different than the human syngap sequence where all of our other experiments were in human material

30:57but to us this was still a nice first result that suggests that this approach is is worth further pursuing in Vivo as

31:05you can see that both the the site 1 and site 2 oligos were capable of significantly increasing syndap1 and

31:12mRNA levels foreign and encouraging I think that’s an

31:19understatement for how we felt in the lab that day um but but to be fair to be a strong

31:25therapeutic lead we really want to do better than this 40 increase in singap

31:31mRNA that you see here um so there’s a lot of optimization to be done from here and I’ll get back to

31:36that in just a moment so I want to highlight or summarize sort

31:42of these more kind of mechanistic studies here and then just talk a little bit at the end but more about ASM Therapies

31:48so sort of summarizing what I’ve shown to date um in case folks got lost

31:54um we see that syngap uh one pre-rna is typically

31:59spliced the most commonly spliced into its productive form to make syngap protein however some portion of that mRNA is

32:07bound by this pdbp RNA binding protein and this promotes the alternative

32:13splicing of Exon 11 to include then what’s called a premature termination

32:18codon that ultimately leads to that RNA being degraded

32:24by placing Asos right where ptbp wants to bind we’re able to redirect this

32:31splicing back down towards this efficient pathway increasing the amount of productive single Gap mRNA and the

32:38amount of singap protein that’s expressedTimeline of ASO development for SYNGAP1

32:43okay so where are we then in sort of a timeline of ASO development for syndek1

32:53so what I showed you today was was really about identifying potential Targets on San gabron and showing some

32:59proof of concept that we can manipulate those targets with Asos to increase syngap1 levels

33:07um what I didn’t show you is is really the work that’s being done in collaboration and at ionis

33:12Pharmaceuticals who is a a partner of ours in these efforts and we’re really

33:17excited to be working with as they are the ASO experts to test some of these Asos we talked

33:23about today as well as others that Target syngap-1 to increase its RNA and protein expression in in human neurons

33:32and now this next stage which is really where we are entering now is where we

33:38will really rely more heavily on ionis’s expertise and their ability to do things

33:44at scale that we can’t do in an academic lab as we need to do really hundreds of

33:50these ASO injections now in mice um to see if they are safe and effective

33:57at increasing syngap expression in Vivo and and because we want Asos that will

34:05work on the human syngap gene to support these experiments we’ve generated a

34:11mouse model where the mouse syngap Gene has been fully replaced with a human

34:18version so we call this dehumanized syngap mouse and these ASO studies we want to be done

34:24in this humanized syngap mouse so this way if we can find a lead that works potently in that Mouse then we have much

34:33more confidence that that that same ASO with that same chemistry can then be moved into a first inhuman application

34:42so finally I just want to add I think it’s really important to not mislead with this slide this this sort of

34:48indicates that we’re sort of four-fifths of the way along this timeline but this timeline is not linearly proportional to

34:55to real time or real challenge at least and and this this last hurdle moving

35:00from here to here of course um is perhaps the largest and and most challenging

35:06um but we think we’re up for it okay so in the last just two slides IQ&A

35:11want to talk and and uh just a bit about what an ASO therapy could look like for

35:16our kids and it’s it’s different pros and cons I think it’s really an exciting time for

35:22ASO therapies and neurodevelopmental disorders um there are multiple Asos that are either already FDA approved or in

35:29clinical trials for the treatment of different neurodevelopmental disorders and and we can learn a lot from this

35:35past and ongoing work so I think the pros right now

35:41um are so far Asos delivered to the central

35:46nervous system they look fairly safe in pediatric populations and and they seem

35:52to be able to work um uh that there’s there’s good demonstrations of efficacy

35:58um uh across these different trials now the other advantage that they have

36:03compared to other genetic therapies is that an ASO is more titratable right because if it’s a drug if you see an

36:11adverse effect you can potentially stop administering it if you see no effect you can increase the dosage you don’t

36:18have this kind of control with other genetic approaches but they’re also really I think

36:24important cons associated with Asos and and most notably is these drugs won’t

36:30cross the blood-brain barrier to get into the brain to get into the neurons so for a CNS application they get

36:38delivered via an intrathecal injection or a lumbar puncture um which which perhaps most of your kids

36:45have had at this point in the mind has had several and it’s certainly not something that we rubbish although it’s

36:51a relatively safe procedure so for example for an ASO that’s in

36:56clinical practice for SMA I believe these injections are repeated maybe three times a year

37:02and so this is a bit of a burden on families um now it has ASO chemistries are

37:08improving these compounds can last longer in the brain and so I think it’s

37:13reasonable to predict that patients may need fewer injections to get a lasting

37:18effect in the future but these injections are still a downside um I think the other important question

37:24for Asos is you know will it be enough in our particular case will it give a big enough increase in syngap and really

37:31will it work for all different types of variants and and for this we need to better understand how different variants

37:37affect the gene processing okay but I wanted to end just on um a more

37:45optimistic note and hopefully left enough time for for for some q a um and this is sort of a so well it’s

37:52not a self-promotion but um I don’t know we’ll call it Shameless but but this is I think a really wonderful opportunity to get involved

37:59and contribute in this space um our our group uh uh has been

38:05participating in What’s called the million dollar bike ride which is a charity fundraising uh bike ride put on

38:11by the orphan Disease Center here at Penn our team is Lulu’s crew Misty xpp1

38:16which is shown on the left and we are indeed the biggest baddest team out there um and and this year we were really

38:22excited to be joined by the srf crew uh participating in their first mdbr this

38:28is actually many of the srf folks back at my house after the after the race and so I just wanted to encourage interview

38:35that may be interested in getting involved this is a very uplifting event it will make you feel like you are doing something because you are and you get to

38:42with a lot of great people and I would encourage you to join us in 2023 and so with that I will close by just

38:49again thanking the many folks that contributed to this work and I’m happy to take

38:54any questions thank you thanks Ben that’s great that looks like a lot of

39:00fun at the at the bike ride yeah you gotta come next to your bubble yeah gotta work on my fitness

39:08um all right let’s jump straight in we got a few questions and I think that’s going to be more coming but I’ll start

39:14with the the one that I think all the parents always want to ask first which is what is your estimation of the time

39:20frame between where we are and where we want to be human trials yeah so this is also uh it’s the most

39:27important question it’s the one that keeps me up at night it’s also the hardest one to answer and maybe the most

39:32dangerous one to answer right because you want to be realistic and optimistic but you also don’t want to give false sex false expectations

39:39so I’ll I’ll say this this is why I got this is why we focused on Asos in the

39:45first place is there potential for Rapid translatability to the clinic so when my

39:51daughter was first diagnosed and we surveyed the therapeutic space and we’ve looked at all the different potential approaches out there there are many that

39:57are promising we went down the ASO path because it has in my opinion the the most rapid potential translate ability

40:04to the clinic and there’s a somewhat famous demonstration of this where an ASO went from sort of inception to human

40:11application in a year and a half now that was for what’s called an NF1 therapy it’s a bit different of a space

40:18um I would to to more clearly answer your question in in our sort of

40:23conversations and I hope I’m I’m reflecting this appropriately but I think the the sort of the 18 month to

40:30two year timeline to a first and human is where we are now if things go as we

40:35hold from here right and so that’s of course always a huge if so what I tell my wife is that you know when she asks

40:42me all the time about where the timeline is for Lucy and for getting the treatment for Lucy I say we’re anywhere from a year and a half to never because

40:49the and that sounds terrible right but the reality is these are so far incurable genetic diseases no one has

40:55cured these and so the uh the any scientist that says oh we’ll have it in

41:01X years you can’t say that with full confidence of course right but

41:07we are optimistic the uh and um we are encouraged including from the early

41:12studies in human neurons and in mice some of which we didn’t have time or that what I was unable to present today

41:18so I’m hoping that we’re on the shorter side of that therapeutic timeline

41:25that’s great thank you that’s what they all you said what the

41:31researchers always say um but that’s that’s great and just to follow up on that you had ionis on that

41:37slide um do you want to just talk briefly about your relationship and how that should hopefully help

41:44so I I think the I mean one we’re really excited to work

41:50with ionis because they’ve helped you know they’ve done it they they have an FDA approved ASO for the treatment for

41:57SMA and many more at various stages of the clinical pipeline that are looking promising for a variety of disorders and

42:03so they really do have this tremendous expertise they can also do things at a scale and a scope that we just can’t do

42:10in an academic lab so in order to optimize those those in Vivo experiments

42:16or those experiments in human neurons which are time consuming lots of reagents lots of injection they can do

42:23these experiments at a scale and scope that we wouldn’t be able to do alone to

42:29really uh uh uh not just identify Asos that are that are effective which I

42:36think we can we can point towards a pretty pretty pretty safely and in an academic setting but really to identify

42:43Asos they’re safe which is which is of course often a harder bar to clear than

42:50efficacy and to do these safety and toxicology studies well um we want an experienced partner

42:58um like ionis and so we work with them in a collaborative setting we meet on a monthly basis and show they’re doing

43:03experiments in-house we do experiments and here we try to capitalize on our strengths and capitalize on their

43:09strengths to together move uh the project forward as quickly

43:15um as we can that’s great yeah I think this the speed factor of having that lined up is is is

43:21really good for the for the community just a couple more questions parent Focus before we jump into the the

43:28sciency ones um people want to know is my kids mutation is this going to work on my

43:34kids mutation and also what about age is there an age cut off for this treatment

43:39just talk a little bit about that yeah absolutely it was a really um important questions

43:45so the um and this comes down in terms of the type of variance that this

43:52therapy would work for um again we are trying we are not

43:57correcting the mutant allele we are targeting the wild type allele to make more syndeme protein

44:04so in a lot of kids kids that would have a nonsense variant a truncating variant a chromosomal deletion where they

44:11clearly are not making syngap protein off of that mutant allele this ASO is

44:16probably not going to affect the mutual in any way and it’s going to increase the production from the wild type and

44:22and and and hopefully increase the total amount of syngap in the neuron and then create a therapeutic benefit

44:29there’s still a question I think in singap community this is true in the sdx pp1 community as well is whether certain

44:35kids with missense variants could be producing a mutants in gap

44:40protein that could be having for example a dominant negative effect and so I think more research is needed

44:48on really defining um whether dominant negative phenotypes

44:53are present in certain syngap populations particularly those with missense variants where it’s a

44:59possibility at least I don’t think it’s a clearly demonstrated it’s not something that we we know right now but

45:06if that was the case if there was a dominant negative mutation then an ASO could both boost the expression from the

45:12wild type but also be making more of that mutant protein and that could have a detrimental effect now there are

45:18potential strategies that you could take around this you can use what we call that that’s silencing ASO right if the

45:25real problem is that the mutant allele is making a protein that’s coming up the works then you design a silencing allele

45:30to try to knock down that mutant protein but it would be a different ASO strategy so what’s what’s clear is in order to

45:38inform those strategies we have to have a better understanding of which particular genotypes lead to which

45:43particular phenotypes and I think that’s still a really important unmet need that the syngap basic research community and

45:49translational research Community um needs to address that was one part of your question Pablo

45:55was there definitely there was a second part that I’ve heard that yeah the age is there an age yeah so this is another

46:01thing that we don’t this is something again I think about all the time as my daughter gets a little bit older and older is therapeutic window

46:08okay I think what we the the crude answer is we don’t know when we need to intervene it common sense suggests that

46:16the earlier that we can intervene in a neurodevelopmental disorder the the the larger uh beneficial effect that we may

46:24be able to have but for both STX bp1 for syngap1 these

46:29are genes that are essential for proper neural function and we would have to imagine that intervening at any stage of

46:37life if you could restore their functional levels I I believe that that would have a a benefit and there’s

46:44there’s good data there is some data to support this or even in mature neurons

46:49or mature uh older mice if you restore the levels of of these these genes of

46:56interest you still get clear functional benefits so um certainly we want to get into kids I

47:02think as early as possible and I just I’m sorry that this is a long-winded answer but I just remember this it’s really

47:08it’s important for me to mention that particularly with this approach that we’re talking about today and for

47:13reasons that I didn’t really detail extensively in this webinar I think this therapy would would have a

47:21its most beneficial effect even if you could do it in development right so if

47:26one day we could do genetic testing and see that there’s a futuresing Appian in utero and you could manipulate these PTP

47:33levels and this in this syngap expression in utero it stands to reason that that is the condition where you

47:39would have the largest effect that doesn’t mean that we couldn’t have an effect later in life but that’s that

47:45that that answer is driven by the fact that ptbp levels are highest during

47:50development and they actually decline over time so you are reducing your opportunity to

47:57Target it um in the postnatal window if that’s if that’s clear yeah that makes sense

48:05okay great um let’s jump into some of the more sciency questions

48:11um there’s one from Hans uh can you can you see them yourself you might yeah I can

48:18open up the Q a

48:24Target out of the neurons and use those altered neurons as a positive control to

48:30see the potential gains of avoiding PTP P2 eventually

48:38okay so there’s a lot in that question that can you basically edit the ptbp2 oh the

48:45target out of neurons so could you yes so what Hans is referring to is if you

48:51could use a base editing strategy which is sort of an evolved crispr Gene editing strategy to

48:58um remove the uh uh Target sites I suppose for where we

49:03think PDP binds out of neurons and [Music]

49:09um and and use those altered neurons as a positive control to see the potential

49:14gains of avoiding ptb2 yeah so and then you can use that to see if we

49:23this is really in a clean experimental setting you you propose that you’re getting rid of all of The ptbp2 Binding

49:30around that Exon 11 which I think is what Hans is getting at so as opposed to an ASO or maybe it’s knocking out of

49:37some portion you just deleted the whole area where p2p2 could bind and actually I’m aware of some work that has not done

49:44this with a base editing strategy but with it with a with a different approach and that does tell you about the

49:50potential gains of avoiding ptp2 and what I can say is that these these these

49:55uh uh this has been done in mice and these mice do have increased expression of of syngak protein

50:03um when they’re born so this does speak to the argument that if you can get rid of this alternatively spliced region of

50:09syngap it’s not done ponds through the exact technique that you’re asking but through I would say an orthogonal approach you do have a stable increased

50:17expression of of of syngap protein um and and I’m not sure if there’s

50:25really been a full functional battery done on those mice to ask what that does and it would have to be done on a syngap

50:33heterozygous background to see is that sufficient for example to create functional rescue on you know metrics of

50:41cognition learning memory Etc in those Mouse models

50:46got it and then there’s a question about biodistribution

50:51um I guess how much of this treatment would have to reach how much of the brain and do you think there’s an issue

50:58potentially of of not good enough biodistribution

51:03yeah so I think this is again something that um we’re learning more about all the

51:10time and it’s evolving so I’ve mentioned early on in a very simplistic way that

51:16Asos have some advantage in their size it’s easier for them to get into cells compared to using perhaps a viral Vector

51:23that would have to be delivered and this can also de-risk the approach that doesn’t mean that biodistribution isn’t still an issue it is still a a

51:30challenge and an open question about getting Asos evenly distributed throughout the brain but I I to me I

51:38don’t see this as a major like uh limiting factor I think the indications from the ongoing clinical trials using

51:46other Asos to Target different neuronal genes um have me feeling optimistic that we

51:52can get sufficient amount of ASO into sufficient neurons to see beneficial and

51:57clinically meaningful outcomes um and so uh I think one of the uh

52:05um uh important questions is still for and

52:12this is you know Gene specific then Are there specific neuronal populations that you need to Target and Are there

52:18specific neuronal populations that an ASO gets into um more than others so for example we

52:24think about this in the STX pp1 Community where we think that stxbp1 deficiency in particular neuronal

52:32populations may be plain and outsized role in the disorder and so we would

52:37want to make sure that the ASO is able to get into those particular neurons I don’t know if we have that level of understanding for syngap in terms of its

52:46its functional roles and how they may differ across different neuronal populations and how that dovetails with

52:53how well Asos get in um but in general I don’t think that ASO

52:59biodistribution across the brain is a major limiting factor

53:04um that would outweigh the therapeutic gain with these approaches

53:09that’s good news and actually speaking of other Asos there’s one question around what’s the

53:16difference between your Technique and what Stoke are doing yeah so that’s a great question

53:21um so what about and you know as we are working on this

53:27particular project a publication came out from Stoke

53:32Therapeutics that also showed that targeting this similar

53:38um site on syngap1 was capable of improving

53:44productive splicing and syngap expression in a non-neuronal Cell line

53:49so the approach I showed you today is very similar the Asos are different and

53:55I think one of the the important differences is that in that particular work

54:01um there was not well it was super helpful in demonstrating sort of the proof of concept of going around this

54:06target there was not a mechanism for housing Gap was alternatively spliced

54:13and so in our study when you when we see that ptbp is what’s regulating this

54:19and then by mapping where ptbp is binding we identified this other site

54:25that is a little bit different than what Stoke was targeting in their published paper so I talked about site one and

54:30site 2 as I went through my presentation those site one oligos and that site one

54:35ptbp binding is very similar to the region that’s still targeted in that publication

54:41site 2 where we actually got our biggest bang for our buck at least in these systems was the one that was really we

54:49only thought about targeting after we saw that it was a hot spot for ptbp binding and so to us this really

54:55emphasized the importance of not just knowing what’s going wrong but understanding the mechanism that’s

55:00driving it right and so by figuring out this mechanism of PTP dependence binding mapping where it is and then guiding the

55:07ASO therapy you may be able to have an improved ASO therapeutic but conceptually it’s very similar to what

55:15we understand Stoke Therapeutics has been going after

55:21okay great all right almost out of time but I think there’s a couple quick ones here

55:28um do you know of any negative implications of producing too much singap one protein yeah that’s a really

55:34good question I I don’t know that that literature well enough off the top of my head and and this is a major concern I

55:41think if you’re talking particularly about like a gene replacement strategy where you can be delivering multiple copies of for example synagogue DNA to

55:48one cell and and well for example with a with a viral delivery you may have 100 copies of one cell right next to a cell

55:54that that has none for example and that you really worry about this over expression and negative effect of

56:00overexpression I I don’t think it’s as big of a concern with an ASO strategy I’m much more concerned about not

56:06getting enough syngap um increase than I am about overshooting and the other advantage of an ASO if you overshoot you

56:12just lower the drug concentration um and so this is again one of these key

56:18advantages is that it’s titratable in that way if we get to the problem where we’re making too much singap one Protein

56:24that’s a great problem to have as we’re trying to move something through as a therapeutic lead yeah yeah and I think if you look at the

56:31design of The Stoke trials you’ll see they’re moving up quite slowly and how much of that is so they’re giving so I

56:38think they’re um trying to hit the same the same Target

56:44um Asos can’t cross the blood brain barrier is there any potential for an oral root drug or is that a dead end

56:52there are I don’t think that there’s a promising lead right now going after the same

56:59Target from an orally bioavailable um therapeutic you can imagine some

57:05small molecules and for example this has happened in in SMA where at first there

57:11was an ASO modulating the splicing of SMA and now there have been some uh

57:17other small molecule drugs that have also been shown to modulate that splicing in a favorable way and are more

57:25moving forward so there’s there’s a precedent for that Tim

57:30um but we don’t have one right now um for cinegap

57:36um I think there’s other routes there’s there’s also work being done for other ways to deliver Asos other than

57:41intrathecal injection and there’s some engineering work being done at Penn along those lines as well for sort of a

57:46slow release distribution um so there may be alternative strategies to administer it right now we

57:51don’t have a small molecule that I know of that manipulates that splicing on the same Gap yeah and and Tim I think you know srf is

58:00trying to fund some of these other you know forward-looking mechanisms we did give a grant for milk access some

58:06research so Jenna suplini he’s you know he’s looking at things like that so we are trying to get to the next

58:13level but as Ben said Asos are something that’s on the market right now and that’s how they’re delivered

58:21um okay uh last one quickly have you looked at locked nucleic acids or other chemical

58:29modifications to increase ASO Affinity yes and and and some of these different

58:35modifications are better for in vitro studies some are better for in Vivo studies and then some we think about in

58:41terms of sort of clinical safety but this is really where we’ll defer to our the experts that for example dionis who

58:49are working on a variety of these different chemistries that are designed to increase lifetimes affinities safety

58:54profile and and um I can’t I don’t I I don’t have that expertise we’re glad

59:00we’re partnering with folks that do [Music] that’s great okay we’re right at the hour Ben thank

59:07you so much this is really exciting news you know this is uh we’re starting to get a taste of of what’s possible here

59:14and and something real for for the children so it’s it’s really exciting and thank you so much for all of your

59:21your tireless work on on this I know you’ve been burning the midnight oil to try to get this stuff done so we really

59:26appreciate what you’re doing thank you guys and I appreciate the very informed and intelligent questions in the Q a as

59:32well so thank you for the opportunity all right have a good one all right take care