Translating Genetic Discoveries Into Targeted Therapies

April 30, 2021

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00:00It's my pleasure to introduce
00:02our final speaker of the day.
00:04Doctor Muenkel,
00:05like he is an assistant professor
00:06in the Department of Genetics.
00:08He got his PhD at the University
00:10of Sydney and did postdoctoral
00:12training at Massachusetts General
00:14Hospital and the Broad Institute.
00:16His lab here at Yale is focused
00:18on the genetics of rare disease,
00:20an understanding genetic mechanisms
00:22of neuromuscular diseases,
00:23and as a patient with a
00:25rare muscular dystrophy,
00:26Dr Lake is very passionate about
00:28translating genetic discoveries
00:29into targeted therapies.
00:30Thank you Doctor
00:31like thank you and thank you for the
00:34kind introduction and thank you for the
00:37organizers for putting together wonderful
00:38workshop and also give me the honor and
00:41opportunity to present on some of the
00:43fantastic work that my lab is working on.
00:46So I'll be talking about translating genetic
00:49discoveries into targeted therapies.
00:51I have no conflicts of interest to disclose,
00:54so my lab works on the full patient journey.
00:57So from the diagnostic Odyssey that the
01:00young we had touched upon all the way to
01:03the development of individualized therapies,
01:05and I can reflect upon this,
01:07this whole patient journey
01:09through reflecting on my own.
01:11So I went on a diagnostic Odyssey,
01:13took over 10 years to actually find the
01:16gene underlying my ran your muscular
01:19disease and you can see on the key.
01:21The gene, the mutation, they fight,
01:24found finally found an and this an.
01:26Nowadays we can actually rapidly shorten
01:28this diagnostic Odyssey time by using
01:30genomic technologies such as xom sequencing,
01:33genome sequencing and RNA sequencing,
01:34and also developing methods to get at that.
01:37And that's one of the things that my
01:40lab focuses on and but still there's
01:42so much work to be done because the
01:45diagnosis rate is still only about
01:4850% for a lot of rare diseases,
01:50and when patients actually.
01:52Find out their genetic diagnosis.
01:54They can work on specific disease
01:56management such as stretching.
01:58As you can see here in this example here,
02:01but one of the most exciting things
02:04that has only become possible in
02:06the last five years or so is there.
02:09The concept of development of
02:11individualized therapies.
02:12I had the good fortune that my
02:15collaborators and colleagues at the
02:17University of Massachusetts Medical
02:18School one dashly work on my mutation,
02:21which was which was.
02:26Oh sorry. Which was working on
02:31the 8 basepair duplication.
02:34And designing a custom crispata target
02:36that and to to cleanly remove one copy
02:39of the eight base pair duplication.
02:42So and they actually perform this on a
02:45skin biopsy I gave and they created.
02:48I PS cell line.
02:49It was able to achieve nearly 80%
02:52correction of my cells and this
02:54was published in Nature in 2019.
02:56So we were inspired by this effort and story.
03:00And we wanted Ashley replicate some of
03:03this idea of the individualized therapy so.
03:06So I'm going to talk for the rest of
03:09touch upon on the rest of the talk.
03:11A story of the patient.
03:13So the patient here is Terry,
03:15who has a rare form of Duchene,
03:17muscular dystrophy and his brother here,
03:19Richt, who created our foundation to
03:21actually find a therapy for his brother.
03:23And you can read a little bit more
03:25about their story on a Harvard
03:27Business School article.
03:28And so we set up on this project
03:31in the summer of 2018.
03:32So this was shortly after I saw
03:35the lab here at Yale.
03:36And this is the genetic report,
03:38and it's very typical for genetic report of
03:41a patient with Duchene muscular dystrophy.
03:43So this patient has an X on one deletion,
03:46so it's it's quite a large deletion
03:48that takes out Exxon one and the
03:51promoter region of the muscle Exxon one.
03:53So the first thing we wanted to
03:55do is a full characterization.
03:57So we performed whole genome sequencing.
03:59So for those that aren't familiar,
04:01follow genome sequencing,
04:03these reads represent.
04:05Next generation sequencing reads and
04:07where they map to the human genome.
04:09And you can see here in this particular
04:14case this is the X chromosome.
04:17And in particular the DMD gene.
04:19And keeping in mind the DMD gene goes,
04:21is on the negative strand,
04:23so goes from right to left.
04:25And here is muscle X on one,
04:27so in the patient we see no
04:29sequencing reads mapping here,
04:30and this indicates that there is
04:32a large deletion an if you look
04:35carefully here at the mother.
04:37You can see the histogram here that
04:40represents coverage that we see this
04:42dip and this dip is approximately 50%
04:44and this represents that the mother is
04:48a heterozygous carrier of this deletion.
04:50We also performed RNA sequencing on
04:53the patient and so for those are not
04:56familiar this is what we call a sashimi plot.
04:59These arcs represent next generation
05:01sequencing reads that span from
05:031X onto another Exxon.
05:04So not surprisingly,
05:05we saw no read support for the muscle,
05:08the muscle isoform, and this is X on one.
05:11But surprisingly we we sorry
05:13support for the cortical isoforms,
05:15so you can see in our that goes from Exxon,
05:18one from the cortical ice form.
05:21*** on two and so forth,
05:22and the the difference between
05:24the cortical eyes from the muscle
05:26isoform is only exon one.
05:27And we've been Exxon one.
05:29Most of it is the untranslated region.
05:31So the coding starts later on next on one.
05:34So there's only a few amino acids
05:36difference between the muscle and
05:38the cortical ice form, and this is
05:40important for the rest of the talk.
05:42But one of the things we hypothesize
05:44is could the upregulation in
05:46switching on the cortical or ice form,
05:48which you typically don't
05:49see in skeletal muscle.
05:51Can this actually be contributing
05:53to some of the delayed progression
05:56of the muscle disease?
05:58We actually seen the patient, so.
06:00When we look at the protein levels,
06:03when you do Western blot you can see
06:06what our collaborators at Boston
06:07Children's Hospital has shown.
06:09Is that the patient does have residual
06:11level of dystrophin protein expression,
06:13and they approximate.
06:14This is about 3% of normal levels,
06:17and when you look at the muscle
06:19biopsy and the Histology of it,
06:21you can see that in control you can
06:24see this nice staining of dystrophin.
06:27Represented by green at the
06:28muscle membrane of these fibers.
06:30While in the patient you do see some
06:33dystrophin but it's patchy standing.
06:35So there's some patching mosaic standing
06:37and this represents what we believe
06:40to be a stochastic random process.
06:42So this is a snapshot in time,
06:44and sometimes you do get the
06:46expression of the cortical isoform,
06:48but you know it's not as strong as a control,
06:52and it's not in each one of the fibers.
06:57And so,
06:58so this shows that the patient is
07:00expressing a quarter Kreisel and when
07:02we look in the literature there are
07:05other patients that were reported in
07:07the late 80s and early 90s that Ashley
07:10have a muscle Exxon one deletion.
07:13However,
07:13the interesting thing about these
07:15patients is that they don't actually
07:17have a skeletal muscle phenotype,
07:19so they don't have dish in muscular
07:21dystrophy or any muscle phenotype at all.
07:24But they still have a cardiac
07:26phenotype and what we believe is
07:28happening here is there's this complex
07:31interplay of enhances surrounding
07:32the muscle Exxon one and also the
07:35cortical Exxon which they've called
07:37the brain isoform in this paper,
07:39and that when you get the
07:42deletion of the muscle X1,
07:44the other enhancers switches on.
07:45And actually turn on the cortical ice
07:48form and they turn it on high enough
07:50in skeletal muscle that these patients
07:52don't actually have a muscle disease.
07:55However,
07:55what we think is happening with the patient
07:58is that he his deletion is a lot larger,
08:01takes out some of these enhancers so
08:04the cortical ice form can switch on,
08:06but not at the levels that is
08:08actually happening in the patient,
08:10so I'm going to stop and pause you
08:13so we have a very good example.
08:15A human example that in the absence
08:18of a muscle isoform,
08:19if you can switch on the cortical
08:21isoform that this could actually perform
08:24a genetic rescue and actually save.
08:26From having a muscle disease
08:28phenotype and this is motivated us
08:30to actually do this for the patient,
08:32will develop a therapy for the
08:35patient so so the
08:36ability to actually switch on and robustly
08:39turn on the cortical ice form in one way.
08:42You can do this is by using CRISPR,
08:45so in this case a dead cast line,
08:48so the ability to home into a particular
08:51place in the genome and bind and.
08:53But in this case not cause a double
08:56stranded break, but bring in.
08:58Inscription activating these transcription
09:00activator allows for the expression
09:02so it's not the best transcription
09:04activated that you could use,
09:06but we had translation in mind.
09:08It was small enough to package into a Navy
09:12which has only a 4.7 KB packaging limit,
09:15so everything we've done has been designed
09:17with translation mine and not rescuing.
09:20Save particularly the cells
09:21or the mouse itself.
09:23But how can this actually translate
09:25to a human clinical trial?
09:27So we also picked.
09:29Skeletal muscle promoter called CCA D
09:31This is their truncated small synthetic
09:34promoter that has also been used in
09:37clinical trials already for mini dystrophin,
09:39so it has a good safety profile.
09:43So next thing we wanted to do is
09:45going over all the possible guides
09:48so upstream of the transcription
09:50start site or the code acquires
09:52form that we could bind and possibly
09:54switch on the cortical eyes form.
09:56So this was done in human cells and we found
10:00that the C7 one of the cortical guides.
10:03One of the guides targeting the cortical
10:06ice form seems to have good upregulation,
10:09so the next thing we wanted
10:12to do is then ask.
10:14How the performance would look like in vivo.
10:17So we're very fortunate 44.
10:18DMD research that there is a range of
10:21great mouse models that you can use,
10:23but the best mouse model you can use for.
10:28Very specific genetic therapies such
10:30as this is a transgenic mouse model,
10:32so this transgenic mouse has
10:34the whole DMD gene,
10:36so a 2.6 megabass fragmente
10:37that not only has the axons,
10:40but also has the introns and also
10:42the flanking intergenic regions.
10:44So guides that we've designed,
10:45and we've proven to work in the human cell
10:49model can also work in this mouse model.
10:52And in an in addition,
10:54this mouse more has the MTX mutation.
10:57So at the mouse locus for the DMD
10:59gene has a nonsense mutation and can't
11:02actually express the mouse dystrophin.
11:04So the only dystrophin this mouse
11:06expresses is the human dystrophin.
11:08And this was this was given to
11:10us by our collaborators at UCLA.
11:13So the mouse study we ran with
11:15was a low dose and a high dose.
11:19At four weeks in eight weeks looking
11:22for the UP regulation as what we saw
11:25in the human cell model and the reason
11:28why we picked this dose is this dose.
11:31Ashley correlates to a human
11:33clinical trial of 3 * 10 to the
11:363rd 14 vector genomes per kilo.
11:38Kilogram is the largest.
11:40The highest dose that has ever
11:43been used safely in delivering
11:45effectors such as this in in a
11:48systemic way to skeletal muscle.
11:50So the results I'm going to show you are
11:53the results from the high dose eight
11:56week mice where we have two untreated
11:59mice and four treated mice and you can
12:02see that we do get on an RNA level.
12:05Looking at qPCR, we do see upregulation
12:08in in cardiac tissue and disappointingly,
12:10the UP regulations in other skeletal
12:13muscles are not just not as high
12:16and in non muscle tissues.
12:18We don't see any regulation or.
12:20And and this was expected because we news.
12:24A skeletal muscle specific ice form.
12:30So what are the lies is so
12:32like preference for harm?
12:34Is that the AAV 9 that will be
12:36using to deliver that therapeutic?
12:38Well? Not surprisingly,
12:40most of that goes to the liver when we
12:44look when we isolate the DNA an look at.
12:47The viral copy numbers,
12:48but also you can see that a lot of
12:51it goes to the cardiac tissue with a
12:53preference toward versus to skeletal muscle,
12:56and this is something that the field has
12:59found that AAV 9 has a preference for heart.
13:03So using this data and other
13:06data that we've generated,
13:07but I don't have time to present
13:10and will very well successful
13:12with a pre Ind application.
13:14And since then we've replicated this
13:16data on multiple other mouse cohorts
13:19and also at other AAV production
13:21facilities producing robust results.
13:23But the future work we have to do after
13:27talking to the FDA is we'd like to
13:30measure increased protein expression.
13:32So what I've shown you?
13:34Is increased in RNA expression,
13:36but we'd also like to show an
13:39increase in protein expression.
13:41One of the challenges is that
13:43the cortical muscle isoforms
13:45are very similar to each other.
13:47It's only a few amino acids at
13:49the intern that Steve different.
13:51We're trying to create also an exon,
13:54one knockout mouse model,
13:55but there's difficulties
13:56naturally creating that.
13:57So what we're working towards is just
14:00showing an increase in overall protein
14:02expression and therefore inferring
14:04that the increase is actually due to
14:06the increase in the cortical ice form.
14:09The other thing we're working on is the
14:12development of a robust patient cell model.
14:14And this is necessarily necessary
14:16to determine off target effects.
14:18So what are the genes weight would?
14:21We may be switching on other
14:23than the dystrophin isoform so,
14:25but we believe that there probably
14:27shouldn't be any off target effects
14:29given that a lot of these off target
14:32sites are actually into Jenny and
14:35shouldn't be switching on genes.
14:37And the last is functional assays
14:39that show the restoration of
14:40dystrophin at the cell membrane in
14:43these patients else comes along with
14:45the restoration of other complexes.
14:47Such as the district liking complex
14:50and also the psych like in complex.
14:53So our lab is very excited that
14:56we're in this new era.
14:58This shift from personalized medicine
15:00to individualized medicine and
15:02the the contrast is an explained
15:05well by Peter Marks from the FDA
15:07is that personalized medicine is
15:09when you profile patient and use
15:12off the shelf drugs and therapies
15:14personalized based on their profile
15:16while individualized medicine.
15:18Is looking at a patient's
15:20mutation and developing a therapy
15:21specific to that mutation,
15:23and so there are two exciting clinical
15:25trials that are going on right
15:27now and using CRISPR technologies.
15:29One is sickle cell which is
15:31using ex vivo approach.
15:33The other one is delivery to the
15:35eye for inherited blindness and
15:37with that I would like to thank
15:39the audience for their attention
15:41and a huge thanks to Karen
15:43K You from my lab.
15:45Have driven a lot of the research that.
15:48I've presented today our funders,
15:50cure a disease,
15:52and also a wonderful collaborators
15:54academic and also industry,
15:56particularly Charles River and our
15:58collaborators at UMass, thanks.