[MUSIC] The story is about modeling
Pitt-Hopkins with stem cells and brain organoids.
This is a recent work in the lab.
Pitt-Hopkins is a neurodevelopmental disorder
caused by a mutation in a gene called TCF4.
It's a transcription factor.
It causes a profound type of autism.
These are situations where
those kids most often do not talk,
have seizures, epilepsy, some of them cannot even move.
So really profound, severe types of autism.
The prevalence is about one in 10,000.
However, we do think that there are
several kids under the spectrum of autism that,
due to the lack of a genetic sequencing,
they do not know that they are
part of the Pitt-Hopkins syndrome,
just because the phenotypes are probably mild and
masked on the heterogeneity of the autism spectrum.
Nonetheless, this is a monogenetic case,
and it's perfect for
disease modeling with brain organoids.
I'm so glad that Alex has introduced
brain organoid technology just briefly.
This is not a new technology.
Some people think that this is quite new.
No. In fact, it was developed by Yoshiki Sasai in 2008.
This is the first description in a stem cell paper that,
if you just neutralize the cells in suspension,
they will self-organize and
form what we call these brain organoids.
That time, they didn't even call them brain organoids,
they have different names,
but the organization of the tissue is
remarkably similar to the human fetal brain.
Many labs, including ours,
have optimized and derived
different protocols to make these more robust,
more reproducible, more reliable,
and even fixing some of
the pitfalls that these organoids have.
For example, you hear from Alex to
create single rosette organoids,
which is more problematic
compared to multiple rosette organoids.
We also developed a very simple and robust protocol
that most of people in my lab is using now.
We start from pluripotent stem cells,
we neuralize by kick-starting
with the [inaudible] inhibitors.
That neutralize the cells,
and we put them in suspension,
they will self-aggregate and form
these spheres that we can stimulate the propagation of
the progenitor cells with addition of
growth factors such as EGF and FGF.
Then we just remove the growth factors and we
let the cells do what they do.
These are only transient exposure
to these ectopic factors.
We start from single cells,
they end up with about five million cells after a while.
A question that I often get is,
what kind of cells you have in these organoids?
As you can see, it's very dynamic process.
The diversity of cells who actually change over time,
it really depends on what stage are you looking at.
I'll give you a snapshot,
at four-months old organoids,
you can see that the vast majority of cells are
glutamatergic, excitatory neurons.
We have the pool of progenitor cells that
continue to produce these neurons.
They are also starting to produce glia cells,
mostly astrocytes, and we start to
see some of the markers for GABAergic neurons.
These are not functionally inhibitory neurons yet.
They will become inhibitory neurons a little
bit down the road when they are six,
seven months of age.
But nonetheless, that's how
dynamically they change the cell populations.
We took this protocol and we
reprogrammed cells from a group of Pitt-Hopkins kid,
about six kids and six controls.
We used the controls,
the none affected father of these kids.
These are all males. Then we look at these organoids.
To our surprise, the phenotypes are quite visible.
They definitely mimic
the microcephalic brains of these kids.
The production of the progenitor cells is reduced,
and the expression of TCF4 in
these cortical organoids is also reduced by half,
which is consistent with the haploid sufficiency,
so one copy of the gene is mutated
while the other copy is not.
Compared to their control parents,
you can see half of the expression of the proteins.
That was interesting.
We decided to see if that
also happens with another type of organoid.
Depending on the factors,
you can manipulate the fate of these organoids.
These are another type of organoids,
we call them glia or GABAergic organoids,
because they're mostly composed by inhibitory neurons.
As you can see, we also
recapitulate the proliferation deficit,
so these organoids are tiny compared to the controls.
Once we have a phenotype that is visible like that,
we always ask, well, what is going on?
Is it the proliferation of the cells?
Are the cells just dying?
What's actually happening?
One easy way to do that is just to slice the organoids,
do some immunostainings and trying to see
the major cell populations, how they behave.
Because these organoids also have
some preserved site architecture,
we can actually see, for example,
number of the rosettes as Alex pointed out.
They normally, as the parental line here,
have multiple rosettes,
and you can just counting them,
and you see a dramatic reduction in
the number of rosettes compared to the mutant cell lines.
Also, the population of SOX2 positive cells,
which is one of the proliferating cell types in
the early stages of brain formation, it is reduced.
Normally, you see a reduction between 4 and 10 weeks,
which is suggesting that the cells
are differentiating to post-mitotic neurons,
but the number that we start with in
the mutant cells is much lower compared to control.
These are proliferating cells that are
just not there anymore.
Why they are not there?
That's an interesting question.
We turn into gene expression,
just performing like global RNA expression,
and looking for potential pathways.
Long story short, there is
several pathways that seems to be affected.
One of them is really suggesting that the type of
cortical neurons that these SOX2 proliferating cells
can make in the mutants are different than the controls.
These are markers for
the telencephalic cortical diversity that we have,
and we see dramatic differences
between the mutant and the control.
We look into validating that with immunostainings,
and these is some markers of different cortical layers,
for example, CTIP2 and SATB2.
You heard from Alex that these
are different cortical layers,
and we quantify them.
Again, we see quite dramatic differences
between the controls and the mutants at
different stages of development.
Basically, these SOX2 positive cells
are not making these type of cortical neurons.
In theory, the patients' brains are
missing some of these layers
in the cortex and that might contribute
to the phenotypes that we have in those kids,
including the cognitive decline and everything else
associated with their conditions and autism.
Always when we get this type of phenotype,
so we look for some in vivo validation.
I must say here that the mouse model for
TCF4 doesn't show dramatic phenotypes,
it does not reproduce the biology that we see in humans.
By the way, for autism
and other neurodevelopmental disorders,
this is quite common.
Most of the time, if you knock out
one copy of the gene in a mouse model,
get like a mild phenotype if any.
For TCF4 is not different,
so the mouse just do normally and might not have
any dramatic behavioral phenotype
that is related to the patients.
What happened here is one of
the patients in our cohort actually passed away.
The family donate the brains
to our labs so we could do the [inaudible] staining,
and look for some of
these markers in the postmortem tissues.
We were able to validate that exactly this subtype of
neurons are also decrease in post-mortem brains,
similar as we anticipated from the cortical organoids.
That gives a reassurance
or validation that the data that we have with
the organoids is exactly mimicking even though we
have a very reductionist modeling in culture here.
Showing phenotypes
in a cellular model is very interesting.
I think especially in
this environment here we're very translational,
we want to not only find phenotypes,
we want to do something about it.
We can translate that to
the families. We thought about that.
TCF4 as I mentioned,
is mutated in one copy of the gene.
The other one is highly functional.
What if we can compensate the happening insufficiency
by overexpression of the version
of the gene that is now mutant.
You can do now something like that using CRISPRa.
We can use CRISPRa activator
that would bind to the promoter of
the TCF4 and force the expression
of the genes so we can compensate that.
In that was very successful.
These are transactivation approach,
it was able to restore the level of
TCF4 inside these organoids.
Another strategy that open;
and these are just the probes that we
use in the promoter region.
This is something that it's empirically defined.
You have to design different probes for your CRISPRa to
bind in this promoter regions to see which one will work.
It's not something that we can really predict it.
I forgot to mention that TCF4 has different isoforms.
Even those isoforms, there is
no consistent agreement in
the literature which ones are important.
We design a different probes for
the CRISPRa to attack
or to express different isoforms here.
Once we have that,
we put the CRISPRa ion, the lentivirus,
then we infect the organoids at
very early stages and we look for potential correction.
To our surprise,
we were able to correct the expression of the TCF4,
as I mentioned, it's a transcription factor,
so it regulates the expression of downstream genes.
We were able to regulate
this downstream genes as well, for example,
this GADD45G or CDKN2A.
All of that we could correct.
We also see the re-expression, for example,
of the SOX2 positive cells that I
told you that was downregulated.
But what I think was amazing is that;
and I didn't highlight that
the mutant organoids have some malformations as well.
There are some clusters of neurons that forms,
some scars inside these organoids.
We don't fully understand.
We see something similar in the postmortem tissues.
If you correct the gene early on,
you can fix even the structure of defect,
which for me as a neuroscientist
was something surprised that during
development you can fix the brain
that was previously damaged.
But yeah, that's what
the science is telling us that you can do that.
There's one major problem
with the CRISPR activation here,
it requires two lentivirus to infect the cells
to activate the endogenous promoters
so you can have the rescue.
That's not a easy task and is not yet translational,
so the technology still have to move forward.
But one thing that we thought, well,
if we showed that we can
overexpress TCF4 and have those rescues.
Why not just clone the cDNA inside an AAV,
which is translational because we already have
several clinical trials with AAV and
propose a straightforward gene therapy approach that
will require a single vector to do the trick.
Gene therapy especially for
neurodevelopmental disorders where the dose
of those genes are quite important,
faces this problem of overexpression.
If you overexpress some
of these neurodevelopmental genes,
you might cause a very similar disease
than the upward sufficiency.
The tight regulation of the gene is quite important here.
This is, for example, a problem.
Take another syndrome; Rett syndrome.
If you overexpress the MCP2 gene,
you can create the MECP2 duplication syndrome,
which is as bad as Rett syndrome.
We don't want to do that.
In manipulating these AAV vectors
to have the right dosage is a challenge.
Our approach here was taken advantage of some of
the binding of the TCF4 in the DNA.
TCF4 binds to these micro E5 motifs
here to regulate the expression of the upstream genes.
We didn't know if that would work or not,
so we clone different sizes of
these micro E5 boxes
upstream to a minimal promoter that would fit
inside an AAV vector and turns out that this
expression we can regulate
really nicely the amount of TCF4.
We end up choosing the right amount
of micro E5 boxes to have
the right amount of expressions that we
see in a y-type or in a parental control.
We use that to construct our AAV vector approach,
and then we build the organoids
and at certain point we infect it with the AAV.
The AAV was very useful
to trans fact the cDNA back and we see
the re-expression of TCF4 to the levels that we
were expecting and similar to the trans activation,
we see the restoration of the SOX2 positive cells,
restoration of the morphology
of the rosettes in the organoids.
More importantly, we start to see back again
the CTIP2 positive cells
that were missing in the first place.
These are more mature organoids.
CTIP2 positive cells are
important in the cortical connections.
It's part of the networks that really
wired the brain at very early stages in development.
One way to measure
the connectivity of those neurons is really to place
the organoids on top of
a multi-electrode array so it can record
the activity of these cortical neurons over time.
This is what we have here.
These are days of the organoids
on top of the microelectrode array in
different readouts that we can
capture from the electrical activity of those neurons,
such as number of
mean firing rates or number of network bursts.
I hope you can appreciate that,
that compared to controls in blue,
you have the mutant on networks that
are reduce in a different time points.
But upon the treatment with the AAV,
we can rescue that.
That was a very interesting way
to show that not only can rescue the expression,
you can rescue the cell or defects,
but you can go to the physiology of
the neurons or physiology of the networks.
That was very interesting approach for TCF4.
We are glad that as soon as we presented these data,
[inaudible] licensed the program.
They're taking these to a clinical trial,
so they are testing now in primates.
We hope that in a year or so
this will be recruiting for the clinical trial.
These are very short story here.
Loss of TCF4 really impair the wind
signaling that controls these population
of SOX2 positive cells.
They decrease proliferation,
changes the composition of the cortical networks,
especially on these CTP2 positive neurons
that we also validate that in
the postmortem tissues and rescue TCF4 expression,
both with CRISPR activation as
well as straightforward gene therapy with an AAV.
As long as you control
the right expression of the TCF4 you can have
a full rescue with
no side effect or predicted side effect.
We don't see any toxicity from these vectors,
so we're quite excited about that.
The project was really led by Fabio Papes,
who was a visiting scholar here in my lab,
and together with several members of my lab,
he really pushed this to happen.
Fabio do have someone in the family
that's affected by Pitt-Hopkins.
So his motivation is way beyond the academic settings.
He really wants to see this beginning in clinics.
I will stop here and maybe we can resume with the panel.
Thank you very much. [APPLAUSE]