(upbeat music)
- It's a pleasure to speak
at the 2020 CIRM Grantee Meeting,
and I should say before I start
that all of the work
that I'll present to you today,
was funded by CIRM over the last many years
and would not have advanced to the point
that I'll show you
in the absence of that funding.
And both stories rely on
a cellular reprogramming approach
to address human forms of heart disease.
And in both cases,
we've leveraged cardiac developmental networks
to either regenerate damaged hearts
by reprogramming resident cardiac fibroblasts
to cardiomyocyte-like cells,
or to understand mechanisms of disease
using human iPS cells,
and then followed by drug discovery.
In the first story, we have leveraged the fact
that the human heart is made up half
of actually cardiac fibroblast
and less than half of myocytes.
And the cardiac fibroblasts are the ones
that support cardiac muscle cells,
but also the ones that are activated
to form scar tissue as you see,
in this section of this heart here.
And because the human heart
and mammalian heart in general,
has very little, if any capacity to regenerate,
once cells are lost after damage, as you see here,
there's no capacity to regenerate.
And so we over the years attempted
to reprogram these resident cardiac fibroblast
into new cardiomyocytes right where they are
in an effort to regenerate damaged hearts.
And to make a long story short,
over the years,
we were able to find that the combination
of these three key developmental transcription factors,
Gata4, Tbx5, and Mef2c were sufficient
to reprogram cardiac fibroblast
into cardiomyocyte-like cells,
that we called induced cardiomyocytes or iCMs.
And this was relatively inefficient
in vitro on plastic, but could be done,
but in vivo in mice,
we found that this was quite efficient,
and these resulting cells were most similar
to adult ventricular heart cells.
They could electrically couple with one another,
which was key for improving cardiac output,
and in fact, when measured by MRI,
these mice did in fact have
a significantly improved cardiac function.
This is an example of what those hearts look like
after coronary ligation
followed by gene therapy mediated
delivery of these three transcription factors.
Following three months later,
one can sacrifice the hearts
and if you look at the apex
cross section around this level,
you see abundance scar and the control
less so at the further up in the heart,
and in comparison,
mice treated with this gene therapy approach
had abundant muscle, even at the apex,
as you can see here,
and we we have fluorescently labeled
with the Cre based system, the fibroblasts,
and can see that all of these
are actually newly formed cardiomyocytes.
We of course asked
will the same combination work
in human cardiac fibroblasts,
and it turns out that
in human cardiac fibroblast replacing
Gata4 with myocardian
a transcriptional coactivator for MEF2C,
actually was sufficient
now to reprogram human cardiac fibroblasts
into cardiomyocyte-like cells,
and you see an example of
a beautifully reprogrammed cell here
with the sarcomeres indicated without
cardiac alpha-actinin.
And so this combination, we then tested in vivo
in pigs to see if they...
which has a heart, more similar
to the size of humans,
where in vitro this combination
also reprogrammed pig cardiac fibroblast.
And using an AAV now,
a vector to deliver these three factors,
we injected these into pig hearts
after coronary occlusion
and co injected them with a retrovirus
expressing dsRed,
because a retrovirus will only infect
dividing cells and myocytes don't divide
so it would allow us to mark
the non myocytes that were infected by virus,
sometimes coinfected with the AAV.
And then we can ask whether they are
dsRed positive cells, that now have sarcomeres
suggesting that these might be newly born cardiomyocytes.
And you can see here in this
high magnification section,
that there are a number of cells
with beautiful sarcomeres that are also red
suggesting that these may be new newly
reprogrammed cardiomyocytes.
And you can see that a little bit more easily here
with the just adjusted dsRed channel in white
and so you can see that this is a
fairly efficient reprogramming event in vivo.
So this was very encouraging
that we might be able to generate enough cardiomyocytes
to actually make a difference,
and this is on the border zone of the damaged area.
And so with this information
all supported by CIRM,
particularly the pig translational studies.
We have put this technology
into a startup company called Tenaya Therapeutics
that was launched with a 50 million series A
financing with the column group in 2016,
followed by a series B event last year,
and they are advancing this towards clinical trials.
And over the years they have refined
the technology and the genetic material,
that would be delivered as well as a vector.
And I'll just show you one slide from Tenaya
with their approval,
that shows you a cohort of pigs around 10 pigs,
either treated with the gene therapy AAV
or control and in blue you can see
that there's a marked improvement
in the ejection fraction of these pigs,
compared to the controls in gray.
And this degree of improvement of 11% absolute,
ejection fraction improvement is really quite significant
and would be clinically meaningful,
particularly for those who might be
waiting for on a transplant list
to potentially be able to avoid
a need for a transplanted heart.
And so we're very excited about
the Tenaya pushing this forward,
and we continue to collaborate with them
to advance this technology.
Now, in the second story,
I wanted share with you as I mentioned,
it's really one of them about
understanding disease mechanism,
and doing drug discovery with iPS cells.
And this story relates to a family shown here
where this multigenerational family
had a very common form of heart disease,
called the calcification of the aortic valve
that looks something like this,
where the aortic valve becomes hard
and calcified, requiring replacement surgically,
and about a 100,000 replacements are done a year
just in the United States,
so it's very common disease.
The etiology hasn't been known,
and there's no currently no medical therapy.
We do know that in cases
where people are born with a congenital anomaly,
where there are only two leaflets
instead of three in the aortic valve
like you see here,
that's something we call a bicuspid aortic valve.
That about a third of those individuals
will develop calcification as they age
into their third, fourth, fifth,
and sixth decades of life.
And this congenital anomaly
is actually the most congenital anomaly of all,
it affects one to 2% of the population.
It turns out this family had both bicuspid
aortic valve and calcification as they got older,
and it's caused by a heterozygous
loss of function mutation,
in the very well studied transcription factor notch one.
And so having identified
the genetic cause of this,
we were able to use a isogenic CRISPR gene edited
gene corrected iPS cells from this family,
to deeply understand the mechanism.
And it turns out that what we found
is that normally NOTCH 1
which sits on the cell membrane
is in a position to sense sheer stress,
and its job is to normally repress osteogenic pathways
in the endothelial lining of the valve.
And we know that the endothelial cells of the valve
can trans differentiate and become
mesenchymal cells and go to the valve,
and there, that's where
this NOTCH is playing a role in preventing
this osteogenic fate.
So essentially what we found is that
in the setting of high flow insufficiency,
what happens is endothelial cells
undergo more EMT and become more osteogenic.
So it's essentially a cellular reprogramming event
from an endothelial cell to a more osteogenic like cell.
And this we were able to discover
through a deep interrogation of these iPS derived
endothelial cells as shown here
that resulted in deep understanding
of the gene network that gets dysregulated.
And it turns out that the network,
it narrows down to three key transcription factors,
SOX7, TCF4 and SMAD 1 that are central players
that then dysregulate a host of other genes
that results in this self fade,
switch if you will.
And so that gave us the thought
that maybe we could drug this process,
and so we screened a library of 1600
highly curated compounds.
And instead of looking for one,
two or three outputs,
we screened for 120 genes in the network
that were dysregulated with each molecule.
And we used a machine learning approach
to classify cells as either normal
or abnormal and asked what drugs might
reclassify abnormal cells?
And from this we found six hits
that actually had resulted
in this change in classification.
I'm just showing you here how,
what this output looks like in blue.
These blue dots are wild type cells, normal cells,
green dots are what the machine learning algorithm
calcified is heterozygous cells.
And in red are what the algorithm classified
as either normal or abnormal,
here you can see that most of the drugs do nothing,
they still cluster with the green,
and so are still heterozygotes.
However, you'll see that there are a few
red dots that are now clustering with the blue dots,
and these are what our hits are,
suggestive of a drug that is shifting
this profile broadly.
And so we have taken these six hits
and tested them in vivo, in a mouse model we generated,
where by shortening the telomeres
and notch when heterozygous mice
to be more like human telomere length,
we can actually recapitulate the human phenotype
of a calcified and obstructed aortic valve.
And so I'm showing you here
by echocardiography in these mice,
the aortic valve peak velocity,
which reflects the aortic valve stenosis
very similar as occurs to in humans.
This is partially penetrant,
but you see in the control,
a number of these mice have acceleration
of the blood flow across the valve
indicative of stenosis,
both of the aortic valve and pulmonary valve.
And one of the six drugs shown here
had a remarkable effect in completely preventing
in a statistically significant way,
aortic valve stenosis in the vast majority of mice,
as you can see here, as well as
preventing pulmonary valve stenosis,
and by histology this drug also prevented
thickening of the aortic valve
and calcification of the aortic valve.
So we think we've got a drug
that we discovered in human iPS cells
that works in IPS cells to alter the dysregulation
and works in vivo.
Now, finally we asked,
would this drug also affect those
who don't have NOTCH1 mutations as a cause?
And to do this, we worked with the group in Russia
who had collected primary aortic valve,
endothelial cells from
ex planted human aortic valve samples,
either with three leaflet calcification
or two leaflet calcification.
And we exposed all of these endothelial cells
to the drug and then did RNA sequencing.
And I'm just showing you here,
the most important result
or the most important factors these three
sort of master transcription factors
that we know were causing the broad gene dysregulation.
And in each case, this drug restored
the corrected the upregulation seen
in the disease valves, endothelial cells to normal,
whether we're looking at SOX7, TCF4 or SMAD1.
And it didn't matter whether their tricuspid
or a bicuspid aortic valves,
we saw the same things in the abnormal cells,
and those were corrected by the drug.
And so we're very excited that through this approach,
we actually have a potential drug candidate,
which would be the first that works in mice
and works in broader populations
of human aortic valve cells.
And we're considering now how to advance this
towards a clinical trial.
So with that, I'll close
and thank the members of my laboratory
that contributed to this work,
including former lab members
and our collaborators
and of course our important funding from CIRM.
And with that, I'll be happy to take questions
at the end of this session.
Thank you very much.
(upbeat music)