(upbeat music)
- I would like to thank CIRM for the opportunity to speak
and share the research that we will be presenting
and that we got funding for.
We will be studying the diversity of COVID-19 lung disease
and variable treatment options in a diverse population
of stem cell induced lung tissue organoids.
COVID-19 disproportionately affects people of color
and minority groups.
There is ample evidence clinically in the hospital system,
as well as the rates of infection.
Black, Indigenous and people of color experience
more severe symptoms and complications from COVID-19,
which is caused by the virus SARS-CoV-2.
This is seen in hospitalizations
and specifically in the lung disease,
acute respiratory distress syndrome or ARDS.
There have been large clinical treatment trials
that aim to treat this disease in the hospital.
But these treatment trials have mainly been done
in large academic centers and lack diversity.
There's been two Remdesivir trials
and one had only included 20% people of color
while the other one actually included even less so.
And therefore these trials really target
only one isolated population of people.
And therefore we don't really know
whether it has the same effects on various races,
ethnicities, as well as genders.
In order to be able to replicate,
not only the infection of SARS-CoV-2
in a lung tissue organoid system,
we can utilize human lung in vitro model systems
in order to study the effects of the infection,
not only on epithelial lung tissue,
but also on the endothelial cells
and the immune cells inherent within the lung tissue.
But not only that,
if we have a human lung in vitro model system,
we can also choose various genders, ethnicities,
as well as HLA types to then be able to determine
the effects of the infection.
And then hopefully utilize repurposed drugs
in the setting of those different genders,
ethnicities and HLA types.
We aim to utilize induced pluripotent stem cell derived
3D lung tissue organoids
to represent a patient specific platform.
So here we have a schematic that shows what we aim to do
for our grant for the next year.
There are multiple published along differentiation protocols
that we're utilizing as well as our own.
And what the workflow is,
is isolating somatic cells from populations
of various genders and diverse ethnicities,
cultivate them in a dish, add reprogramming factors
to then reprogram them into fully functional
induced pluripotent stem cells.
And then add small molecules and growth factors to push
the pluripotent stem cells
into three-dimensional lung organoids,
endothelial cells and immune cells.
And then finally using these co-cultured lung tissue in
vitro models,
we can then push them through a high throughput mechanism
to determine drug treatment options.
So this is a schematic of our directed differentiation
into multicellular, 3D lung organoids.
So these are just the isolated 3D lung organoids.
We start with our STEM cell population
and then push them towards an endodermal germ layer
going through the definitive endoderm,
anterior foregut endoderm and the lung progeneter
or ventralized anterior foregut endoderm.
And this is all done in a mono layer.
And then finally, we push them into actual lung organoids
by passaging them into 3D Transwell with maitre gel in them.
What we're also able to do
and that's very innovative and novel is actually direct
the differentiation of these lung organoids
into three different populations.
We can add variable small molecules and growth factors
to push them into a more upper airway
or approximal lung phenotype and more parenchymal
or alveolar distal lung phenotype,
or a combination of both
and to represent an entire whole lung phenotype.
These are schemata of different phase contrast images of
the various lung tissue cells that we can make.
Here we show a bright field of the proximal lung organoids
that we can derive.
Here is a schemata of NKX2-1 GFP receptor phenotype,
which indicates a more distal alveolar type.
And here we have actually have a video of the whole lung
24 hours after the addition of a DCI cocktail or a
Dexamethazone Cyclic AMP and isobutal xanthine,
which actually causes these branching organoids
to start to fully mature and become more of these
sphere like organoids.
What we are also aiming to do is
to co-culture these lung organoids
with isogenic pluripotent stem cell derived endothelial
and macrophage cells.
Here in the Snyder Lab, they've already devised a,
a routine protocol that targets hiPSCs into CD31
in lectin expressing vascular three-dimensional
endothelial cells.
And with a collaborator at SBP,
we are also able to differentiate IPSCs into macrophages
and co-culture them with the lung organoids
here seen in red.
Finally, after we have derived
these isogenic lung tissue organoids,
we aim to utilize high throughput drug screening
to assess cytotoxicity from SARS-CoV-2 infection.
And this has done in collaboration with Dr. Sumit Chanda,
who has published a screen of over 12,000 late stage,
clinical stage or FDA approved drugs, or his reframe study.
He used primary screen
actually utilized immortalized cell lines
and not only human ones, but also kidney monkey ones.
And therefore we aim to utilize a more clinically relevant
human lung system
in order to fast track promising drugs to patients.
And these will not only incorporate epithelial
and mesenchymal cell populations,
which we have in our lung organoids,
but also incorporate the endothelial
and immune populations as well.
And finally, as I introduced in the background,
these must include a diversity of genders and ethnicities in
order to truly understand
the effects of not only SARS-CoV-2 on lung tissue,
but also the effects of the different therapeutic options
that we will be trialing.
So this is our project timeline in a Gantt chart.
And just to split it up quickly,
and then go into more detail, we're gonna utilize,
we're initially gonna differentiate the hiPSCs into lungs,
macrophages and vasculature,
characterize them.
Then we're going to get baseline molecular characterization
using single cell RNAseq and cytokine proteomics.
Finally, we're going to infect them with both
the SARS-CoV-2 pseudotyped spike
and the authentic live virus.
And then we'll determine
the efficacy of two protease inhibitors
that we have determined may be important
in reducing cytotoxicity from these viruses.
This is our panel of the various hiPSC derived organoids
that we will be utilizing.
We will be obtaining male and female hiPSCs from Caucasian,
African American and that next populations,
as well as the HLA B4601 type,
in collaboration with Dr. Maria Bettinotti.
So milestone one,
we have obtained the hiPSCs from the table
that I've just presented.
We have been differentiating them into 3D lung organoids,
as well as endothelial cells.
We're starting to differentiate them
into alveolar macrophages.
And we are starting to characterize the cell populations.
Here's a schematic of our co-culture systems
with our lung organoids expressed with cytotracker red
and the endothelial cells tagged with cytotracker green.
And you could see here that they're co-localizing nicely
in a three dimensional confirmation in maitre gel.
And they're probably even secreting
a little bit of mucus surfactant,
which is kind of nice.
Milestone two is to determine
the baseline molecular characterization
using single cell RNAseq proteomics.
And we have already done this
with our proximal lung organoid system.
We have performed single cell RNAseq,
and we will be performing the same types of experiments
in collaboration with Dr. Gene Yao
and the proteomics with Dr. Carl Ware.
Milestone three will be infecting
our lung tissue organoids with SARS-CoV-2 pseudotyped
and authentic virus.
Here, we show our preliminary data with
the pseudotyped virus, which also is expressing GFP
once it enters the cell and infects.
And you could see it infects our organoids
in three-dimensional formation, as well as the mono layer
in a lower and higher magnification.
And here we show that the virus is targeting goblet cells,
which express Mac5 AC
and definitely targets the H2 receptor.
But what's interesting is we're also finding
that SARS-CoV-2 pseudotyped virus can also infect cells
that in fact, do not express the H2 receptor.
And that's why it's important to study medications
and compounds that may not actually utilize
the temperance to H2 receptor entrance.
Finally, this is our data for infecting our lung organoids
with authentic live virus.
I do have BSL-3 access and have been infecting
the lung organoids with the live virus.
Now here we see our intact whole lung organoids infect
by showing positivity for spike, as well as nuclear capsid,
as well as our proximal lung organoids.
Also our positive population.
We have determined the most infective MOI.
And finally, we've also run some compounds just to show
that these lung organoids can in fact, be infected
and show a response to various treatment options.
Milestone four will determine the action
and efficacy of one of the protease inhibitors, ONO5334.
This is a potent non-lysosomotropic inhibitor
of cathepsin K protease.
Its initial use was in a phase two trial
in menopausal women.
And it was used in oral doses,
which is preferred over IV
and initially utilized for inhibiting osteoporosis.
It inhibits viral entry and vital enzymes
that destroy connective tissue in the interstitial matrix.
And therefore it may actually protect alveolar
and bronchial lung epithelium.
And here we show our preliminary data using
the pseudotyped virus along with treatment with ONO5334.
And a negative and a positive control of DMSO
and Remdesivir
and showing that it does actually decrease infectivity.
Finally, milestone five will be to assess
the action and efficacy of another protease inhibitor
of VBY825.
This is more of a pancathepsin protease inhibitor.
And it has been already tested for toxicity
and efficacy in multiple pre-clinical models.
And it's initial indication for use was
for autoimmune disorders,
which actually may be effective against the immune
and inflammatory responses triggered by SARS-CoV-2,
which is why it's important to actually have an immune cell
within the modeling system.
Finally, milestone six,
if everything goes well and according to plan
and milestones four and five suggest efficacy,
then we can actually schedule an INTERACT or pre-IND meeting
for the two different molecules.
And we're already in a collaborating with IQVIA
in order to make sure that this process is seamless.
And then hopefully our work and our model system
will then be able to lead towards clinical trials
utilizing these molecules.
So in conclusion, using lung tissue organoid co-cultures
to just study disease disparity from COVID-19
is extremely important
and also allows feasibility and facility
by utilizing patient specific lines,
including those of different genders,
ethnicities and HLA types.
And utilizing the different cells from
the same isogenic pluripotent stem cells,
which retains that population's genetics,
as well as epigenetics.
We can also mimic the human organ in a dish.
And instead of awaiting a lung biopsies from donors,
we can have an infinite supply
that we can continue to differentiate,
passage, as well as, cryopreserve.
And here again is our panel of the hiPSC derived organoids
that we will be differentiating and a co-culturing.
Finally, if genetics is not the reason for disparity
and it's more of an environmental or epigenetic phenomenon,
we can actually expose these lung tissue organoids
to either nicotine or vaping.
And that's in collaboration with our,
with a lab that studies lung cancer in
the setting of smoking and tobacco.
And we can actually study various environmental factors
to determine if they are the cause for worse outcomes.
And finally, the use for drug screening
in our lung organoid model is great
because you can actually avoid the animal model.
As we know, all know that there are multiple drugs
that may be efficacious in animals,
but then ultimately fail in large human clinical trials.
We can directly touch human cells for effect
and human cells are important because they're the ones
that express the different receptors and entry points for
the virus that may not actually be utilizing
specific receptors that animals may not have.
And finally, patient-specific treatment regimes
as we've talked about before.
So I just want to acknowledge Dr. Evan Snyder
who's the PI on this grant, as well as of the grad students,
postdocs and all research assistants in the lab
who are helping with all the differentiation and infections
as well as all of our collaborators.
And of course the funding from CIRM.
Thank you.