[APPLAUSE]
>> Sarah, thank you for the introduction.
So just a couple of things.
While CRISPR is allowing us to
manipulate genes with precision,
we're not quite in-patients yet.
So what I'm gonna talk to you
today about is our progress of
bringing this technology as
a new therapeutic to patients.
And yes, my dad was a professor here, and
it was at the old business school
which I think is right over there.
But he is happily enjoying retired life.
I was told by the organizers
that that's the social media
link if you guys wanna go on.
And so, the title of my talk is CRISPR,
Genome Editing and Deadly Diseases.
And I've kinda co-opted a term here called
precision medicine, that you hear widely.
Because what I think I'll try
to describe to you today is for
many diseases we know the exact cause,
and yet
we don't have precision medicine
that is based on that exact cause.
What I'm gonna show you I hope, is that
with this new tool called CRISPR-Cas9,
we're able to change DNA sequences
that we know cause disease.
And one day we hope what we can do is use
that technology to change mutations or
change DNA variants that cause
disease in patient's own stem cells,
give them back to the patient and
cure them of the disease.
So we're in Silicon Valley, and so
I'm gonna start with a quote and
end with a quote.
So the starting quote is from
somebody named K.R. Sridhar.
I actually have no idea who he is,
but I like the quote.
And it says that, supposedly on Wikipedia
he's some sort of famous entrepreneur,
is a belief that finding innovative ways
to make the world better is important.
A mind in search of better ideas,
even if they sound radical,
is more likely to stumble across one.
And I think that sort of mantra is not
only something that drives Silicon Valley,
but certainly drives research in my lab.
So I actually, as you heard,
I grew up in the area, went away and
came back as an associate professor
over at the School of Medicine.
And after I got here on the MDPHD program,
has a seminar series called
Medical Mysteries and they said Matt why
don't give a talk on sickle cell disease.
And I thought [SOUND] and I'll tell you
a lot more about sickle cell disease.
And it was a little confusing to me
because we know a lot about sickle cell
disease.
It's not a medical mystery.
So what I came up with is a title
102 years later: How come we don't
have a cure for sickle cell disease yet?
And so, why 102?
So I gave this talk in 2012.
So I would actually have to title it 106
years ago: How come we don't have a cure?
And the reason I used 102 years
is that it was in 1910 that Dr.
James Hera looked at the blood
smear of a dental student of his,
or a dental student at University of
Chicago, named Walter Noel, unfortunately
there's no picture of him, and he saw this
abnormal blood smear under the microscope.
Normally our red blood cells
look like this one here.
Actually, not even like this one.
More like these up here.
Round, they have what we call
a bioconcave shape and what he was seeing
was these abnormally shaped cells and so
he wrote a paper and this what he wrote.
So there's many sort unique
things about this paper.
One, is that that's a single author paper.
In science nowadays,
it's almost impossible to publish a paper
in which there's only a single author.
Many of my papers have 10,
15, 20 authors on it.
And the other thing is this,
the language of the times.
So he describes, this case is reported
because of the unusual blood findings,
no duplicate of which
I have ever described.
Whether the blood picture represents
a merely freakish, poikilocytosis,
which just means abnormally shaped cells,
or dependent on some peculiar physical or
chemical condition of the blood, or is
characteristic of some particular disease,
I cannot present answer.
I can tell you if I tried to write that in
the second sentence of my paper saying,
I don't know what's going on here but
there's something weird and
I think you guys should all know about it,
it wouldn't get past anybody.
But he got away with it.
And then, of course, the fact that in the
history he had to describe the patient as
an intelligent negro of 20.
Also, I think, points to our
times have fortunately changed.
Now, of course, this disease,
sickle cell disease,
was long known before 1910 in Africa.
And it goes by many different names,
none of which I can say.
I mean, I just can't say them.
I could say them.
But they have this feature in which they
have repeating high pitched vowel sounds.
And what these words
translate as is "beaten up",
"body biting", "body chewing".
So they're describing a disease
that is characterized by
frequently painful crisis.
Where these people get beaten up.
And so, nothing much happened for
39 years.
And then, in 1949, Linus Pauling,
only person to win two Nobel prizes,
took blood from a patient
with sickle cell disease and
somebody who has sickle cell trait,
I'll describe to you what that is,
and somebody who doesn't
have sickle cell disease.
And ran it on a gel and separated
the proteins in the red blood cells,
the hemoglobins in the red blood cells.
And what he found is that
the normal hemoglobin
in the red blood cell ran with
this characteristic shape, and
the sickle cell hemoglobin ran
at a slightly different rate.
And you could see that a 50-50 mixture of
A and B, you could see the separation.
And so, he deduced from this small
shift in migration on a gel,
that this molecule had two to four net
positive charges than this molecule.
It turns out he was right.
There's two more positive
charges that's it.
And so, this has been called
the first molecular disease.
Eight years later, Vernon Ingram who was
then in Cambridge, England who is now
a professor of at MIT, actually took
the hemoglobin molecule from sickle
cell patients and non sickle cell patients
and separated the amino acids on a gel.
And was able to deduce that
the single change was a glutamic
acid that was converted to a valine.
So on one protein, one amino acid change,
turns out the hemoglobin molecule has
two copies of this protein, and so
that's what gives the two charge change.
And [COUGH] then just a short time later,
Makio Murayama showed that what happens
with sickle hemoglobin under certain
conditions the molecule polymerases.
So normally hemoglobin is, can be
tightly packed into red blood cells.
It's normally a crystal
in concentration but
when sickle hemoglobin looses it's oxygen,
and I'll show you schematic of this,
it creates a hydrophobic surface,
a surface on the surface of the protein.
That allows it to polymerize with itself.
And it creates these stiff polymers.
And it's those stiff polymers that
turn the squishy red blood cell into
that stiff sickle red blood cell
that gives the disease its name.
And finally, in 1977, after the discovery
of recombinant DNA down here at Stanford,
and we won't mention
the contributions of Berkeley,
>> [LAUGH]
>> The actual sequence change that caused
this glutamic acid to
valine was identified.
And it's a single nucleotide change,
an adenine to a thymidine.
And as I mentioned,
let me just skip forward one slide, so
what happens then is we have this single A
to T change that creates the sickle gene,
that leads to an RNA sequence
that differs by one nucleotide.
But that one nucleotide changes the code
from a glutamic acid to a valine.
And this is a hydrophobic residue,
and that allows the plumerzation,
which converts this soft red blood
cell into this sickle red blood cell.
Let's see if I,
we don't need to go through that.
So why does this cause a disease?
Well, it causes a disease
through this process of vaso or
veno occlusion or vaso-occlusive disease.
And what happens is,
these stiff sickle red blood cells
get trapped in the small
blood vessels of our body.
They're not able to transverse
through these small red blood vessels.
There's a characteristic that, actually,
the lining of the blood vessels is
actually inflamed because of
the damage that sickling induces.
And so, you create these blockages for
blood getting to the distil tissues.
And so, the most common manifestation of
oxygen not getting to the distil tissues
is acute pain in the bones.
And I think we, as doctors,
are only starting to understand how
frequent these bony crises are.
And there have been recent studies that
have shown that sickle cell patients
are taking pain medicines two or
three times a week for these bony crises.
Sometimes they become so severe that
they need to come into the hospital and
get our strongest pain medicines.
And they sit in the hospital for
two to three weeks at a time.
And this was a picture that
was drawn by a teenage girl,
as she's trying to depict
the pain she was in.
And [COUGH] I first got a sense
of how much pain this was,
when I was listening to somebody from
the Sickle Cell Disease Association,
talk about these painful crises.
And he had the disease himself.
And he said, it's like having
your hand slammed in a car door.
Except, that instead of the pain lasting
for a few seconds, no, it lasts for weeks.
And so, yeah,
I think, that gives you a sense of
what these people are going through.
But the problem isn't just that it
causes recurrent bony painful crises.
The problem is that it
causes the inability of
oxygen to get to all the tissues.
So we don't get enough oxygen to our
kidneys and it causes kidney damage.
We don't get enough oxygen to
the lungs and it causes lung damage.
And importantly,
you don't get enough oxygen to the brain.
And you end up with silent strokes and
a decrease in IQ.
So not only is there significant
morbidity through life,
this then leads to early mortality.
And 30 years ago, well,
actually 20 years ago.
But a study that was started 30 years ago,
Laura Platt, a pediatrician in Boston.
Showed that on average, people with
sickle cell disease, both men and women,
were only living until their mid 40s.
Now, with contemporary care, now,
people with sickle cell disease are living
to adulthood just like everybody else.
So the expectation was, is that if
we got people to adulthood better,
maybe we would extend their lifespan.
But it turns out that,
that has not happened.
And this recent study that came out of USC
has shown that the average life span for
people with sickle cell disease,
still remains in the mid 40s.
I'm not gonna tell you whether that
means I should be alive or not.
So what that means though, is so
that here is the sequence of the protein,
in the most common variant of beta globin.
And you have no sickle cell disease, and
you have one copy of A, and one copy of A.
You'll have two copies of S, so
this is a recessive disease.
You get the sickle cell disease.
And if you have one copy of A,
and one copy of S,
you have no sickle cell disease,
in what we call sickle cell trait.
And so this is all genetics.
But one of the interesting things
about this disease, is that change.
Is change from the E to the V
has occurred four separate times
during human evolution.
So it occurred by looking at the variance
around the change and genome.
We can deduce that this change
occur four separate times.
And has persisted through human evolution.
Once here, in what we call the Senegal
variant, the Benin variant,
the CAR variant, and the Asian variant.
So this is interesting, we have a mutation
that causes people to die early.
That has occurred several different times
and persisted in the human population.
And the reason for that is it turns out
that patients who have sickle cell trait.
So one S and one A,
have a higher resistance to malaria.
And that was first hypothesized when
people looked at where people with sickle
cell lived.
Which is here in Africa and
India and parts of Saudi Arabia.
And you overlap that
with where malaria is.
And you can see there's
a striking overlap.
Now, there are parts of the world
with malaria that don't have sickle
cell anemia.
Turns out, they have other mutations
that confer protection against malaria.
And [COUGH] so, that was suggestive and
the proof has come in many different ways.
But the first sort of proof that
sickle cell trait is protective,
was shown by Tony Allison.
And what he showed is,
when he looked at survival.
Is that patients with SA had
a higher survival than patients
with AS in a setting.
I mean, sorry,
this was this person's data,
whereas patients with SS
had the lowest survival.
So this minor survival advantage
by being AS sickle cell trait over AA,
is why this allele has persisted.
Because malaria puts such
pressure on the human population.
So now, we can go back and say, aha,
AA doesn't give you sickle cell disease.
But you have a higher
risk of severe malaria.
AS does not give you
sickle cell disease and
you have a lower risk of severe malaria.
Unfortunately, if you have
sickle cell disease and
you get malaria, you die like crazy.
Now, one other thing
that I wanna point out,
is those survival curves are based
on sickle cell patients in the US.
In Africa, right now, where obviously,
medical therapy is not as strong.
The average life expectancy is on
the order of five to seven years.
So these patients are dying young and
yet, that allele still stays in
the population because of this
protection against malaria.
So what we have now, is a 6 billion
base pair DNA sequence that codes for
how our cells function.
And there is a single typographical
error that is causing the disease.
The idea is, can we fix that error?
Of course, these diseases, sickle cell
disease is not the only disease like this.
And there's probably close
to 10,000 such diseases.
And they span all aspects of medicine,
from cystic fibrosis,
to hemophilia, to bubble boy disease.
To different diseases of the heart,
different diseases of the skin,
the muscles, and the brain.
Okay, so
the idea is if we can cure one, maybe we
would have a mechanism to cure them all.
[COUGH] So the disease lies out
here in the red blood cell.
The problem is, that the red blood
cell only lives for 100 days.
So if we could fix the red blood cell,
that would only give benefit to
the patient for about 100 days.
And we can fix red blood cells
by giving blood transfusions.
And so, some of the ways we treat sickle
cell disease patients who are having sever
manifestation.
We give them repeated
blood transfusions and
replace their red blood cells with
red blood cells without sickle.
But as I've said,
that's only a temporarily solution.
So if we really wanna fix this disease for
the long term.
What we have to do is go back and
fix the hematopoietic stem cell.
The stem cell that gives rise to all
the different types of blood in the body,
and this cell lasts a lifetime.
And there's a picture,
electromicrograph of this round bowling
ball that sits in the bone marrow.
And the progeny of this
cell makes red blood cells,
platelets, your immune system and
different aspects of your immune system.
So if we could give fixed stem cells,
we could cure this disease.
So one way of getting fixed stem
cells is taking the bone marrow.
The stem cells from someone
who doesn't have the disease.
And transplanting them into
somebody who does have the disease.
And so, the way this is done is we
take the patient into hospital.
We give them high doses of
chemotherapy to get rid
of all their own stem cells
in their bone marrow.
And then, we harvest the blood
stem cells from the donor.
And we infuse those stem
cells through an IV.
Those stem cells float through the blood.
Find their homes in the bone,
their niches and
then are able to reconstitute
the blood system.
And this was first done to cure
sickle cell disease in 1984.
And actually, what was interesting is
that this was a little girl who had
leukemia and needed a bone marrow
transplant to cure her leukemia.
And so she got a transplant from her
brother who had sickle cell trait.
And what you can see is this
the mixture between A and
S in the donor was about 60% A and 40% S.
And then about seven months after the
transplant, the patient actually developed
the same hematologic parameters,
and was cured of her disease.
And so after this,
this approach has been broadly utilized.
And it turns out that if
you have a good donor.
By a good donor I mean, a sibling who is
an immune match to you, but doesn't have
the disease, the cure rate is now around
95% with a bone marrow transplant.
And after the bone marrow transplant
these patients are cured of the disease.
Now unfortunately, this is,
most patients only about 10
to 15% of patients actually
have one of these donors.
And so really we need to find
now something better for
all the other patients.
And so the idea is that instead of using
corrected cells from somebody else,
is can we take the patients own
stem cells and correct them?
And to be very simplistic about this,
would be the idea of taking a 57
Chevy that had two broken headlights.
And somehow being able to fix one of the
headlights because we know that would be
sufficient to cure the disease.
And we call the fixing of headlight,
we can fix these genes by a process
called homologous recombination.
And the idea of using
homologous recombination
was first described about 30 years ago.
Now, unfortunately,
while he, Oliver Smithies
showed that you could use homologous
recombination to fix the mutation.
He can only achieve it in
about one in a million cells,
which was not gonna be sufficient.
And so that brings us to genome editing.
How do we increase this frequency
of homologous recombination?
How do we increase this frequency
of correcting mutations
to a high enough level that we can use it?
And the way we do that is
through the following process.
Is we design a specific protein,
that is designed to bind the DNA
at a very one site in the genome.
And then attached to it
is an enzymatic activity,
a nuclease that will create
a double-stranded break at that site.
And if there's a break
created at that site,
the cell says I have a broken piece
of DNA, I need to fix that DNA.
One of the ways that can fix the DNA is by
just stitching the two ends back together.
And we use that sometimes and
through that a process of breaking the DNA
and stitching it back together over and
over again, you can end up with
mutations at the side of the break.
And so, this is a good way of
activating genetic element.
So it's not something that we'd
wanna do for sickle cell disease but
something that's useful
as an experimental tool.
But the other way cells repair
a double-stranded break is by using this
Homologous Recombination process.
So if we give an extra piece of DNA that
shares homology or shares identity to
the where the break is but
we've introduced small changes in the DNA.
The cell will use this piece of DNA
through a copy and paste mechanism, and
fix the break and
introduce this new sequence.
And so
sort of going back to the car analogy,
what we're doing is we're
taking a sledgehammer.
And we're busting up the broken headlight.
And that's inducing the cell,
then, to fix the headlight so
at least one headlight is fixed.
What's interesting about this
way of breaking the DNA and
then that allows us to rearrange the DNA
is actually nature does that itself.
And there's multiple different processes
in which nature wants to rearrange the DNA
in the cell in different ways
to create different activities.
And nature does this by creating a protein
that makes a double-stranded break.
And then either repairing it by that
non-homologous stitching mechanism or
by homologous recombination.
And one of the things that we can
use is study these to try to better
understand how to increase the frequency
of the process when we wanna use it.
I'm gonna go back just a couple slides.
So there's lots of different ways of
engineering proteins that can make
a double-stranded break.
But what has turned out to transform the
field is this new class of proteins called
the CRISPR/Cas9 protein.
And by the way, my lab has worked on all
of, well we haven't worked on those but
we've worked on all of these.
And these we now use exclusively
to CRISPR/Cas9 platform.
Now where did this platform come from?
It's really a fascinating story.
So it turns out that we know
we have an immune system.
So a system that allows us to prevent
being infected by bacteria and viruses and
other things that wanna invade us.
Well, it turns out that bacteria
have to do with the same sort of
invasive particles.
They're viruses for
bacteria or DNA for bacteria.
And for the viruses for
bacteria, we call them phage.
And it turns out that when
a phage infects a bacteria,
the bacteria have this CRISPR/Cas9
system that will take the incoming DNA,
chop it into little bits,
integrate it into an array.
And then express little fragments
of this array as an RNA molecule
that complexes with Cas9, a protein.
And so that then next time the phage
comes in that guide RNA molecule
complexed to Cas9 will
find that piece of DNA,
will find the phage DNA and
chop it into little bits.
And so
that's a really interesting discovery.
But what has sort of
transformed my research
is that you can adopt that
system to work mammalian cells.
So now, what we do is we synthesize
a piece of RNA that we've
engineered to recognize a specific
DNA site in the genome.
So we're gonna engineer
what we call a guide RNA.
That is going to bind to
the beta-globin gene,
the gene that's involved
in sickle cell disease.
We're gonna take that guide RNA and
complex it to the Cas9 protein.
And it's this Cas9 protein that
then makes the break in the DNA.
And amazingly, this simple
bacterial system of Cas9 protein
with a guide RNA works
in a mammalian cell.
It can find that target site and
it can break that target
site incredibly efficiently.
So now we have some tools.
So some of, now we need to
understand how to use those tools.
A couple things that we've
learned is that, in order to get
high frequencies of editing in a cell, you
need to get a lot of nuclease in the cell.
So if you only give a little
bit of nuclease to the cell,
in the green bar here,
you only get a little bit of editing.
And if you give a lot of nuclease to
the cell, you can get a lot of editing.
So when we find that we're not getting
a lot of editing in cells, what we have to
focus on is how do we get more nucleus
into the cell to create that break and
to create the edits?
If we're creating the break, then how
can we get the break to get repaired
by correcting the break rather than
repairing the break by enjoining?
And the simplest way we found to bias
the way the break gets repaired is by
delivering lots of that donor DNA.
So if we swamp the cell with donor DNA,
the cell for
reasons we don't fully understand,
will more often use that donor DNA to fix
the break than just
putting it back together.
And if we measure the ratio
of how many times it fixes it
using our donor DNA versus just
breaks using the other pathway.
When we give lots of donor
DNA we can find that
we get over one of one HR
of M per every NHEJ event.
And this worked great in cell lines.
So cancer cells lines
are easy to work with but
they of course are not what
we wanna modify in patients.
And so when we took the CRISPR system and
we moved it into the cells we wanted to
modify, we found that it didn't work.
And what we thought is that we
weren't getting this crisper system
expressed at high enough levels
in the hematopoietic stem cells,
in those stem cells,
to get the activity we wanted.
So we began a collaboration
with Agilent Technologies,
a biotech company right down the road,
and they have the ability to
synthesize this RNA molecule in a test
tube and make large quantities of it.
And so we made some different
flavors of this large RNA molecule.
And we put modifications on the end
to protect that RNA molecule from
being degraded.
And we made different sorts of
modifications where there was just one
modification, or two or three, with the
idea of creating more and more resistance
so that the molecule, once we introduced
it into the cell, would not be chewed up.
And we said, okay, will this finally
allow us to modify the stem cells?
And it did.
And so, at two different genes, that gene
involved with sickle cell disease and
the gene involved with bubble boy disease,
what we found is,
is that if we delivered
the Cas 9 as a MRNA, and
we delivered the guide RNA as
the stabilized forms of that small RNA.
So, Cas 9 MRNA will get
translated into protein,
the protein will then complex
with the guide RNA, and
what we find is that we're able to get
insertions and deletions at our gene.
So our first step,
we can make breaks at our target gene.
So then the next question is, is how
do we get our donor DNA into the cell?
It turns out that if we take naked DNA and
put it into our cells, those cells see
that naked DNA as an invading pathogen and
they'll actually react to it and
secrete all the things that make us
feel sick as stink when we get the flu.
So they make tons of Interferon and
they make themselves sick
because they don't want the invading
pathogen to take over the cell.
So when we introduced DNA into these
cells, we were making them sick, and
that wasn't working.
So what we had to do was figure out how
to get this donor DNA into our stem cells
without making them sick.
And the way we've done that is we now
packaged our donor DNA into a virus,
a recombinant adeno associated virus.
This is what my colleague, Mark at
Standard calls nature's nano particle,
cuz it is evolved as a way of delivering
DNA into the nucleus of cells
without activating that
interferon response.
So what we do is we take millions of
these cells, we put them on a little.
We mix it with our Cas9-guide RNA complex,
and we zap that mixture,
we create holes in the cell's membrane.
This will float into the cell and
get to the nucleus.
And then we pull it out the cuvette and
we add our virus, and
then the virus delivers the donor DNA.
And so, the first thing we did is
just to see how well this would work,
is we made a virus in which we were
gonna insert what we call a cassette
that would turn the cell green.
So we were gonna insert a promoter and
the GFP gene so
it'd integrate into the beta globin gene.
And if it integrated into
the beta globin gene,
it would turn our not-green
cell into a green cell,
which we can measure by microscopy or
flow cytometry, or other techniques.
And what we found is,
is that using this strategy,
we could get 30% of our
stem cells to turn green.
So now we have an ability of making
DNA changes at the betaglobin gene.
Now, of course,
turning stem cells green is cool, but
it's not actually gonna
cure sickle cell disease.
So how are we gonna actually
cure sickle cell disease?
Well, first we have to show that this same
process of turning cells green in wild
type stem cells will work in stem cells
that come from a sickle cell patient.
It could be that the sickle cell
patient's stem cells are different and
everything we worked out in
normal stem cells won't apply.
But fortunately,
we found that that was not true.
And what we found is, is that using that
same system I just described to you, using
sickle cell, what we call CD34 stem cells,
we get 40% of the cells to turn green.
So not even 30%, but 40%.
Now, again, that's not gonna do, I mean,
again, we can make green cats and
green animals, but what we really wanna
do is correct that sickle cell mutation.
And so now, instead of making a donor
DNA that made cells green, is now,
we had a donor DNA that
only had a few DNA changes,
the most important of which is that
was gonna change that T back to an A.
So I'm gonna just switch
that one nucleotide back.
Now, for technical reasons we introduce
these other changes, and they allowed us
to monitor it, and they allowed us to
increase the efficiency of the process.
And then we took sickle
cell stem cells and
we introduced our nuclease, and
we introduced our donor DNA.
And then we measured what happened
to all the beta globin genes?
10% of the beta globin
genes were unchanged,
they didn't do anything to it to them,
okay?
40% of the beta globin genes,
the nuclease came,
it cut the gene, and the gene mutated.
So we broke the gene even more.
But 50% of the beta
globin genes were fixed.
So we've now turned 100% of the beta
globin genes into 50% fixed,
40% broken even more, and 10% unchanged.
And then, what if we took
this population of cells and
analyzed individual cells and said what
happened to the two genes in each cell?
We got this distribution.
We got a distribution in which about 2%
of the cells had neither gene change.
But importantly is we go about
50% of the cells in which at
least on of the sickle genes was
converted back to the hemoglobin A gene.
It was fixed.
Okay, now sometimes both
of them were fixed.
Sometimes it was A over S.
So we created that sickle trait, and
sometimes it was A over
the further broken.
And by the way, when we break it further,
that creates a disease called beta
thalassemia, but we know that
A over beta thalassemia is good.
So we know that 50% of the stem cells
have at least one gene corrected.
And when we turn those stem
cells into red blood cells,
what we find is that 84% of the hemoglobin
coming out of those stem cells
making the hemoglobin A, and
we're only getting 16% hemoglobin S.
Whereas in the unmodified sickle stem
cells, 100% of the hemoglobin is S.
And the important thing
to know is that when we
keep the level of Hemoglobin S below 30%,
we're here at 16%,
we keep it below 30%,
patients are cured of their disease.
So we've hit a number that we think
would cure patients of the disease.
This was all done in a test tube, so
a really important aspect
of all this is did we
create stem cells that can actually make
blood if you put it back into an animal?
So we can't put it back into patients yet,
we're still in the laboratory.
So what we did is we take the human
stem cells and we put them into a mouse
that has been engineered to accept
human stem cells, all right?
The mouse immune system has been ablated,
and it allows our human cells to graft,
and we can see if our human stem cells
behave like human blood stem cells.
And what we find is, is this that they do.
So our input correction
frequency is about 44%.
And when we look at three mice that
had gotten these cells, on average,
the correction frequency was 40%.
So, now we have an ability to
correct the mutation of stem cells,
we can show those stem cells make blood,
and we can show those stem cells
act like stem cells when we
put them into an animal model.
So that puts us in the position to
actually think about taking this process
that we're doing at about
a million cells per time and
actually scaling it up Into a patient.
And so
we can define sort of some criteria.
So we will harvest the stem
cells from a patient, and
we're not gonna do babies first.
The FDA, the regulatory agencies,
will say they're too special.
You're gonna have to do adults.
But babies are cuter.
So we'll harvest the stem cells when we
know we have to harvest a lot of them on
the order.
We're gonna have to, for an adult,
we're probably gonna have to harvest on
the order of five hundred
million stem cells.
And then we're gonna have to bring them
into a specialized facility that is
designed to allow you
to manufacture cells.
And we know we're gonna have
to make casein protein and
the guide RNA and the virus and
we'll have to have a delivery
device that all have been qualified
as appropriate ways to modify a cell
that you would give back to a patient.
And then we would analyze this
modified cell population and
make sure that we achieve
the right correction frequency.
Make sure that we didn't do
anything bad to the cells like
create a translocation or
something that might harm the patient.
And then we transplant these
cells back into the patient
after giving the patient chemotherapy
to make room for our corrected cells.
So now instead of using corrected
cells from someone else,
we would use the patient's
own corrected cells.
And so that really puts us right here
at what we call The Valley of Death.
Is how do we go from here, and
that is what my lab looks like.
It's not my lab, but I can tell you
that's what it looks like, a bloody mess.
But it means stuff is going on.
And how do we bring across so
we can get these cells to a patient?
And we're doing something at Stanford
that has not been done at Stanford,
which is let's try to
do it all at Stanford.
So normally what we would do is what I
would do is I'd say we've proved that
we can cure, we can do this in a model.
I will then license it to a company,
or I'll start my own company.
And we'll do it through a company,
and then I give up everything.
And instead what we're gonna try to do,
or we're doing,
is creating a new infrastructure here
at Stanford called the Center for
Definitive and Curative Medicine.
And the idea is to create
an infrastructure that will allow
people like myself and
others to bring their discoveries and
not have to license them out to a company.
But bring these discoveries and
develop them and
bring them to the patients right at
Stanford, so in a wholly internal system.
And so the idea is that we
have discovery research and
then there's pre-clinical development and
clinical development.
And we wanna be able to do everything up
to these first phase one to the first
in human clinical trials.
And if those work then idea is to say,
okay, big pharmaceutical company,
we've shown that it works.
Now you figure out how to bring it to
the hundreds of thousands of patients.
Sorry, I didn't tell you this.
So there's about 100,000 patients
in the US with sickle cell disease.
There's about 100,000 patients in Western
Europe with sickle cell disease, but
there are tens of millions of patients in
places like Ghana, Nigeria, India, and so
on and so forth.
[COUGH] So we need to have that
facility to manufacture those cells,
and Stanford has built a facility
in the last year to do that.
Let's see,
California Avenue is that direction.
It was an old research building,
and they remodeled i.
And it's now got special air filters,
and there's ISO 9 and ISO 8, which just
means that there's only one dust particle
per ten to the ninth particles of air.
It's run by David DiGiusto.
And so now it gives us the infrastructure
to actually manufacture these cells.
And so what we're hoping over the next
year and a half is to take that
process that we developed in the lab and
move it into this facility.
Such that we can create a large number of
cells that have been done in what we call
GMP grade fashion, good manufacturing
practice fashion, so we can give them to
patients in the new children's
hospital that is being built as well.
So to end I just wanna end on a few slides
discussing the ethics of genome editing
because it's been in a lot in the press.
So to me, one of the big
ethics is it's all well and.
So by the way, this is a picture
of Packard Children's Hospital,
which is where I work when I'm not
in the lab or here talking to you.
And I think that we will be able to
do this very sophisticated
manufacturing process.
By the way,
the GNP facility costs $10 million.
Actually it cost $9,999,999 because
if it went over $10,000,000 it
was gonna need the trustees approval, and
no one wanted to go through that process.
And so
they kept the budget $1 under 10 million.
So you guys all know how Stanford works.
But it's really,
I showed you that the patients,
most of the patients with sickle cell
disease are here and here and here.
And places in the world where they're
not gonna build a $10 million facility.
So how are we gonna take
a complex process done in
some place like Palo Alto and Stanford,
California, and bring it to this patients.
And of course I have ideas, but
I don't know the complete solution.
And so
whenever I talked to undergraduates and
high school students and
medical students, that's your job.
Figure it out.
I think we can do it,
but it's gonna be hard.
Now the second thing about genome editing
that I want to bring up is it's been
incredibly powerful.
The CRISPR system has
been incredibly powerful
at creating genetically
modified organisms.
So we can take yeast or
cells in a tissue culture dish or worms or
flies or mice and rats and pigs and
dogs and monkeys, non human primates.
And we can use the CRISPR system to
make mutations in their DNA that mimic
human disease.
And so don't have much problem creating
mutations in these things or these things.
But I think as you go up
the mammalian landscape, and
perhaps I'm primate centric here.
I think we all have to ask ourselves
is that while a disease like
Huntington's disease is devastating in
humans and we need to find a cure for it,
is it ethically right to create
a monkey with Huntington's disease to
allow us to find cures for human disease?
And I'll leave it at that for
you guys to come up with your own answers.
Finally, the other thing about genome
editing is because we have found
that there is an efficiency to be able to
create genetically modified organisms,
it raises the question of how far
should we go with this technology?
So I just talked to you about using
somatic cell editing to cure disease.
And so we would take a patient's own
somatic cells out, we'd modify them,
give them back to cure a disease.
Those somatic cells won't get passed
on to future generations, and
they will cure disease.
So I put that in green.
I don't think there's much
ethical problems with that.
Where people are starting to get into
really have discussions about this
is what about using somatic
cell editing for enhancement.
So let's say, we've identified a gene,
let's say erythropoietin.
We know erythropoietin.
If you have more erythropoietin,
you make more red blood cells.
You make more red blood cells,
your endurance will be better, and
you have a better chance of winning a gold
medal, or winning the Tour de France,
or any other things
that require endurance.
And so if we get really good at this, what
do you think about engineering the EPO
gene, or the growth hormone gene,
to allow people to be Enhanced?
And as you can see,
I sort of think that's maybe not so good.
But other people say, why not?
Let's make the human race better.
We're not perfect people.
Let's figure out how to
make our physiology better.
And then there's this
big divide right here.
So this is still somatic cells so
it wouldn't get passed on
to future generations.
What about editing the zygote?
Or the stem cell that gives
rise to sperm and egg?
And so therefore, the change would be
passed along to future generations.
Should we use that to cure disease?
So for example there are diseases
that are so disseminated in the body
it is unlikely that we'd be able
to use somatic cell editing.
To get at all those tissues and
cure the disease.
And perhaps the only way to cure that
disease is by doing editing much earlier,
that would result in passing on of
that change into future generations.
Now, one would argue, we don't need
to do that, there's a process called
pre-implantation genetic diagnosis
where you create a bunch of embryos.
And you screen for
the ones that don't have the disease, and
plant those that don't, and
that people have done that.
It's not paid for by insurance in the US,
so I'm talking to patients
with Huntington's disease, which is
an autosomal dominant neurologic disease.
They said they have a 50% chance of
passing on their disease to their
children, and they don't have
the resources or the funding to pay for
PGD out of their own pocket,
and so they just take a chance.
And you can imagine the angst
that puts people through.
And then finally, we get into this
lower quadrant here about we do
heritable editing for
enhancement to create a super human race.
And I put that in bright red.
But there are other people out there who
said, no, we should actually do that,
we should make the human race better.
So anyway, I wanna thank you for
your attention and
the opportunity to
discuss some of our work.
I get to come up here and
talk in front of you all.
But to have a great group of people in
the lab who'll actually do the work,
great collaborators both within my
clinical division at our new GMP facility,
collaborators around the country.
We can't do this without money and
multiple different funding sources,
including the federal government, the
California Institute for General Medicine,
and some great philanthropy
that has supported the lab.
We all know that Stanford
is a great place.
Silicon Valley is a great place
to work as is Stanford as is
Packard Children's Hospital.
Here's the Medical School.
Here's the low-key building for
stem cell research where my lab is.
Here's some of the people,
a part of the team.
I told you I'd end with a quote, so
this is the quote I wanna end with.
So this is a picture taken.
That's my dad, that's me,
my brother, and my sister,
when my dad was on
sabbatical in Australia.
It turns out my brother and
his family were here yesterday but
didn't have this picture to recreate it.
So anyway, but I like this picture because
it highlights a quote from Kurt Vonnegut
that says, I want to stay as close to
the edge as I can without going over.
Out on the edge, you can see all kinds
of things you can't see from the center.
And I think that's what drives us to
think about trying to cure diseases
in ways that have never been cured.
And with that, I'd be happy to
take any questions upon us.
Thank you very much.
>> [APPLAUSE]
>> So
there's microphones up here for
those of you who have loud voices.
>> When I first read about the science
news a while back, they were talking about
the possibility of taking the mosquito
that carries Malaria and modifying it so
that all of its prodigy would also
have that thing to not carry Malaria.
>> Yeah.
>> And they're concerned
about all the repercussions.
Can you address that?
>> Yeah, so I can a little bit.
So that would be the ethics
of genome editing for.
It has more to do with ecology, I think,
although it is focused on disease.
I think if you could, yeah,
it's complicated, right?
So I think, technically,
we could engineer.
So there's been two thoughts about this.
One is there's a specific type of mosquito
that carries malaria, and people have
proposed, why don't we use the CRISPR
system to create a bunch of infertile
males that will breed with females and
will just wipe out the species?
And so we'll no longer have mosquitoes
that can carry malaria around.
And you can imagine,
the way I'm saying it is I don't know what
the ecologic ramifications of wiping out
an entire species that's
actually low on the food chain.
So therefore, who knows what
their ramifications would be?
But people said, look, malaria is an awful
disease, this should be worth killing.
What you're proposing is another, and
so I think that's technically possible.
Once that species is out in the
environment, there's no bringing it back.
There's no way we can suck all those
genetically modified mosquitoes back.
Same thing with the idea of,
well, we're not gonna make
the mosquitoes infertile,
we're not gonna the mosquitoes.
But we'll make them resistant to malaria,
so they can't pass along the disease.
That seems like a little
more reasonable approach.
Falciparum malaria, no one really
knows what it's good for, but
again, what would happen
if we wiped out falciparum?
Hard to know what that would happen.
I think that would be
a more cautious approach.
Maybe a more reasonable approach because
this malaria still is an awful disease.
I think, again though, the issue is that,
how could you do it in a way that if
there were adverse consequences,
you could contain the process?
And unfortunately, mosquitoes are not
gonna respect national borders.
All of you guys fly on planes.
So containment, as you understood whether
you've created something devastating,
is something we really
have to think about.
I don't know if I fully
answered your question,
I probably would never answer your
question so really that quick.
>> [INAUDIBLE]
>> Exactly.
I think technically,
it is possible, exactly.
But there's a question of I think we've
gone beyond the can we to the should we.
>> Is it possible to harvest
a small number of stem cells and
then let them grow and divide until you
have the half a billion that you need?
>> So the question is this.
>> [INAUDIBLE]
>> Yeah, so I've talked about taking
a large number of stem cells and
modifying the bulk population and
then infusing that large
population in the cells.
But another strategy would be to take
a small number of stem cells, modify them,
and then expand those up.
What's the pros and cons?
So for hematopoietic stem cells,
it turns out that we don't yet
know how to expand them.
So for that type diseases in which we need
to transplant hematopoietic stem cells,
that's not a possibility.
But there are other stem cells
that we can grow and expand.
Mesenchymal stem cells, airway stem cells,
pluripotent stem cells,
even neural stem cells.
We can grow in a dish and expand them.
So there's a possibility.
Adipose stem cells, which are a variant
of mesenchymal stem cells.
Yep, yep, all those we can do.
Now, what's the potential
downside of doing it?
Well, it turns out that every time a cell
divides in culture, it requires mutations.
And the question would be if you started
from a small number of cells and
you ended up growing into several billion
cells, would you then have created
a population that has acquired so many
mutations that one of them may go bad?
No one knows that.
But that's the potential downside of
starting from a small number of cells and
expanding up to a large number of cells.
So I think it's important to look at what
is the distribution of mutations at that
end product, and how would that compare
to the distribution of mutations
that might occur by just modifying
a large number of cells all at once?
Yup?
Yeah, yeah.
So I didn't show the slide,
cuz one of the issues in
the CRISPR technology is
while it's designed to make a break in a
very specific site, it's about chemistry.
And it has a probability of making
a break at a site you don't want and
creating mutations there.
And so I spend time at meetings, and
I will be at a meeting next week
where we have active discussions about,
how much should we worry about that?
I don't worry about as much
as others because when we
measure those off target
mutations they're very low.
But also I don't worry about as
much because I contrast that
to a number of mutations that
are occurring in us all the time.
So on average, every time a cell divides
in our body It acquires one new mutation.
So if we have 40 trillion cells, and
maybe 10% of them are dividing every day,
we're acquiring 10 trillion
mutations per day.
If we give somebody, I am sure that
there's no one in this audience
who smokes, so I don't even wanna
talk about that, By for instance.
If you go out on sunlight,
you are acquiring 10,000 DNA,
10,000 DNA legions in your
skin per second from sunlight.
So now our cells have evolved,
we've evolved in suns.
We know how to fix most of them.
The point is, is that our cells
are very good at fixing DNA damage.
So I think putting this expansion or
the CRISPR off target
effects into the context of our ongoing
mutation frequency is really important.
And then I'll step back and
I know there's another question, and
then is we're all born with
an incredible number of differences.
So, people at Stanford here,
but others have done the same thing, where
they've sequenced mom, dad, and a kid and
it turns out each of those people have
3,500,000 variants from each other.
So, we're all born with an incredible
number of variants to begin with
So again is putting the initial variation,
the ongoing variation, into the context
of what we're willing to tolerate as
we're trying to engineer therapies.
Yeah, great question.
So adeno-associated virus,
there's actually a lot of
different flavors of AAD.
There's one through nine, and
then I think we're creating more flavors.
And the different flavors, different
serotypes, have different tropisms for
different cells.
And so what I didn't go into is,
we're using a serotype that has
the particular ability to get
into hematopoietic stem cells.
It actually has the ability to get
into a lot of different cells.
It seems to not have a lot
of specificity so yes,
you need to find the right flavor
to get into your cell type.
And different cell types
will need different flavors.
So, one of the reasons that we like
the AEV, is that when we've looked at
the cells after they've been exposed
to the AEV and said, how did you react?
Did you change the way
expressed different genes?
There's some changes in gene
expression but not very much and
when we look into the cells divide and
replicate and
signal like their sick they have none
of the signatures of being sick.
After the recombination process,
there is no viral DNA left in the cell.
It's just the changes we left in and
the other pieces of DNA have disappeared.
And that's why we like this
genome editing approach
to modifying cells rather then
using other viruses which do leave
some of the viral DNA into the genome of
the cell that will stay there permanently.
Well I'll look into it.
Thank you for letting me know and no
were always looking for ways of tweaking
the system, and I showed you
the summation of a lot of small.
There were some bigger improvements but
really it was a lot of little improvements
as we went from 1% to 4%, to 8%,
to 12% and eventually getting the 50%.
So little improvements all the way along.
We're always looking, so
we're always on the lookout.
So I'll look into that, thanks.
>> Using this technology to cure disease
is really exciting, but is there anything
we can do to try to slow the possibility
of somebody using this for bad purposes?
>> Yeah [LAUGH].
Yeah, Well, I mean,
you guys probably have just as many
good ideas and maybe better than I.
So I think one thing, I've been
fortunate enough to be on a committee,
the National Academy of
Science is how a diverse
community that's coming
together will put out some
recommendations about how to address some
of these issues that I've brought up.
So I think first thing you can do,
is you can start to define how
this process should be regulated.
So that anybody who wants to work
in the bounds of regulation and
society will follow that.
How do you prevent somebody going rogue?
That's a tough one.
I will say that as much as
I made this sound simple,
maybe it doesn't sound simple,
it's not really that simple.
I know that you can buy crisper kits
that you're supposed to be able to use
in your garage to really
engineer stem cells, and
do this in a way, that's not so easy.
So, there is some
technical hurdles to this.
And then the question is, and
so then the other thing is that
the Department of Defense is
very concerned about this issue.
And has put out what
they call requests for
applications, which means send us
a grant proposal about just this.
How do we regulate it,
how would we make it safer,
how would we detect if somebody
was genetically engineered?
So, we'll see.
And I think it's complicated, there's no
one answer, so it's a great question.
Somebody up here, you had a question,
yeah, or comment yeah?
Also we didn't expect, so
GFP is a piece of DNA from jellyfish and
we put it into human cells.
Now we don't intend to use that
therapeutically, we use that as
a tool to allow us to measure it's
easy to see green cells or not.
But yeah, I think people
are thinking about taking pieces of
DNA from one species and put in into human
cells and I'm gonna give you an example.
And maybe you're gonna throw food at me.
And so turns out that HIV which affects
30 million people around the world.
There's no cure,
no vaccine we have some good drugs.
Doesn't effect, doesn't infect monkeys and
people have identified genes in monkeys
that are similar to the genes in humans
but they confirm resistance to HIV.
And so what we and
others have thought about doing is
taking those monkey variants, or
the small changes that were in the monkey
gene and putting them into the human cell.
So now we create an immune
system that is resistant to HIV.
And so that would fall into the rubric
of what people call synthetic biology
in which we're engineering cells to
adopt new features and phenotypes and
where you get that DNA from then is
complicated we are actually using.
Viruses, so not genome medicine, but
viruses that contain pieces of DNA from
other species and
introducing them into human cells.
And since they don't seem to have
any problems, they stay there.
So, yes?
>> I have a very basic
question about genome editing.
So how does when you modify a cell or a
subset set of cells, how does it actually
spread throughout the body, because
the goal is to get it everywhere right?
>> Right, so what we do is we take
cells outside the body and modify them.
And the only way it'll spread
is if that cell divides and
its progeny spread throughout the body.
But other people are working on can you
deliver the crisper editing machinery
to cells within the body and
modify the cells directly in the body and
we'll spend some progress on that too.
You won't be able to actually edit
like all the cells in the body, right?
You still depend on it being spread.
>> I think,
as I said there's 40 trillion cells in us.
I don't think we can get
at 40 trillion cells.
So as we think about using a secure
disease, we have to understand which cells
and at what frequency and what way can
we use editing on to cure that disease.
Different diseases will have different
frequencies that will move together.
Turns out, one of the diseases,
bubble SCID,
bubble boy disease, we might only need
to correct a small number of cells,
whereas other diseases we might need
to correct a large number of cells.
So every disease,
I think is gonna have different criteria.
>> Hi, assuming that your CRISPR
technology is successfully applied
to human beings, how soon can we
expect a cure for sickle cell?
>> So what I will say is taking,
we're shooting to take what the data
I just showed and scaling it up and
doing safety studies that would
satisfy the FDA, and
start a clinical trial in early 2018.
And then,
they won't allow us to treat 100 patients.
We'll have to treat one patient and
make sure that person does okay and
then treat another.
But I would expect that granted me
that everything would work, all right?
I'm gonna assume everything works, that
I would expect, that say, in five to ten
years, we'll be at a point where we're
starting to treat hundreds of patients.
And maybe in ten years, thousands
of patients and so on and so forth.
So I think this is, I'm biased, right?
Go to Midas, get a muffler.
Go to somebody who does cell and
gene therapy, I'm gonna tell you cell and
gene therapy is great.
But I do think that this is one of
the next new horizons in medicine,
the ability to get modified cells to cure
disease rather than a drug or a biologic,
really opens up a whole host of diseases
to be treated in ways we never thought of
before.
>> Unfortunately, we only have time for
one more question.
>> Yeah, and I'm happy stay after and
answer questions as well.
>> My question is about
the target audience for
the sickle cell therapy.
If sickle trait provides some
protection against malaria,
and you administer this, and
it's successful in Africa or
India, do you then put the people at risk?
>> Yeah, yeah.
I mean, I think that's a great question.
So let me just go back here.
Remember, we're gonna end up
creating a population of cells
in which the patient will
have some sickle trait cells,
some not sickle cells, some sickle fat,
I mean, some beta thal cells.
These cells are also resistant to malaria.
So I don't know, now,
the best way of not getting malaria,
is to sleep with a bed net at night.
So perhaps, if we're gonna give
someone a therapy that costs $100,000,
we might be able to find $2.50 to
have them sleep in a bed net and
make sure they don't get
bitten by a mosquito.
Sorry, I mean,
there could be simple solution.
But I think that the disease
is severe enough.
Look, as I said kids in Africa are dying
at age five with sickle cell disease.
Let's get them to 25 with bed nets and
good antimalarial drugs, and
we'll deal with that slight
change in mortality.
Remember, we're not changing it
because we're doing this semantically.
We're not changing the frequency that will
be passed onto future generations as well.
So it will still be in
the human population.
All right, well I'm happy to take any
more more questions, and thank you for
your great questions and attention.
>> [APPLAUSE]
