Gene editing for cure of persistent viral infections

Gene editing for cure of persistent viral infections


[MUSIC PLAYING] KEITH JEROME: All right,
it’s a pleasure to be here. It’s always fun to come,
I always say, back to sort of the mother ship here because
I do spend a lot of time off campus. The basic science
research you’ll see is done in laboratory
space at the Fred Hutch, and then everything
that’s an assay and involves
measurement of a virus is in the medicine
laboratory at 1616 Eastlake. So it’s fun to be here. It’s also especially
fun to do this because I had no
idea that Sean’s so good at introductions. I really liked that,
so thank you very much. It felt really nice. It is a pleasure to talk
about the idea of curing persistent viral infections. And that is one thing
that we’ve tried to do, and I think pretty successfully
over the past 5 or 10 years– is really change the
discussion around these viruses from things that people
resign themselves to living with for a
lifetime to afflictions that there is a
prospect of actual cure. And I think it has
been a paradigm shift, and now cure is actually a
major component of NIH funding, particularly for HIV, but now
increasingly for hepatitis B. And we hope to change that for
herpes simplex virus infections as well. So right up front, a
couple of disclosures– I’ve done a bit of consulting
for gene-editing company called Editas based out of Boston
and have obtained reagents called meganucleases from a
French company, Cellectis, in Paris. So I spend a lot of my
time at a cancer center, and as such, you have to justify
why you’re there, particularly having a joint appointment. And so it turns out that
persistent viral infections are actually major causes
of cancer, and depending on how you look at it, they
might be responsible for up to a quarter of all
the human cancers. I think people are familiar
with human papillomavirus, which causes cervical cancer,
causes other cancers as well, including an increasing epidemic
of head and neck cancers. Hepatitis B is the major cause
of hepatocellular carcinoma, an extremely serious and
typically fatal cancer. HIV increases a person’s
risk for a number of lymphomas, for Kaposi’s
sarcoma and other cancers many manyfold. And the other virus–
and in fact the virus I’ll spend the most time
talking about today– herpes simplex, although
it was originally a culprit in cervical
cancer, turned out not to be the direct
cause, but actually is an indirect cause of cancer. HSV infection raises the
relative risk of acquiring HIV by about two-fold. That doesn’t sound like
much, but the prevalence is so high in HIV-endemic
areas that almost half of all HIV cases can actually be
attributed to pre-existing HSV infection, so indirectly a
major cause of cancer as well. And we have ways of
addressing these infections, and the typical way
we’d think about doing that is preventing them, right? So we’ll make a vaccine. And there is a great vaccine
for human papillomavirus, and in the US, it’s being
increasingly brought into play. There’s a great vaccine
for hepatitis B virus that certainly
all of us who work on viruses or diagnosis
of infectious materials have likely had. We desperately need
a vaccine for HIV, and the prospects
are mixed at best. There may be a vaccine. There’s lots of work, but it’s
an extremely challenging virus. And herpes simplex
is actually sort of been the black hole
of vaccine development and has actually led to the
demise of several substantially well-funded companies
because it just has proven to be extremely difficult. So now we have antivirals,
and we can actually suppress all these infections. But none of them do
we actually cure. So for HIV, the
infection’s sort of gone from this essential death
sentence to an infection that people can live with
for a normal lifespan in excellent health, simply by
taking now typically one pill a day. In hepatitis B, which is often
treated with repurposed HIV drugs, you can suppress
viral replication. You can lower viral loads. You can even reverse
some liver damage, but you don’t cure
the infection. Acyclovir can reduce
recurrences, reduce shedding, and it decreases the risk
of transmission of HSV to a new partner by about 50%. But it’s still not a cure. And so really, I think
of this metaphorically as sort of you’re in your
garden, in your yard. And you’ve got these dandelions,
and you’re plucking the tops off of them, right? And you keep doing that, and
as long as you keep doing that, your yard looks great. But if you stop, all
that pops back up, OK? So all of our current
treatments fail to get at the root cause
of these infections. And the reason they
fail is because each of these infections have
some sort of long-lived DNA form that gets into cells
and stays there, OK? And it sort of rests. It hides. It might go latent and really
become almost invisible. And despite all the
therapies that we use, they don’t go away. So if you stop, the
infection will come back. So to really address
these infections, we need to get rid
of the root cause. So we can get rid of
the infected cells. You might be able to
get rid of T cells, for example, maybe a fair
number of your pericytes, but you can’t get rid
of your neurons, which is where herpes lives. We’ll talk about that. So killing the cells is
not such a good idea, but maybe you could just
get at the long-lived form, leave the cell alone,
but destroy that form. And that’s really what
we’re trying to do. So I want to spend a
couple of minutes early on just talking about the
biology of herpes simplex virus so that we understand
what it is. First of all, it’s a
very common infection. It’s one of the most common
infections in humanity. This virus has
co-evolved with us. It’s very well adapted
to human beings. So about half the
people in the world have infection with either
or both HSV-1 and HSV-2. So for HSV-1, about
half people have it. In the United States, the
most recent nationwide survey says about 12% of adults
have HSV-2 infection. And the virus infects
at a mucosal surface, and it gets just below
the epithelial surface and finds the nerve endings
that innervate that area. And there, it jumps on the
molecular motors that carry it all the way down to
the nerve body, which could be in a ganglion,
OK, a trigeminal ganglion in the head or neck
or a dorsal root ganglion along
the spinal column, and that’s where it
establishes a latent form. The virus actually goes
there and goes to sleep. But periodically, that
virus can reactivate. It jumps on an alternative
set of molecular motors and comes back out,
recedes to the periphery where it begins to
replicate, and this causes viral shedding, which
could infect a new person. Or if it’s big enough and not
well controlled very quickly, it can lead to
ulceration and a lesion that people would
notice clinically. So ulcers are the most common
manifestation of HSV infection, but it can also lead
to encephalitis. It can lead to
keratitis, which is a major cause of
infectious blindness, and then it can be
devastating in neonates. Now, some people who
are infected with HSV have no idea they’re infected. They never have a lesion. They have no problems with it. It’s kind of be irrelevant
to their health. Other people have
recurrences very frequently, once a month or more. And typically, the people
who have frequent occurrences are bothered by them. And originally in justifying
this work, what can I point to say this is a
problem due to a grant review or somebody looking at a paper? How can I impress upon
them that the people living with these infections
care about it? And so one thing I used
to say was, well, 1994, which was the last year that
acyclovir was on patent– so that’s kind of the
mainstay drug that we use– people spent $1.4
billion in 1994 money on this, which was
quite a lot of money, even though that drug’s
not all that good. For a recurrent lesion, it might
shorten duration of ulceration by a day or so, so it’s
not that great a treatment. And yet people used a whole
lot of it, so they cared. And that got us a
little ways on this. So let’s talk a little bit
about the latency of the virus. So I mentioned that latency
is established in ganglia along the spinal
cord in the head. It can be in a sensory ganglion. It can be in an
autonomic ganglion, and you’ll hear me talk mainly
about the trigeminal ganglion today as well as the
superior cervical ganglion. Those are both in the
head and neck area. Typically, ganglion
contains 10,000– we’ll call it 10,000 neurons
to make the math easy, OK? So there’s 10,000
nerve bodies there, and this is why HSV
is a great target for the sorts of therapies I’m
going to talk about because we know exactly where it is. There’s not very
much of it we need to get at that’s causing all the
disease that people deal with. So there’s 10,000 neurons
in a typical ganglion. 10% of those have herpes in
them, so we’re in 1,000 cells. And each one of
those might contain– we’ll call it 10 copies, OK? So maybe there’s
10,000 copies of HSV that’s causing all the
disease people worry about. We also know that the burden
of herpes within that ganglion is a major determinant
of how bad disease is, how frequently one recurs, how
severe those recurrences are. So by knowing where
it is and knowing that there’s not much of it, we
might have a shot at curing it. So I mentioned trying to justify
why we’re working on cure. And we finally got tired of
quoting the business literature from 1994, and we actually
took a cue from our work in HIV disease in
which people were asked, what aspects of living
with HIV don’t you like? And what would you
like to see in a cure? What do you find about cure
that is compelling to you? And so we took that
sort of methodology and applied it to HSV
disease, and we simply asked, of all the
things that we think a cure might do for you,
what do you find desirable? And the answer was pretty much
anything we could think of. People said, yeah,
that’d be great. I’d like that. But the biggest one with an
amazing degree of unanimity– I can think of very few things
where you can get 96% of people to agree on a 5-point scale
that this is fantastic, and that’s to eliminate
the risk of transmitting the infection to a new partner
to a neonate, to someone else. OK, so lots of reasons cure is
extremely desirable to people living with the virus,
and not only that, people are willing to
take part in trials. OK, so even early phase
trials that may or may not benefit a given person directly,
there’s a sense of altruism, and people say, I might
consider doing that. And so one thing I’ve
been very gratified that we sort of hit on early
the talk is the conversation around this has changed from– originally, I’d
get grant reviews that said, why in
the world would you work on a cure for herpes? It’s just a nuisance. Don’t bother about it– to now
people go, oh, people care, and American taxpayers care. This is something we
should consider funding. The approach that we’ve been
using for HSV and for hepatitis B that I’ll talk about today
as well is gene editing. This is in the papers a lot. You’ve probably all
read about this. Just for completeness, I’ll
give you a very quick overview. The idea is that we have
some sort of enzyme that essentially interrogates DNA
and is looking for a very specific sequence of DNA. Typically for the
enzymes that we use, it’s a long sequence of 16
or 20 base pairs of DNA. And if it finds
the exact sequence that it’s looking for– not something close,
but the exact sequence– it’ll bind to DNA here and
induce a double-strand break. Cells have to repair
double-strand breaks. Cells stop everything
they’re doing when there’s a
double-stranded DNA break, and they look to repair that. And if they don’t,
they’ll typically die, so they’re very good
at repairing it. There’s two ways it can happen. One is called
homology-driven repair, HR. That doesn’t happen– that’s
not favored in mammalian cells. What typically
happens is something called non-homologous
end joining, which is essentially
the two broken ends are bound by a series of proteins. And they’re brought
together, and those ends are just stuck back
together and repaired. And typically, this is
a very precise thing, so you get exactly the
sequence you started with. But of course, if we do that
and our enzyme is there, we restore the target site. So it’s cleaved, and
it gets repaired. And it gets cleaved and repaired
and cleaved and repaired until something goes wrong. And now we have maybe a deletion
or an insertion of something, and so we’ve made a mutation
there called an indel, right, an insertion deletion. And so the money aspect
of this for all the gene editing, whether it’s for what
we’re doing or someone else is, you’ve changed the
sequence of the gene. You’ve knocked the gene
out functionally, OK? So if we do that in something
that a virus needs– an essential viral gene–
the virus can no longer have whatever essential function. It can’t replicate. It can’t cause disease. So essentially we’re trying
to attack these long-lived DNA forms, damage them, or maybe,
as I’ll talk about later, make them go completely away. Now, I said this. We use these enzymes. What are they? There’s a bunch of different
enzymes you can use. Probably 95% of you have
heard about something called CRISPR-Cas9. That’s the one on
the very right, and that is, in
many people’s minds, synonymous with gene editing. It’s not. It’s a tool that we
use for gene editing. It’s the most common tool
of use for gene editing, but in every application,
it may not be the best. So we’ve worked with
all four classes. I’ll show you some
data in the last third of the talk about CRISPR-Cas9,
but for the herpes work, we’ve mainly use this class of
enzyme called a meganuclease. They’re sometimes called
homing endonucleases. It’s the same thing. And these two things
have some characteristics that make one or the other
better than the other. Cas9’s great if you’re
in the research lab, and today, I want to
target this sequence. But tomorrow, I want to target
that one, and the next day, I want to target this
one because all you need to do for Cas9 to tell
it to target a different site is to give it a little tiny RNA
that matches the site you’re interested in, OK? So if we change that RNA,
which is super easy to do, you can have a new enzyme. So if you tell me you want
to work on this tomorrow, literally, we can be doing
an assay with Cas9, OK? Just change the RNA. The downside of Cas9 is
it’s a great big protein, and these pictures are roughly– not quite, but
roughly– the scale, OK? So big protein equals
big coding sequence equals a lot to put into
a gene therapy vector. And in fact, it is
quite a challenge to put these things into
the gene therapy vectors that we use. And we end up having
to make a lot of– take some shortcuts, cut some
corners just to make sure everything will fit. And then we can’t optimize
everything just like we’d like. Conversely, meganucleases
have this wonderful advantage that they’re tiny, OK? They’re really small. They fit into any gene therapy
vector you can think of, and you can use any
promoter you want. And you can put in other
things to help it work better. So it’s wonderful, right? Except these are really,
really difficult to redirect toward new specificities. They exist in nature
in yeast, and they’re selfish genetic elements. They have a sequence
they recognize. And if we want to
change something that exists in nature into
something that recognizes the herpes sequence,
we literally have to change
the protein itself so that the protein DNA
interactions work, OK? And that’s a huge challenge. It’s easy to make
these things by DNA, but since the DNA binding
and the DNA cleavage is [INAUDIBLE] by
single protein domain, everything you’re changing
to change specificity tends to change
activity as well. So to make both of those
things work is difficult. So I can give you one of these
for a new sequence tomorrow. I can say, for
this, I’ll give you a 50-50 chance I’ll give
you one in six months, OK? So you can see why we talk
about this all the time, but if you have a target
like herpes simplex that has very little sequence
diversity, that is extremely stable genetically, and has
very high fidelity replication, you only have to do that once. And once you have the
enzyme, you’re set, OK? And then you get
to take advantage of all the other things,
including the small size. So can we use these
sorts of things against viral infections? And there’ve been a ton
of papers that basically take a virus, put it
on a culture in a dish, and then throw an enzyme
at it and go, hey, I can cleave a virus. We’ve published those papers,
and lots of other people have as well. So there’s no doubt
that can be done now, but the question is, how do you
transfer that into something that you can do in an organism? Can you do that in a mouse? Can you do it in a guinea pig? Can you do it in a human being? And really, there’ve
been only a couple of studies that have done this
in any kind of actual animal model. There have been a
couple of papers in HIV, and then we have
published a paper that I’ll talk about briefly
in herpes simplex as well. So I mentioned we have
these nucleases for HSV. I’ll predominately
show you two of them. We have a third, and these
tend to be our best enzymes. One is called HSV M5,
and one is called HSV M8. And each of them target a
specific gene in herpes simplex is essential for replication
of the virus even in cultures. These are very important. M5 targets the major
capsid protein, and M8 targets the catalytic
subunit of the DNA polymerase. We also have some controls
that we’ll mention, and so we express these
sometimes with helper [INAUDIBLE] as well. But the idea is we’re
going to induce mutations in those essential parts of
the virus and knock it out. So this is a paper– I won’t go into
the data, but this is a paper we published
five years ago now showing that in
this culture dish, these enzymes are pretty good
at inducing indels in HSV. And we also started knocking
out the ability of the virus to replicate. So then the challenge was,
how do we move this in vivo? And we took this
into a mouse model. We did that for
a couple reasons. First of all, mice,
as animals go, are reasonably
easy to work with, and you can infect a
mouse with herpes simplex. And you put it on
the eye, basically, and they get a little infection. And they show a
lesion, basically, that lasts for a week or so. And then it heals up,
and the mouse is fine. But the virus has made that trip
down the axon to the ganglion in the head to the
trigeminal ganglion to the superior
cervical ganglion. And it establishes
latency there, and the latency
program that it runs is exactly like it does in
a human in that it makes one gene product called LAT. Actually, it’s one
gene transcript. It doesn’t even make a protein. It makes one mRNA,
and that’s it. So everything’s completely
normal in the mouse, except that it
doesn’t reactivate in the mouse spontaneously, so it
doesn’t make that round trip. But we have that
latent infection, and we can work on ways to
actually attack it in latency. So these are the AAV
constructs that we use, so we use adeno-associated
virus vectors to deliver these. So essentially, you make
a construct like this, and I’ll show you a little bit
more about AAV in a minute. But these are just little
empty virus vectors where we replace all
the working parts of this helper dependent
virus with the genes we want to express. And we put them into– in these experiments– the
whisker pad of the mouse, so right there in the face. That innervates all the same
places that the eye does, and it turns out that AAV gets
on those same molecular motors and goes down to the ganglion. And you can see nice high
titers there in the ganglion, so we can get a transgene there. So what happens now if, in
these latently infected mice, we send our nuclease down there? Can it edit HSV? And the answer is yes. Now, we published this
now three years ago, and we were really excited
about this at the time. There’s sort of two
ways to look at this– the optimist says, well,
this is really exciting because this is the
first demonstration of gene editing
of an established viral infection in an animal
that was ever done, OK? So that’s pretty cool. Of this entire field
of looking at doing this for HIV or hepatitis or
human papillomavirus or HSV, this is the first time it
was shown successfully. The pessimist can say,
well, that’s all great, but in fact, the mutagenesis
frequency is 2% to 4%. So of all the herpes in
there, we mutated at best 4%. So yeah, it’s a
demonstration of principle. I doubt that reducing
someone’s HSV burden by 4% is going to do very much, so
the challenge at that point and since has been to increase
the efficiency of all of this. But I’ll say one nice
thing that came out this study is all this
seems to be really safe. I mean, we still
do have concerns. You have these enzymes
that are modifying DNA. You’re putting them into cells. Is it going to target
something that we don’t expect? Is it going to cause an
inflammatory response? Is it going to kill neurons? The answer seems to be
no in every way we look. These are just H&E
sections of the ganglia, and you can see expression
of our transgene there. But there’s no evidence
of any inflammation. There’s no neuronal loss. The mice act completely normal. You can’t tell whether the
mouse has been actually treated with this or not. So it’s at least well tolerated
and safe as far as we can tell, and in fact, there doesn’t
seem to be any genotoxicity. So here, actually,
with Alex’s help, we looked at the target site
that we wanted to cleave, so you can see the red bar
just says, wow, here in herpes, we’re getting that, in
this case, 2% mutation that I told you about. But if we look at the most
closely related genomic sites– so these are the sites
in the mouse genome that are as close as
possible to the target site. Typically, they’ll have
either three or four nucleotides that are different
from the herpes recognition site– and we sequence
those and compare those to the frequency
of alterations that are in the controls, none
of them show any difference, OK, so that there’s no evidence
of any increased mutagenesis at these sites
compared to controls. So we don’t even
see genotoxicity that we can detect. So how do we make
this go better? And we decided that the
first thing we should do is try to take advantage of
this quality of meganucleases is that they’re really tiny. And the fact they’re
really tiny allows you to do a little AAV trick
that Dan Stone in the lab educated me about. So this is a little
more realistic view of what AAV looks
like, so AAV itself would have two genes
in here, rep and cap, that allow it to replicate if
there’s a helper virus present. It needs to have
adenovirus, hence the name, or another helper virus with it. So in our vectors,
we take all of this out and put our gene
in there, but it has this area of
single-stranded DNA and then these inverted
terminal repeats. And so when this goes into a
cell, this can’t do anything, and it won’t do anything until
the second strand is filled in, OK? And this happens
from cellular genes. It’s a very slow process
where this will happen. And once the second
strand is synthesized– that might take a week
or two weeks or a month– then you get an
episome like this, and then gene
expression can begin. Alternatively, if enough
AAV gets into the same cell, one of these can
find another one, and they can bind together
and make something like this. And it can start to
replicate as well. But if your gene
payload is small, you can do a cute
little trick, which is you can put it in
a reverse orientation, put it in twice
into your vector. So when this goes
into the cell– this anneals to this– you get an
intermolecular annealing, which happens almost
instantly in the cell, and you get immediate
high-level gene expression. So now we can get
a lot of enzyme really quickly in the cell. Maybe that’ll work better. This is a trick that
works in meganucleases because they’re small. No possible way you could
fit two copies of Cas9 into an AAV vector just
because of the size. So this just shows you how much
better expression you can get. Here, we’re looking at
a trigeminal ganglion. The middle panels here show
you where the neuron cell bodies are, so there’s
a lot of fibers coming in that appear in gray. But these dark
things are actually where the neuronal bodies are. With a single-stranded AAV, you
can see some rare expression of a transgene. You can maybe see these
little dark dots here, and if this were blown
up, you could see that. But I think you can sense that
in this self-complementary, the scAAV, we have much
rapid and much more intense staining and many,
many more cells. So we thought this might
help gene editing happen substantially better. And in fact, the answer in
our very first experiment was, yeah, this helps
things work a lot better. So in an essentially unoptimized
experiment, we already– simply by going to a
self-complementary AAV– doubled the frequency
of gene editing, and some animals were
showing over 8% gene editing. So we felt like we were on the
right track, and we could– still probably not where we’d
have therapeutic benefit, but we’re moving in
the right direction. So we had another
insight, and I’ve mentioned these two ganglia,
the trigeminal ganglion and the superior
cervical ganglia. Trigeminal’s a sensory ganglion. The superior cervical is
an autonomic ganglion. It turns out that
herpes actually prefers to go to the
trigeminal ganglion. It goes to both,
but if you just look at the burden per
100 neurons, there’s probably seven to tenfold more
herpes in the TG than the SCG. But the converse is true
for AAV, it turns out, at least for many
of the serotypes that we’ve been using. You’ll hear that word
lot, “serotypes.” There’s basically a
lot of flavors of AAV. Some appear in nature. Some have been designed
rationally by scientists, and they’ll have
different receptor tropisms and different
fates once they enter cells. So they behave
really differently. You’ve got to figure
out what’s the best one for what you want to do. But it turns out that AAV
likes to go to the SCG, and what that means is you
can have one type of ganglion with a lot of herpes and only
a so-so amount of your gene therapy vector. You can have another
one with less herpes, but a ton of vector. Maybe the outcome of gene
editing is different in those, right? Maybe more is better. It turns out that that
prediction is exactly true. In the same experiment,
while we might have here 2% gene editing in the
trigeminal ganglion, here we have 8% to
10% in the SCG, OK? So optimization of
delivery turns out to be something
that’s very important. We need to get a
good dose of enzyme to the places of latency. So we’ve spent a lot of time
optimizing this process. How do we get it there? What kind of AAV
serotype do we use? How do you deliver that AAV? I mentioned we were putting
it in the whisker pad. Turns out for a lot of AAV
serotypes, the best thing to do is not to put in
the whisker pad. It’s to inject it into the vein. That’s really nice. Surprisingly enough, people
feel uncomfortable with the idea about injecting something
just below the skin, but everyone’s comfortable
with an IV injection. But if you’ve had like
a tuberculin skin test, it’s a pretty minimal thing. But anyway, it turns out that
some of them were great IV, and it’s probably
our best enzymes. And if you find an
AAV that’s actually quite good at getting
to these ganglia, you can not only get higher
levels of mutagenesis– here’s an animal, for example,
with a serotype called rh10. Really great at going to
superior cervical ganglion, and we’ve got 30%
mutagenesis, OK? So in this process, we’re
getting closer and closer. And this is every time we keep
optimizing these experiments, it gets better, so
maybe 30% gene editing. But even more
impressively, it turns out that under these
conditions where you see a lot of
gene editing, we also start to see actual
loss of herpes genomes. That is, the burden of
herpes in the ganglion is actually starting to go down. And we hypothesize
that what’s happening is these episomes
are being broken. They’re being opened up. The cell’s generally repairing
them, and we’re getting indels. But occasionally,
that’s failing, and the cell’s sensing free DNA. And it’s simply degraded. It’s being lost. And actually, that’s
a great outcome. You can tell somebody,
hey, I’m going to inactivate your herpes,
and you’ll still have it. But it won’t be able
to activate anymore because the viral
polymerase– they’ll be, what are you talking about? But if I say, hey,
it’s going away. I’m getting rid of it,
people like that, right? And it’s pretty rational. So in this experiment,
we have about a 60% loss of virus in SCG, so now
we’re not talking about 30%. We’re talking about 60%
that’s actually gone. Not going to hurt you if it’s
gone, and much of what remains is actually mutagenized. It’s been altered, and
it can’t actually recur. And then we had one more major
insight that actually came out of the HIV field. People are, as I mentioned,
doing this sort of approach. We’ve targeted HIV genes
right in the middle and knocked things out. Another group was
targeting what we call the “LTRs,” long
terminal repeats that are at the end of
the integrated virus. And so if you do that– there’s one at each end, right? So a single enzyme
cleaves the thing twice. And what they started
to notice was, yeah, they were getting
these indels like we did. But a lot of times, it looked
like the virus was just being, they would say, excised. It was being lost. So the cell
repaired, but it just took the two free ends
of the chromosome, put them together, and
let the virus go away. So we thought, well,
what would happen if we cleaved herpes twice? So if you think about,
you cleave it once, right? You’ve got this opening, and
the cell’s trying to repair it. That’s probably a
pretty good chance. Maybe you guys who’ve done
old-time molecular cloning stuff, you know it’s an
intramolecular repair. It’s pretty efficient, right? You can close a
plasmid pretty easily. What happens if we cut it twice? Those two pieces start
to float around freely. Maybe it’s unlikely that
the two pieces can actually be repaired. Maybe we’ll have
more degradation. And in fact, that turns out
to be the case that if we do that now, we can
take two enzymes, put them in using
one of our AAV types. This is AAV8, and now you
can see a 90% reduction in the SCG and viral load. These are our controls. This is single
enzyme treatments, and here’s the double. And that’s highly
statistically significant. It’s about a 90% reduction. And even in the TG, we get
a statistically significant reduction. Typically there, we’re going
to be on the range of about 50% or 60% reduction. So it seems like
using two enzymes with the right delivery tools
is really the key to making this work well. So how do we move all this
past this sort of– now, if we’re at 90%– obviously, I’d love
to be at 99%, right? Or I want to get the trigeminal
ganglion from 50% to 90%. Why are we there? Is the enzyme getting in there? And it’s trying
to cleave herpes, and it simply can’t do it? Or is it a delivery problem? And so we turn to
single-cell sequencing, RNA sequencing to address
this with the hypothesis that maybe different
types of AAV differ in their ability to get
to different kinds of neurons. I mentioned there
are sensory neurons. There’s autonomic neurons. There’s subsets within
those definitions. And we did an experiment like
this in which, essentially, we made single-cell suspension of
neurons from treated animals. And they’re encapsulated
into droplets together with beads
that are barcoded so you can tell exactly
which cell each of these RNAs came from. Then you sequence them. You identify what they
are, and you link them back to the cell of origin. And then you get a
snapshot of that cell. You can tell all the RNAs
that are being expressed in that cell, so you know
that’s an autonomic neuron of this subclass. And then we’ll know, oh,
does it have AAV in it? Does that herpes in it, OK? And we did it like this. Essentially, animals were
injected with one of four of– and at the time, our
favorite AAV types. And each one had a
different transgene that we were essentially
using as a barcode. It was a fluorescent protein. We can use the
colors if we want, but generally, we just use this
for sequence identification. Pool everything and ask what
kind of neurons are they in? So the first thing that
falls out if you do this– you can divide neurons
into clusters of identity. This is a tSNE depiction of
the clusters that are defined. So the first thing you see is
that superior cervical ganglia neurons cluster
completely differently from trigeminal ganglia. And again, these are
sensory versus autonomic, so not too surprising. SCG neurons tend to be more
homogeneous than TG neurons, and it may make sense. We have proprioceptors. We have pain sensors. We have all these things. Maybe they’re all
different sorts of neurons. Oh, and then it turns
out, we can do this right because it’s reproducible
within our study. The clusters define
themselves very well, but also agrees quite well
with three previous papers. They’re kind of just
neurobiology papers, but it had defined neuronal
subsets within ganglia. And ours look reasonably
close to those, so we knew we were on a
reasonable pathway on this. But now we can ask, where
do our AAV types go, and where is herpes? So we were a little surprised
that we could actually find lots of HSV LAT in these cells. I mentioned this is the
one transcript that’s made during latency. So we have HSV reads in
our sequence analysis. 99.5% of them or so are HSV LAT. The rest might represent
reactivating virus. We don’t know. But in complete agreement
with the digital PCR data that I showed you before,
herpes prefers to go to the TG. Some of it goes the SCG,
but it’s a lot less. But you can also see that the
AAV serotypes vary tremendously in where they like to go, right? Like AAV1 seems to really
like the TG reasonably well, certainly better
than it does the SCG. AAV8 that I showed you great
results in the SCG with– yeah, well, here’s why. It really does a great job
transducing those cells and so forth. Here’s rh10. That’s pretty good for both. And you can actually
cross-reference those and say, of my herpes-infected
cells, how many of them have AAV? And you have to do a little bit
of math on this, but if you do, you can generate this
sort of bar graph where of all the herpes-infected
cells, how many have AAV of a given serotype? So if we use AAV8
or rh10, about 80% of the herpes-infected neurons
have detectable AAV transcript in them. So this is why we can get
up to those levels, right? And those levels are
substantially worse with the TG. These are even
lower than what we were getting by gene editing,
so gene editing is probably a slightly more sensitive
readout, actually, of the presence of these. But it kind of
tells you why we’re doing better for SCG than TG. And kind of the implication of
that work is, at least to date, we don’t have a
single AAV serotype that goes to all the
places we need to go. So we hypothesized we
needed to use combinations, so here we took three different
types that we now consider some of our very best– rh10, AAV, and one called DJ/8. We do those as single AAV
types, combinations of two, or a combination of all three. And in this experiment,
to make things simple, all you need is a single enzyme,
so we kind of stacked the deck against ourselves, right? We’re not doing the
two cuts, just the one. But if we do that and then look
in TG, which is a worse site– if you do that,
the only place you can see a statistically
significant reduction but with a single enzyme– a reduction of almost
60% of viral load– is with the triple
AAV therapy, OK? So the ongoing
experiments now are to take this triple AAV therapy,
combine it with the two cuts, and see if we can get the
TG all the way up to 90% or so where we are with the SCG. OK, let’s take 10 minutes
and talk about hepatitis B. So I’ve mentioned what a
major health problem worldwide hepatitis B is. 250 million people
are chronically infected with hep B. A
substantial number will go on to die of complications
of their infection. This is not a solved
problem at all despite the vaccine and
treatments that we have. So HPV has a replication cycle. It infects a cell, a hepatocyte. Essentially, it has this
partially double-stranded genome. This comes into the nucleus
and is filled in and makes this molecule called cccDNA. It stands for “covalently
closed circular DNA,” and this is the long-lived form. It’ll stay in a hepatocyte
for months or years. This is not latent. It’s not completely quiet. It continues to replicate,
and it replenishes itself even under most of
our therapies now. There’s sort of a replication
and replenishment cycle. And all of our drugs just kind
of stop or slow this cycle. So there’s a bunch of
different drug classes, but they don’t cure. They just slow this down,
so we want to attack cccDNA very specifically. Another molecule called
“relaxed circular DNA”– that’s that partially
double-stranded form. This tends to, in an
untreated individual, be a couple logs more
plentiful than this, but it is just an intermediate
and is not actually making new gene products. It’s not the long-lived form. OK, so for hepatitis B,
we’ve worked with Cas9 that I mentioned before. So here now, we’ll
be able to just do this in a single-stranded AAV. But the nice thing
is because Cas9 is driven by just
the guides, you can’t put two guides
together with your Cas9 into one construct
and fit in AAV. So with one vector,
you can get two cuts. And so this is just showing
some of the screening that we went to find
really strong guides. These are guide RNAs that target
regions of hepatitis B that are highly conserved, that span
multiple open-reading frames so they’re very bad hits on
the virus when they happen. And they also happen to be
in regions of open chromatin that we felt was important for
accessibility of the enzyme to actually be able to
get down to the DNA. And again, we wanted
to do this in vivo, so we used a mouse model. Now, the mice that we use
for herpes are a strain called Swiss Webster. You can get them
from Charles River. They cost about $3
a mouse, so we’re able to do a lot of studies. And what we learn in
this set of studies, we can apply in the next
one and make it better, and you can see this
progress that I showed you. Instead of $3, a mouse
for hepatitis B work cost $3,500 for one mouse, OK? And the reason for that is
they’re very complicated. It’s an immunodeficient
mouse that’s been crossed with
a background that has a genetic
lesion in its liver that’s going to cause its liver
to die slowly after birth, OK? So it’s actually not compatible
with life in these mice unless you give them
exogenous hepatocytes, and since it’s
immunodeficient, you can give it human hepatocytes. And so if you give
them human hepatocytes, they’ll start to grow up while
the mouse hepatocytes are dying, and
essentially, you end up with a mouse that’s
got a human liver. Then you can infect those
with hepatitis B or hepatitis C, whatever you’re
interested in and study them. They’re pretty sick mice. They’re hard to handle. Most academic labs haven’t been
able to successfully do this. So you end up working
with a company, and you pay enormous
sums to do this. But the company is very
responsive and great to work with. So they have these nodules
of human hepatocytes. This is standing
with human albumin, and you can see the areas
of mouse albumin, which are negative here, but green. So you’ve got these
parts that are human. And then we used an AAV
serotype called LK03. It was developed by Mark
Kay down at Stanford. And it was actually developed
in this mouse model– in this very mouse model– to
go to human hepatocytes, but not mouse hepatocytes. It was very specific, and it
actually works quite well. And so this just
shows that we have GFP transgene in these green
that colocalizes really nicely with our human albumin, and
that’s why we get yellow. So it really looks quite good. And we did an
experiment like this. We actually got a bit
of a deal on the mice because they had
been previously used. We actually got used
mice, yeah, which was great because the
company, Seventh Wave, we worked with was
very responsive and gave them to us at cost. The flipside is they were very
old, and they were very sick. And so we were hurrying to
get this experiment done before the end of
their natural lives. But essentially, they were
humanized and infected with hepatitis B. They’d
gone through a lot of things and had hep B for a long time. So we did this experiment. We treated them with a
drug called entecavir, repurposed HIV drug, so
it’s a reverse transcriptase inhibitor. It knocks down HPV replication. It doesn’t stop
it in these mice, but it knocks it down so
that we’re slowing down the replenishment,
the refeeding of stuff into the liver because
we thought that might make it harder to do this. So we gave them three
weeks of entecavir, kind of lead in, and then
we treated them with our AAV vectors that either contained
guides against hepatitis B or guides for
[INAUDIBLE] control, anti-GFP, just as a
control that there’s no GFP sequence in these mice. This should do nothing. So entecavir was kept
on for four more weeks, and then at the end of
that time, some of the mice were sacrificed and evaluated
for what had happened during the entecavir therapy. In the remainder, entecavir
was withdrawn, and we asked, did we have any effect on
the rebound of hepatitis B? Again, extremely well tolerated. We’ll go into this, but
there’s absolutely no evidence of any sickness in these mice. So it’s wonderful that,
again, we have safety, and we have a lot of
histology and things on this. So they look great. And do we get gene editing? Yes, so we saw gene editing
in five of eight treated mice. So we have eight mice
in our treated groups. We have some controls. Two of the eight animals showed
gene editing, indel formation at both cleavage sites
within hepatitis B, but again, the frequencies
were very low, OK? One of the best
ones was 2/10 of 1%. Here’s one– almost 0.4, OK,
so even worse than herpes. And honestly, we sort of
let this data set around for a while because we were
pretty discouraged by that. That’s not very good. And then we got all
this herpes stuff, so everything’s out of
order as I’m presenting it. But then we get all this herpes
stuff, and we said, well, gosh, we can get a lot of
loss of herpes genomes even if we don’t have
very much mutation. Maybe this is actually
doing something. Let’s look at these
animals a little bit more. And I’m glad we did,
and it goes back to this single-cut
versus two-cut idea. Remember, we have
two guides in here, so we’re making these
two cuts in hepatitis B. And the first thing we did
was look at DNA levels, so just to orient you for this,
the A groups are the controls. And the B groups are
the treated animals. So if you look at
the total HPV DNA, there’s really not
much of a difference between any of those groups. Remember, we’re mostly looking
at this relaxed circular form, and whenever the reservoir
is being replenished, this is coming in, so not
too surprising that we don’t see much there. But if we look specifically
at cccDNA, the long-lived form that takes a long
time to actually be made, both at the early time
points and late time points, we have anywhere between
about a 65% and 50% reduction in cccDNA load in
the hepatocytes. Now, I told you we have
eight treated animals, so as you can imagine,
those sorts of things don’t reach statistical
significance in this. So we could have
beta error, or we could have a spurious
finding that’s not true. And I can’t tell you which
of those is accurate. But we did wonder,
OK, if this is true, would this manifest any way
clinically with the mice, anything we can look at? We said, well, the hepatitis B
genotype they use, genotype C, is a really bad genotype. It’s very cytotoxic. As HPV genotypes go,
it’s the one that will kill hepatocytes the most. So is there any effect
on hepatocyte survival? And in fact, when
we looked at that, we achieved really dramatic
and statistically significant results. So here, we’re actually
quantitating human versus mouse hepatocytes
within the liver, OK, because there’s this
competition between the two of them. So the mouse hepatocytes
are generally dying, and the human ones
are trying to live. But hepatitis B is trying
to kill the human ones, and it doesn’t infect
the mouse ones, right? So in our control animals, the
human hepatocytes turned out to not be doing very well, OK? The percentage of
human hepatocytes ranges anywhere from
about 6% up to about 20%. But in both of our
treated groups, we’re seeing approximately
40% of the hepatocytes are human, OK? And in both the early
and late time points, that’s highly
statistically significant. There really seems to be a
pro-survival advantage that seemed to be resultant
of our therapy, so this suggests to us that
maybe this reduction in cccDNA that we’re seeing is real. Clearly, we need to do
more experimentation, and we’re currently
setting up to do that. So if you reduce the cccDNA
by 50%, do you change rebound? No. These animals aren’t cured. This is the rebound. One of these lines– the red one is the
control animals. The blue one is the
treated animals– no evidence that reducing
viral load by 50%, at least in an immunodeficient
mouse, prevents recurrence. Probably some partial
incomplete reduction to hepatitis B of A log or
two in the presence of a fully intact immune system might
actually prevent recurrence, but we’re not there yet. And these mice don’t have a
functioning immune system. All right, so that’s pretty much
the story I want to tell you. I’ll leave you with just
a couple of thoughts. They can kind of be
the take-home message if you need just a couple
of things to remember. It’s very clear from
our work that you can perform gene
editing successfully of latent and persistent
viruses in vivo, and it can be really quite
an efficient process. Ranges anywhere from over
90% in SCG to maybe 50% trigeminal ganglion,
and in hepatitis B, we clearly can promote the
survival of human hepatocytes. There’s a lot of
different enzyme classes that can do this. Don’t give up on Cas9. It clearly seems to be working
for us in hep B, but remember, there are other tools as well. And for the right application,
they may be superior. We really like
single-cell RNA sequencing to understand gene therapy. It’s a really powerful
tool for optimizing. And we think that
what this is really telling us is we need to have
combinations of multiple AAV serotype to cover all
the target cells, ideally with a couple of different
cuts, and then we’re going to be able to achieve
levels of results that we think will lead to
therapeutic benefit. With that, I want to thank
everybody who did this work. Martine Aubert has led
the herpes simplex work. Michelle and Nori,
who are here, have done a tremendous amount
of our animal work, so thank you so much for them. Dan’s our AAV guru. Pavitra, who’s faculty
within the department, does all of our informatics,
and the super laboratory work by Meei-Li, many of you know. I want to thank
Alex and his group. Oh, two Dans– thanks
to both of them, and I mentioned Cellectis
and other folks in the lab. A lot of funders– and they’re listed here. Now, the NIH is a big
supporter of this, so we’re glad that
that’s come along and they recognize the
importance of the infection. I want to call out
for the first time because then it’s going
to end up in YouTube– this work’s actually
been supported by over 200 individuals who have
given small funding for this, and some of it’s not what
we’d really consider small. It’s incredibly generous. And I mentioned the
limitations of the mouse model, and because of this funding
from these 208 individuals, we’re actually able to move
into a new model, guinea pigs, where the virus actually
spontaneously recurs and actually has
lesions that look almost exactly like a human lesion. So we’re going to be
able to ask the question, if I reduce the
viral load by 90%, do I cause clinical benefit? Do I prevent shedding so
that people won’t transmit? Do I prevent lesions? These are the things
people care about. So that work would be at
least a year away by the time we go through the NIH
process to get that funded. And that’s happening now, and
it’s thanks to those folks. So I thank them. I thank you for your
attention, and I’m happy to take questions. [APPLAUSE] Sean? SEAN: Great talk,
very inspiring. My question is, it seems like
the viral load is like a moving target, right? You have a certain number
of ganglia and neurons that are infected, so is there
a threshold below which– getting to zero would be great. But is there a
threshold below which you won’t have recurrence? You won’t shed at a level
that would transmit? Some of these might be
unknowable questions. And to get there, can
you reuse the AAVs? Can you give it again
and again, or do you get antivector response? KEITH JEROME: Yeah, those
are great questions. So we did a lot of
that work before we got to where we are now. So I work with a lot of people– Josh Schiffer at
the Hutch, who’s a mathematical
modeler, who’s built a lot of models
for HSV recurrence and lesions and shedding. And that work, together with
some pre-existing literature, suggests to me that the
magic number for sort of having clinical benefit is
90%, which is where we are, OK? So I think we’re
there, but that’s extrapolation from other
kinds of experiments. So the cool thing is now– hopefully, we’re there. We don’t know yet that we’re
getting those sorts of numbers in guinea pigs. We may have to reoptimize. We’ll see. But assuming we can get there,
we can ask exactly that. It’ll turn up those models
well, and we’ll see. I suspect, if we
continue to optimize, I think we’re going to
get past that, honestly. I would like to only
have to treat once. You definitely get
an anti-AAV response. We probably get anti-payload
responses as well. You can certainly treat twice
within about a two-week window as that immunoresponse revs up. After that, you
have to do tricks. You can immunosuppress. We’ve worked with [INAUDIBLE]
mice and immunosuppression, and you can get AAV in. That gets to be a
little more invasive, and so I’d like to
have it just be once. It’s easier to perform
clinically, anyway, but again, we’ll see. Jeff? JEFF: I had that question, but
a related question to that, too, which is– I’m trying to remember the math. If there was 1,000 infected
neurons in the ganglion and you knock out 900 of
them, say, there’s 100 left. Do we know why those
100 are special? Are there ways that things are
resistant to being AAV knocked out? Are they different,
or is it just statistics and just the dose? And it just didn’t
get to everything. KEITH JEROME: Well, I think
there are aspects of– they’re special in that
our AAV types just aren’t good at getting to
certain types of neurons right now. So we’re working through that
to look at Additional AAV serotypes. I’ve shown you some of them. We have additional ones. Some of it, though, I
think is just stochastic, that it’s just luck or not. And that may be why we
actually have a room to go up with our AAV dose, actually. We can go up another
log, actually, with safety and approvals. It’s just a lot of AAV to
make, but we can do that. So that may help. We may want to come in twice
within that two-week window or just come in later. Or you might say,
well, I’m going to treat, see how we’re doing,
and if I need to come back, I might have a different
set of serotype where the immunity
I’ve generated won’t prevent
those from working. I think there’s a
number of things. Again, we kind of just
need to do the experiments and see exactly what
we need to tackle. Yeah, in the back. INTERVIEWER 1: Do you know
if any of your AAV serotypes target the dorsal root ganglia? KEITH JEROME: Oh,
great question. Yeah, so working with the
DRG is kind of tough in mice. It’s not impossible. So we haven’t done
a ton of stuff yet. We’re going to do a lot of
that with the guinea pigs. The limited work
that we’ve done– and there’s one other lab in
Florida who’s done some work with delivery to DRG– it seems to be
pretty, quote, “easy.” It seems to be more
like SCG than TG, so it’s not really even
broken down by serotype so much where you try things. You can just take
a single serotype and get like 90% transduction. So I’m hopeful that that’s
not going to be a problem, but it’s not a ton of robust
data like I’ve shown you. It’s based on a relative
handful of experiments. Mark? MARK: Keith, great talk. I’m interested in the potential
for an immune response caused by the double-stranded
DNA itself, since double-stranded DNA
can serve as an adjuvant. What’s the fate of
the cleaved DNA? Is it broken down
intracellularly? Is it released from
the cell, and can that either enhance the
immune response against the infected cells or
potentially cause autoimmunity? What’s your thoughts about that? KEITH JEROME: Mark,
I really don’t know. To tell you the
truth, I’ve sort of assumed it was degraded
intracellularly. We have no evidence
that the neuron itself is being damaged or destroyed. We’ve never seen any hepatitic
markers, no neuronal loss. Could it be released? I don’t know. The anti-DNA response
is actually interesting. Maybe I’ll talk
with you some more after this to think about
ways we might look at that. We are starting
to get to a point where we’re starting to think
about clinical translation here, so we’re starting to
think about safety issues more and more. We sort of felt philosophically
like the first thing for us to do is just show you
can do this, right, and get it to work pretty well. And we’re sort of there,
and so we’re definitely beginning to think about how
we’re going to translate this into human studies. And the more safety data we
can generate now, the better, so let’s talk about
that a little bit. That’s a long way of saying,
I don’t know, but thank you. INTERVIEWER 2: But
Keith, wouldn’t you be ready maybe after
the guinea pigs to move into nonhuman primates? KEITH JEROME: Yes, definitely. I think that’s an important
step along the way. All right, thanks very much. [APPLAUSE] [MUSIC PLAYING]

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