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A discussion of Alnylam's RNAi therapeutic called patiseran for identification of biomarkers associated with the knockdown of a disease-causing protein transthyretin.
Proteomics in Proximity discusses the intersection of proteomics with genomics for drug target discovery, the application of proteomics to reveal disease biomarkers, and current trends in using proteomics to unlock biological mechanisms. Co-hosted by Olink's Dale Yuzuki, Cindy Lawley and Sarantis Chlamydas.
Welcome to the
Proteomics in Proximity podcast, where
your cohosts Dale Yuzuki,
Cindy Lawley, and Sarantis Chlamydis from
Olink Proteomics talk about
the intersection of proteomics with
genomics for drug target discovery,
the application of proteomics to reveal
disease biomarkers, and current
trends in using proteomics to
unlock biological
mechanisms. Here we have your hosts,
Dale, Cindy, and Sarantis.
Welcome to the Proteomics and Proximity
podcast. And today we're going to
be talking about
hATTR and
Amylodoisis. What is
that, Cindy? So
we're specifically, we've
narrowed down this really,
I think, a fantastic paper from, uh,
Samina Takao and
Paul Nioi,
who
looked at essentially a broad
proteomic profiling of samples
that they just had sitting in their
freezers. And so Evan
Mills on our team,
has often said that
these samples that have completed
clinical trials and are sitting in the
freezer might have, some
nuggets, that
we might be able to mine by doing
a broad scale approach in proteomics. And
that's exactly what this team did.
And they came out of it with a,
biomarker that shows
potential for not only
diagnosing a hard to diagnose
genetic disease,
it shows promise as monitoring,
disease progression, as well
as response to the therapy that
was part of that clinical trial. So,
yeah, this is an exciting paper. So, to
back up a little bit, what is
hATTR. This is a rare
disease, I understand. Yeah, that's
right. It's a hereditary
transthyretin amyloidosis. So the
TTR is transthyretin gene,
which is... a protein.
So there's a mutation in the
protein for TTR,
and it results in
some amyloid. We know
amyloid from our
discussions
around plaques,
particularly in Alzheimer's disease.
Correct. So here you have these
aggregations of proteins
that result in some
challenging polyneuropathy
that is progressive,
and fatal. So
you can imagine being someone who's been
tested for this and they find out they have
it and they're just got a ticking bomb.
They've just got to wait and test and wait
to see if it shows up, if it
ever revolves. It does.
It sounds awful because this
is a progressive, debilitating
disease, because it affects your nervous
system, but it also affects other organs. Is
that correct?
As I understand it, it
affects the, um,
ability of the
schwann cells, I think, to
give,
their signals. I think it's
interfering with the signals in the
musculature. I
remember my high school physiology.
Let's see, Schwann cells is one of
the types of nerve cells, is
that correct? Yeah. In
sending signals. Yeah. I
remember reading something about the
proposed mechanism of
the disease. Uh,
and of course, as most disease,
uh, there's
heterogeneity, which makes it challenging
also to stratify and get through
a successful clinical trial. I see. And
thus the need for this kind of study. Can
you describe what they did?
Yeah, sure. So,
uh, they did a cross sectional
arm as well as a longitudinal arm.
So essentially
they essentially looked at
uh, their cases where
they're giving the therapy over
baseline at nine months and 18
months. And they looked at
their placebo and it was
randomized, double blind I
believe. /and
that was the component that was
longitudinal. So again, three time points
over the course of treatment.
And then they had a cross
sectional aspect of just healthy
controls that they monitored over that
same period of time. So this is
a clinical trial that they were
conducting, like phase two or phase three?
Yeah, I
presume it was phase three.
yes,
it looked like a
Phase three trial. I don't remember
them stating that in the paper, but they
probably did. It's called the Apollo Trial.
It's a pretty well known trial because it's
an RNAi therapeutic.
So it's an interfering
with RNA.
So RNAi
therapeutics. Help me with this. Is
that like gene therapy? Is this like
a vaccine? We've talked
about mRNA vaccines.
Is it like that?
To my knowledge, there are very few
drugs that they are FDA approved for
that target the silencing of genes
at the end. What happens with this,
let's say pathologies, we have a mutated
version of the TTR protein, and also
what type version of TTR
kind of overexpression of the
protein. In that case, they try to
target the expression of
the, of the mRNA post-
transcriptionally. In the cytoplasm, they inject
double-stranded RNA targeting the 3-prime
untranslated region of the
gene. This is the RISC complex
with the DICER, Argonaut
that helps the targeting actually
of the antisense strand,
on the complementary
region of the gene. And then
degradation of the mRNA with
Argonaut, that is the end of
the endonuclase in the system. So that's pretty
much the mechanism of action. Okay. With
DICER and Argonaut. Understood.
Backing up a little bit,
right. These COVID vaccines,
right, from Pfizer
BioNTech and Moderna... I mean
these were mRNA vaccines
that were used to actually
produce spike protein. But
here what you're talking about. RNAi
is similar right... They're
injecting RNA, they
inject double-stranded RNA targeting the
gene. Interesting, that's pretty much what
happened. And then it actually then turns
down the expression
of the gene and they're tackling the
mutant form. Is that correct? The one that's
messing up, they got them both. Because
actually this region that they are target is
for both the mutant and the wild-type.
And they target both. They down-regulate
both, but the
disease problem... The patients, they have,
they have two alleles,
the mutant and the other the wild type,
usually, most of the cases. And the whole
problem is it's just overexpression
kind of. Right?
Because you have the overexpression of the
mutated version as well. Right. Uh, you
have the presence of the wild type and the
presence of a new protein that is
misfolded and aggregated. I
see. So then they develop this drug,
what is this drug called?
Patiseran. Okay, so
Alnylam produces this
drug, Patiseran. This
drug actually is RNA
for RNA
interference. ...And
we're talking Cindy then also about
the middle of this clinical trial, and
they're using healthy versus
uh, affected
patients, then what do they find?
Yeah, so
in their... cross sectional
arm, they found
that an expected
protein sorry, the
longitudinal arm. So in the aspect
without the healthy control. So just looking
at the placebo and the
treated over baseline
nine months and 18 months. So in that
longitudinal arm, they looked
at... the
time progression of
different proteins as
patiseran was given. Right. So they're
looking for signals in various
proteins, that
are popping up above
um, significance
for being
differentially,
expressed. And
they found two of note, they found,
I think, 66 total
that were, providing
significant differences. But the
two that really popped out,
and especially in those
volcano plots, are
NT-proBNP, which was
an
expected signal. And in fact, I
think that was part of the clinical trial
using NT-
proBNP. But what was
unexpected was nFL. And of course that's
part of... the title of the paper Neuro-
Filament Light Chain is a biomarker of
Hereditary Transthyretin-
mediated Amyloidosis. And
so with nFL,
they were finding that the
placebo group uh,
uh,
progressively increased in their
nFL levels over the course of the
and that they could compare. So once
they got those signals in the
longitudinal arm, now they could actually
put in the nFL levels for
the healthy controls. And look at healthy
controls relative to those
placebo treated. Those are going to be the
ones that aren't going to be getting any
better and they're going to be comparing the
healthy controls to those that are treated
with Patiseran. And what they saw
was that the placebo,
...samples,
were increasing in
neurofilament light over the course of
the trial, whereas those
treated with Patiseran
were trending toward
healthy controls over the course of the
trial. Now, as
far as in the results of the trial, the
people that were on patiseran
got better or didn't
get worse, is that correct? That's
correct. And in fact,
um, there's a
statement around
the current methods by
which we monitor
for progression of polyneuropathy in
these patients or in even initial diagnosis
for these patients. And so there's
a neuropathy impairment
score that they used and in fact,
they used this to stratify and
figure out who are the patients that are
relevant for the clinical trial. You
want ones that have active disease,
obviously. And they
were able to see that
the impairment score,
improved as the
neurofilament light levels, uh, went
toward healthy controls. So that's a
nice
suggestion that
you might be able to use neurofilament
light to actually identify
uh, when that impairment
is happening. The challenge with I think
that impairment score is it was
described as burdensome to administer. I'm
guessing with some of these
scores where
they're doing a movement ah
requirement, they can be a
bit objective, hard to really nail down
across different doctor's offices. I'm just
making that assumption. That's what they
mean. You're talking about a subjective
measurement of some ability to do
something right in terms of nervous
activity. And here, well,
backing up, how did they discover
neurofilament light? I mean, this is
using Olink panels then. Yeah,
good point. So
we should probably emphasize that a little
bit.
they used our
tool. That was the
broadest look at the,
proteomic... the proteome
that we had at the time. So they essentially
used all of the unique
proteins we had in our library across, I
think it was 13 or 14
different panels. One of
our 14 human panels
is actually, redundant
with the others. So I think it's
an Olink Target 96.
Yeah, 13. Yeah, exactly. qPCR
panels. And so each of those
qPCR panels have 92
proteins, uh, along with controls.
So you can run 96
samples against 96 proteins at a
time, uh, including those
controls. So, yeah, they did a broad
look, as broad as they could
with what was available at the time. I think
it would be interesting to see
what they could do now with, uh, just
under 3000 proteins. Right. We've
more than doubled the library since
then on the NGS readout.
Right. So out of these, what, over a
thousand proteins, they were able to
you mentioned find 66,
and you mentioned the two of note the NT pro-
BNP. Sounds familiar. Isn't that
a cardiac marker?
I, I would
ask the man who is the specialist on
the "affairs of the heart"
Cardiac marker
as well.
So why do they
find, Cindy, you mentioned
that the proBNP was
expected. I guess that was
already known with this, uh,
amylodois's condition.
Yeah. Any thoughts why you have a
marker for heart health
that crosses over to
basically neurological disease?
I think that happens a
lot. Right. That's where the
excitement is, where our bodies
are incredibly good at
repurposing, uh,
different things that
we all thought that
there would be this really simple
smoking gun in the genetic world. Right.
Once we were able to look at genetics, we
thought that we would have like, one, uh,
mutation for each disease. And now we find
that there's actually lots of different
mutations that lead to, uh, death by
for various diseases, like
cardiac disease and stuff like that. So
I think the overlap of
how these different diseases
have the same pathways is going to tell us
something about mechanism But
I think the speculation,
I don't remember if they speculated in
the paper, Dale, but I'm guessing you might
have some ideas. But I would push it over
to our Sarantis
"affairs of the heart". I'm taking the difficult
part right? I don't know, I mean I have seen
the bit in the literature that this amyloid
could be formed also with different tissues
like cardiac and people, they have
a combination of disease like
neuropathy, cardomeyopathies,
and usually these amyloids, they
target cells that they are not mitotic
active, like, cardiac
cells.They are polynucleated. And
they are not mitotic active. They target
these (cells). That's a fascinating point.
That's an explanation, but of course
they don't go
through the details and they don't try to
give an explanation. Yeah I think they just
stated as fact, right, that this is not a
surprising finding. The
fact that they got not only these
two markers, but another
That seems to be quite a
lot of markers. Right. They're
not going to chase after each of them.
But what can you say about well,
naturally, the title is around one marker.
Is there value in looking at more than one?
Yeah, I'll just jump in and say
this group is part of
the UK Biobank
Pharma Proteomics project now. So
they joined in kind of a second
wave of additions to
that proteomics project. Initially it was
ten partners, now it's 13.
And Alnylam was one
of them.
So probably
these multi analyte signatures
are something that
they may be pursuing
internally, but not
publishing on. And in fact, I will
say it's pretty rare
that
a team within
a pharmaceutical company
is able to publish on
findings like this. I was very excited to
see that they took the time to put
it out into the literature to help
others understand this mechanism. Well like
we were talking about how difficult it is to
assess the diagnosis.
Then one thought, right, is that the
important findings is
neurofilament light as a diagnostic
marker, is that right? Yeah, yeah
exactly as a diagnostic marker. And one that
can monitor disease
progression and
response to therapy. Right.
So what do you mean by go ahead
(Dr.) Paul Noe, who
was the
principal investigator on this
paper, the final author
has a long history
with deCODE and Amgen; very
strong genetics
specialist. He's
quite accomplished
as a scientist. And then
Simone Ticau is a
principal scientist, of course, at
Alnylam. And as far as then,
you mentioned disease
progression. What do you mean by that?
So if I understood correctly,
the people with disease with
placebo, right, they had this rising
level of nFL and
people that were on the
medication stayed
constant. What will
a biomarker do with regard
to telling
how far you were coming along with the
disease? You
mentioned disease progression. Actually, as
far as I understand, it's not like a
constant. I think that they are decreasing
and reaching out to the levels of the
healthy controls because they are ranked
healthy controls. Right. And then you see
that initially healthy controls with the
disease, they are like four times
lower, roughly. And
by having the
patiseran right, they see that levels of
the patients, the
patient samples, the levels of an effective
goal, like, towards, leans
towards the healthy conditions. Right. That
was the case and for which it was
responsive to the therapy at the end.
Right. I think that was a really nice thing
with his protein. I see. Yeah. And I
think if I'm correct on
this topic. Yeah, I agree.
I think that the levels,
trended toward healthy
samples in the patiseran-treated
and they reached,
a level that they stayed at
from nine months to 18 months.
At least that's what it looks like in the
significance levels and the
graphs that essentially
they're saying, I think
that they're able to,
have a quick response
and that those patients appear to
stay steady at least over those 18 months.
Right. But that longer
studies, are needed to really
nail down the value of that as a
marker, over that long
period of time. Now, if
I understand correctly,
Sarantus, you mentioned this patiseran
drug is actually double stranded
RNA. It's silencing,
uh, these particular, uh,
transthyretin genes that are
aberrantly mutated, what have
you. Is it just one
dose? Do they have to take multiple
doses? This I
don't know. And actually,
I think it's one dose they are
injectable. It happens to the liver,
targeted to the liver.
But I don't
know if there's a second dose or third
dose... It is intravenous and does
target liver.
Thinking about it, it probably does
make sense that they're a regular
dosage. I was thinking about
sort of after the trial was over. Right.
They used the broadest
panel that they could with the Olink
target 96, the 13 or 14 of them.
But that was for discovering more. Right.
Wasn't the goal of that
just to look at mechanism
of action in terms of how patiseran was
working? I
think it's not like how patiseran is
working. Uh, it's like how the
silencing of the gene affects
other proteins that could be potential
biomarkers. I see. Because at the end, the
patiseran targets the gene,
TTR gene. It doesn't target any other gene.
It targets this gene, then what we
are screening, what they actually are
screening is the effect on other proteins
that they are may be connected... upon,
some similarities. Got it.
Well, the system's biology of it
gets pretty complicated because you're just
talking about reducing the expression of
one gene. But we're a
system.
But we can speculate what they're
doing internally, but we're not going to
know if they're not publishing on it. Right.
Yeah,
absolutely. Sure. Absolutely.
Well, Cindy, you
mentioned some interesting
things related to multiple sclerosis
and with
neurological disorders,
I mean, coming from the genomics
perspective. I myself know
very little about neurological
disease like multiple sclerosis. But you
mentioned a conversation about
Epstein Bar virus and multiple
sclerosis. Do you mind sharing
that? Yeah. Sure. So uh,
uh, I think it's quite the buzz in the
Twitter-verse, and uh,
several people I
trust have um,
been really impressed with this paper, and
we can put it in the show notes around
really assigning causality to a subset of MS
patients. And MS is of course, very
different from hereditary,
transthyretin amyloidosis. It's
an autoimmune
disease. But um, that
Epstein Bar has
been identified causally, uh,
uh, as
causing Multiple
Sclerosis in a subset of those patients,
which that's been
a postulate
in Parkinson's disease, of course, as
well. That virus can turn
on something that's sort of hidden in your
genome, uh, and make it
active. Um,
in that case, I
haven't done much reading on Parkinson's
disease or where we're at with that
hypothesis, but I certainly know,
people with Parkinson's disease
who can tell me stories
about having had ah, a
viral infection very soon
before, very recently in the
past, before their
onset of symptoms. So, it's
a compelling idea,
and understanding the mechanism of that
would be incredibly important.
Now, Epstein Barr virus, isn't
this one of those viruses that's
endemic in the population?
Meaning it's very, very common,
Okay? And
if you have Epstein Bar virus, it
might present itself as a
mild cold or something, right? You wouldn't
know that you had Epstein Bar
virus. But what you're saying is people
that get, or maybe I'll put it this
way, there are a subset of
multiple sclerosis patients who are
triggered by getting infected with
EBV, is that correct? That's right. Yep.
Where they could find the link to EBV
infection. So, um,
the numbers I'm seeing from Cleveland Clinic
are an estimated 50% of all children that
are up to five years in
age have been exposed to
EBV. And 95% of
adults experience an EBV
infection in their lifetime.
Okay. And they
experience an infection, they recover, like
from any other virus. Right?
And they go on with their life. But then
there's that subset of so. They don't know
what the mechanism right. They don't
know what the mechanism is. Right. Because
we're talking about a complex autoimmune
disorder,
and now we're getting into
the nuances of
autoimmunity. Right? Yeah,
exactly. And I think I've heard
it linked, although I don't know the
scientific case for it, but I've heard
people claim that it's linked to
fibromyalgia or
um, what's the other
disease, um, that's been
compared to long COVID.
Mike Snyder is doing
a study, I think looking
or he proposed a study
looking at the difference between chronic
fatigue
And, uh, long COVID. Just to
understand what what are the differences,
and, um yeah, it might be interesting
to see what the link? Well,
getting back to the EBV and
multiple sclerosis story,
is there any,
um, I don't
know, development
preventative that could be
prevent S for these
subset of people?
Yeah. Like I said,
I haven't read that paper
directly. I just know it's quite the buzz
from people that I trust. Yeah,
interesting... man. And then you
mentioned that EBV may be implicated
in some of these other neurological
disorders, but if people
adults being exposed,
what can you do? Right.
Do we go back to wearing
masks all the time? No, I'm not
suggesting that. I'm just thinking
about the sort of practical
implications. Well, Epstein Bar, remember,
is a herpes virus. Right? So
those are very hard to
avoid. Yeah.
This battle
right. Between infectious organisms
and humans. That's right. It's been
going on for a while. That's
right. Well, Sarantis, do you want to
close with any thoughts?
No, I think
the folks and the nice take home headimgs, take
home message for this type of papers is
like, we hope you're going to see double-
stranded RNA therapies coming. We are in
the RNA era and therapies from vaccines, uh,
to double-stranded RNA and
the proteomics can really be a nice
tool to assess, uh, biomarkers.
Right. Like endpoint,
uh, endpoint effects. So that's that's pretty
much what I see what excitement is in this paper.
That's great. Well, thank you for the time
today, friends and cohosts. Thank you very
much. Okay. Thank you very much. Bye bye
bye bye bye bye bye.
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