Rational therapeutics for genetic conditions – Hal Dietz


Hal Dietz:
Good morning. It’s really a pleasure to be here. Thank you for that kind introduction.
So I have a tall task today, and that is to discuss treatment of genetic disorders in
general. I’m going to approach this by presenting many small vignettes that reveal both the
power and pitfall of some new approaches that are being used that have the potential to
be very powerful. So when we’re first thinking about a disease
— I’m sorry — often we feel stranded on a desert island. We really have no compass
and we can look in 360 degrees without clear conviction about what direction to go in.
Now genetics has received a lot of hype, appropriately so, for its ability to allow you to make giant
leaps in understanding without taking each small derivative step along the way. You can
learn about a new disease gene and suddenly be firmly within a pathway that you never
would have anticipated. And initially when you get there, things look very appealing.
But once you get your bearings, you often learn that you’re no better off than where
you started and that it takes a lot of hard work to reach the promised shore. So initially when people think about treatment
of genetic diseases, often gene therapy comes to mind first. It’s a fairly straightforward
strategy: if the body is missing a gene product because of a defective gene, you simply use
a virus to reinsert the normal gene into the cells of interest, which will then make the
protein and restore function. Gene therapy can also be used to treat patients’ cells
that have been removed from the body, again giving them the normal gene and allowing them
to go back into the patient to restore function. There are many obstacles to gene therapy,
including an immune response to viral proteins. There’ve been a number of very catastrophic
cases of failure of gene therapy due to this effect. And also because of disruption of
essential genes when the virus inserts the gene into the host genome, for example, causing
leukemia. But despite this there’ve been many startling recent successes of gene therapy
for conditions such as adenosine deaminase deficiency, causing immunodeficiency, for
Leber’s congenital amaurosis, causing blindness, and also for hemophilia B. So I’m not going
to spend a lot of time on gene therapy. There is a reference that is provided in the — will
be provided with the slide set that goes over that topic in detail. Rather, I want to cover the basic concept
that it takes a village to really bring a new concept to a patient. And then it really
requires a confluence of and synergy between both basic and clinical sciences to develop
a full mechanistic understanding of a disease process, and in that manner, to derive novel
and rational treatment strategies. So the first story I want to tell you concerns the
so-called lysosomal storage diseases. A lot of very well-known names here such as Hunter
disease, or Hurler disease, or Pompe disease. All of these conditions are unified by the
toxic accumulation of lysosomal substrates due to a deficiency of a lysosomal enzyme.
So, real progress in this area actually derived from an accident in a laboratory, in Liz Neufeld’s
laboratory. For this particular example, I’m going to
show a green cell as a normal cell that has a full complement of enzymes and normal function
and various flavors of red cells to show a defective cell lacking a lysosomal enzyme.
And you can see in this hypothetical example that in this cell there’s no Enzyme A, shown
by a yellow circle, causing lysosomal storage disease 1. In this cell there’s no Enzyme
B, causing a different lysosomal storage disease. The accident came when a technician in the
Neufeld lab accidentally mixed cells from two different patients with two different
lysosomal storage diseases. And the remarkable result is that when they were mixed, all the
cells adopted normal function; suddenly they worked, everything worked. So this is called
a complementation, where one cell will complement the deficiency of another cell, and it really
herald the concept of treatment for lysosomal storage diseases simply by infusing the defective
enzyme or the deficient enzyme. So in this example, once again, this cell does not make
the enzyme shown in yellow, this cell does not make the enzyme shown in blue. What the
Neufeld lab learned is that each cell secretes the enzyme that it makes outside of the cell.
And then the other cell can take up that enzyme and in fact complement its deficiency by taking
up the enzyme that its neighbor made. And again this leads to cells that have both enzymes
and normal function. So everyone is understandably excited about
the prospect of simply infusing enzymes, but then there was a pretty startling and disturbing
result that if you made the enzymes that are defective in these patients, and simply put
them in the culture medium of the cells, the cells failed to take up that enzyme and there
was no complementation. And the obvious question is why, what’s missing? So the answer came
when the Neufeld lab studied a different disease. This disease is called I-Cell Disease. And
what they learned is that cells from patients with I-Cell Disease make all the appropriate
enzymes, they secrete all those enzymes outside of the cell, but that other cells lack the
ability to take up those enzymes that were secreted. And what ultimately they learned
is that in I-Cell Disease, there’s deficiency of a specific enzyme called N-acetylglucosamine-1-phosphotransferase
that modifies all these enzymes by attaching a chemical called Mannose-6-Phosphate. And
it was learned that a recipient’s cells, in order to take up an enzyme, have to have a
Mannose-6-Phosphate receptor that will then bind the enzymes in its environment and facilitate
cellular uptake. So now there’s a complete strategy for enzyme replacement therapy. You
could synthesize these enzymes in the laboratory, but you had to add this modification, the
Mannose-6-Phosphate Group. So does it work? The answer is yes. So here’s an example in treatment of Hurler
Syndrome with recombinant enzyme alpha-l-Iduronidase. You can see that all of these patients at
the start of therapy had rather high New York Heart Association classifications, but during
the course of therapy, about 50 weeks of therapy, all of them showed a clinical improvement
and many of them showed a very striking clinical improvement down to the best level. If you
look at other markers such as liver size — the toxic substrates and lysosomal storage diseases
often accumulate in the liver — you can see that liver size decreases dramatically during
the course of infusion of the deficient enzyme. And you can also look at skeletal performance.
Patients typically would develop contracture and you can see that there’s increased movement
at the shoulder, at the elbow, and at the knee while these children are receiving infusion
of the enzyme. So I don’t expect you to be able to read anything
on this slide, but the basic concept is that there are now many enzyme replacement therapies
for many lysosomal storage diseases that are either in late-phase clinical trial or that
are FDA-approved to use for treatment of these children. Is this the whole answer? Is this
the cure for lysosomal storage diseases? Unfortunately, there are some obstacles. In the example that
I showed you, the enzyme with its Mannose-6-Phosphate group is free to interact with cells in their
Mannose-6-Phosphate receptor, leading to functional recovery. But it was recognized that some
patients over time develop antibodies that attack the enzyme that’s being replaced. And
what distinguishes these two groups of patients, those without and with antibodies, is the
ones without antibodies make some small level of the enzyme that’s being replaced. It’s
not enough to achieve the function that is necessary, but it’s enough for the body to
say this enzyme is okay, it’s not foreign, I’m not going to attack it. However, in patients
that make no enzyme naturally, their body has never seen that enzyme. So it’s recognized
as foreign and therefore attacked by the immune system. Another obstacle is the so-called
blood-brain barrier. The micro-vessels within our brain contain a very solid barrier that
prevents diffusion of multiple substances from the circulation into brain tissue. And
unfortunately, this blood-brain barrier is impermeable to all the enzymes used in enzyme
replacement therapy. So currently, there’s excellent utility of
enzyme replacement therapy in conditions such as Maroteaux-Lamy where there’s no central
nervous system manifestations, but there’s really very poor performance of enzyme replacement
therapy in diseases such as Gaucher disease type 2 or 3, where there’s very severe brain
manifestations and the enzyme simply can’t get there to help. There are some potential
solutions. You know, just like children receive allergy shots to teach their body to tolerate
something that’s recognized as foreign, similar approaches are being used to induce tolerance
to enzyme replacement therapy. There’s alternative targeting procedures that are being explored
that might allow these molecules to get past the blood-brain barrier to get into the tissue.
And there’s also intensive exploration of complementary therapeutic regimens that use
drugs rather than antibodies, and drugs have a greater ability to diffuse into the brain. There are many different classes of drugs
that are being explored. In this scheme that I’m showing you here, I show you a normal
protein that’s folded properly, it’s transported appropriately within the cell, it has its
intended activity breaking down some substrate into a metabolite. On the bottom I’m showing
you a patient that has a mutation or a change in that protein causing the protein to fold
abnormally. Sometimes these abnormally-folded proteins traffic properly to the right place
in the cell, but they simply can’t work. Other times unfolded proteins are degraded within
the cell. So one way to approach this would be to just ignore the enzyme completely and
come up with an alternative strategy to remove the toxic substrate that’s accumulating. So
this is called substrate reduction therapy. Another possibility is to simply block the
toxic effects of this substrate that’s accumulated within the body, something that’s called a
pathogenic modulator. There are other flavors of drugs that are
being explored. For example, drugs that bind the abnormal protein and allow it to fold
properly, something called a chaperone drug. There are also drugs that can cause proper
folding and restore the activity of the enzyme. And finally, there are stabilizer drugs that
prevent the cell from breaking down abnormally-folded proteins. And all of these classes of drugs
can lead to some enzyme activity and therefore a reduction in the amount of the toxic chemical.
And once again, just for illustration, there are many of these drugs that are in either
clinical trial or are already FDA-approved for some of these conditions. So now I want to turn to a different disease,
and that is cystic fibrosis. I’m sure you’re all familiar with this condition. These children
have mutations in a protein called CFTR and that leads to things like chronic lung disease,
it leads to pancreatic insufficiency, it leads to multiple problems with intestinal performance.
So ultimately, these individuals typically die of their condition. It used to be that
they would die in early childhood. Now with just supported therapy, many are living to
mid-adult life. But again there’s the question, “Can we do better?” There are many possible
problems that can occur from the — if you begin in the nucleus with the DNA that encodes
this channel, the CFTR channel, all the way to the protein being at the cell surface and
having its function. You can have problems producing the channel protein. You can have
problems processing and trafficking the protein within the cell. You can have problems regulating
its activity; it’s either inappropriately on or off. Or you can simply have problems
allowing chloride to move through the channel. So this is perhaps the most exciting story,
in my mind, in therapy for genetic disorders that’s come out recently. And it all stems
from a public-corporate partnership between the Cystic Fibrosis Foundation and a drug
company called Vertex Pharmaceuticals. This partnership set its sights high but focus
narrow. So what they wanted to do was develop a drug therapy for people with the class III
mutation G551D. This means that the glycine at position 551 in the protein is changed
to an aspartic acid. And you might ask, why so narrow? The chance of finding a drug that
would address all the problems, all the potential problems in making this channel, trafficking
this channel, and regulating this channel was really low. By definition, a drug that
binds to the CFTR protein improves the function of this particular mutant form might have
potential to bind other mutant forms. So let’s start with this mutation, but then we can
see whether this drug works for many different people with many different mutations. And
even at a minimum, a drug for this particular mutation would address about 4 percent of
all people with cystic fibrosis. This happens to be a fairly common mutation. So how do they go about finding a drug for
a patient with a specific mutation or change in a specific protein? They used a process
that’s called small molecule screening. So what you would do is you’d take a plate and
you’d put patient cells into each of these welds and you’d also introduce some sort of
marker, some sort of, often a florescent marker that will only glow when activated by chloride
conductance when the performance of the channel has been restored. And then you take a library
of small molecules. These could include hundreds of thousands of small molecules, often includes
established FDA-approved drugs to see if they’ll have an effect. And in each weld of this plate
you’d add a different drug. So you’re basically screening all the drugs within this library
to see if they have any beneficial effect on restoring how the channel works. You then
read the marker, the fluorescent marker for example, and you score the performance of
different compounds within this library. You look for the welds that glow the brightest,
for example. You might have a reasonable compound, something that’s doing pretty well, but not
a perfect compound. Then you can take one of your hits from the screen, you can tweak
that compound a little bit using medicinal chemistry, and then put it back through this
whole process to come up with the best drug for this particular purpose. And this led
to the identification of a drug for this particular mutation causing cystic fibrosis. And again,
when I read this paper, it was one of the few times that I sat back in my chair and
smiled. I mean, the results were just so remarkable. So here we’re looking at patients who were
either not treated with this drug with CF or people that were treated with this drug.
And you can see that you’re seeing changes in the sweat chloride, the amount of chloride
that’s moving through the channel. So these people that were taking the drug dropped their
sweat chloride significantly. And in fact, the absolute sweat chloride level went within
the normal range. So it didn’t only nudge this in the right direction, it shoved it
in the right direction. They then looked at performance of these patients and they saw
that by about two weeks, there was already this dramatic elevation of FEV1, so a marker
of lung function. There was a significant drop in the number of serious events like
pulmonary exacerbations that these patients were having. Their respiratory score increased
dramatically. And they showed abrupt and sustained weight gain over a full year while taking
this medication. The one question was, “Well, do they cherry pick their patients?” Was it
just the most mild patients that would respond? And the answer was no. The response was dramatic,
regardless of initial severity, regardless of geographic location, regardless of gender,
and regardless of age. So it really seemed to work for the full spectrum of CF patients
with this particular mutation. The drug was also tolerated extremely well. There was a
significant reduction of serious adverse events when you compared the treatment group to the
placebo group. So the conclusions of this remarkable study
was that this drug, now called ivacaftor, was associated with improvement in lung function
at two weeks and that this improvement was sustained through 48 weeks. But there was
substantial improvements also in the risk of pulmonary exacerbations, patient-reported
respiratory symptoms, weight, and concentration of sweat chloride, and that ivacaftor was
not associated with increased incidence of adverse events when compared to placebo. This
drug was FDA-approved on January 31 of 2012 and it represents the first and only drug
that is approved for the treatment of cystic fibrosis. Currently in children older than
the age of 6, with this particular mutation, but again, the value of this drug is now being
explored for other patients. Yes? Male Speaker:
This is fantastic, but just for economics, what is a ballpark figure of what it costs
per year of this [inaudible] Hal Dietz:
Unfortunately, I don’t know that answer, but I can find it out. So I’m going to turn to a different disease
now, again, a very common condition known as Duchenne muscular dystrophy. These individuals
show loss or significant impairment of muscle function usually with a diagnosis at around
the age of 5. Usually these patients are wheelchair-dependent by their teens, and death occurs due to respiratory
insufficiency by young adult life. In contrast, there’s a different condition that’s called
Becker muscular dystrophy. And these people are typically not diagnosed until their teenage
years, are not wheelchair-dependent until mid-adulthood, and if death occurs, it’s generally
delayed until the fourth to fifth decade of life. The remarkable observation is that both
of these conditions, very severe and mild, are caused by mutations or changes in the
same gene, the dystrophin gene. So, you know, an obvious question is, “What’s underlying
this clinical difference?” If you look at the muscle and you use an antibody that reacts
with the dystrophin protein, in normal muscle you see that every muscle fiber is surrounded
by a mantle of dystrophin. In Duchenne muscular dystrophy, generally the protein is absent.
And you can see in Becker muscular dystrophy, while not normal, there is at least some preservation
of dystrophin expression. What does dystrophin do? Well, it serves as
a link between molecules deep in the cell and a cluster of molecules that are at the
cell’s surface, you know, establishing a bridge between what’s called the cytoskeleton of
the cell and the extracellular matrix outside the cell. In normal people, not only dystrophin,
but all the other proteins within this complex are easily seen at the cell surface. If you
simply take away dystrophin, you lose this whole complex, the entire complex is destabilized.
So you can see from this diagram that dystrophin plays an important bridging role and you might
infer from this that dystrophin needs its head to attach to proteins within the cell;
it needs its tail to attach to proteins at the cell surface. But the obvious question
is, does it need all of its middle? Is it important exactly how long this middle segment
is? So we’re going to have to look at this in a little bit more detail to understand
what’s going on. As with all other protein-encoding genes,
the dystrophin gene is initially copied in the cell to a molecule that’s called precursor-messenger
RNA. And this precursor-messenger RNA includes blocks of sequence that encode the protein
called exons and intervening junk sequence that’s called introns. And ultimately, all
of these introns are removed by a process that’s called mRNA splicing. So you end up
with all these coding blocks all in a row. And there’s a signal that tells the ribosome
where to start making protein, that’s called the start signal. And there’s also a signal
that tells the ribosome to stop making protein, that’s called the termination codon. So in
this normal example, a ribosome can come along and it’s going to read the triplet code of
a messenger RNA. Every three base pairs encodes a specific amino acid. So the ribosome will
move along, you’ll end up with a full-length protein, and you’ll end up with a normal muscle
phenotype. Now, in Duchenne muscular dystrophy, the most
common cause is that one of the exon blocks is skipped during splicing. And the important
fact is that in Duchenne muscular dystrophy, the block that is skipped is not an even multiple
of three. So now when you splice these two exons together that don’t belong together,
you’re going to shift this triplet reading frame. Everything downstream of that point
is just going to be nonsense. And this invariably leads to what we call a premature termination
codon, something that will tell the ribosome to stop too early. So again, the ribosome
will latch on, it’ll move along this RNA, but it will stop early. And you might guess,
“Well, that’s going to cause a truncated protein missing its tail.” Now I’ve already told you
the tail is very important. In fact, when RNA has a premature stop signal, the most
important consequence is that the cell simply gets rid of that RNA for a process that’s
called nonsense-mediated mRNA decay. So you don’t have any potential to make protein from
these prematurely terminated transcripts. And the answer is you make no dystrophin and
you get Duchenne muscular dystrophy. So what’s going on in the mild form, the Becker
muscular dystrophy? Well, here you also commonly have skipping of an exon, but in this circumstance,
the blocks that are skipped are in even multiple of three nucleotides. So your reading frame
is going to remain preserved. You’ve got a piece missing right here, but you don’t have
any premature stop signal, the code is all intact. So again, the ribosome latches on,
it reads through to the end, and it makes a largely normal dystrophin protein that’s
missing a central block, but it’s got both its head and its tail to latch onto the appropriate
protein complexes, and that’s what causes the more mild Becker muscular dystrophy. So
the obvious question, is there anything that we can do to turn this into this? That is
a goal: let’s make this more mild. All right, so in order to explain how this
happens, I have to tell you a little bit more about splicing. And the only important point
is that this is a very tightly orchestrated process, it’s not random. You have your precursor-messenger
RNA that has different signals embedded within it that tells protein complexes where to bind.
And these protein complexes ultimately define what’s the beginning of the junk and what’s
the end of the junk, so it tells the cell what to get rid of and what to splice together.
And after this you end up with the appropriate splicing event joining Exon 1 to Exon 2, and
then this continues down the length of the messenger RNA. So what if you wanted to get rid of exon2?
You thought that that might be a good thing. So what can be done is you can introduce into
cell a small little artificial piece of RNA or DNA that we call an antisense oligonucleotide.
You can make it so it attacks just this spot at the beginning of Exon 2, for example. What
would be the consequence of that? Well, some of these protein complexes would continue
to bind, but the protein complex that was supposed to bind here is blocked. So this
is no longer recognized as important sequence and that would lead to skipping of Exon 2
and then you’d have the messenger RNA having Exon 1 to Exon 3. How is that helpful? Why
would that be a good thing? I’m going to tell you now about a convention
that I’m going to use for the next couple of slides. So, these little exons that are
shown as puzzle pieces show how the exons are supposed to fit together to maintain the
triplet code along the whole sequence. So if the puzzle pieces fit together like this,
everything is A-okay. You’re, you’ve got a good messenger RNA with an open reading frame
for the ribosome, and you’ll get your protein. Now let’s take an example where a patient
has skipped Exon 45. You can see that when Exon 44 splices to Exon 46, the puzzle pieces
don’t fit. You’re going to get one of your premature stop codons, you’re not going to
make dystrophin, and you’ll get Duchenne muscular dystrophy. Now in this same patient that skipped
Exon 45, what would happen if you used antisense oligonucleotides to tell the cell to also
skip Exon 46? Now, suddenly 44 and 47 fit together, the puzzle pieces attach. So you’ve
preserved the reading frame for the ribosome, you’ll be missing a central chunk but you’ll
make a protein that still has both its head and its tail, and therefore some function.
Now, you might say, “Well, this is a nightmare. Okay, I understand it might work, but you’re
going to have to come up with a different method for every patient because everyone’s
going to skip a different exon. So you’re going to have to optimize this for hundreds
and hundreds of different circumstances.” Well, that doesn’t turn out to be the case
because it’s been recognized that about 60 percent of all muscular dystrophy mutations
occur within this central block of exons, from Exon 45 to 55. And someone came up with
the brilliant idea of coming up with a method of causing all of these exons to skip. So
you’d use a pool of your antisense oligonucleotides, targeting them all. Exon 44 fits together
nicely with Exon 56, the puzzle works. So this would be a potential cure for about two
thirds of patients with Duchenne muscular dystrophy, just a single cocktail of antisense
oligonucleotides. So how does this work? Well, I hear we’re
looking at local delivery of antisense oligonucleotides by injecting them directly into a muscle group
in the foot of Duchenne muscular dystrophy patients. And what the investigators saw is
that adding the antisense oligonucleotide indeed causes the exons to skip. In each case,
you get a smaller product. And suddenly, you go from no dystrophin protein to lots of dystrophin
protein in all of these patients. And it’s not just little tiny spots. If you look at
the entire biopsy of the muscle, you can see that there’s widespread dystrophin expression.
So this is looking really good. Of — Male Speaker:
How long does this take? Hal Dietz:
I’m sorry? Male Speaker:
How long does this take after treatment? Hal Dietz:
It takes somewhere between one to two weeks to see robust expression. Now, an important question is how would you
need to deliver this to make a difference to an individual? You can’t just inject each
muscle group. So instead, please have been infusing this antisense oligonucleotides into
the circulation. These are early days, but in some patients, just putting it into the
circulation causes significant improvement in dystrophin expression. In other cases,
there’s perhaps a subtle effect. You know, I think that this is still within the phase
of optimization and the very important issues are going to be to address how to best deliver
these antisense oligonucleotides so they get to all muscle groups and how do you improve
the stability of these small, little antisense oligonucleotides in the circulation? Are they
simply just being degraded too quickly? But I would say an extremely promising approach,
for a very important condition. Now I’m going to change gears and talk about
a very rare, but very interesting condition called Hutchinson-Gilford progeria, a premature
aging syndrome that causes young children to be — to start an accelerated aging process
in early childhood at around age 3 to 5 or so. They rapidly show loss of hair, they show
wrinkling of skin, atrophy of fat, osteoporosis, coronary artery disease, and are basically
dead of old age by the time they are about 15 to 20; really a devastating condition.
So Francis Collins’ group a few years ago described the cause of Hutchinson-Gilford
progeria, and it’s due to a single point mutation in a protein that’s called lamin A. So here
on top I’m showing you normal lamin A. In patients with Hutchinson-Gilford progeria,
there’s an altered splicing event that causes the skipping of a small chunk of lamin A shown
in green. So the mutant lamin A protein in these patients has both a head and a tail
but it’s missing a central bit. And it turns out that this central bit that it’s missing
is really important. So normally what happens is, when the cell makes this lamin A protein,
that protein is modified by a process that’s called farnesylation. And this causes the
protein to attach to the nuclear membrane. But that little bit that’s missing in progeria
is so essential because ultimately enzymes have to come along and cleave the protein
right at that site to cause it to release from the nuclear membrane. In patients with
progeria, because they’re missing that cleavage site, this lamin A protein remains stuck to
the nuclear membrane. It can’t get off. And so that led to the question, what would happen
if you just prevented this farnesylation process in the first place, using a class of drugs
called farnesyl transferase inhibitors? If it can’t get on, it’s not going to get stuck
there, and it doesn’t matter that it can’t get off. That’s the logic. So the, initially, there was a need for a
marker to say are we doing something good or not? And the marker that was selected is
called nuclear blebbing. If you have lamin A stuck to the nuclear membrane as patients
with progeria have, the nucleus becomes very misshapen. So that was initially used as the
readout. So here are cells from patients with Hutchinson-Gilford progeria in the absence
of any treatment and you can see the obvious altered contour of the nucleus. If you give
a little bit of a farnesyl transferase inhibitor that prevents the protein from getting stuck,
you already see improvement. If you give a little bit more, the improvement is striking;
they look like normal nuclei. And here’s that quantified: in someone that doesn’t have progeria,
there’s very little nuclear blebbing. In three people that do have progeria, there’s a lot
of nuclear blebbing in the absence of drug. Give a little bit, of drug it gets better;
get a lot of drug, it gets even better. So this, as a marker of progeria, this suggests
that this treatment is working beautifully. So now there has been, there have been trials
of farnesyl transferase inhibitors in patients and in mouse models with progeria. I’m going
to show you the results from a mouse model with the same type of lamin A mutation because
it’s much further along. So here we’re looking at both female and male mice. You can see
that the patients, or mice that have progeria, have very — sorry, I’m having trouble seeing
that. So their body weight is very low compared to normal individuals. If you give them this
farnesyl transferase inhibitor, there’s a significant increase in body weight. You can
also see that mice with, that are treated with this farnesyl transferase inhibitor,
both female and male show a significant improvement in grip strength of the muscle, that there
is delay of death in mouse model of progeria, and there’s also a dramatic reduction in rib
fractures. So again, just by understanding where the
mutation is, a little bit about the protein, you can come up with a completely novel hypothesis.
I mean, no one would have possibly imagined that this class of drugs would have anything
to do with treating progeria. What’s really interesting is that it’s not only relevant
to these children with this rare, devastating disease. What’s been shown is that as we get
older, our bodies get a little bit sloppy at how they make lamin A. And some of that
lamin A looks exactly like the lamin A in progeria. So the concept is that this accumulation
over years and years and years of this mutant progerin protein may be contributing to our
aging, and that treatments under development for this rare condition might be relevant
to the broader population. Now I’m going to end by telling you a little
bit about my own work which has focused on a condition called Marfan syndrome. So Marfan
syndrome is a disorder of the body’s connective tissue: the material between the cells that
give the tissues form and strength. It’s a very complex and variable condition, but the
main features include dislocation of the lens of the eye that shifts out of place, overgrowth
of the bones and low fat stores, and also, and most importantly, progressive dilatation
of the root of the aorta just as it’s leaving the heart that will lead to aortic tear, rupture,
and early death if left untreated. In 1991 we were able to show that Marfan syndrome
is caused by mutations in the gene that encodes the connective tissue protein fibrillin-1.
So what do we know about fibrillin-1? Well, we knew that it aggregates outside of the
cell to form these very complex structures called microfibrils and that these microfibrils
cluster around the maturing ends of an elastic fiber during embryonic growth. So this simple
spatial and temporal relationship led to the absolute conclusion that you need a lattice
of microfibrils to make an elastic fiber during embryogenesis and that in people with Marfan
syndrome this never happens; there’s inadequate elastic fibers. Game over. If you think about
it, that really boded poorly for the development of productive treatments. It suggested that
children with Marfan syndrome are born with inadequate elastic fibers and that there’s
nothing that you could do after birth to improve the situation. So I remember the day and even the moment
that I walked into a patient room, saw these exceptionally long fingers just like this,
and thought to myself, “This just doesn’t make sense. Why would weakness of the tissues
cause the bones to overgrow? For that matter, why would weakness of the tissues cause the
facial features of Marfan syndrome like downward slanting eyes, flat cheekbones, and a small
chin?” Each of these findings was more suggestive of altered cellular behavior rather than simple
tissue weakness. Now, to make a long story short, we learned that microfibrils that are
composed of fibrillin-1 serve a second important function. They’re not just glue, but rather
they bind to the inactive complex of a growth factor called TGF-beta. It’s a molecule that
tells cells how to behave. And what we learned is that in Marfan syndrome, where you have
inadequate microfibrils, you have failed matrix sequestration of latent TGF-beta, and that
leads to too much TGF-beta activation and activity. They’re over — this molecule is
now over-stimulating the cells. This sets in motion a cascade of events inside the cell.
One important event, and the only event that I want you to notice here, is there’s a molecule
called phosphorylated SMAD2. That’s going to be our marker of how much TGF-beta activity
is going on. And we were ultimately able to show that this excess of TGF-beta stimulation
of cells had consequences in Marfan syndrome including emphysema, mitral valve prolapse,
aortic aneurysm, and skeletal muscle myopathy. The way that we proved that is we made Marfran
mice that were deficient in fibrillin-1, still had bad glue between the cells, and then inject
them — injected them with a TGF-beta-blocking antibody, and we found that virtually all
of these conditions were prevented by simply blocking TGF-beta in Marfan mice. So we then asked, “Well, is there a drug,
and even better an FDA-approved drug, that might mimic this protection?” And our attention
got turned to a very common antihypertensive medication called losartan that lowers blood
pressure, something we think is good for people with aneurism, but had also been shown to
block TGF-beta in mouse models of kidney disease. So we wondered whether this might be a magic
bullet having two different effects. So we know that angiotensin II, a molecule that
regulates blood pressure, acts by working through both a type 1 receptor and type 2
receptor, and it’s the type 1 receptor that simulates the TGF-beta pathway. So it’s also
known that angiotensin II, working through its type 2 receptor, can suppress all the
events that are caused by the type 1 receptor. So in this view, AT1 is bad. That’s the culprit.
And AT2 might actually be protective. So we reason that if you use an ACE inhibitor that
stopped the production of angiotensin II, you’d be limiting signaling through both the
culprit and the potentially protective pathway. But if you selectively block the AT1 cascade
with a drug like losartan, you might actually stimulate signaling through the protective
pathway. So we did a clinical trial in our mouse model
of Marfan syndrome. Normal mice show very slow rate of growth of their aortic root.
Marfan mice treated with placebo showed this very accelerated aortic growth. If we gave
a drug such as propranolol that only lowered blood pressure but did not address TGF-beta,
the aortic growth was decreased to an extent. But if we treated these mice with losartan,
we saw that they showed absolutely normal aortic growth for a full lifetime. And even
more important, if you looked at the aorta under the microscope, no observer could distinguish
between the losartan-treated animal Marfan mice from normal mice by any parameter. So this, there’s a large clinical trial that’s
now ongoing, but we felt compelled to treat a subset of children with the most severe
form of Marfan syndrome. These kids show unrelenting growth of the aorta despite maximal treatment
with beta blockers or ACE inhibitors reaching death or surgical endpoints in early childhood.
So here’s the aortic growth curves for the first two such children we treated with losartan,
and you can see that there was a dramatic plateau, no further growth of the aortic root
once losartan was started. In this child, this plateau has remained, now with eight
years of follow-up. There still are some things that require improvement. We found that some
children show a relative response to losartan, but then the aortic growth starts creeping
up again. And what we’ve learned is that other drugs in the class of angiotensin receptor
blockers, such as irbesartan, with ultra-high dosing can allow you to achieve a plateau
in aortic growth even in these most severe children. So we again wanted to try to understand more
about this pathway. I’ve told you about TGF-beta activating the so-called SMAD pathway, but
it’s also known that TGF-beta can activate other pathways. And the one that I want you
to pay attention to is particularly this one: the so-called ERK pathway. When we looked
at the aorta of our Marfan mice, we saw that the SMAD pathway was excessively activated
compared to normal mice or what we call “wild-type mice,” but look at ERK activation. It’s dramatic
in the Marfan mice and almost non-existent in the normal mice. So our attention turned
to the significance of this ERK activation. We went on to partner with the Therapeutics
for Rare and Neglected Disorders Program at the NIH and they were able to provide a compound
called RDEA-119 that’s a potent inhibitor of ERK activation. When we treated our Marfan
mice with this ERK inhibitor, not only did we greatly diminish abnormal aortic growth,
we actually caused regression in the size of the aneurism over time. It got smaller
over time, the first time that we’ve ever seen that. So now ERK is firmly on the radar
screen. You know, having — finding genes and making
animal models allows you to find new therapies. It also allows you to evaluate the performance
of existing therapies. And currently calcium channel blockers are considered the second-line
treatment for patients with Marfan syndrome that can’t tolerate beta blocker medications.
But there’s really not a lot of evidence that they work, and there’s not even a lot of evidence
that they’re safe. So we decided to test this on our mouse model. We were initially very
optimistic because there was some work in the literature showing that calcium channel
blockers can blunt ERK activation in at least some cell types. So we did a trial of amlodipine
in our Marfan mice. Now, here I’m showing you the heart and ascending heart of a normal
or wild-type mouse. In a mouse with our Marfan mutation we see that there is flaring at the
base of the aorta. If you give a normal mouse amlodipine you don’t see much. Perhaps the
aorta is looking slightly generous, but it’s subtle. Virtually 100 percent of Marfan mice
given amlodipine develop these massive ascending aortic aneurisms. And this occurs, the aortic
size triples, within three weeks of starting the medication. And the mice are dying due
to aortic dissection within four weeks of starting the medication. If I show you this quantitatively, a placebo-treated
Marfan mouse shows accelerated growth of the aortic root. Look what happens when you add
amlodipine. The rate of aortic growth, root growth, doubles. If you look further up the
aorta where Marfan mice normally don’t have an aneurism, you now see that amlodipine is
causing this dramatic aortic growth. In this Marfan mouse model shown in red, there is
typically no death if you treat with placebo. But if you treat the mice with amlodipine,
death due to aortic dissection starts within five weeks and then accelerates quickly. We
are able to see that, contrary to hypothesis, amlodipine is actually accentuating ERK activation
rather than blunting ERK activation. And now we found the identical results for all calcium
channel blockers. This is not specific to amlodipine, it’s a class effect. Okay. So we want to try to figure this out. And
one thing that we did was ask, well, what happens if you give amlodipine but also block
ERK with our ERK-blocking drug? And the answer is that you prevent all of this detrimental
effect. So it is ERK activation that’s responsible and gives us greater confidence that this
is a great therapeutic target. We also, in our mice that are dying due to aortic dissection
with amlodipine, if you give the ERK antagonist you again see no death due to aortic dissection.
So, not only do you suppress aortic growth, but you prevent aortic tear. So I think it’s
pretty remarkable that seven years ago we had the model that there’s weak tissues in
Marfan syndrome at birth and there’s nothing you can do about it. Currently we have over
seven different medical treatment strategies that have shown remarkable effectiveness in
our mouse model and that we are moving forward to people. Just as important as finding new
effective drugs is learning about things that might be detrimental, such as calcium channel
blockers. In the last two minutes I’m just going to
tell you that what we’ve learned about Marfan syndrome is not just relevant to that condition.
It extends to many causes of aortic aneurism. About six years ago now my colleague Bart
Loeys and I recognized and described a new aortic aneurism syndrome that has many features
of Marfan syndrome like curvature of the spine, long fingers, and aortic aneurism, but also
many unique features like widely-spaced eyes, a cleft palate, or a bifid uvula. Most importantly
these patients don’t just have aneurisms at the root of the aorta but rather all through
the arterial tree. So much more aggressive condition. And these aneurisms rupture at
young ages, as young as six months, and have smaller dimensions when compared to Marfan
syndrome. So based upon what we had learned about Marfan
syndrome, we bet that this condition that’s called Loeys-Dietz syndrome would also relate
to TGF-beta. And in fact the very first two genes we looked at are the two genes that
encode the TGF-beta receptor, and we learned that all of these patients have mutations
in the receptor for TGF-beta. They also show high TGF-beta signaling looking at our old
friend phosphorylated SMAD2, in the nucleus of cells in the aorta. We went on to make
mouse models. We saw that these mice have horrible-looking aortas. Their aortas grow
really fast, but if we treat them with losartan, aortic root growth returns to normal and aortic
wall architecture returns to normal. We go from this picture with all these fractured
elastic fibers to a very normal-looking aorta. Currently there is now a new class of conditions
that are called the TGF-beta vasculopathies, that are shown here, all aortic aneurism conditions
that now have been associated with high TGF-beta. So these data suggest that altered TGF-beta
signaling is a common pathway to aneurism and that treatments under development for
Marfan syndrome may find broad application. My last slide, the conclusions, are that the
study of rare Mendelian disorders is both an obligation and an opportunity. The obligation
stems from the fact that while they are individually rare, these conditions are personally burdensome
and collectively common, and also that patients with rare genetic disorders have really fueled
progress in the field of molecular therapeutics. They’ve given of themself, they’ve accepted
risk, they’ve allowed us to learn, so there’s a real personal cost, despite a remote chance
of personal advantage. The opportunity relates to the single-gene basis of the defect. What
we know when we find a gene for a Mendelian disorder is that a defect in this pathway
is sufficient to cause this condition, this disease phenotype, and therefore it tells
us that these pathways are inherently attractive therapeutic targets. If you can nudge them
in the right direction, they might make a big difference. And such therapies can then
be explored in more common conditions, like emphysema rather than the lung disease in
Marfan syndrome. I’d like to end by acknowledging the truly
remarkable young people that I have the privilege of working with every day, my collaborators
at other institutions, and my funding sources. And I’d be happy to answer questions. Thank
you for your attention. [applause] Yes, sir. Male Speaker:
[inaudible] Is there any evidence of a new response then to the new protein? Hal Dietz:
So again it depends on whether someone normally makes a little bit of dystrophin or whether
they’re completely na久e for dystrophin. But so far, to my knowledge, that has not
turned out to be a problem. And it seems that everyone, even with the most severe form of
DMD, is making enough protein to elicit tolerance so the immune system doesn’t react. Yes. Male Speaker:
It will take me weeks to absorb all this. But I do treat osteoporosis, and you mentioned
in progeria and Marfan that there was osteoporosis. Is that just disuse or is it because the bone
is much different from some of the other tissues? Hal Dietz:
Yeah, it is not a disuse situation. We’ve been able to, at least in Marfan syndrome,
tie the osteoporosis to the same TGF-beta cascade. And our collaborator in New York,
Francesco Ramirez, in culture systems, has shown dramatic responses to some of the TGF-beta
modulating agents. So I think it’s going to teach us something about common osteoporosis.
Yes, sir? Male Speaker:
Are adults with Marfan all treatable? Hal Dietz:
Yeah, so that’s a great question. The question is, are adults with Marfan all treatable or
is the window of opportunity to make a difference over in childhood? At least in our mice, we
can allow them to become mature adults. They’re sexually mature at about two months, by six
months of age they are sort of mid-adult life, and by a year of age they are old mice. And
whether we start treatment right after birth, in the middle of that sequence, or at the
end, we see the same kind of benefits. So we think that the window doesn’t close, that
there is an opportunity even later in life. Male Speaker:
Is there a test that can determine this embryonically? Hal Dietz:
Yes, so the question is, is there a test that can determine these diagnoses as a fetus is
developing? Yes, for all the conditions that I discussed the gene is known so you would
be able to do prenatal diagnosis and know that a fetus has this predisposition. Male Speaker:
Sir, you didn’t mention the so-called readthrough drugs for in-frame premature termination codons.
Do you have a comment on those? Hal Dietz:
Yes, I actually have slides for those, but I just didn’t have time to cover them. So
there are drugs that are called readthrough agents. Basically they make the ribosome sloppy.
So the ribosome reaches a stop codon, but it just marches through and inserts any amino
acid at that position. There is a company called PTC Therapeutics that has developed
a drug called PTC124 that makes the ribosome sloppy, so the whole idea is that the ribosome
will march through an early stop codon and make a full-length protein. There is a clinical
trial, or was a clinical trial, for muscular dystrophy with PTC124, but that trial was
stopped because they did not reach endpoints. There was some suggestion that there might
have been a subtle benefit at a lower dose as opposed to a higher dose. So that’s being explored, but there are many
potential problems with the readthrough approach. You know, one potential problem is that most
of the RNAs are degraded by nonsense-mediated decay. So there’s not many transcripts left
that are engaged by ribosomes where you could have readthrough. Also the context of the
premature termination codon defines the efficiency of readthrough and there’s only one context
that’s really potent that allows a lot of readthrough to happen. We also know that inserting
any amino acid where a stop codon was is often not good enough. What’s needed at that position
is the intended amino acid rather than any amino acid for the protein to have its function.
So I think it’s a really exciting idea. I think it potentially could treat a broad spectrum
of genetic diseases, but I think the idea is in need of refinement. One potential refinement
would be to both block this decay pathway, this nonsense-mediated decay pathway, while
also stimulating readthrough. I know that a number of companies are looking at that
closely. Yes? Male Speaker:
[inaudible] Hal Dietz:
Yeah, so the question is, what’s actually activating TGF-beta that doesn’t bind to the
matrix, for example in Marfan syndrome. There are many, many activators of TGF-beta, including
many proteases, including integrins, including low pH. There is some evidence that in Marfan
syndrome a molecule called the MMP9 or matrix metallopeptidase 9, may be particularly important
in activating TGF-beta. There are some trials going on with different inhibitors of TGF-beta
activators but there simply aren’t results yet to share. So it is a developed concept
though to go after these activators. Male Speaker:
What do you think about the gene correction using [inaudible] technology? Hal Dietz:
Yeah, so there are a number of very interesting technologies that are being considered to
actually correct the mutant sequence within your gene, within the native gene. And some
of them have shown promise in cell culture systems. To my knowledge none have shown sufficient
efficiency to suggest that they may work in vivo. So I think it’s again a very exciting
concept. It would be curative for many genetic, if not most genetic conditions, but I just
don’t think it’s far enough along. There are some still leaps in technology that will be
needed to bring that to fruition. All right, well, thank you again for your
attention. [applause]